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1.  Tobacco Counseling Experience Prior to Starting Medical School, Tobacco Treatment Self-Efficacy and Knowledge among First-Year Medical Students in the United States 
Preventive medicine  2015;73:119-124.
To explore students’ tobacco dependence counseling experiences prior to medical school and their associations with tobacco counseling self-efficacy, and familiarity with and perceived effectiveness of tobacco dependence treatment among first-year medical students in the United States.
In 2010, 1266 first-year medical students from 10 U.S. medical schools completed a survey reporting their clinical experiences with specific tobacco counseling skills (e.g., 5As) prior to medical school. The survey also included questions on tobacco counseling self-efficacy, perceived physician impact on smokers, and familiarity and effectiveness of tobacco-related treatments.
Half (50.4%) reported some tobacco counseling experiences prior to medical school (i.e. at least one 5A). Students with prior counseling experiences were more likely to have higher tobacco counseling self-efficacy, and greater familiarity with medication treatment, nicotine replacement treatment, and behavioral counseling for smoking cessation, compared to those with no prior experiences. Perceived physician impact on patient smoking outcomes did not differ by prior tobacco counseling experiences.
Many first-year medical students may already be primed to learn tobacco dependence counseling skills. Enhancing early exposure to learning these skills in medical school is likely to be beneficial to the skillset of our future physicians.
PMCID: PMC4378235  PMID: 25666737
Tobacco dependence treatment; self-efficacy; perceived effectiveness; medical students
2.  A mutant (‘lab strain’) of the hyperthermophilicarchaeonPyrococcusfuriosus, lacking flagella, has unusual growthphysiology 
A mutant(‘lab strain’) of the hyperthermophilicarchaeonPyrococcusfuriosusDSM3638 exhibited an extended exponential phase andatypical cell aggregation behavior. Genomic DNA from the mutant culturewas sequenced and compared to wild-type (WT) DSM3638, revealing145genes with one or more insertions, deletions, or substitutions (12 silent, 33amino acid substitutions, and 100 frame-shifts). Approximately half of the mutated genes weretransposases or hypothetical proteins.The WT transcriptomerevealednumerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observedprolonged exponential phase. Targeted gene deletions,based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutantgenome negatively impacted transcription ofa flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopyofPF0331-PF0337 deletionsin P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore,indicatedthat flagella play a key role, beyond motility, in thegrowthphysiology ofP. furiosus.
PMCID: PMC4342268  PMID: 25472011
Pyrococcusfuriosus; hyperthermophile; archaeon; growth physiology; flagella
3.  Medical school curriculum characteristics associated with intentions and frequency of tobacco dependence treatment among 3rd year U.S. medical students 
Preventive medicine  2015;72:56-63.
Physicians play a critical role in addressing tobacco dependence, yet report limited training. Tobacco dependence treatment curricula for medical students could improve performance in this area. This study identified student and medical school tobacco treatment curricula characteristics associated with intentions and use of the 5As for tobacco treatment among 3rd year U.S. medical students.
Third year medical students (N=1065, 49.3% male) from 10 U.S. medical schools completed a survey in 2009-2010 assessing student characteristics, including demographics, tobacco treatment knowledge, and self-efficacy. Tobacco curricula characteristics assessed included amount and type of classroom instruction, frequency of tobacco treatment observation, instruction, and perception of preceptors as role models.
Greater tobacco treatment knowledge, self-efficacy, and curriculum-specific variables were associated with 5A intentions, while younger age, tobacco treatment self-efficacy, intentions, and each curriculum-specific variable was associated with greater 5A behaviors. When controlling for important student variables, greater frequency of receiving 5A instruction (OR = 1.07; 95%CI 1.01-1.12) and perception of preceptors as excellent role models in tobacco treatment (OR = 1.35; 95%CI 1.04-1.75) were significant curriculum predictors of 5A intentions. Greater 5A instruction (B = .06 (.03); p< .05) and observation of tobacco treatment (B= .35 (.02); p< .001) were significant curriculum predictors of greater 5A behaviors.
