Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness

The Centers for Disease Control and Prevention and United States Army Research Institute for Infectious Diseases have developed real-time PCR assays for the detection of bioterrorism threat agents. These assays all rely on a limited number of approved real-time PCR master mixes. Because the availability of these reagents is a critical element of bioterrorism preparedness, we undertook a joint national preparedness exercise to address the potential surge needs resulting from a large-scale bio-emergency. We identified 9 commercially-available potential alternatives to an existing approved master mix (LightCycler FastStart DNA Master HybProbes): the TaqMan Fast Universal PCR master mix, OmniMix HS, FAST qPCR master mix, EXPRESS qPCR SuperMix kit, QuantiFast Probe PCR kit, LightCycler FastStart DNA MasterPLUS HybProbe, Brilliant II FAST qPCR master mix, ABsolute Fast QPCR Mix and the HotStart IT Taq master mix. The performances of these kits were evaluated by the use of real-time PCR assays for four bioterrorism threat agents: Bacillus anthracis, Brucella melitensis, Burkholderia mallei and Francisella tularensis. The master mixes were compared for target-specific detection levels, as well as consistency of results among three different real-time PCR platforms (LightCycler, SmartCycler and 7500 Fast Dx). Realtime PCR analysis revealed that all ten kits performed well for agent detection on the 7500 Fast Dx instrument; however, the QuantiFast Probe PCR kit yielded the most consistently positive results across multiple real-time PCR platforms. We report that certain combinations of commonly used master mixes and instruments are not as reliable as others at detecting low concentrations of target DNA. Furthermore, our study provides laboratories the option to select from the commercial kits we evaluated to suit their preparedness needs

Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness

The Centers for Disease Control and Prevention and United States Army Research Institute for Infectious Diseases have developed real-time PCR assays for the detection of bioterrorism threat agents. These assays all rely on a limited number of approved real-time PCR master mixes. Because the availability of these reagents is a critical element of bioterrorism preparedness, we undertook a joint national preparedness exercise to address the potential surge needs resulting from a large-scale bio-emergency. We identified 9 commercially-available potential alternatives to an existing approved master mix (LightCycler FastStart DNA Master HybProbes): the TaqMan Fast Universal PCR master mix, OmniMix HS, FAST qPCR master mix, EXPRESS qPCR SuperMix kit, QuantiFast Probe PCR kit, LightCycler FastStart DNA MasterPLUS HybProbe, Brilliant II FAST qPCR master mix, ABsolute Fast QPCR Mix and the HotStart IT Taq master mix. The performances of these kits were evaluated by the use of real-time PCR assays for four bioterrorism threat agents: Bacillus anthracis, Brucella melitensis, Burkholderia mallei and Francisella tularensis. The master mixes were compared for target-specific detection levels, as well as consistency of results among three different real-time PCR platforms (LightCycler, SmartCycler and 7500 Fast Dx). Realtime PCR analysis revealed that all ten kits performed well for agent detection on the 7500 Fast Dx instrument; however, the QuantiFast Probe PCR kit yielded the most consistently positive results across multiple real-time PCR platforms. We report that certain combinations of commonly used master mixes and instruments are not as reliable as others at detecting low concentrations of target DNA. Furthermore, our study provides laboratories the option to select from the commercial kits we evaluated to suit their preparedness needs

Psychological and Genetic Predictors of Pain Tolerance

Previous studies have shown associations between genetic polymorphisms and pain tolerance, but psychological evaluations are seldom measured. The objective of this study was to determine the independent effects of demographic, psychological, and genetic predictors of cold noxious pain tolerance. Healthy subjects (n = 89) completed the Pain Catastrophizing Scale (PCS) and Fear of Pain Questionnaire (FPQ-III), underwent genotyping for candidate single nucleotide polymorphisms (SNPs), and completed a cold-pressor test in a 1-2 degrees C water bath for a maximum of 3 minutes. The primary outcome measure was pain tolerance, defined as the maximum duration of time subjects left their nondominant hand in the cold-water bath. Cox proportional hazards regression indicated that female sex, Asian race, and increasing PCS and FPQ-III scores were associated with lower pain tolerance. No candidate SNP was significantly associated with pain tolerance. Future genetic studies should include demographic and psychological variables as confounders in experimental pain models.Open access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Characterizing the limited use of point-of-care ultrasound in Colombian emergency medicine residencies

