3D-Printed Carbon Nanoneedle Electrodes for Dopamine Detection in Drosophila.

In vivo electrochemistry in small brain regions or synapses requires nanoelectrodes with long straight tips for submicron scale measurements. Nanoelectrodes can be fabricated using a Nanoscribe two-photon printer, but annealed tips curl if they are long and thin. We propose a new pulling-force strategy to fabricate a straight carbon nanoneedle structure. A micron-width bridge is printed between two blocks. The annealed structure shrinks during pyrolysis, and the blocks create a pulling force to form a long, thin, and straight carbon bridge. Parameterization study and COMSOL modeling indicate changes in the block size, bridge size and length affect the pulling force and bridge shrinkage. Electrodes were printed on niobium wires, insulated with aluminum oxide, and the bridge cut with focused ion beam (FIB) to expose the nanoneedle tip. Annealed needle diameters ranged from 400 nm to 5.25 µm and length varied from 50.5 µm to 146 µm. The electrochemical properties are similar to glassy carbon, with good performance for dopamine detection with fast-scan cyclic voltammetry. Nanoelectrodes enable biological applications, such as dopamine detection in a specific Drosophila brain region. Long and thin nanoneedles are generally useful for other applications such as cellular sensing, drug delivery, or gas sensing.

Microdosing ketamine in Drosophila does not block serotonin reuptake, but causes complex behavioral changes mediated by glutamate and serotonin receptors.

Microdosing ketamine is a novel antidepressant for treatment-resistant depression. Traditional antidepressants, like selective serotonin reuptake inhibitors (SSRIs), inhibit serotonin reuptake, but it is not clear if ketamine shows a similar mechanism. Here, we tested the effects of feeding ketamine and SSRIs to Drosophila melanogaster larvae, which has a similar serotonin system to mammals and is a good model to track depressive behaviors, such as locomotion and feeding. Fast-scan cyclic voltammetry (FSCV) was used to measure optogenetically stimulated serotonin changes, and locomotion tracking software and blue dye feeding to monitor behavior. We fed larvae various doses (1-100 mM) of antidepressants for 24 h and found that 1 mM ketamine did not affect serotonin, but increased locomotion and feeding. Low doses (≤10 mM) of escitalopram and fluoxetine inhibited dSERT and also increased feeding and locomotion behaviors. At 100 mM, ketamine inhibited dSERT and increased serotonin concentrations, but decreased locomotion and feeding because of its anesthetic properties. Since microdosing ketamine causes behavioral effects, we further investigated behavioral changes with a SERT16 mutant and low doses of other NMDA receptor antagonists and 5-HT1A and 2 agonists. Feeding and locomotion changes were similar to ketamine in the mutant, and we found NMDA receptor antagonism increased feeding, while serotonin receptor agonism increased locomotion, which could explain these effects with ketamine. Ultimately, this work shows that Drosophila is a good model to discern antidepressant mechanisms, and that ketamine does not work on dSERT like SSRIs, but effects behavior with other mechanisms that should be investigated further.

Electrochemical and biosensor techniques to monitor neurotransmitter changes with depression.

Depression is a common mental illness. However, its current treatments, like selective serotonin reuptake inhibitors (SSRIs) and micro-dosing ketamine, are extremely variable between patients and not well understood. Three neurotransmitters: serotonin, histamine, and glutamate, have been proposed to be key mediators of depression. This review focuses on analytical methods to quantify these neurotransmitters to better understand neurological mechanisms of depression and how they are altered during treatment. To quantitatively measure serotonin and histamine, electrochemical techniques such as chronoamperometry and fast-scan cyclic voltammetry (FSCV) have been improved to study how specific molecular targets, like transporters and receptors, change with antidepressants and inflammation. Specifically, these studies show that different SSRIs have unique effects on serotonin reuptake and release. Histamine is normally elevated during stress, and a new inflammation hypothesis of depression links histamine and cytokine release. Electrochemical measurements revealed that stress increases histamine, decreases serotonin, and leads to changes in cytokines, like interleukin-6. Biosensors can also measure non-electroactive neurotransmitters, including glutamate and cytokines. In particular, new genetic sensors have shown how glutamate changes with chronic stress, as well as with ketamine treatment. These techniques have been used to characterize how ketamine changes glutamate and serotonin, and to understand how it is different from SSRIs. This review briefly outlines how these electrochemical techniques work, but primarily highlights how they have been used to understand the mechanisms of depression. Future studies should explore multiplexing techniques and personalized medicine using biomarkers in order to investigate multi-analyte changes to antidepressants.

