tmRNA - a novel high-copy-number RNA diagnostic target - its application for Staphylococcus aureus detection using real-time NASBA

A real-time nucleic acid sequence-based amplification assay, targeting tmRNA, was designed for the rapid identification of Staphylococcus aureus. The selectivity of the assay was confirmed against a panel of 76 Staphylococcus strains and species and 22 other bacterial species. A detection limit of 1 cell equivalent was determined for the assay. A chimeric in vitro transcribed internal amplification control was developed and included in the assay. Application of the assay in natural and artificially contaminated unpasteurized (raw) milk enabled detection of 1-10 CFUS. aureus mL(-1) in 3-4 h, without the need for culture enrichment. Staphylococcus aureus was detected in all artificially contaminated milk samples (n=20) and none of the natural milk samples (n=20). Microbiological analysis of the natural milk samples was performed in parallel according to ISO 6888-3 and confirmed the absence of S. aureus. The method developed in this study has the potential to enable the specific detection of S. aureus in raw milk in a significantly shorter time frame than current standard methods. The assay further demonstrates the usefulness of tmRNA/ssrA as a nucleic acid diagnostic target

Lights, camera, action: Microbiology laboratory teaching in the spotlight

[EN] Broadening of access to higher education, leading to increasing class sizes, presents particular challenges in teaching specialised, laboratory-based subjects such as Microbiology. The Republic of Ireland has seen a 20% increase in undergraduate student numbers in the past ten years, with this trend set to continue in the near future. To complement traditional learning approaches, we have designed, produced and trialled a comprehensive suite of teaching videos that demonstrate common techniques taught in our Microbiology degree programme. The 42 videos, each of 4-9 minutes duration, were filmed in-house using a professional film maker. Videos were designed for viewing before linked laboratory sessions to increase student engagement, assist learners with little prior technical experience to process core concepts, and improve the quality of hands-on practical training in the laboratory. Student reaction to a pilot release was exceptionally positive, underlining the videos’ effectiveness for visual learners and the added value of the content due to its bespoke nature. The complete video collection will be amalgamated into our B.Sc. programme in 2019-20. The initiative is expected to enhance students’ experience in hands-on laboratory sessions, promote active learning by blending video into traditional teaching programmes, and support reflective study through their availability.The NUI Galway Student Project Fund is thanked for financial support.Lacey, K.; Wall, JG. (2019). Lights, camera, action: Microbiology laboratory teaching in the spotlight. En HEAD'19. 5th International Conference on Higher Education Advances. Editorial Universitat Politècnica de València. 939-946. https://doi.org/10.4995/HEAD19.2019.9429OCS93994

High Sensitivity DNA Detection Using Gold Nanoparticle Functionalised Polyaniline Nanofibres

Polyaniline (PANI) nanofibres (PANI-NF) have been modified with chemically grown gold nanoparticles to give a nanocomposite material (PANI-NF–AuNP) and deposited on gold electrodes. Single stranded capture DNA was then bound to the gold nanoparticles and the underlying gold electrode and allowed to hybridise with a complementary target strand that is uniquely associated with the pathogen, Staphylococcus aureus (S. aureus), that causes mastitis. Significantly, cyclic voltammetry demonstrates that deposition of the gold nanoparticles increases the area available for DNA immobilisation by a factor of approximately 4. EPR reveals that the addition of the Au nanoparticles efficiently decreases the interactions between adjacent PANI chains and/or motional broadening. Finally, a second horseradish peroxidase (HRP) labelled DNA strand hybridises with the target allowing the concentration of the target DNA to be detected by monitoring the reduction of a hydroquinone mediator in solution. The sensors have a wide dynamic range, excellent ability to discriminate DNA mismatches and a high sensitivity. Semi-log plots of the pathogen DNA concentration vs. faradaic current were linear from 150 × 10−12 to 1 × 10−6 mol L−1 and pM concentrations could be detected without the need for molecular, e.g., PCR or NASBA, amplification

Federal Research, Additional Actions Needed to Improve Public Access to Research Results: Report to Congressional Requesters [with Responses from Federal Agencies]

