Postnatal neuronal Bace1 deletion impairs neuroblast and oligodendrocyte maturation.

Beta amyloid cleaving enzyme 1 (BACE1) is largely expressed by neurons and is the sole ß-secretase for initiating the production of neuronal ß-amyloid peptides (Aß). To fully understand the physiological functions of neuronal BACE1, we used mouse genetic approach coupled with unbiased single nucleus RNA sequencing (snRNAseq) to investigate how targeted deletion of Bace1 in neurons, driven by Thy-1-Cre recombinase, would affect functions in the nervous system. Our transcriptome results revealed that BACE1 is essential for maturation of neural precursor cells and oligodendrocytes in mice. RNA velocity analysis confirmed deficit in the trajectory of neuroblasts in reaching the immature granule neuron state in young Bace1fl/fl; Thy1-cre mice. Further analysis of differential gene expression indicated changes in genes important for SNARE signaling, tight junction signaling, synaptogenesis and insulin secretion pathways. Morphological studies revealed a hypomyelination in Bace1fl/fl;Thy1-cre sciatic nerves, but no detectable myelination changes in the corpus callosum, despite clear reduction in myelination proteins in the brain. Functional studies showed reduction in long-term potential, defects in synaptogenesis and learning behavioral. Altogether, our results show that neuronal BACE1 is critical for optimal development of central and peripheral nervous system, and inhibition of neuronal BACE1 will result in deficits in synaptic functions and cognitive behaviors.

ER-stress response in retinal Müller glia occurs significantly earlier than amyloid pathology in the Alzheimer's mouse brain and retina.

Alzheimer's Disease (AD) pathogenesis is thought to begin up to 20 years before cognitive symptoms appear, suggesting the need for more sensitive diagnostic biomarkers of AD. In this report, we demonstrated pathological changes in retinal Müller glia significantly earlier than amyloid pathology in AD mouse models. By utilizing the knock-in NLGF mouse model, we surprisingly discovered an increase in reticulon 3 (RTN3) protein levels in the NLGF retina as early as postnatal day 30 (P30). Despite RTN3 being a canonically neuronal protein, this increase was noted in the retinal Müller glia, confirmed by immunohistochemical characterization. Further unbiased transcriptomic assays of the P30 NLGF retina revealed that retinal Müller glia were the most sensitive responding cells in this mouse retina, compared with other cell types including photoreceptor cells and ganglion neurons. Pathway analyses of differentially expressed genes in glia cells showed activation of ER stress response via the upregulation of unfolded protein response (UPR) proteins such as ATF4 and CHOP. Early elevation of RTN3 in response to challenges by toxic Aß likely facilitated UPR. Altogether, these findings suggest that Müller glia act as a sentinel for AD pathology in the retina and should aid for both intervention and diagnosis.

The CX3CL1 intracellular domain exhibits neuroprotection via insulin receptor/insulin-like growth factor receptor signaling.

CX3CL1, also known as fractalkine, is best known for its signaling activity through interactions with its cognate receptor CX3CR1. However, its intrinsic function that is independent of interaction with CX3CR1 remains to be fully understood. We demonstrate that the intracellular domain of CX3CL1 (CX3CL1-ICD), generated upon sequential cleavages by α-/ß-secretase and γ-secretase, initiates a back signaling activity, which mediates direct signal transmission to gene expression in the nucleus. To study this, we fused a synthetic peptide derived from CX3CL1-ICD, named Tet34, with a 13-amino acid tetanus sequence at the N terminus to facilitate translocation into neuronal cells. We show that treatment of mouse neuroblastoma Neuro-2A cells with Tet34, but not its scrambled control (Tet34s), induced cell proliferation, as manifested by changes in protein levels of transcription factors and progrowth molecules cyclin D1, PCNA, Sox5, and Cdk2. Further biochemical assays reveal elevation of phosphorylated insulin receptor ß subunit, insulin-like growth factor-1 receptor ß subunit, and insulin receptor substrates as well as activation of proliferation-linked kinase AKT. In addition, transgenic mice overexpressing membrane-anchored C-terminal CX3CL1 also exhibited activation of insulin/insulin-like growth factor-1 receptor signaling. Remarkably, we found that this Tet34 peptide, but not Tet34s, protected against endoplasmic reticulum stress and cellular apoptosis when Neuro-2A cells were challenged with toxic oligomers of ß-amyloid peptide or hydrogen peroxide. Taken together, our results suggest that CX3CL1-ICD may have translational potential for neuroprotection in Alzheimer's disease and for disorders resulting from insulin resistance.