Microdosing ketamine in Drosophila does not inhibit SERT like SSRIs, but causes behavioral changes mediated by glutamate and serotonin receptors.

Recently, the FDA approved microdosing ketamine for treatment resistant depression. Traditional antidepressants, like serotonin selective reuptake inhibitors (SSRIs), block serotonin reuptake, but it is not clear if ketamine blocks serotonin reuptake. Here, we tested the effects of feeding ketamine and SSRIs to Drosophila melanogaster larvae, which has a similar serotonin system to mammals, and is a good model to track depression behaviors, such as locomotion and feeding. Fast-scan cyclic voltammetry (FSCV) was used to measure optogenetically-stimulated serotonin changes, and locomotion tracking software and blue dye feeding to monitor behavior. We fed larvae various doses (1-100 mM) of antidepressants for 24 hours and found that 1 mM ketamine did not affect serotonin, but increased locomotion and feeding. Low doses (≤ 10 mM) of escitalopram and fluoxetine inhibited dSERT and also increased feeding and locomotion behaviors. At 100 mM, ketamine inhibited dSERT and increased serotonin concentrations, but decreased locomotion and feeding due to its anesthetic properties. Since microdosing ketamine causes behavioral effects, we also investigated behavior changes with low doses of other NMDA receptor antagonists and 5-HT1A and 2 agonists, which are other possible sites for ketamine action. NMDA receptor antagonism increased feeding, while serotonin receptor agonism increased locomotion, which could explain these effects with ketamine. Ultimately, this work shows that Drosophila is a good model to discern antidepressant mechanisms, and that ketamine does not work on dSERT like SSRIs at microdoses, but affects behavior with other mechanisms.

Emerging healthcare interventions: Patient-Centered Outcomes Research Institute's programmatic initiative.

The Patient-Centered Outcomes Research Institute (PCORI) is a nonprofit, nongovernmental organization established by the U.S. Congress to fund comparative clinical effectiveness research focusing on patient-centered outcomes through the engagement of stakeholders. Evaluation of emerging healthcare innovations is one of PCORI's five National Priorities for Health. One such initiative is PCORI's Emerging Technologies and Therapeutics Reports program, established to provide timely overviews of evidence on new drugs and other healthcare technologies. This article provides an overview of completed and ongoing Emerging Technologies and Therapeutics Reports including lessons learned to date. In addition to systematic searches, systematic selection of studies, and transparent reporting of the available evidence, informed by a select number of stakeholders (i.e., key informants), these reports focus on contextual factors shaping the diffusion of emerging technologies that are often not reported in the medical literature. This article also compares processes and methodologies of health technology assessments (HTAs) from a selected number of national and international publicly funded agencies with a goal toward potential future enhancement of PCORI's Emerging Technologies and Therapeutics Reports program. HTAs vary considerably in terms of funding, types of assessments, the role of manufacturers, stakeholder engagement, timeline to complete from the start to the finish of a draft report publication, and communication of uncertainty for informed decision making. Future Emerging Technologies and Therapeutics Reports may focus on rapid reports to support a more expedient development of evidence. Future research could explore the role of contextual factors identified in these reports on targeted evidence generation.

How an international research funder's forum developed guiding principles to ensure value and reduce waste in research.

