Reporting standard for describing first responder systems, smartphone alerting systems, and AED networks.

Standardized reporting of data is crucial for out-of-hospital cardiac arrest (OHCA) research. While the implementation of first responder systems dispatching volunteers to OHCA is encouraged, there is currently no uniform reporting standard for describing these systems. A steering committee established a literature search to identify experts in smartphone alerting systems. These international experts were invited to a conference held in Hinterzarten, Germany, with 40 researchers from 13 countries in attendance. Prior to the conference, participants submitted proposals for parameters to be included in the reporting standard. The conference comprised five workshops covering different aspects of smartphone alerting systems. Proposed parameters were discussed, clarified, and consensus was achieved using the Nominal Group Technique. Participants voted in a modified Delphi approach on including each category as a core or supplementary element in the reporting standard. Results were presented, and a writing group developed definitions for all categories and items, which were sent to participants for revision and final voting using LimeSurvey web-based software. The resulting reporting standard consists of 68 core items and 21 supplementary items grouped into five topics (first responder system, first responder network, technology/algorithm/strategies, reporting data, and automated external defibrillators (AED)). This proposed reporting standard generated by an expert opinion group fills the gap in describing first responder systems. Its adoption in future research will facilitate comparison of systems and research outcomes, enhancing the transfer of scientific findings to clinical practice.

Are first responders first? The rally to the suspected out-of-hospital cardiac arrest.

BACKGROUND: Time is the crucial factor in the "chain of survival" treatment concept for out-of-hospital cardiac arrest (OHCA). We aimed to measure different response time intervals by comparing emergency medical system (EMS), fire fighters and smartphone aided volunteer responders. METHODS: In two large Swedish regions, volunteer responders were timed from the alert until they arrived at the scene of the suspected OHCA. The first arriving volunteer responders who tried to fetch an automated external defibrillator (AED-responder) and who ran to perform bystander cardiopulmonary resuscitation (CPR-responder) were compared to both the first arriving EMS and fire fighters. Three-time intervals were measured, from call to dispatch, the unit response time (from dispatch to arrival) and the total response time. RESULTS: During 22 months, 2631 suspected OHCAs were included. The median time from call to dispatch was in minutes 1.8 (95% CI = 1.7-1.8) for EMS, 2.9 (95% CI = 2.8-3.0) for fire-fighters and 3.0 (95% CI = 2.9-3.1) for volunteer responders. The median unit response time was 8.3 (95% CI = 8.1-8.5) for EMS, 6.8 (95% CI = 6.7-6.9) for fire fighters and 6.0 (95% CI = 5.7-6.2) for AED-responders and 4.6 (95% CI = 4.5-4.8) for CPR-responders. The total response time was 10.4 (95% CI = 10.1-10.6) for EMS, 10.2 (95% CI = 9.9-10.4) for fire fighters, 9.6 (95% CI = 9.1-9.8) for AED-responders and 8.2 (95% CI = 8.0-8.3) for CPR-responders. CONCLUSION: First arriving volunteer responders had the shortest unit response time when compared to both fire fighters and EMS, however this advantage was reduced by delays introduced at the dispatch center. Earlier automatic dispatch should be considered in further studies.

Characteristics and motivational factors for joining a lay responder system dispatch to out-of-hospital cardiac arrests.