Genetic variants in the bipolar disorder risk locus SYNE1 that affect CPG2 expression and protein function.

Bipolar disorder (BD) is a common mood disorder characterized by recurrent episodes of mania and depression. Both genetic and environmental factors have been implicated in BD etiology, but the biological underpinnings remain elusive. Recently, genome-wide association studies (GWAS) of neuropsychiatric disorders have identified a risk locus for BD containing the SYNE1 gene, a large gene encoding multiple proteins. The BD association signal spans, almost exclusively, the part of SYNE1 encoding CPG2, a brain-specific protein localized to excitatory postsynaptic sites, where it regulates glutamate receptor internalization. Here we show that CPG2 protein levels are significantly decreased in postmortem brain tissue from BD patients, as compared to control subjects, as well as schizophrenia and depression patients. We identify genetic variants within the postmortem brains that map to the CPG2 promoter region, and show that they negatively affect gene expression. We also identify missense single nucleotide polymorphisms (SNPs) in CPG2 coding regions that affect CPG2 expression, localization, and synaptic function. Our findings link genetic variation in the CPG2 region of SYNE1 with a mechanism for glutamatergic synapse dysfunction that could underlie susceptibility to BD in some individuals. Few GWAS hits in human genetics for neuropsychiatric disorders to date have afforded such mechanistic clues. Further, the potential for genetic distinction of susceptibility to BD from other neuropsychiatric disorders with overlapping clinical traits holds promise for improved diagnostics and treatment of this devastating illness.

Recent advances in pre-clinical diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia with currently no known cures or disease modifying treatments (DMTs), despite much time and effort from the field. Diagnosis and intervention of AD during the early pre-symptomatic phase of the disease is thought to be a more effective strategy. Therefore, the detection of biomarkers has emerged as a critical tool for monitoring the effect of new AD therapies, as well as identifying patients most likely to respond to treatment. The establishment of the amyloid/tau/neurodegeneration (A/T/N) framework in 2018 has codified the contexts of use of AD biomarkers in neuroimaging and bodily fluids for research and diagnostic purposes. Furthermore, a renewed drive for novel AD biomarkers and innovative methods of detection has emerged with the goals of adding additional insight to disease progression and discovery of new therapeutic targets. The use of biomarkers has accelerated the development of AD drugs and will bring new therapies to patients in need. This review highlights recent methods utilized to diagnose antemortem AD.

Activated CX3CL1/Smad2 Signals Prevent Neuronal Loss and Alzheimer's Tau Pathology-Mediated Cognitive Dysfunction.

Neurofibrillary tangles likely cause neurodegeneration in Alzheimer's disease (AD). We demonstrate that the CX3CL1 C-terminal domain can upregulate neurogenesis, which may ameliorate neurodegeneration. Here we generated transgenic (Tg-CX3CL1) mice by overexpressing CX3CL1 in neurons. Tg-CX3CL1 mice exhibit enhanced neurogenesis in both subgranular and subventricular zones. This enhanced neurogenesis correlates well with elevated expression of TGF-ß2 and TGF-ß3, and activation of their downstream signaling molecule Smad2. Intriguingly, the enhanced adult neurogenesis was mitigated when Smad2 expression was deleted in neurons, supporting a role for the CX3CL1-TGF-ß2/3-Smad2 pathway in the control of adult neurogenesis. When Tg-CX3CL1 mice were crossed with Alzheimer's PS19 mice, which overexpress a tau P301S mutation and exhibit age-dependent neurofibrillary tangles and neurodegeneration, overexpressed CX3CL1 in both male and female mice was sufficient to rescue the neurodegeneration, increase survival time, and improve cognitive function. Hence, we provide in vivo evidence that CX3CL1 is a strong activator of adult neurogenesis, and that it reduces neuronal loss and improves cognitive function in AD.SIGNIFICANCE STATEMENT This study will be the first to demonstrate that enhanced neurogenesis by overexpressed CX3CL1 is mitigated by disruption of Smad2 signaling and is independent of its interaction with CX3CR1. Overexpression of CX3CL1 lengthens the life span of PS19 tau mice by enhancing adult neurogenesis while having minimal effect on tau pathology. Enhancing neuronal CX3CL1, mainly the C-terminal fragment, is a therapeutic strategy for blocking or reversing neuronal loss in Alzheimer's disease or related neurodegenerative disease patients.