Background: Emergency medicine (EM) is a growing specialty in Colombia with five residency programs in the country. EM leadership is interested in incorporating point-of-care (POC) ultrasound into a standardized national EM residency curriculum. This study is a nationwide survey of Colombian EM residents designed to explore the current state of POC ultrasound use within EM residencies and examine specific barriers preventing its expansion. Methods: We conducted a mix-methodology study of all available current EM residents in the five EM residencies in Colombia. The quantitative survey assessed previous ultrasound experience, current use of various applications, desire for further training, and perceived barriers to expanded use. Focus group discussions (FGDs) were conducted with current EM residents to gather additional qualitative insight into their practice patterns and perceived barriers to clinician-performed ultrasound. Results: Sixty-nine EM residents completed the quantitative survey, a response rate of 85% of all current EM residents in Colombia; 52% of resident respondents had previously used ultrasound during their training. Of these, 58% indicated that they had performed 40 scans. The most frequently used applications indicated by respondents were trauma, obstetrics, and procedures including vascular access. A quarter indicated they had previously received some ultrasound training, but almost all expressed an interest in learning more. Significant barriers included lack of trained teachers (indicated by 78% of respondents), absence of machines (57%), and limited time (41%). In FGDs, the barriers identified were inter-specialty conflicts over the control of ultrasonography, both institutionally and nationally, and program-specific curriculum decisions to include POC ultrasound. Conclusion: While currently limited in their access, EM residents in Colombia have a strong interest in integrating POC ultrasound into their training. Current barriers to expanded use include traditional barriers such as a lack of equipment seen in many developing countries, as well as inter-specialty conflicts typical of developed countries. Further collaboration is underway to help overcome these obstacles and integrate POC ultrasound into Colombian EM residency training

Characterizing the limited use of point-of-care ultrasound in Colombian emergency medicine residencies

Background: Emergency medicine (EM) is a growing specialty in Colombia with five residency programs in the country. EM leadership is interested in incorporating point-of-care (POC) ultrasound into a standardized national EM residency curriculum. This study is a nationwide survey of Colombian EM residents designed to explore the current state of POC ultrasound use within EM residencies and examine specific barriers preventing its expansion. Methods: We conducted a mix-methodology study of all available current EM residents in the five EM residencies in Colombia. The quantitative survey assessed previous ultrasound experience, current use of various applications, desire for further training, and perceived barriers to expanded use. Focus group discussions (FGDs) were conducted with current EM residents to gather additional qualitative insight into their practice patterns and perceived barriers to clinician-performed ultrasound. Results: Sixty-nine EM residents completed the quantitative survey, a response rate of 85% of all current EM residents in Colombia; 52% of resident respondents had previously used ultrasound during their training. Of these, 58% indicated that they had performed <10 scans and 17% reported >40 scans. The most frequently used applications indicated by respondents were trauma, obstetrics, and procedures including vascular access. A quarter indicated they had previously received some ultrasound training, but almost all expressed an interest in learning more. Significant barriers included lack of trained teachers (indicated by 78% of respondents), absence of machines (57%), and limited time (41%). In FGDs, the barriers identified were inter-specialty conflicts over the control of ultrasonography, both institutionally and nationally, and program-specific curriculum decisions to include POC ultrasound. Conclusion: While currently limited in their access, EM residents in Colombia have a strong interest in integrating POC ultrasound into their training. Current barriers to expanded use include traditional barriers such as a lack of equipment seen in many developing countries, as well as inter-specialty conflicts typical of developed countries. Further collaboration is underway to help overcome these obstacles and integrate POC ultrasound into Colombian EM residency training. © 2014 Henwood et al.; licensee Springer

Dolores articulares y osteomusculares en practicantes de capoeira

La Capoeira es un arte marcial y danza ambientada en ritmos afro-brasileros. Existen pocos estudios que evalúen la presencia patologías, factores de riesgo desencadenantes de lesiones y elementos biomecánicos asociados a este arte marcial. El Dolor es un síntoma guía que permite detectar la presencia de lesiones y/o secuelas, determina en ciertos casos la morbilidad en practicantes de artes marciales. Por lo anterior, este texto pretende recopilar información que favorezca en un futuro, reducción de la incidencia de lesiones, control adecuado del dolor y finalmente mejoría en el rendimiento de los practicantes de capoeira. La capoeira es realizada mediante un ritual colectivo, donde un grupo de personas forman un círculo en el cual dos participantes del grupo dedican a demostrar su destreza física y técnica, guiados por la música. Tiene 2 estilos principales a lo largo de su historia, dentro de los cuales la modalidad “regional” es la más marcial. Los deportes de contacto y las artes marciales por su naturaleza predisponen al desarrollo de lesiones, eso incluye a la capoeira. Los grupos musculares más utilizados en un arte marcial se corresponden con las regiones con mayor número de lesiones, en la capoeira se hace referencia al miembro inferior. La capoeira tiene un potencial para predisponer a lesiones, que pueden dejar varias secuelas relacionadas con dolor y discapacidad. Se sugiere la investigación a profundidad de la capoeira en relación con el desarrollo lesiones y el dolor crónico, pues que se evidencia escasez en este campo.PregradoMedic