Greater exposure to tobacco treatment teaching during medical school is associated with both greater intentions to use and practice tobacco 5As. Clerkship preceptors, or those physicians who provide training to medical students, may be particularly influential when they personally model and instruct students in tobacco dependence treatment.
PMCID: PMC4562320  PMID: 25572623
tobacco dependence treatment; medical school curriculum and education; medical students
4.  Spatial variation in risk and consequence of Batrachochytrium salamandrivorans introduction in the USA 
Royal Society Open Science  2016;3(2):150616.
A newly identified fungal pathogen, Batrachochytrium salamandrivorans(Bsal), is responsible for mass mortality events and severe population declines in European salamanders. The eastern USA has the highest diversity of salamanders in the world and the introduction of this pathogen is likely to be devastating. Although data are inevitably limited for new pathogens, disease-risk assessments use best available data to inform management decisions. Using characteristics of Bsalecology, spatial data on imports and pet trade establishments, and salamander species diversity, we identify high-risk areas with both a high likelihood of introduction and severe consequences for local salamanders. We predict that the Pacific coast, southern Appalachian Mountains and mid-Atlantic regions will have the highest relative risk from Bsal. Management of invasive pathogens becomes difficult once they are established in wildlife populations; therefore, import restrictions to limit pathogen introduction and early detection through surveillance of high-risk areas are priorities for preventing the next crisis for North American salamanders.
PMCID: PMC4785982  PMID: 26998331
fungal pathogen; amphibians; urodeles; Caudata; invasive species; disease
5.  Molybdenum Availability Is Key to Nitrate Removal in Contaminated Groundwater Environments 
Applied and Environmental Microbiology  2015;81(15):4976-4983.
The concentrations of molybdenum (Mo) and 25 other metals were measured in groundwater samples from 80 wells on the Oak Ridge Reservation (ORR) (Oak Ridge, TN), many of which are contaminated with nitrate, as well as uranium and various other metals. The concentrations of nitrate and uranium were in the ranges of 0.1 μM to 230 mM and <0.2 nM to 580 μM, respectively. Almost all metals examined had significantly greater median concentrations in a subset of wells that were highly contaminated with uranium (≥126 nM). They included cadmium, manganese, and cobalt, which were 1,300- to 2,700-fold higher. A notable exception, however, was Mo, which had a lower median concentration in the uranium-contaminated wells. This is significant, because Mo is essential in the dissimilatory nitrate reduction branch of the global nitrogen cycle. It is required at the catalytic site of nitrate reductase, the enzyme that reduces nitrate to nitrite. Moreover, more than 85% of the groundwater samples contained less than 10 nM Mo, whereas concentrations of 10 to 100 nM Mo were required for efficient growth by nitrate reduction for two Pseudomonas strains isolated from ORR wells and by a model denitrifier, Pseudomonas stutzeri RCH2. Higher concentrations of Mo tended to inhibit the growth of these strains due to the accumulation of toxic concentrations of nitrite, and this effect was exacerbated at high nitrate concentrations. The relevance of these results to a Mo-based nitrate removal strategy and the potential community-driving role that Mo plays in contaminated environments are discussed.
PMCID: PMC4495186  PMID: 25979890
6.  Heterologous Production of an Energy-Conserving Carbon Monoxide Dehydrogenase Complex in the Hyperthermophile Pyrococcus furiosus 
Carbon monoxide (CO) is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a CO dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na+/H+ antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na+ motive force that is used to conserve energy by a Na+-dependent ATP synthase. Herein we used a bacterial artificial chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100°C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80°C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms.
PMCID: PMC4731540  PMID: 26858706
hyperthermophile; archaea; hydrogen; carbon monoxide; energy; anaerobic respiration; Thermococcales
7.  Light Sheet Fluorescence Microscopy (LSFM) 
The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light Sheet Fluorescent Microscopy (LSFM), a century old idea (Siedentopf and Zsigmondy, 1902) made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light sheet based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements.