Postoperative Complications and Outcomes Associated With a Transition to 24/7 Intensivist Management of Cardiac Surgery Patients.

OBJECTIVES: Nighttime intensivist staffing does not improve patient outcomes in general ICUs. Few studies have examined the association between dedicated in-house 24/7 intensivist coverage on outcomes in specialized cardiac surgical ICUs. We sought to evaluate the association between 24/7 in-house intensivist-only management of cardiac surgical patients on postoperative complications and health resource utilization. DESIGN: Before-and-after propensity matched cohort study. SETTING: Tertiary care cardiac surgical ICU. PATIENTS: Patients greater than 18 years old who underwent cardiac surgery between January 1, 2006, and April 30, 2013 (nighttime resident model), were propensity-matched (1:1) to patients from August 1, 2013, to December 31, 2014 (24/7 in-house intensivist model). INTERVENTIONS: Cardiac surgical ICU coverage change from a nighttime resident physician coverage model to a 24/7 in-house intensivist staffing model. MEASUREMENTS AND MAIN RESULTS: The primary outcome of interest was a composite of postoperative major complications. Secondary outcomes included duration of mechanical ventilation, all-cause cardiac surgical ICU readmissions, and surgical postponements attributed to lack of cardiac surgical ICU bed availability. A total of 1,509 patients during the nighttime resident model were matched to 1,509 patients during the intensivist model. The adjusted risk of major complications (26.3% vs 19.3%; odds ratio, 0.73; 95% CI, 0.36-0.85; p < 0.01), mean mechanical ventilation time (25.2 vs 19.4 hr; p < 0.01), cardiac surgical ICU readmissions (5.3% vs 1.6%; odds ratio, 0.31; 95% CI, 0.19-0.48; p < 0.01), and surgical postponements (3.4 vs 0.3 per mo; p < 0.01) were lower with the intensivist model. CONCLUSIONS: A transition to a 24/7 in-house intensivist care model was associated with a reduction in postoperative major complications, duration of mechanical ventilation, cardiac surgical ICU readmissions, and surgical postponements. These findings suggest that 24/7 intensivist physician care models may improve patient outcomes and health resource utilization in specialized cardiac surgical ICUs.

Using biophysical models to manage nitrogen pollution from agricultural sources: Utopic or realistic approach for non-scientist users? Case study of a drinking water catchment area in Lorraine, France.

The objectives of this comparison of two biophysical models of nitrogen losses were to evaluate first whether results were similar and second whether both were equally practical for use by non-scientist users. Results were obtained with the crop model STICS and the environmental model AGRIFLUX based on nitrogen loss simulations across a small groundwater catchment area (<1 km(2)) located in the Lorraine region in France. Both models simulate the influences of leaching and cropping systems on nitrogen losses in a relevant manner. The authors conclude that limiting the simulations to areas where soils with a greater risk of leaching cover a significant spatial extent would likely yield acceptable results because those soils have more predictable leaching of nitrogen. In addition, the choice of an environmental model such as AGRIFLUX which requires fewer parameters and input variables seems more user-friendly for agro-environmental assessment. The authors then discuss additional challenges for non-scientists such as lack of parameter optimization, which is essential to accurately assessing nitrogen fluxes and indirectly not to limit the diversity of uses of simulated results. Despite current restrictions, with some improvement, biophysical models could become useful environmental assessment tools for non-scientists.