Background: When health-related research funding agencies choose to fund research, they balance a number of competing issues: costs, stakeholder views and potential benefits. The REWARD Alliance, and the related Lancet-REWARD Campaign, question whether those decisions are yielding all the value they could. Methods: A group of health-related research funding agencies, organisations that represent health-related research funding agencies and those that inform and set health-related-research funding policy from around the world have come together since 2016 to share, learn, collaborate and influence emerging practice. This group meets under the name of the Ensuring Value in Research Funders' Forum (EViR Funders' Forum). The EViR Funders' Forum worked together to develop a set of ten Guiding Principles, that if funders adhered to would reduce research waste and ensure value in research. Results: The EViR Funders' Forum has previously agreed and published a Consensus Statement. The Forum has agreed on a set of ten Guiding Principles to help health-research funders to maximise the value of research by ensuring that: research priorities are justifiable; the design, conduct and analysis of research minimise bias; regulation and management are proportionate to risks; methods and findings are accessible in full; and findings are appropriately and effectively disseminated and used. Conclusions: When setting research funding policy, we must balance multiple stakeholders' needs and expectations. When funders do this well, they maximise the probability of benefits to society from the research they support - when funders do this badly, they passively allow or actively contribute to research waste. These challenges must be resolved by funders either working together or in conjunction with other actors in the research ecosystem.

SSRI antidepressants differentially modulate serotonin reuptake and release in Drosophila.

Selective serotonin reuptake inhibitor (SSRI) antidepressants are commonly prescribed treatments for depression, but their effects on serotonin reuptake and release are not well understood. Drosophila melanogaster, the fruit fly, expresses the serotonin transporter (dSERT), the major target of SSRIs, but real-time serotonin changes after SSRIs have not been characterized in this model. The goal of this study was to characterize effects of SSRIs on serotonin concentration and reuptake in Drosophila larvae. We applied various doses (0.1-100 µM) of fluoxetine (Prozac), escitalopram (Lexapro), citalopram (Celexa), and paroxetine (Paxil), to ventral nerve cord (VNC) tissue and measured optogenetically-stimulated serotonin release with fast-scan cyclic voltammetry (FSCV). Fluoxetine increased reuptake from 1 to 100 µM, but serotonin concentration only increased at 100 µM. Thus, fluoxetine occupies dSERT and slows clearance but does not affect concentration. Escitalopram and paroxetine increased serotonin concentrations at all doses, but escitalopram increased reuptake more. Citalopram showed lower concentration changes and faster reuptake profiles compared with escitalopram, so the racemic mixture of citalopram does not change reuptake as much as the S-isomer. Dose response curves were constructed to compare dSERT affinities and paroxetine showed the highest affinity and fluoxetine the lowest. These data demonstrate SSRI mechanisms are complex, with separate effects on reuptake or release. Furthermore, dynamic serotonin changes in Drosophila are similar to previous studies in mammals. This work establishes how antidepressants affect serotonin in real-time, which is useful for future studies that will investigate pharmacological effects of SSRIs with different genetic mutations in Drosophila.

Health funders' dissemination and implementation practices: results from a survey of the Ensuring Value in Research (EViR) Funders' Forum.

BACKGROUND: A significant gap persists between evidence from research and its use in practice. Research funders, important actors in the health research system, can help reduce this gap by initiating dissemination and implementation (D&I) activities. The specific types of D&I activities funders currently lead have not been explored thoroughly. The Ensuring Value in Research (EViR) Funders' Forum-an international collaboration of health-related research funders-was established in 2017 to address research waste issues and increase the value of research. The Forum surveyed funders to learn about their D&I practices and challenges. METHODS: We distributed a five-item exploratory survey to participating funders in August 2018. The results informed the development of a survey instrument, distributed in June 2019. The survey instrument contained 15 items prompting respondents to categorize and describe their level of effort in six practice areas: release of findings, dissemination, knowledge exchange/partnering, implementation, building capacity, and implementation research. In addition, funders were asked to describe examples of their practices in detail. Thirty-one funders completed the survey instrument, a 58% response rate. RESULTS: Most funders regard D&I as a high priority, but funders vary in levels of activity per practice area. Over half of respondents reported that they have at least some activity in all D&I practice areas surveyed, with the exception of implementation research. The vast majority indicated some or significant activity in release of findings (97%) and dissemination (87%). Nearly one-fifth of funders (19%) indicated that implementation is outside their remit, and 26% indicated that implementation research is outside their remit. Survey respondents shared a broad range of examples of activities in each practice area. Lack of evidence for successful approaches and measuring impact were named frequently as challenges and as potential areas for collaboration. CONCLUSIONS: Although models of dissemination and implementation vary across organizations, the majority of funders indicated that D&I of research findings is a priority. Funders indicated a need for evidence on effectiveness of various approaches to D&I. Increased collaboration between funders, including sharing good practices, will increase our collective learning and knowledge development.