PMCID: PMC4294425  PMID: 25559221
Light Sheet Fluorescence Microscopy (LSFM); Selective Plane Illumination Microscopy (SPIM); Inverted Selective Plane Illumination Microscopy (iSPIM); Multi-view Selective Plane Illumination Microscopy (mSPIM); Bessel Beam Super-Resolution Structured Illumination Microscopy (BB-SR-SIM); Stimulated Emission Depletion Selective Plane Illumination Microscopy (STED-SPIM); 3D imaging; 4D imaging; developmental imaging; embryogenesis
8.  Urocortin3 mediates somatostatin-dependent negative feedback control of insulin secretion 
Nature medicine  2015;21(7):769-776.
The peptide hormone Urocortin3 (Ucn3) is abundantly expressed by mature beta cells, yet its physiological role is unknown. Here we demonstrate that Ucn3 is stored and co–released with insulin and potentiates glucose–stimulated somatostatin secretion via cognate receptor on delta cells. Further, we found that islets lacking endogenous Ucn3 demonstrate fewer delta cells, reduced somatostatin content, impaired somatostatin secretion and exaggerated insulin release, and that these defects are rectified by synthetic Ucn3 in vitro. Our observations indicate that the paracrine actions of Ucn3 activate a negative feedback loop that promotes somatostatin release to ensure the timely reduction of insulin secretion upon normalization of plasma glucose. Moreover, Ucn3 is markedly depleted from beta cells in mouse and macaque diabetes models and in human diabetic islets. This suggests that Ucn3 is a key contributor to stable glycemic control whose reduction during diabetes aggravates glycemic volatility and contributes to the pathophysiology of this disease.
PMCID: PMC4496282  PMID: 26076035
Biological sciences/Physiology/Metabolism/Homeostasis; Health sciences/Endocrinology; Health sciences/Diseases/Endocrine system and metabolic diseases/Diabetes/Type 2 diabetes; Biological sciences/Physiology/Metabolism/Metabolic diseases/Diabetes/Type 2 diabetes
9.  Rise in DPA Following SDA-Rich Dietary Echium Oil Less Effective in Affording Anti-Arrhythmic Actions Compared to High DHA Levels Achieved with Fish Oil in Sprague-Dawley Rats 
Nutrients  2016;8(1):14.
Stearidonic acid (SDA; C18:4n-3) has been suggested as an alternative to fish oil (FO) for delivering health benefits of C ≥ 20 long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA). Echium oil (EO) represents a non-genetically-modified source of SDA available commercially. This study compared EO and FO in relation to alterations in plasma and tissue fatty acids, and for their ability to afford protection against ischemia-induced cardiac arrhythmia and ventricular fibrillation (VF). Rats were fed (12 weeks) diets supplemented with either EO or FO at three dose levels (1, 3 and 5% w/w; n = 18 per group). EO failed to influence C22:6n-3 (DHA) but increased C22:5n-3 (DPA) in tissues dose-dependently, especially in heart tissue. Conversely, DHA in hearts of FO rats showed dose-related elevation; 14.8%–24.1% of total fatty acids. Kidney showed resistance for incorporation of LC n-3 PUFA. Overall, FO provided greater cardioprotection than EO. At the highest dose level, FO rats displayed lower (p < 0.05) episodes of VF% (29% vs. 73%) and duration (22.7 ± 12.0 vs. 75.8 ± 17.1 s) than the EO group but at 3% EO was comparable to FO. We conclude that there is no endogenous conversion of SDA to DHA, and that DPA may be associated with limited cardiac benefit.
PMCID: PMC4728628  PMID: 26742064
n-3 fatty acids; fish oil; Echium oil; stearidonic acid; docosapentaenoic acid; docosahexaenoic acid; eicosapentaenoic acid; cardiac arrhythmia; rat
10.  Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO2-based 3-hydroxypropionate production 
Biotechnology and bioengineering  2015;112(8):1533-1543.
Metabolically engineered strains of the hyperthermophile Pyrococcus furiosus(Topt 95-100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO2 using enzymes from the Metallosphaera sedula (Topt73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formation catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas-liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/hr to 11 mg/L/hr, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. The results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures.
PMCID: PMC4664069  PMID: 25753826
3-Hydroxypropionate; CO2 fixation; Metallosphaera sedula; Pyrococcus furiosus
11.  Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals 
Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high temperature industrial biotechnology.