BACKGROUND: There has been in increase in the use of systems for organizing lay responders for suspected out-of-hospital cardiac arrests (OHCAs) dispatch using smartphone-based technology. The purpose is to increase survival rates; however, such systems are dependent on people's commitment to becoming a lay responder. Knowledge about the characteristics of such volunteers and their motivational factors is lacking. Therefore, we explored characteristics and quantified the underlying motivational factors for joining a smartphone-based cardiopulmonary resuscitation (CPR) lay responder system. METHODS: In this descriptive cross-sectional study, 800 consecutively recruited lay responders in a smartphone-based mobile positioning first-responder system (SMS-lifesavers) were surveyed. Data on characteristics and motivational factors were collected, the latter through a modified version of the validated survey "Volunteer Motivation Inventory" (VMI). The statements in the VMI, ranked on a Likert scale (1-5), corresponded to(a) intrinsic (an inner belief of doing good for others) or (b) extrinsic (earning some kind of reward from the act) motivational factors. RESULTS: A total of 461 participants were included in the final analysis. Among respondents, 59% were women, 48% between 25 and 39 years of age, 37% worked within health care, and 66% had undergone post-secondary school. The most common way (44%) to learn about the lay responder system was from a CPR instructor. A majority (77%) had undergone CPR training at their workplace. In terms of motivation, where higher scores reflect greater importance to the participant, intrinsic factors scored highest, represented by the category values (mean 3.97) followed by extrinsic categories reciprocity (mean 3.88) and self-esteem (mean 3.22). CONCLUSION: This study indicates that motivation to join a first responder system mainly depends on intrinsic factors, i.e. an inner belief of doing good, but there are also extrinsic factors, such as earning some kind of reward from the act, to consider. Focusing information campaigns on intrinsic factors may be the most important factor for successful recruitment. When implementing a smartphone-based lay responder system, CPR instructors, as a main information source to potential lay responders, as well as the workplace, are crucial for successful recruitment.

Copepods drive large-scale trait-mediated effects in marine plankton.

Fear of predation may influence food webs more than actual predation. However, the mechanisms and magnitude of nonconsumptive predator effects are largely unknown in unicellular-dominated food webs such as marine plankton. We report a general mechanism of chemically induced predator effects in marine plankton. Copepods, the most abundant zooplankton in the oceans, imprint seawater with unique polar lipids-copepodamides-which trigger toxin production and bioluminescence in harmful dinoflagellates. We show that copepodamides also elicit defensive traits in other phytoplankton, inducing the harmful algal bloom-forming diatom Pseudo-nitzschia seriata to produce 10 times more toxins, and colony-forming diatoms to decrease colony size by half. A 1-year study in the northeast Atlantic revealed that natural copepodamide concentrations are high enough to induce harmful algal toxins and size reduction in dominant primary producers when copepods are abundant. We conclude that copepodamides will structure marine plankton toward smaller, more defended life forms on basin-wide scales.

Microplastics in a freshwater mussel (Anodonta anatina) in Northern Europe.

Alarming amounts of microplastics have recently been shown to accumulate in the environment. Recent focus has been on synthetic material contaminating the marine environment, while effects on freshwater habitats and organisms have received less attention. We here confirm and analyse occurrence of microplastics in the duck mussel, Anodonta anatina, in a Swedish river. All analysed mussels contained microplastics, and the number of microplastic debris found in the mussels increased with mussel size. In addition, we demonstrate higher concentrations of microplastics downstream urban areas with wastewater treatment plants compared to a rural upstream location. Both fibres and particles were found in the mussels, indicating that the emissions of these pollutants may have varying origin. Our study indicates that microplastics can be suspended in the water column in streams and that concentrations are higher downstream anthropogenic activity. We discuss our results in light of potential pathways in rural versus surrounding arable land, and highlight a number of required research directions in the aquatic system.

Ex Vivo Generation of Donor Antigen-Specific Immunomodulatory Cells: A Comparison Study of Anti-CD80/86 mAbs and CTLA4-lg Costimulatory Blockade.