Improving serotonin fast-scan cyclic voltammetry detection: new waveforms to reduce electrode fouling.

Serotonin is a neuromodulator implicated in depression that is often measured in real-time by fast-scan cyclic voltammetry (FSCV). A specialized "Jackson" waveform (JW, 0.2, 1.0 V, -0.1 V, 0.2 V, 1000 V s-1) was developed to reduce serotonin fouling, but the 1.0 V switching potential limits sensitivity and electrodes still foul. The goal of this study was to test the effects of extending the FSCV switching potential to increase serotonin sensitivity and decrease fouling. We compared the Jackson waveform, the dopamine waveform (DA, -0.4 V, 1.3 V, 400 V s-1), and two new waveforms: the extended serotonin waveform (ESW, 0.2, 1.3, -0.1, 0.2, 1000 V s-1) and extended hold serotonin waveform (EHSW, 0.2, 1.3 (hold 1 ms), -0.1, 0.2, 400 V s-1). The EHSW was the most sensitive (LOD = 0.6 nM), and the JW the least sensitive (LOD = 2.4 nM). With the Jackson waveform, electrode fouling was significant with repeated injections of serotonin or exposure to its metabolite, 5-hydroxyindoleacetic acid (5-HIAA). Using the extended waveforms, electrodes fouled 50% less than with the Jackson waveform for both analytes. No electrode fouling was observed with the dopamine waveform because of the negative holding potential. The Jackson waveform was the most selective for serotonin over dopamine (800×), and the ESW was also highly selective. All waveforms were useful for measuring serotonin with optogenetic stimulation in Drosophila larvae. These results provide new FSCV waveforms to measure dynamic serotonin changes with different experimental requirements, like high sensitivity (EHSW), high selectivity (ESW, JW), or eliminating electrode fouling (DA).

Examining the role of funders in ensuring value and reducing waste in research: An organizational case-study of the Patient-Centered Outcomes Research Institute.

International experts have recommended actions that funders can take to improve the value of research investments. They state that self-assessment and public sharing are the basis for accountability and improvement. We examined our policies and practice to determine the extent to which the Patient-Centered Outcomes Research Institute's (PCORI) policies and practices as a research funder align with international best practice recommendations. A self-audit of current policies and practice against 17 recommendations and 35 sub-recommendations representing five major stages of research production, based on adapted methods used for self-assessment by another funder, was performed.  Fit of existing PCORI policies and practices with 35 sub-recommendations, qualitative assessment of adequacy (area of strength; area of partial strength; area of growth; not applicable) for 17 recommendations for five stages of research production was assessed. Of the 17 recommendations, 15 were applicable to PCORI's research mission and focus.  PCORI has policies and practices in place for all elements of six recommendations ("area of strength") and policies that address each element but with some still in active development for three ("area of partial strength"). PCORI is partially addressing six of the 15 relevant recommendations ("area of growth"). Areas for growth include making study protocols publicly available, improving policies on data sharing, and enhancing collaboration with other funders to reduce redundant funding. A voluntary consortium of international funders is underway to encourage further progress, including additional self-assessment and public sharing for accountability. These findings indicate PCORI has undertaken efforts to align its funding practices with international recommendations to ensure the value of public dollars invested in research.  Further efforts will likely require additional coordination and collaboration between funders and stakeholders.

Noncommercial US Funders' Policies on Trial Registration, Access to Summary Results, and Individual Patient Data Availability.