Why GAO Did This Study Research and development helps catalyze breakthroughs that improve the overall health and wellbeing of our society. Federal research and development expenditures averaged about $135 billion annually for fiscal years 2015 to 2017. According to OSTP, providing free public access to federally funded research results can improve both the impact and accountability of this important federal investment. In February 2013, OSTP directed federal agencies with more than$100 million in annual research and development expenditures to develop a plan to support increased public access to the results of federally funded research. GAO was asked to examine public access to federally funded research results. This report examines the extent of agencies’ (1) progress implementing plans to increase public access to federally funded research results and (2) coordination on public access plan implementation. GAO administered a questionnaire to 19 federal agencies selected based on annual research and development expenditure amounts, among other criteria; reviewed agency documents; and interviewed officials from 11 agencies, OSTP, and 21 stakeholder organizations. What GAO Found The 19 agencies that GAO reviewed have made progress implementing their plans to increase public access to federally funded research results (publications and data), as called for in a 2013 Office of Science and Technology Policy (OSTP) memorandum. However, some agencies have not fully implemented some aspects of their plans, in particular those related to data access and mechanisms to ensure researchers comply with public access requirements. Agencies are coordinating with each other and with nonfederal stakeholders to implement public access plans, including through an interagency group led by OSTP and five other agencies. However, the group has not fully implemented selected leading practices identified by GAO that can enhance and sustain interagency collaboration, such as defining and articulating common outcomes. For example, according to OSTP staff, key outcomes have not yet been decided upon. Agency officials and stakeholders identified several challenges to implementing public access plans that interagency coordination might help them address, such as • Absence of common standards in several areas; • Measuring effectiveness of public access plan implementation; and • Balancing providing public access with safeguarding sensitive information. By taking steps to fully implement relevant leading collaboration practices, the interagency group could help agencies better marshal their collective efforts to address common challenges to public access plan implementation. What GAO Recommends GAO is making 37 recommendations to 16 agencies to promote full and effective implementation of agency public access plans. For example, GAO recommends that OSTP and 5 agencies leading a public access interagency group take steps to fully implement selected leading collaboration practices. Of the 16 agencies, 15 agreed with GAO’s recommendations while 1 (OSTP) disagreed. GAO continues to believe the recommendation to OSTP is warranted

Injecting New Knowledge: A Statistical Analysis of Safe Injection Sites and Harm Reduction

Objective Safe injection sites (SIS) are proven to reduce overdoses, crime, and fatal blood-borne infections. Despite those benefits, no sites exist in Windsor, ON., likely due to the abundance of fear-mongering and misinformation. We critically analyzed statistical methods used in dozens of quantitative studies and found critical gaps in peer-reviewed literature worldwide, substantially affecting intravenous drug users and communities around them. Methods We systematically analyzed existing studies’ statistical methods to appraise investigations of SIS and harm reduction. To our knowledge, ours is the first academic analysis of statistical methods for SIS and harm-reduction. Statistical rigor in studies regarding SIS and harm reduction vary in methodology and conclusions, presenting a conundrum for government authorities and clinicians alike. Results We found alarming methodological issues with SIS studies, such as poor precision and rigor, in addition to failure to adhere to research reporting standards. Such a dearth of attention to detail on this topic warrants more granular study, especially in the Windsor, ON., area. Our recommendations provide reliable basis for government decision-making based on facts, not fear, that will help foster healthy and safe communities

Reusable Surface Plasmon Resonance Assay for the Specific Detection of Streptococcus Pneumoniae tmRNA.

A capture oligonucleotide-based surface plasmon resonance assay was developed for the specific detection of tmRNA from the gram-positive pathogen Streptococcus pneumoniae. Candidate species-specific probes were initially screened on microarrays using labeled in vitro transcribed tmRNA beforebeing subjected to further analysis on a Biacore 2000 biosensor using a variety of unlabeled tmRNA-containing samples. Previous work had demonstrated that there is approximately 1,000 copies/cfu of tmRNA in S. pneumoniae. The natural amplification of this target has the potential to improve the detection capability of a nucleic acid diagnostics assay. In this study, S. pneumoniae-specific probes were designed that enabled the detection of 10 exp12 cRNA copies of the tmRNA target on the Biacore instrument. When evaluated in combination with nucleic acid sequence-based amplification, specific detection of tmRNA from a single S. pneumoniae cell was demonstrated

Tmrna â a novel high-copy-number rna diagnostic target â its application forstaphylococcus aureusdetection using real-time nasba

A real-time nucleic acid sequence-based amplification assay, targeting tmRNA, was designed for the rapid identification of Staphylococcus aureus. The selectivity of the assay was confirmed against a panel of 76 Staphylococcus strains and species and 22 other bacterial species. A detection limit of 1 cell equivalent was determined for the assay. A chimeric in vitro transcribed internal amplification control was developed and included in the assay. Application of the assay in natural and artificially contaminated unpasteurized (raw) milk enabled detection of 1-10 CFUS. aureus mL-1 in 3-4 h, without the need for culture enrichment. Staphylococcus aureus was detected in all artificially contaminated milk samples (n=20) and none of the natural milk samples (n=20). Microbiological analysis of the natural milk samples was performed in parallel according to ISO 6888-3 and confirmed the absence of S. aureus. The method developed in this study has the potential to enable the specific detection of S. aureus in raw milk in a significantly shorter time frame than current standard methods. The assay further demonstrates the usefulness of tmRNA/ssrA as a nucleic acid diagnostic target