In Vitro and In Vivo Characterization of the Alkaloid Nuciferine

RationaleThe sacred lotus (Nelumbo nucifera) contains many phytochemicals and has a history of human use. To determine which compounds may be responsible for reported psychotropic effects, we used in silico predictions of the identified phytochemicals. Nuciferine, an alkaloid component of Nelumbo nucifera and Nymphaea caerulea, had a predicted molecular profile similar to antipsychotic compounds. Our study characterizes nuciferine using in vitro and in vivo pharmacological assays.MethodsNuciferine was first characterized in silico using the similarity ensemble approach, and was followed by further characterization and validation using the Psychoactive Drug Screening Program of the National Institute of Mental Health. Nuciferine was then tested in vivo in the head-twitch response, pre-pulse inhibition, hyperlocomotor activity, and drug discrimination paradigms.ResultsNuciferine shares a receptor profile similar to aripiprazole-like antipsychotic drugs. Nuciferine was an antagonist at 5-HT2A, 5-HT2C, and 5-HT2B, an inverse agonist at 5-HT7, a partial agonist at D2, D5 and 5-HT6, an agonist at 5-HT1A and D4 receptors, and inhibited the dopamine transporter. In rodent models relevant to antipsychotic drug action, nuciferine blocked head-twitch responses and discriminative stimulus effects of a 5-HT2A agonist, substituted for clozapine discriminative stimulus, enhanced amphetamine induced locomotor activity, inhibited phencyclidine (PCP)-induced locomotor activity, and rescued PCP-induced disruption of prepulse inhibition without induction of catalepsy.ConclusionsThe molecular profile of nuciferine was similar but not identical to that shared with several approved antipsychotic drugs suggesting that nuciferine has atypical antipsychotic-like actions

The Problem of Experience in the Study of Organizations

This paper deals with the fact that we cannot experience large organizations directly, in the same way as we can experience individuals or small groups, and that this non-experientiability has certain implications for our scientific theories of organizations. Whereas a science is animated by a constructive interplay of theory concepts and experience concepts, the study of organizations has been confined to theory concepts alone. Implications of this analysis for developing a science of organizations are considered.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/68303/2/10.1177_017084069301400102.pd

VPS29 Is Not an Active Metallo-Phosphatase but Is a Rigid Scaffold Required for Retromer Interaction with Accessory Proteins

VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn2+ and Zn2+ in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies

Structural basis of the filamin A actin-binding domain interaction with F-actin

Cryo-EM reconstructions were deposited in the Electron Microscopy Data Bank with the following accession numbers: F20-F-actin-FLNaABD, EMD-7833; F20-F-actin-FLNaABD-Q170P, EMD-7832; F20-F-actin-FLNaABD-E254K, EMD-8918; Krios-F-actin-FLNaABD-E254K, EMD-7831. The corresponding FLNaABD-E254K filament model was deposited in the PDB with accession number 6D8C. Source data for F-actin-targeting analyses (Figs. 2c,d,g,h, 3b,c,e,f, 4d,e, 5c,d, and 6a,b) and co-sedimentation assays (Figs. 5g and 6d) are available with the paper online. Other data are available from the corresponding author upon reasonable request. We thank Z. Razinia for generating numerous FLNa constructs, S. Wu for expertise in using the Krios microscope, J. Lees for advice on model refinement, and M. Lemmon for helpful comments in preparing the manuscript. We also thank the Yale Center for Research Computing for guidance and use of the Farnam Cluster, as well as the staff at the YMS Center for Molecular Imaging for the use of the EM Core Facility. This work was funded by grants from the National Institutes of Health (R01-GM068600 (D.A.C.), R01-NS093704 (D.A.C.), R37-GM057247 (C.V.S.), R01-GM110530 (C.V.S.), T32-GM007324, T32-GM008283) and an award from American Heart Association (15PRE25700119 (D.V.I.)).Peer reviewedPostprin