Combining beef cattle and sheep in an organic system. II. Benefits for economic and environmental performance.

Combining several animal species to optimise the performance of the whole farming system is one of the core tenets of agroecology. Here, we associated sheep with beef cattle (40-60% livestock units (LU)) in a mixed system (MIXsys) and compared its performances to those of a specialised beef cattle-only system (CATsys) and a specialised sheep-only system (SHsys). All three systems were designed to have identical annual stocking rates and similar farm areas, pastures and animals. The experiment was conducted for four campaigns (2017-2020) in an upland setting exclusively on permanent grassland under certified-organic farming standards. The young animals were fattened almost exclusively with forages: at pasture for lambs and indoors with haylage in winter for young cattle. Abnormally dry weather conditions led to hay purchases. We compared between-system and between-enterprise performances based on technical, economic (gross product, expenses, margins, income), environmental (greenhouse gas emissions (GHG), energy consumption) and feed-food competition balance indicators. The mixed-species association only benefited the sheep enterprise, with +17.1% meat production per LU (P < 0.03), -17.8% concentrate used per LU (P < 0.02), +10.0% gross margin (P < 0.07) and +47.5% income per LU (P < 0.03) in MIXsys vs SHsys, as well as environmental performance benefits via a reduction of 10.9% in GHG emissions (P < 0.09) and 15.7% in energy consumption (P < 0.03), and a 47.2% improvement in feed-food competition (P < 0.01) in MIXsys vs SHsys. These results are due to both better animal performance and lower concentrate consumption in MIXsys, as presented in a companion paper. These benefits outweighed the additional costs of the mixed system, especially for fencing, in terms of net income per sheep LU. There were no between-system differences in productive and economic performance (kilos live-weight produced, kilos concentrate used and income per LU) for the beef cattle enterprise. Despite good animal performances, the beef cattle enterprises in both CATsys and MIXsys had poor economic performance due to large purchases of conserved forages and difficulty selling the animals, which were ill-adapted to the traditional downstream sector. This multiyear study at the farming-system level, which has thus far been underresearched for mixed livestock farming systems, highlighted and quantified the benefits for sheep when combined with beef cattle on economic, environmental, and feed-food competition performance.

Combining beef cattle and sheep in an organic system. I. Co-benefits for promoting the production of grass-fed meat and strengthening self-sufficiency.

Numerous advantages of combining cattle and sheep have been demonstrated at the grazing-season level, but the effects of this practice on system self-sufficiency require system-level and longer-term studies. We established three grassland-based organic systems as separate farmlets: one mixed system combining beef cattle and sheep (MIX) and two specialised systems, beef cattle (CAT) and sheep (SH), to serve as reference points. These farmlets were managed for 4 years, to assess the benefits of combining beef cattle and sheep in promoting the production of grass-fed meat and strengthening system self-sufficiency. The ratio of cattle to sheep livestock units in MIX was 60:40. The surface area and stocking rate were similar across all systems. Calving and lambing were adjusted to grass growth to optimise grazing. Calves were pasture-fed from 3 months old on average until weaning in October, fattened indoors with haylage and slaughtered at 12-15 months. Lambs were pasture-fed from 1 month old on average until slaughter; if lambs were not ready for slaughter when the ewes mated, they were stall-finished with concentrates. The decision to supplement adult females with concentrate was based on the achievement of a target body condition score (BCS) at key periods. The decision to treat animals with anthelmintics was based on mean faecal egg excretion remaining below a certain threshold. A higher proportion of lambs were pasture-finished in MIX vs SH (P < 0.001) due to a higher growth rate (P < 0.001) which led to a lower age at slaughter (166 vs 188 days, P < 0.001). Ewe prolificacy and productivity were higher in MIX vs SH (P < 0.02 and P < 0.065, respectively). The levels of concentrate consumption and number of anthelmintic treatments in sheep were lower in MIX vs SH (P < 0.01 and P < 0.08). Cow productivity, calf performance, carcass characteristics and the level of external inputs used did not differ between systems. However, cow BW gain during the grazing season was higher in MIX vs CAT (P < 0.05). These outcomes validated our hypothesis that the association of beef cattle and sheep promoted the self-sufficient production of grass-fed meat in the sheep enterprise. It also promoted better ewe and cow BCS and BW at key stages of the reproduction cycle and better development of the females used for replacement, which may enhance animal and system resilience.