Adoptive transfer of alloantigen-specific immunomodulatory cells generated ex vivo with anti-CD80/CD86 mAbs (2D10.4/IT2.2) holds promise for operational tolerance after transplantation. However, good manufacturing practice is required to allow widespread clinical application. Belatacept, a clinically approved cytotoxic T-lymphocyte antigen 4-immunoglobulin that also binds CD80/CD86, could be an alternative agent for 2D10.4/IT2.2. With the goal of generating an optimal cell treatment with clinically approved reagents, we evaluated the donor-specific immunomodulatory effects of belatacept- and 2D10.4/IT2.2-generated immunomodulatory cells. Immunomodulatory cells were generated by coculturing responder human peripheral blood mononuclear cells (PBMCs) (50 × 106 cells) with irradiated donor PBMCs (20 × 106 cells) from eight human leukocyte antigen-mismatched responder-donor pairs in the presence of either 2D10.4/IT2.2 (3 µg/106 cells) or belatacept (40 µg/106 cells). After 14 days of coculture, the frequencies of CD4+ T cells, CD8+ T cells, and natural killer cells as well as interferon gamma (IFN-γ) production in the 2D10.4/IT2.2- and belatacept-treated groups were lower than those in the control group. The percentage of CD19+ B cells was higher in the 2D10.4/IT2.2- and belatacept-treated groups than in the control group. The frequency of CD4+CD25+CD127lowFOXP3+ T cells increased from 4.1±1.0% (preculture) to 7.1±2.6% and 7.3±2.6% (day 14) in the 2D10.4/IT2.2- and belatacept-treated groups, respectively (p<0.05). Concurrently, delta-2 FOXP3 mRNA expression increased significantly. Compared with cells derived from the no-antibody treated control group, cells generated from both the 2D10.4/IT2.2- and belatacept-treated groups produced lower IFN-γ and higher interleukin-10 levels in response to donor-antigens, as detected by enzyme-linked immunospot. Most importantly, 2D10.4/IT2.2- and belatacept-generated cells effectively impeded the proliferative responses of freshly isolated responder PBMCs against donor-antigens. Our results indicate that belatacept-generated donor-specific immunomodulatory cells possess comparable phenotypes and immunomodulatory efficacies to those generated with 2D10.4/IT2.2. We suggest that belatacept could be used for ex vivo generation of clinical grade alloantigen-specific immunomodulatory cells for tolerance induction after transplantation.

Physiologically based pharmacokinetic modeling in regulatory decision-making at the European Medicines Agency.

Physiologically based pharmacokinetic (PBPK) modeling is a valuable tool in drug development and regulatory assessment, as it offers the opportunity to simulate the pharmacokinetics of a compound, with a mechanistic understanding, in a variety of populations and situations. This work reviews the use and impact of such modeling in selected regulatory procedures submitted to the European Medicines Agency (EMA) before the end of 2015, together with its subsequent reflection in public documents relating to the assessment of these procedures. It is apparent that the reference to PBPK modeling in regulatory public documents underrepresents its use. A positive trend over time of the number of PBPK models submitted is shown, and in a number of cases the results of these may impact the decision-making process or lead to recommendations in the product labeling. These results confirm the need for regulatory guidance in this field, which is currently under development by the EMA.

Electrochemical Measurements of Optogenetically Stimulated Quantal Amine Release from Single Nerve Cell Varicosities in Drosophila Larvae.

The nerve terminals found in the body wall of Drosophila melanogaster larvae are readily accessible to experimental manipulation. We used the light-activated ion channel, channelrhodopsin-2, which is expressed by genetic manipulation in Type II varicosities to study octopamine release in Drosophila. We report the development of a method to measure neurotransmitter release from exocytosis events at individual varicosities in the Drosophila larval system by amperometry. A microelectrode was placed in a region of the muscle containing a varicosity and held at a potential sufficient to oxidize octopamine and the terminal stimulated by blue light. Optical stimulation of Type II boutons evokes exocytosis of octopamine, which is detected through oxidization at the electrode surface. We observe 22700±4200 molecules of octopamine released per vesicle. This system provides a genetically accessible platform to study the regulation of amine release at an intact synapse.

microRNA expression signatures of gastrointestinal stromal tumours: associations with imatinib resistance and patient outcome.

BACKGROUND: Gastrointestinal stromal tumour (GIST) is mainly initialised by receptor tyrosine kinase gene mutations. Although the tyrosine kinase inhibitor imatinib mesylate considerably improved the outcome of patients, imatinib resistance still remains a major therapeutic challenge in GIST therapy. Herein we evaluated the clinical impact of microRNAs in imatinib-treated GISTs. METHODS: The expression levels of microRNAs were quantified using microarray and RT-qPCR in GIST specimens from patients treated with neoadjuvant imatinib. The functional roles of miR-125a-5p and PTPN18 were evaluated in GIST cells. PTPN18 expression was quantified by western blotting in GIST samples. RESULTS: We showed that overexpression levels of miR-125a-5p and miR-107 were associated with imatinib resistance in GIST specimens. Functionally, miR-125a-5p expression modulated imatinib sensitivity in GIST882 cells with a homozygous KIT mutation but not in GIST48 cells with double KIT mutations. Overexpression of miR-125a-5p suppressed PTPN18 expression, and silencing of PTPN18 expression increased cell viability in GIST882 cells upon imatinib treatment. PTPN18 protein levels were significantly lower in the imatinib-resistant GISTs and inversely correlated with miR-125a-5p. Furthermore, several microRNAs were significantly associated with metastasis, KIT mutational status and survival. CONCLUSIONS: Our findings highlight a novel functional role of miR-125a-5p on imatinib response through PTPN18 regulation in GIST.