PMCID: PMC4633485  PMID: 26594201
extreme thermophiles; metabolic engineering; bio-based chemicals; genetics; biotechnology
12.  Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus 
Archaea  2015;2015:912582.
Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity's growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophile Pyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed.
PMCID: PMC4620386  PMID: 26543406
13.  Annulus Fibrosus Can Strip Hyaline Cartilage End Plate from Subchondral Bone: A Study of the Intervertebral Disk in Tension 
Global Spine Journal  2015;5(5):360-365.
Study Design Biomechanical study on cadaveric spines.
Objective Spinal bending causes the annulus to pull vertically (axially) on the end plate, but failure mechanisms in response to this type of loading are poorly understood. Therefore, the objective of this study was to identify the weak point of the intervertebral disk in tension.
Methods Cadaveric motion segments (aged 79 to 88 years) were dissected to create midsagittal blocks of tissue, with ∼10 mm of bone superior and inferior to the disk. From these blocks, 14 bone–disk–bone slices (average 4.8 mm thick) were cut in the frontal plane. Each slice was gripped by its bony ends and stretched to failure at 1 mm/s. Mode of failure was recorded using a digital camera.
Results Of the 14 slices, 10 failed by the hyaline cartilage being peeled off the subchondral bone, with the failure starting opposite the lateral annulus and proceeding medially. Two slices failed by rupturing of the trabecular bone, and a further two failed in the annulus.
Conclusions The hyaline cartilage–bone junction is the disk's weak link in tension. These findings provide a plausible mechanism for the appearance of bone and cartilage fragments in herniated material. Stripping cartilage from the bony end plate would result in the herniated mass containing relatively stiff cartilage that does not easily resorb.
PMCID: PMC4577330  PMID: 26430588
intervertebral disk; tension; herniation; hyaline cartilage; end plate; subchondral bone
14.  Placental Growth Factor Influences Maternal Cardiovascular Adaptation to Pregnancy in Mice 
Biology of reproduction  2014;92(2):44.
In healthy human pregnancies, placental growth factor (PGF) concentrations rise in maternal plasma during early gestation, peak over weeks 26–30, then decline. Since PGF in non-gravid subjects participates in protection against and recovery from cardiac pathologies, we asked if PGF contributes to pregnancy-induced maternal cardiovascular adaptations. Cardiovascular function and structure were evaluated in virgin, pregnant and postpartum C56BL/6-Pgf−/− (Pgf−/−) and C57BL/6-Pgf+/+ (B6) mice using plethysmography, ultrasound, qPCR and cardiac and renal histology. Pgf−/− females had higher systolic blood pressure in early and late pregnancy but an extended, abnormal midpregnancy interval of depressed systolic pressure. Pgf−/− cardiac output was lower than gestation day (gd)-matched B6 after mid-pregnancy. While Pgf−/− left ventricular mass was greater than B6, only B6 showed the expected gestational gain in left ventricular mass. Expression of vasoactive genes in the left ventricle differed at gd8 with elevated Nos expression in Pgf−/− but not at gd14. By gd16, Pgf−/− kidneys were hypertrophic and had glomerular pathology. This study documents for the first time that PGF is associated with the systemic maternal cardiovascular adaptations to pregnancy.
PMCID: PMC4490891  PMID: 25537372 CAMSID: cams4691
Cardiac remodeling; Cardiovascular risk; Fetal growth; Placenta; Ultrasound
15.  Nuclear resonance vibrational spectroscopy (NRVS) of rubredoxin and MoFe protein crystals 
Hyperfine interactions  2012;222(2 Suppl):77-90.
We have applied 57Fe nuclear resonance vibrational spectroscopy (NRVS) for the first time to study the dynamics of Fe centers in Fe-S protein crystals, including oxidized wild type rubredoxin crystals from Pyrococcus furiosus, and the MoFe protein of nitrogenase from Azotobacter vinelandii. Thanks to the NRVS selection rule, selectively probed vibrational modes have been observed in both oriented rubredoxin and MoFe protein crystals. The NRVS work was complemented by extended X-ray absorption fine structure spectroscopy (EXAFS) measurements on oxidized wild type rubredoxin crystals from Pyrococcus furiosus. The EXAFS spectra revealed the Fe-S bond length difference in oxidized Pf Rd protein, which is qualitatively consistent with the X-ray crystal structure.