Reproductive performance in two organic sheep farming systems differing by the number of mating sessions in and out of the breeding season.

Hormonal treatments to control ovulation and the intensification of reproduction rhythm are usual strategies to increase ewe productivity. However, in organic farming the use of hormones is banned, making any increase of reproduction rhythm an inappropriate practice. This work tested if increasing the number of mating sessions per year could improve ewe annual productivity, while maintaining a non-intensive reproduction rhythm of one lambing per ewe per year. Reproductive performance was studied over five years in two organic sheep farming systems differing by the number of mating sessions (MS) per year, two (2MS) or four (4MS). The 4MS system included two mating sessions in the breeding season (September and November) and non-breeding season (April and June). The 2MS system included mating in November and April. Non-pregnant ewes were recycled one (2MS) or two times (4MS system) on consecutive mating sessions. Considering all mating attempts per ewe individually (n = 1366), the time elapsed from mating to lambing (159 ± 0.2 days), fertility (86%), prolificacy (166%) and productivity (143%) did not differ between the systems (P > 0.05). Fertility, prolificacy and productivity were higher in the breeding than the non-breeding season (P ≤ 0.05), and in adults than young ewes (P ≤ 0.05), without interaction with the systems (P > 0.05). In the non-breeding season, fertility, prolificacy and productivity did not relate to ewe cyclic status (P > 0.05), whereas the time elapsed from mating to lambing was shortened in cyclic ewes (P ≤ 0.05). Good ewe body condition at lambing and mating and dynamic gain in body condition from drying to mating improved productivity (P ≤ 0.05) by increasing fertility or prolificacy regardless of the system (P > 0.05). On a yearly scale, the number of lambed ewes was higher in the 4MS than the 2MS system (91% vs 85%; P ≤ 0.05) leading to higher ewe annual productivity (154% vs 141%; P ≤ 0.05). The percentage of ewes that were recycled on consecutive mating sessions was higher in the 4MS than the 2MS system (24% vs 18%; P ≤ 0.05), possibly explaining the 4MS increase of ewe annual productivity by extending mating opportunities for the ewes. The increase in the number of mating sessions per year is a hormone-free strategy suitable for organic sheep farming systems to improve annual ewe productivity on a basis of one lambing per ewe per year. This approach proved beneficial to compensate for lower fertility in the non-breeding season, especially for young ewes.

Potentially Inappropriate Medication Dispensing in Outpatients: Comparison of Different Measurement Approaches.

Purpose of the Research: This paper aims at comparing different approaches to measure potentially inappropriate medication (PIM) with routinely collected data on prescriptions, patient age institutionalization status (ie in nursing home or in the community). A secondary objective is to measure the rate and prevalence of PIM dispensing and to identify problematic practices in Switzerland. Material and Methods: The studied population includes about 90,000 insured over 17 years old from a Swiss health maintenance organization in 2019 and 2020. We computed and compared the number of PIM per patient for Beers criteria, Priscus list, Laroche, NORGEP and Prescrire approaches. We also created a composite indicator that accounts for the specificities of the Swiss context (adaptation to the Swiss drugs' market, recommendations in force related to sleeping pills, anxiolytics and NSAIDs). We also stratified the analysis per physician, including initiation and cessation of PIM prescription. Results: Our comparison revealed similarities between the approaches, but also that each of them had specific gaps that provides further motivation for the development of a composite approach. PIM rate was particularly high for sleeping pills, anxiolytics, NSAIDs, even when analyses were limited to chronic use. Drugs with anticholinergic effect were also frequently prescribed. Based on our composite indicator, 27% of insured over 64 years old received at least one PIM in 2020, and 8% received more than one. Our analyses also reveal that for sleeping pills and anxiolytics, half of the volume (or prevalence?) occurs in the <65 population. We observed strong variations between physicians and a significant proportion of new users among patients with PIM. Conclusion: Our results show that PIMs prescribing is very frequent in Switzerland and is driven mostly by a few drug categories. There is important physician variation in PIM prescribing that warrants the development of intervention targeted at high PIM-prescribers.