Evaluation of various static in vitro-in vivo extrapolation models for risk assessment of the CYP3A inhibition potential of an investigational drug.

Nine static models (seven basic and two mechanistic) and their respective cutoff values used for predicting cytochrome P450 3A (CYP3A) inhibition, as recommended by the US Food and Drug Administration and the European Medicines Agency, were evaluated using data from 119 clinical studies with orally administered midazolam as a substrate. Positive predictive error (PPE) and negative predictive error (NPE) rates were used to assess model performance, based on a cutoff of 1.25-fold change in midazolam area under the curve (AUC) by inhibitor. For reversible inhibition, basic models using total or unbound systemic inhibitor concentration [I] had high NPE rates (46-47%), whereas those using intestinal luminal ([I]gut) values had no NPE but a higher PPE. All basic models for time-dependent inhibition had no NPE and reasonable PPE rates (15-18%). Mechanistic static models that incorporate all interaction mechanisms and organ specific [I] values (enterocyte and hepatic inlet) provided a higher predictive precision, a slightly increased NPE, and a reasonable PPE. Various cutoffs for predicting the likelihood of CYP3A inhibition were evaluated for mechanistic models, and a cutoff of 1.25-fold change in midazolam AUC appears appropriate.

Capillary electrophoresis-mass spectrometry-based detection of drugs and neurotransmitters in Drosophila brain.

Capillary electrophoresis coupled to mass spectrometry has been used to determine the in vivo concentrations of the neuroactive drug, methylphenidate, and a metabolite in the heads of the fruit fly, Drosophila melanogaster . These concentrations, evaluated at the site of action, the brain, have been correlated with orally administrated methylphenidate. D. melanogaster has a relatively simple nervous system but possesses high-order brain functions similar to humans; thus, it has been used as a common model system in biological and genetics research. Methylphenidate has been used to mediate cocaine addiction due to its lower pharmacokinetics, which results in fewer addictive and reinforcing effects than cocaine; the effects of the drug on the nervous system, however, have not been fully understood. In addition to measurements of drug concentration, the method has been used to examine drug-dose dependence on the levels of several primary biogenic amines. Higher in vivo concentration of methylphenidate is observed with increasing feeding doses up to 25 mM methylphenidate. Furthermore, administrated methylphenidate increases the drug metabolism activity and the neurotransmitter levels; however, this increase appears to saturate at a feeding dose of 20 mM. The method developed for the fruit fly provides a new tool to evaluate the concentration of administered drug at the site of action and provides information concerning the effect of methylphenidate on the nervous system.

Transporter studies in drug development: experience to date and follow-up on decision trees from the International Transporter Consortium.

The International Transporter Consortium (ITC) organized a second workshop in March 2012 to expand on the themes developed during the inaugural ITC workshop held in 2008. The final session of the workshop provided perspectives from regulatory and industry-based scientists, with input from academic scientists, and focused primarily on the decision trees published from the first workshop. These decision trees have become a central part of subsequent regulatory drug-drug interaction (DDI) guidances issued over the past few years.

Oral administration of methylphenidate blocks the effect of cocaine on uptake at the Drosophila dopamine transporter.