PMCID: PMC4453832  PMID: 26052177
57Fe; nuclear resonant scattering; nuclear resonant vibrational spectroscopy; NRVS; Mössbauer; synchrotron radiation; EXAFS; normal mode analysis; nitrogenase; rubredoxin
16.  Inhibiting the progression of arterial calcification with vitamin K in HemoDialysis patients (iPACK-HD) trial: rationale and study design for a randomized trial of vitamin K in patients with end stage kidney disease 
Cardiovascular disease, which is due in part to progressive vascular calcification, is the leading cause of death among patients with end stage kidney disease (ESKD) on dialysis. A role for vitamin K in the prevention of vascular calcification is plausible based on the presence of vitamin K dependent proteins in vascular tissue, including matrix gla protein (MGP). Evidence from animal models and observational studies support a role for vitamin K in the prevention of vascular calcification. A large-scale study is needed to investigate the effect of vitamin K supplementation on the progression of vascular calcification in patients with ESKD, a group at risk for sub-clinical vitamin K deficiency.
We plan a prospective, randomized, double-blind, multicenter controlled trial of incident ESKD patients on hemodialysis in centers within North America. Eligible subjects with a baseline coronary artery calcium score of greater than or equal to 30 Agatston Units, will be randomly assigned to either the treatment group (10 mg of phylloquinone three times per week) or to the control group (placebo administration three times per week). The primary endpoint is the progression of coronary artery calcification defined as a greater than 15% increase in CAC score over baseline after 12 months.
Vitamin K supplementation is a simple, safe and cost-effective nutritional strategy that can easily be integrated into patient care. If vitamin K reduces the progression of coronary artery calcification it may lead to decreased morbidity and mortality in men and women with ESKD.
Trial registration
NCT 01528800.
PMCID: PMC4465015  PMID: 26075081
Vitamin K; End stage kidney disease; Hemodialysis; Coronary artery calcification; Randomized controlled trial
17.  Resident Duty Hours in the Outpatient Electronic Health Record Era: Inaccuracies and Implications 
The Accreditation Council for Graduate Medical Education expects resident duty hours to be monitored, yet no previous studies have examined the effect of after-hours electronic health record (EHR) use on resident hours or burnout.
We assessed internal medicine residents' perceived and actual time spent on after-hours outpatient EHR use and calculated increased duty hours if after-hours EHR use were included; we also assessed its effect on resident burnout.
We retrospectively aggregated time spent logged on to the outpatient EHR for residents in a general internal medicine clinic for 13 weeks in 2011. Residents completed a survey on EHR use, which was correlated with objectively recorded data on EHR usage. We compared actual and self-reported EHR time and identified violations that would be generated if these hours were included in reported duty hours. We also correlated resident after-hours EHR use with responses to an internally developed burnout survey.
The 44 residents in this study overestimated time spent on the ambulatory EHR (they spent 3.03 hours/week on after-hours use compared with a recorded 1.20 hours/week). In total, 190 duty hour violations (mean duration of violation  =  37 minutes) would have been generated if after-hours EHR usage were included in residents' reported duty hours.
Resident estimates of EHR use by residents were not accurate; including after-hours EHR use would increase the number of reported duty hour violations. There was no association between after-hours EHR use and resident burnout.
PMCID: PMC3963775  PMID: 24701327
18.  Teaching tobacco dependence treatment and counseling skills during medical school: rationale and design of the Medical Students helping patients Quit tobacco (MSQuit) group randomized controlled trial 
Contemporary clinical trials  2014;37(2):284-293.
Physician-delivered tobacco treatment using the 5As is clinically recommended, yet its use has been limited. Lack of adequate training and confidence to provide tobacco treatment are cited as leading reasons for limited 5A use. Tobacco dependence treatment training while in medical school is recommended, but is minimally provided. The MSQuit trial (Medical Students helping patients Quit tobacco) aims to determine if a multi-modal and theoretically-guided tobacco educational intervention will improve tobacco dependence treatment skills (i.e. 5As) among medical students.
