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

The neurodevelopmental implications of hypoplastic left heart syndrome in the fetus

Abstract As survival after cardiac surgery continues to improve, an increasing number of patients with hypoplastic left heart syndrome are reaching school age and beyond, with growing recognition of the wide range of neurodevelopmental challenges many survivors face. Improvements in fetal detection rates, coupled with advances in fetal ultrasound and MRI imaging, are contributing to a growing body of evidence that abnormal brain architecture is in fact present before birth in hypoplastic left heart syndrome patients, rather than being solely attributable to postnatal factors. We present an overview of the contemporary data on neurodevelopmental outcomes in hypoplastic left heart syndrome, focussing on imaging techniques that are providing greater insight into the nature of disruptions to the fetal circulation, alterations in cerebral blood flow and substrate delivery, disordered brain development, and an increased potential for neurological injury. These susceptibilities are present before any intervention, and are almost certainly substantial contributors to adverse neurodevelopmental outcomes in later childhood. The task now is to determine which subgroups of patients with hypoplastic left heart syndrome are at particular risk of poor neurodevelopmental outcomes and how that risk might be modified. This will allow for more comprehensive counselling for carers, better-informed decision making before birth, and earlier, more tailored provision of neuroprotective strategies and developmental support in the postnatal period

Paper Session II-D - Project Vision (Very Intensive Scientific Intercurricular Onsite Education): A Partnership Among NASA/John F. Kennedy Space Center, Florida International University, Universidad del Turabo, Dade County Public Schools, and the Caguas/Gurabo Public Schools.

Project VISION is a joint effort among NASA/John F. Kennedy Space Center, Florida International University, Universidad del Turabo, Dade County Public Schools and the Caguas/ Gurabo Public Schools. The project’s main mission is to institutionalize change among the 7th grade science and mathematics teachers at participating public middle schools. A further aspect of the mission is to enhance the science and math education of the public middle school students during the phase of institutionalization. Project VISION will not need to generate any new educational materials to fulfill its mission. Rather than generating new materials, Project VISION will use the vast quantities of high quality learning modules, lessons, hands-on experiments and other educational materials available at NASA and other scientific depositories. The project will identify, adopt and then adapt these learning modules or learning materials to best meet the needs and capabilities of the target student and teacher populations. A further goal of this project lies within the realm of NASA’s Mission - to specifically focus our activities on middle schools that serve socially and economically disadvantaged students. Additionally, the project will invite members of the private and public sectors to serve as lecturers, mentors and role models. The project will perform program evaluations to measure the levels of success and accomplishments of each of the proposed activities

Field-deployable, quantitative, rapid identification of active Ebola virus infection in unprocessed blood

The West African Ebola virus outbreak underlined the importance of delivering mass diagnostic capability outside the clinical or primary care setting in effectively containing public health emergencies caused by infectious disease. Yet, to date, there is no solution for reliably deploying at the point of need the gold standard diagnostic method, real time quantitative reverse transcription polymerase chain reaction (RT- qPCR), in a laboratory infrastructure-free manner. In this proof of principle work, we demonstrate direct performance of RT-qPCR on fresh blood using far-red fluorophores to resolve fluorogenic signal inhibition and controlled, rapid freeze/thawing to achieve viral genome extraction in a single reaction chamber assay. The resulting process is entirely free of manual or automated sample pre-processing, requires no microfluidics or magnetic/mechanical sample handling and thus utilizes low cost consumables. This enables a fast, laboratory infrastructure-free, minimal risk and simple standard operating procedure suited to frontline, field use. Developing this novel approach on recombinant bacteriophage and recombinant human immunodeficiency virus (HIV; Lentivirus), we demonstrate clinical utility in symptomatic EBOV patient screening using live, infectious Filoviruses and surrogate patient samples. Moreover, we evidence assay co-linearity independent of viral particle structure that may enable viral load quantification through pre-calibration, with no loss of specificity across an 8 log- linear maximum dynamic range. The resulting quantitative rapid identification (QuRapID) molecular diagnostic platform, openly accessible for assay development, meets the requirements of resource- limited countries and provides a fast response solution for mass public health screening against emerging biosecurity threats

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

Engineered nonlinear materials using gold nanoantenna array

Gold dipole nanoantennas embedded in an organic molecular film provide strong local electromagnetic fields to enhance both the nonlinear refractive index (n(2)) and two-photon absorption (2PA) of the molecules. An enhancement of 53x for 2PA and 140x for nonlinear refraction is observed for BDPAS (4,4'-bis(diphenylamino) stilbene) at 600 nm with only 3.7% of gold volume fraction. The complex value of the third-order susceptibility enhancement results in a sign change of n(2) for the effective composite material relative to the pure BDPAS film. This complex nature of the enhancement and the tunability of the nanoantenna resonance allow for engineering the effective nonlinear response of the composite film8119Agências de fomento estrangeiras apoiaram essa pesquisa, mais informações acesse artig

Binding of molecules to DNA and other semiflexible polymers

A theory is presented for the binding of small molecules such as surfactants to semiflexible polymers. The persistence length is assumed to be large compared to the monomer size but much smaller than the total chain length. Such polymers (e.g. DNA) represent an intermediate case between flexible polymers and stiff, rod-like ones, whose association with small molecules was previously studied. The chains are not flexible enough to actively participate in the self-assembly, yet their fluctuations induce long-range attractive interactions between bound molecules. In cases where the binding significantly affects the local chain stiffness, those interactions lead to a very sharp, cooperative association. This scenario is of relevance to the association of DNA with surfactants and compact proteins such as RecA. External tension exerted on the chain is found to significantly modify the binding by suppressing the fluctuation-induced interaction.Comment: 15 pages, 7 figures, RevTex, the published versio

Cigarette Smoke Induces Intestinal Inflammation via a Th17 Cell-Neutrophil Axis

Epidemiological evidence finds cigarette smoking is a common risk factor for a number of diseases, not only in the lung but also in other tissues, such as the gastrointestinal tract. While it is well-documented that smoking directly drives lung inflammatory disease, how it promotes disease in peripheral tissues is incompletely understood. In this study, we utilized a mouse model of short-term smoke exposure and found increased Th17 cells and neutrophilia in the lung as well as in the circulation. Following intestinal inflammatory challenge, smoke exposed mice showed increased pathology which corresponds to enhanced intestinal Th17 cells, ILC3 and neutrophils within intestinal tissue. Using cellular depletion and genetic deficiencies, we define a cellular loop by which IL-17A and downstream neutrophils drive cigarette smoke-enhanced intestinal inflammation. Collectively, cigarette smoke induced local lung Th17 responses lead to increased systemic susceptibility to inflammatory insult through enhanced circulating neutrophils. These data demonstrate a cellular pathway by which inflammatory challenge in the lung can sensitize the intestine to enhanced pathological innate and adaptive immune responses