Although our understanding of the actions of cocaine in the brain has improved, an effective drug treatment for cocaine addiction has yet to be found. Methylphenidate binds the dopamine transporter and increases extracellular dopamine levels in mammalian central nervous systems similar to cocaine, but it is thought to elicit fewer addictive and reinforcing effects owing to slower pharmacokinetics for different routes of administration between the drugs. This study utilizes the fruit fly model system to quantify the effects of oral methylphenidate on dopamine uptake during direct cocaine exposure to the fly CNS. The effect of methylphenidate on the dopamine transporter has been explored by measuring the uptake of exogenously applied dopamine. The data suggest that oral consumption of methylphenidate inhibits the Drosophila dopamine transporter and the inhibition is concentration dependent. The peak height increased to 150% of control when cocaine was used to block the dopamine transporter for untreated flies but only to 110% for methylphenidate-treated flies. Thus, the dopamine transporter is mostly inhibited for the methylphenidate-fed flies before the addition of cocaine. The same is true for the rate of the clearance of dopamine measured by amperometry. For untreated flies the rate of clearance changes 40% when the dopamine transporter is inhibited with cocaine, and for treated flies the rate changes only 10%. The results were correlated to the in vivo concentration of methylphenidate determined by CE-MS. Our data suggest that oral consumption of methylphenidate inhibits the Drosophila dopamine transporter for cocaine uptake, and the inhibition is concentration dependent.

Freeze-drying as sample preparation for micellar electrokinetic capillary chromatography-electrochemical separations of neurochemicals in Drosophila brains.

Micellar electrokinetic capillary chromatography with electrochemical detection has been used to quantify biogenic amines in freeze-dried brains of Drosophila melanogaster. Freeze-drying samples offers a way to preserve the biological sample while making dissection of these tiny samples easier and faster. Fly samples were extracted in cold acetone and dried in a rotary evaporator. Extraction and drying times were optimized in order to avoid contamination by red pigment from the fly eyes and still have intact brain structures. Single freeze-dried fly brain samples were found to produce representative electropherograms as a single hand-dissected brain sample. With utilization of the faster dissection time that freeze-drying affords, the number of brains in a fixed homogenate volume can be increased to concentrate the sample. Thus, concentrated brain samples containing five or fifteen preserved brains were analyzed for their neurotransmitter content, and four analytes; N-acetyloctopamine, N-acetylserotonin, N-acetyltyramine, and N-acetyldopamine were found to correspond well with previously reported values.

Predicting drug interaction potential with a physiologically based pharmacokinetic model: a case study of telithromycin, a time-dependent CYP3A inhibitor.

Telithromycin is a substrate and an inhibitor of cytochrome P450 3A (CYP3A4), with dose- and time-dependent nonlinear pharmacokinetics (PK). We hypothesized that the time-dependent inhibition (TDI) of CYP3A4 was responsible for the nonlinear PK of telithromycin and then used physiologically based PK (PBPK) modeling and simulation to verify this mechanism. Telithromycin PBPK models integrating in vitro, in silico, and in vivo PK data ruled out the contribution of enzyme/transporter saturation and suggested that TDI is a plausible mechanism for PK nonlinearity. The model successfully predicted the clinical interaction with the CYP3A4 substrate midazolam, as verified by external data not used for the model-building (intravenous (i.v.) and oral (p.o.) midazolam area under the concentration-time curve (AUC) ratio with/without concurrent telithromycin administration: 3.26 and 6.72 predicted vs. 2.20 and 6.11 observed, respectively). Models assuming reversible inhibition failed to predict such strong CYP3A4 inhibition. In the absence of in vitro TDI data, a PBPK model can be used to incorporate TDI mechanisms based on nonlinear PK data to predict clinical drug-drug interactions.

Digital gene expression profiling of primary acute lymphoblastic leukemia cells.

We determined the genome-wide digital gene expression (DGE) profiles of primary acute lymphoblastic leukemia (ALL) cells from 21 patients taking advantage of 'second-generation' sequencing technology. Patients included in this study represent four cytogenetically distinct subtypes of B-cell precursor (BCP) ALL and T-cell lineage ALL (T-ALL). The robustness of DGE combined with supervised classification by nearest shrunken centroids (NSC) was validated experimentally and by comparison with published expression data for large sets of ALL samples. Genes that were differentially expressed between BCP ALL subtypes were enriched to distinct signaling pathways with dic(9;20) enriched to TP53 signaling, t(9;22) to interferon signaling, as well as high hyperdiploidy and t(12;21) to apoptosis signaling. We also observed antisense tags expressed from the non-coding strand of ~50% of annotated genes, many of which were expressed in a subtype-specific pattern. Antisense tags from 17 gene regions unambiguously discriminated between the BCP ALL and T-ALL subtypes, and antisense tags from 76 gene regions discriminated between the 4 BCP subtypes. We observed a significant overlap of gene regions with alternative polyadenylation and antisense transcription (P<1 × 10(-15)). Our study using DGE profiling provided new insights into the RNA expression patterns in ALL cells.