Can emergency physicians predict severity and time away from work?

BACKGROUND: Emergency and primary care physicians are often asked to estimate patients' likely duration of sickness absence or temporary disability following work-related injury or illness. However, return to work is a complex interaction of multiple factors and often difficult to predict accurately. AIMS: To compare physician estimates of expected time away from work and severity of injury, made at the time of the initial presentation, with actual duration of temporary disability following work-related shoulder or knee injury. METHODS: Patients aged 18-65 with work-related shoulder or knee injuries who attended one of three Edmonton Emergency Departments were recruited. For each participant the treating physician made an estimate of severity and expected time before they would return to their work. This was compared with information on actual temporary disability (TDdays) obtained from the Alberta Workers' Compensation Board (WCB) data. RESULTS: Over the study period, 443 (88%) of 501 patients were enrolled into the study; however, only 177 (35%) agreed to linking their data with WCB. Median TDdays increased with the physicians' estimates of both severity and likely temporary disability. Physicians tended to underestimate time off work for those with long duration of TDdays, but overestimated this for those with short durations. CONCLUSIONS: Emergency physicians' estimates of expected lost work time and severity of injury were correlated with actual temporary disability, although their accuracy was fairly low. Further work to define why differences between estimated and actual temporary disability occur could help physicians and others planning return to work.

BACE-1 inhibition facilitates the transition from homeostatic microglia to DAM-1.

BACE-1 is required for generating ß-amyloid (Aß) peptides in Alzheimer's disease (AD). Here, we report that microglial BACE-1 regulates the transition of homeostatic to stage 1 disease-associated microglia (DAM-1) signature. BACE-1 deficiency elevated levels of transcription factors including Jun, Jund, Btg2, Erg1, Junb, Fos, and Fosb in the transition signature, which transition from more homeostatic to highly phagocytic DAM-1. Consistently, similar transition-state microglia in human AD brains correlated with lowered levels of BACE-1 expression. Targeted deletion of Bace-1 in adult 5xFAD mice microglia elevated these phagocytic microglia, correlated with significant reduction in amyloid plaques without synaptic toxicity. Silencing or pharmacologically inhibiting BACE-1 in cultured microglia-derived cells shows higher phagocytic function in microglia. Mechanistic exploration suggests that abolished cleavage of IL-1R2 and Toll-like receptors via BACE-1 inhibition contributes to the enhanced signaling via the PI3K and p38 MAPK kinase pathway. Together, targeted inhibition of BACE-1 in microglia may offer AD treatment.

Dynactin 6 deficiency enhances aging-associated dystrophic neurite formation in mouse brains.

Formation of Reticulon 3 (RTN3)-immunoreactive dystrophic neurites (RIDNs) occurs early during the growth of amyloid plaques in Alzheimer's disease (AD) brains. We have shown that RIDNs in AD and aging mouse brains are composed of abnormally clustered tubular endoplasmic reticulum (ER) and degenerating mitochondria. To understand RTN3-mediated abnormal tubular ER clustering, we aimed to identify proteins that interact with RTN3 and impact accumulation of tubular ER in RIDNs. We found that the N-terminal domain of RTN3, which is unique among RTN family members, specifically interacted with dynactin 6 (DCTN6), a protein involved in dynein-mediated retrograde transport of cargo vesicles. DCTN6 protein levels decrease with aging in the hippocampal regions of WT mice. We found that DCTN6 deficiency enhanced RTN3 protein levels, high molecular weight RTN3 levels, and hippocampus-specific RIDN formation in aging brains of transgenic mice overexpressing RTN3. Our results suggest that the DCTN6-RTN3 interaction mediates tubular ER trafficking in axons, and a DCTN6 deficiency in the hippocampus impairs axonal ER trafficking, leading to abnormal ER accumulation and RIDN formation in brains of aging mice.

CPG15/Neuritin Mimics Experience in Selecting Excitatory Synapses for Stabilization by Facilitating PSD95 Recruitment.