10 U.S. medical schools were pair-matched and randomized in a group-randomized controlled trial to evaluate whether a multi-modal educational (MME) intervention compared to traditional education (TE) will improve observed tobacco treatment skills. MME is primarily composed of TE approaches (i.e. didactics) plus a 1st year web-based course and preceptor-facilitated training during a 3rd year clerkship rotation. The primary outcome measure is an objective score on an Objective Structured Clinical Examination (OSCE) tobacco-counseling smoking case among 3rd year medical students from schools who implemented the MME or TE.
MSQuit is the first randomized to evaluate whether a tobacco treatment educational intervention implemented during medical school will improve medical students’ tobacco treatment skills. We hypothesize that the MME intervention will better prepare students in tobacco dependence treatment as measured by the OSCE. If a comprehensive tobacco treatment educational learning approach is effective, while also feasible and acceptable to implement, then medical schools may substantially influence skill development and use of the 5As among future physicians.
PMCID: PMC4048818  PMID: 24486635
tobacco dependence treatment and counseling; 5As; medical school education; medical students; tobacco control; group randomized controlled trial
19.  Sensorimotor function of the cervical spine in healthy volunteers 
Sensorimotor mechanisms are important for controlling head motion. However, relatively little is known about sensorimotor function in the cervical spine. This study investigated how age, gender and variations in the test conditions affect measures of position sense, movement sense and reflex activation in cervical muscles.
Forty healthy volunteers (19M/21F, aged 19–59 years) participated. Position sense was assessed by determining repositioning errors in upright and flexed neck postures during tests performed in 25%, 50% and 75% cervical flexion. Movement sense was assessed by detecting thresholds to passive flexion and extension at velocities between 1 and 25°s− 1. Reflexes were assessed by determining the latency and amplitude of reflex activation in trapezius and sternocleidomastoid muscles. Reliability was evaluated from intraclass correlation coefficients.
Mean repositioning errors ranged from 1.5° to 2.6°, were greater in flexed than upright postures (P = 0.006) and in people aged over 25 years (P = 0.05). Time to detect head motion decreased with increasing velocity (P < 0.001) and was lower during flexion than extension movements (P = 0.002). Reflexes demonstrated shorter latency (P < 0.001) and greater amplitude (P = 0.009) in trapezius compared to sternocleidomastoid, and became slower and weaker with age. None of the measures were influenced by gender. Reliability was good for movement sense measures, but was influenced by the test conditions when assessing position sense.
Increased repositioning errors and slower reflexes in older subjects suggest that sensorimotor function in the cervical spine becomes impaired with age. In position sense tests, reliability was influenced by the test conditions with mid-range flexion movements, performed in standing, providing the most reliable measurements.
•Sensorimotor function in the cervical spine was assessed in healthy volunteers.•Cervical spine position sense was affected by posture, movement direction and age.•Cervical spine movement sense was influenced by velocity and direction of movement.•Reflex activation of cervical muscles became slower and weaker with age.•Position sense and movement sense showed no correlation with reflex responses.
PMCID: PMC4372261  PMID: 25686675
Cervical spine; Neck muscles; Proprioception; Position sense; Movement sense; Reflex activation
20.  Secretory Competence in a Gateway Endocrine Cell Conferred by the Nuclear Receptor βFTZ-F1 Enables Stage-specific Ecdysone Responses Throughout Development in Drosophila 
Developmental biology  2013;385(2):253-262.
Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETH). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor βFTZ-F1. Selective RNA silencing of βftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in βftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted βFTZ-F1 expression. Moreover, premature βftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a “gateway cell”, whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed βFTZ-F1 expression late in the molt as steroids decline.
PMCID: PMC3900412  PMID: 24247008
Drosophila; ecdysis; ecdysis triggering hormone (ETH); Inka cells; βFTZ-F1; RNA-silencing; secretory competence
21.  Mannosylglycerate and Di-myo-Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress 
Applied and Environmental Microbiology  2014;80(14):4226-4233.
Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.