Applications of physiologically based pharmacokinetic (PBPK) modeling and simulation during regulatory review.

Physiologically based pharmacokinetic (PBPK) modeling and simulation is a tool that can help predict the pharmacokinetics of drugs in humans and evaluate the effects of intrinsic (e.g., organ dysfunction, age, genetics) and extrinsic (e.g., drug-drug interactions) factors, alone or in combinations, on drug exposure. The use of this tool is increasing at all stages of the drug development process. This report reviews recent instances of the use of PBPK in decision-making during regulatory review. The examples are based on Center for Drug Evaluation and Research reviews of several submissions for investigational new drugs (INDs) and new drug applications (NDAs) received between July 2008 and June 2010. The use of PBPK modeling and simulation facilitated the following types of decisions: the need to conduct specific clinical pharmacology studies, specific study designs, and appropriate labeling language. The report also discusses the challenges encountered when PBPK modeling and simulation were used in these cases and recommends approaches to facilitating full utilization of this tool.

Biogenic amines in microdissected brain regions of Drosophila melanogaster measured with micellar electrokinetic capillary chromatography-electrochemical detection.

Micellar electrokinetic chromatography with electrochemical detection has been used to quantify biogenic amines in microdissected Drosophila melanogaster brains and brain regions. The effects of pigment from the relatively large fly eyes on the separation have been examined to find that the red pigment from the compound eye masks much of the signal from biogenic amines. The brains of white mutant flies, which have characteristically low pigment in the eyes, have a significantly simplified separation profile in comparison to the red-eyed, wild-type, Canton S fly. Yet, the white mutant flies were found to have significantly less amounts of dopamine, l-3,4-dihydroxyphenylalanine (L-DOPA), salsolinol, and N-acetyltyramine in their dissected brains when compared to dissected brains of Canton S flies. In addition, significant variation has been observed in the dissected brains between individual flies that might be related to changes in neurotransmitter turnover. The transgenic GFP fly line (TH-GFP), for which the overall profile of biogenic amines is not found to be significantly different from Canton S, can be used to visualize the location of dopamine neurons. Biogenic amines were then quantified in three brain regions observed to have dopamine levels, the central brain, optic lobes, and posterior superiormedial protocerebrum (PPM1) region.

Determination of salsolinol, norsalsolinol, and twenty-one biogenic amines using micellar electrokinetic capillary chromatography-electrochemical detection.

Micellar electrokinetic chromatography coupled to amperometric electrochemical detection was used to resolve and then quantify biogenic amines and metabolites within the fruit fly Drosophila melanogaster. A new separation scheme was devised to allow resolution of 24 compounds of interest. This was accomplished by precisely controlling the amount of base added to the background buffer, optimizing the resolution of the separation, and then calculating the pH. Here we focused on measurements of six of the analytes that are thought to be involved in the response to alcohol, dopamine, salsolinol, norsalsolinol, N-acetyloctopamine, octopamine, and N-acetyldopamine. These were identified and quantified within the fly head. We believe that the identification of salsolinol and norsalsolinol in the fly brain is novel.

Micellar capillary electrophoresis--electrochemical detection of neurochemicals from Drosophila.

The fruit fly is one of the most heavily studied model organisms for genetics research and has significantly contributed to the molecular, cellular, and evolutionary understandings of human behavior. Recent research in the analytical chemistry of the fruit fly has focused on developing methods to obtain highly sensitive chemical quantification information of Drosophila melanogaster, especially looking at the nervous system. We provide a brief overview of work in the area of CE of the fly head and brain.