Immunomodulatory effect of cryopreserved platelets: altered BDCA3(+) dendritic cell maturation and activation in vitro

Cryopreservation of platelets (PLTs) is useful in remote areas to overcome logistic problems associated with supply and can extend the shelf life to 2 years. During cryopreservation, properties of PLTs are modified. Whether changes in the cryopreserved PLT (CPP) product are associated with modulation of recipients' immune function is unknown. We aimed to characterize the immune profile of myeloid dendritic cells (mDCs) and the specialized blood DC antigen (BDCA)3(+) subset after exposure to CPPs.Using an in vitro whole blood model of transfusion, the effect of CPPs on mDC and BDCA3(+) DC surface antigen expression and inflammatory mediator production was examined using flow cytometry. In parallel, polyinosinic:polycytidylic acid (poly(I:C)) or lipopolysaccharide (LPS) was utilized to model processes activated in viral or bacterial infection, respectively.Cryopreserved PLTs had minimal impact on mDC responses but significantly modulated BDCA3(+) DC responses in vitro. Exposure to CPPs alone up regulated BDCA3(+) DC CD86 expression and suppressed interleukin (IL)-8, tumor necrosis factor (TNF)-α, and interferon-γ inducible protein (IP)-10 production. In both models of infection-related processes, exposure to CPPs down regulated BDCA3(+) DC expression of CD40, CD80, and CD83 and suppressed BDCA3(+) DC production of IL-8, IL-12, and TNF-α. CPPs suppressed CD86 expression in the presence of LPS and IP-10 and IL-6 production with poly(I:C).Cryopreserved PLTs may be immunosuppressive, and this effect is more evident when processes associated with infection are concurrently activated, especially for BDCA3(+) DCs. This suggests that transfusion of CPPs in patients with infection may result in impaired BDCA3(+) DC responses

Federal Research, Additional Actions Needed to Improve Public Access to Research Results: Report to Congressional Requesters [with Responses from Federal Agencies]

Why GAO Did This Study Research and development helps catalyze breakthroughs that improve the overall health and wellbeing of our society. Federal research and development expenditures averaged about $135 billion annually for fiscal years 2015 to 2017. According to OSTP, providing free public access to federally funded research results can improve both the impact and accountability of this important federal investment. In February 2013, OSTP directed federal agencies with more than$100 million in annual research and development expenditures to develop a plan to support increased public access to the results of federally funded research. GAO was asked to examine public access to federally funded research results. This report examines the extent of agencies’ (1) progress implementing plans to increase public access to federally funded research results and (2) coordination on public access plan implementation. GAO administered a questionnaire to 19 federal agencies selected based on annual research and development expenditure amounts, among other criteria; reviewed agency documents; and interviewed officials from 11 agencies, OSTP, and 21 stakeholder organizations. What GAO Found The 19 agencies that GAO reviewed have made progress implementing their plans to increase public access to federally funded research results (publications and data), as called for in a 2013 Office of Science and Technology Policy (OSTP) memorandum. However, some agencies have not fully implemented some aspects of their plans, in particular those related to data access and mechanisms to ensure researchers comply with public access requirements. Agencies are coordinating with each other and with nonfederal stakeholders to implement public access plans, including through an interagency group led by OSTP and five other agencies. However, the group has not fully implemented selected leading practices identified by GAO that can enhance and sustain interagency collaboration, such as defining and articulating common outcomes. For example, according to OSTP staff, key outcomes have not yet been decided upon. Agency officials and stakeholders identified several challenges to implementing public access plans that interagency coordination might help them address, such as • Absence of common standards in several areas; • Measuring effectiveness of public access plan implementation; and • Balancing providing public access with safeguarding sensitive information. By taking steps to fully implement relevant leading collaboration practices, the interagency group could help agencies better marshal their collective efforts to address common challenges to public access plan implementation. What GAO Recommends GAO is making 37 recommendations to 16 agencies to promote full and effective implementation of agency public access plans. For example, GAO recommends that OSTP and 5 agencies leading a public access interagency group take steps to fully implement selected leading collaboration practices. Of the 16 agencies, 15 agreed with GAO’s recommendations while 1 (OSTP) disagreed. GAO continues to believe the recommendation to OSTP is warranted