Importance: Incomplete information about existing research is an underlying cause of research waste. National and international initiatives and requirements have been launched to address this issue. Objectives: To characterize current clinical trial transparency policies among the largest noncommercial US funders and examine whether the policies are concordant with international funders. Design, Setting, and Participants: This retrospective review of public information used methods developed for documenting funder policies internationally; 2 researchers searched each funder's website and Google between May and November 2018 to locate trial transparency policies for 10 top US funders. Key informants at each funding organization were contacted by email and given 3 or more weeks to review and confirm or correct the findings. Nonresponders were contacted 2 or more additional times. Descriptive statistics were calculated to summarize the findings. The study was conducted using publicly available policy information with findings confirmed by funder representatives where possible. Participants included top 10 noncommercial US health research funders with the highest reported investment in health research (2013 dollars) who fund clinical trials. Data analysis was conducted from November 6, 2018, to November 23, 2018. Exposures: Availability of policies addressing each of the 3 key trial transparency domains as specified by the World Health Organization in 2017. Main Outcomes and Measures: Independent assessment by 2 investigators of availability (yes or no) of a policy addressing registration for trials, sharing of summary results, and individual participant data sharing activities; requirements (yes, no, or supportive statement) of these policies in terms of completeness, timeliness, public access, and provision of additional technical or financial support to meet data sharing requirements; description (yes or no) of internal monitoring for policy adherence. Results: All 10 funders acknowledged the outreach. One funder who indicated that less than 1% of their research funding goes to clinical trials was removed. Six (67%) of the remaining 9 top US funders have a publicly available written policy for all 3 major trial transparency domains. The most comprehensive trial transparency practice among US funders addresses summary results sharing as follows: 8 of 9 US funders (89%) have a policy, 5 of 9 US funders (56%) require reporting of summary results within 1 year, and 6 of 9 US funders (67%) monitor compliance with their summary results sharing policy. For clinical trial registration, 7 of 9 US funders (78%) have a policy and 5 of 9 US funders (56%) require registration and monitor trial registration to measure adherence to the policy. Conclusions and Relevance: In this study, overall the proportion of US funders with policies and practices to support trial transparency in this sample was similar or compared favorably with the larger international sample of noncommercial funders recently reported.

Methodological considerations for detection of terrestrial small-body salamander eDNA and implications for biodiversity conservation.

Environmental DNA (eDNA) can be used as an assessment tool to detect populations of threatened species and provide fine-scale data required to make management decisions. The objectives of this project were to use quantitative PCR (qPCR) to: (i) detect spiked salamander DNA in soil, (ii) quantify eDNA degradation over time, (iii) determine detectability of salamander eDNA in a terrestrial environment using soil, faeces, and skin swabs, (iv) detect salamander eDNA in a mesocosm experiment. Salamander eDNA was positively detected in 100% of skin swabs and 66% of faecal samples and concentrations did not differ between the two sources. However, eDNA was not detected in soil samples collected from directly underneath wild-caught living salamanders. Salamander genomic DNA (gDNA) was detected in all qPCR reactions when spiked into soil at 10.0, 5.0, and 1.0 ng/g soil and spike concentration had a significant effect on detected concentrations. Only 33% of samples showed recoverable eDNA when spiked with 0.25 ng/g soil, which was the low end of eDNA detection. To determine the rate of eDNA degradation, gDNA (1 ng/g soil) was spiked into soil and quantified over seven days. Salamander eDNA concentrations decreased across days, but eDNA was still amplifiable at day 7. Salamander eDNA was detected in two of 182 mesocosm soil samples over 12 weeks (n = 52 control samples; n = 65 presence samples; n = 65 eviction samples). The discrepancy in detection success between experiments indicates the potential challenges for this method to be used as a monitoring technique for small-bodied wild terrestrial salamander populations.

Gatekeepers for pragmatic clinical trials.