PMCID: PMC4068688  PMID: 24795373
22.  Phosphate: an old bone molecule but new cardiovascular risk factor 
Phosphate handling in the body is complex and involves hormones produced by the bone, the parathyroid gland and the kidneys. Phosphate is mostly found in hydroxyapatite. however recent evidence suggests that phosphate is also a signalling molecule associated with bone formation. Phosphate balance requires careful regulation of gut and kidney phosphate transporters, SLC34 transporter family, but phosphate signalling in osteoblasts and vascular smooth muscle cells is likely mediated by the SLC20 transporter family (PiT1 and PiT2). If not properly regulated, phosphate imblanace could lead to mineral disorders as well as vascular calcification. In chronic kidney disease-mineral bone disorder, hyperphosphataemia has been consistently associated with extra-osseous calcification and cardiovascular disease. This review focuses on the physiological mechanisms involved in phosphate balance and cell signalling (i.e. osteoblasts and vascular smooth muscle cells) as well as pathological consequences of hyperphosphataemia. Finally, conventional as well as new and experimental therapeutics in the treatment of hyperphosphataemia are explored.
PMCID: PMC3895346  PMID: 23506202
cardiovascular disease; chronic kidney disease; hyperphosphataemia; phosphate; SLC20 (PiT1 and 2); vascular calcification
23.  A Bio-Hybrid Tactile Sensor Incorporating Living Artificial Skin and an Impedance Sensing Array 
Sensors (Basel, Switzerland)  2014;14(12):23781-23802.
The development of a bio-hybrid tactile sensor array that incorporates a skin analogue comprised of alginate encapsulated fibroblasts is described. The electrical properties are modulated by mechanical stress induced during contact, and changes are detected by a ten-channel dual-electrode impedance sensing array. By continuously monitoring the impedance of the sensor array at a fixed frequency, whilst normal and tangential loads are applied to the skin surface, transient mechanotransduction has been observed. The results demonstrate the effectiveness and feasibility of the preliminary prototype bio-hybrid tactile sensor.
PMCID: PMC4299087  PMID: 25615726
bio-hybrid sensors; bioimpedance; haptics; tactile sensors; artificial touch; artificial skin; microfluidics
24.  Functional Phylogenetics Reveals Contributions of Pleiotropic Peptide Action to Ligand-Receptor Coevolution 
Scientific Reports  2014;4:6800.
The evolution of peptidergic signaling has been accompanied by a significant degree of ligand-receptor coevolution. Closely related clusters of peptide signaling molecules are observed to activate related groups of receptors, implying that genes encoding these ligands may orchestrate an array of functions, a phenomenon known as pleiotropy. Here we examine whether pleiotropic actions of peptide genes might influence ligand-receptor coevolution. Four test groups of neuropeptides characterized by conserved C-terminal amino acid sequence motifs and their cognate receptors were examined in the red flour beetle (Tribolium castaneum): 1) cardioacceleratory peptide 2b (CAPA); CAPAr, 2) pyrokinin/diapause hormone (PK1/DH); PKr-A, -B, 3) pyrokinin/pheromone biosynthesis activating hormone (PK2/PBAN); PKr-C, and 4) ecdysis triggering hormone (ETH); ETHr-b. Ligand-receptor specificities were established through heterologous expression of receptors in cell-based assays for 9 endogenous ligands. Based on ligand-receptor specificity analysis, we found positive pleiotropism exhibited by ETH on ETHR-b and CAPAr, whereas PK1/DH and CAPA are more highly selective for their respective authentic receptors than would be predicted by phylogenetic analysis. Disparities between evolutionary trees deduced from receptor sequences vs. functional ligand-receptor specificities lead to the conclusion that pleiotropy exhibited by peptide genes influences ligand-receptor coevolution.
PMCID: PMC4210869  PMID: 25348027
25.  Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway 
The extremely thermoacidophilic archaeon Metallosphaera sedula (optimum growth temperature, 73°C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle. This cycle adds two CO2 molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA β-ketothiolase (Msed_0656), were produced recombinantly in Escherichia coli, combined in vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2 fixation and central metabolism were examined using a gas-intensive bioreactor system in which M. sedula was grown under autotrophic (CO2-limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates in M. sedula metabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO2 fixation and central metabolism in M. sedula.
PMCID: PMC3993168  PMID: 24532060

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