A key feature of brain plasticity is the experience-dependent selection of optimal connections, implemented by a set of activity-regulated genes that dynamically adjust synapse strength and number. The activity-regulated gene cpg15/neuritin has been previously implicated in stabilization and maturation of excitatory synapses. Here, we combine two-photon microscopy with genetic and sensory manipulations to dissect excitatory synapse formation in vivo and examine the role of activity and CPG15 in dendritic spine formation, PSD95 recruitment, and synapse stabilization. We find that neither visual experience nor CPG15 is required for spine formation. However, PSD95 recruitment to nascent spines and their subsequent stabilization requires both. Further, cell-autonomous CPG15 expression is sufficient to replace experience in facilitating PSD95 recruitment and spine stabilization. CPG15 directly interacts with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on immature dendritic spines, suggesting a signaling mode for this small extracellular molecule acting as an experience-dependent "selector" for spine stabilization and synapse maturation.

Pyrilamine and O-desmethylpyrilamine detection in equine serum and urine.

Pyrilamine (mepyramine) is an H1-receptor antagonist used in human and veterinary medicine. It has the potential to produce central nervous system effects in horses and therefore may have some impact on an outcome of a horse race. A single oral dose of pyrilamine (300 mg/horse) was given to three animals. Serum samples were collected before drug administration and at 0.25, 0.5, 1, 2, 4, 6, 24, 48, 72, 96, 120, and 144 h, and 7, 8, 9, 10, 11, 12, and 13 days post-administration. Urine samples were collected at 0-1, 1-2, 2-4, 4-6, 24, 48, 72, 96, 120, and 144 h, and 7, 8, 9, 10, 11, 12, 13 days post-administration. Urine and serum samples were initially screened by the pyrilamine enzyme-linked immunosorbent assay (ELISA) kit with subsequent confirmation and quantitation utilizing a newly developed and validated gas chromatography-mass spectrometry (GC-MS) method for pyrilamine and its major metabolite O-desmethylpyrilamine with chlorpromazine as an internal standard. Prior to the basic extraction, urine specimens were hydrolyzed using beta-glucuronidase. The urine extracts as well as the serum samples were then subjected to solid-phase extraction on Bond Elut LRC-PRS columns. Pyrilamine was not found in any of the urine samples but it was present in serum in low concentrations (4-123 ng/mL) up to 6 h after drug administration. The limit of detection and limit of quantitation for the GC-MS method for pyrilamine in serum were 1.5 and 3.1 ng/mL, respectively, and for O-desmethylpyrilamine in urine were 5 and 6.2 ng/mL, respectively. Pyrilamine concentration in serum peaked at 15 min, 30 min, and 1 h in horse #1, #2, and #3, respectively. Urine specimens were screened positive for pyrilamine and its metabolites using ELISA for extended periods of time (4 days in one horse and 9 days in two other animals). Using GC-MS, O-desmethylpyrilamine was detected in urine for 11 days in horse #1, 4 days in horse #2, and 9 days in horse #3. While pyrilamine was eliminated from the bloodstream rather quickly, the metabolite level remained in the urine for days after administration. When evaluating laboratory results, regulators must take into account that a urine sample positive for O-desmethylpyrilamine does not necessarily indicate that the drug remains active in the horse's system, possibly affecting the outcome from the race.

RECOTOX, a French initiative in ecotoxicology-toxicology to monitor, understand and mitigate the ecotoxicological impacts of pollutants in socioagroecosystems.

RECOTOX is a cross-cutting initiative promoting an integrated research to respond to the challenges of monitoring, understanding, and mitigating environmental and health impacts of pesticides in agroecosystems. The added value of RECOTOX is to develop a common culture around spatial ecotoxicology including the whole chain of pressure-exposure-impact, while strengthening an integrated network of in natura specifically equipped sites. In particular, it promotes transversal approaches at relevant socioecological system scales, to capitalize knowledge, expertise, and ongoing research in ecotoxicology and, to a lesser extent, environmental toxicology. Thus, it will open existing research infrastructures in environmental sciences to research programs in ecotoxicology of pesticides.