To successfully implement a pragmatic clinical trial, investigators need access to numerous resources, including financial support, institutional infrastructure (e.g. clinics, facilities, staff), eligible patients, and patient data. Gatekeepers are people or entities who have the ability to allow or deny access to the resources required to support the conduct of clinical research. Based on this definition, gatekeepers relevant to the US clinical research enterprise include research sponsors, regulatory agencies, payers, health system and other organizational leadership, research team leadership, human research protections programs, advocacy and community groups, and clinicians. This article provides a framework to help guide gatekeepers' decision-making related to the use of resources for pragmatic clinical trials. Relevant ethical considerations for gatekeepers include (1) concern for the interests of individuals, groups, and communities affected by the gatekeepers' decisions, including protection from harm and maximization of benefits; (2) advancement of organizational mission and values; and (3) stewardship of financial, human, and other organizational resources. Separate from these ethical considerations, gatekeepers' actions will be guided by relevant federal, state, and local regulations. This framework also suggests that to further enhance the legitimacy of their decision-making, gatekeepers should adopt transparent processes that engage relevant stakeholders when feasible and appropriate. We apply this framework to the set of gatekeepers responsible for making decisions about resources necessary for pragmatic clinical trials in the United States, describing the relevance of the criteria in different situations and pointing out where conflicts among the criteria and relevant regulations may affect decision-making. Recognition of the complex set of considerations that should inform decision-making will guide gatekeepers in making justifiable choices regarding the use of limited and valuable resources.

Capacity of, and Demand for, Pediatric Nurse Practitioner Educational Programs: A Missing Piece of the Workforce Puzzle.

BACKGROUND: In contrast to family nurse practitioners and other adult nurse practitioners, the percentage of new pediatric nurse practitioners (PNPs) graduating each year has not increased. PURPOSE: The aim of this study was to determine whether the marginal increase in the pipeline for PNPs is related to a limit in the capacity of educational programs or whether unfilled student openings exist. METHODS: Self-administered survey of program directors at all recognized PNP educational programs in the United States. RESULTS: Approximately 10% of PNP programs in the United States were either closed, put on hold, or did not have new graduates in the last 3 years. Even with these closures, over 25% of active programs did not fill all available positions for the class entering in 2012. CONCLUSION: Despite evidence that demonstrates plans by employers to hire a greater number of PNPs in a variety of clinical venues including pediatric hospitals, primary care and subspecialty pediatric practices, the PNP pipeline has remained relatively stagnant. More than one third of program directors do not believe that their PNP program is currently at capacity, indicating that underutilized capacity to educate PNPs is a hindrance to meeting the current and future demands for these professionals.

Capacity of, and Demand for, Neonatal Nurse Practitioner Educational Programs: A Missing Piece of the Workforce Puzzle.

BACKGROUND: Studies have demonstrated a dramatic increase in the number of new nurse practitioners (NPs) overall completing NP education each year. However, NPs who provide specialized care to children have not experienced increases in their pipeline at all. This has resulted in shortages of neonatal nurse practitioners (NNPs). PURPOSE: The aim of this study was to gain a greater understanding of the NNP pipeline and potential for increasing the number of new NNPs graduating each year. METHODS: Telephone survey of all NNP educational programs. DISCUSSION: Approximately one fourth of all NNP education programs had closed over the past several years. This is despite a strong job market, planned increases in hiring NNPs, and a seemingly growing shortage of NNPs. CONCLUSION: Problems with the NNP pipeline are not due to a lack of capacity of existing programs, but rather to difficulties in increasing the enrollment demand.

Pediatric nurse practitioners: influences on career choice.

BACKGROUND: The demand for hiring pediatric nurse practitioners (PNPs) is strong. However, the number of newly educated PNPs has remained relatively flat during the past several years. Understanding the rationale and timing for the decision to pursue this profession is essential to having a positive impact on increasing the future workforce. METHODS: A mail survey of all new PNPs certified between January 2009 and July 2011 (N = 1040) was conducted. RESULTS: The response rate was 79.9%. Nearly half of all respondents (45%, N = 314) reported that they work in outpatient general pediatrics, 26% (N = 184) in outpatient subspecialty pediatrics, and 22% (N = 152) in inpatient settings. More than one third (36%, N = 253) spend most of their time in a private practice. Forty percent (N = 307) reported that they decided to pursue education as an advanced practice nurse while in practice as a registered nurse (RN), and 38% (N = 289) made the decision before pursuing RN education. CONCLUSIONS: Efforts to increase the PNP pipeline will need to be directed both to students during their RN education and to creating opportunities for current RNs to pursue advanced practice nurse education that is focused on children.