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Background: Fentanyl has emerged as the leading cause of fatal drug overdoses in the U.S. Individuals misusing drugs may not always be aware of exposure to fentanyl. Objectives: To determine the prevalence of fentanyl use and extent of awareness of fentanyl exposure among a national sample of treatment-seeking individuals with opioid use disorder (n = 1098). Methods: Participants provided oral fluid and urine specimens, which were tested for drugs by liquid chromatography/tandem mass spectrometry. Participants also provided self-reports of fentanyl use. Results: 49.5% tested positive for fentanyl in oral fluid, urine, or both. Of those testing positive for fentanyl, 29.8% were unaware that they had been exposed to fentanyl. Participants testing positive for opioids methadone, and specifically 6-monoacetylmorphine (6-MAM), a unique metabolite of heroin, were significantly more likely to be unaware of fentanyl exposure than participants testing negative for these substances, with a similar trend for oxycodone and tramadol. Conclusions: These findings may be due to fentanyl's effect being difficult to distinguish from that of other opioids, whereas when other types of drugs are adulterated with fentanyl, the differences in effects are likely to be readily discernable. These results support the importance of expanded drug-checking services. [ABSTRACT FROM AUTHOR]

Xylazine has emerged in recent years as a dangerous adulterant in illicit fentanyl use, and methods for the detection of xylazine in toxicology panels are still lagging. We developed methods for the screening and quantitation of xylazine in oral fluid (OF), a popular testing medium due to its ease of collection and reflection of presence in blood for many classes of drugs. Enzyme-linked immunosorbent assays were employed for the rapid screening of xylazine directly from the collection device buffer with a cutoff of 1 ng/mL. Solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry facilitated the confirmation and quantification of xylazine as low as 0.1 ng/mL and a dynamic range of 0.1-25 ng/mL. Selectivity, ionization suppression, processed sample stability, and dilution effect were also assessed. The method was validated through the American National Standards Institute/American Academy of Forensic Sciences Standards Board (ANSI/ASB) Standard 036, first edition from 2019, and found to be accurate, precise, and robust. Living human subject OF samples collected within substance use disorder and therapeutic drug monitoring clinics received between September 2023 and January 2024, with the specific request to test for xylazine (n = 57), were screened. Presumptive positive samples were confirmed using the validated method. Xylazine confirmed living human subject OF sample concentrations ranged from 1.2 to 23.3 ng/mL., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

The standard protocol in addiction treatment/pain management is to conduct immunoassay screens for major drugs subject to misuse, followed by confirmatory testing of positive results. However, this may miss unscreened or rarely screened drugs that could pose risks, especially to polydrug users. We sought to determine the prevalences of unscreened/rarely screened drugs in a sample of individuals misusing drugs in 7 U.S. states, and to compare the results of urine vs. oral testing for these drugs by direct-to-definitive liquid chromatography/tandem mass spectrometry (LC-MS-MS). The five drugs with the highest prevalences were: gabapentin (16.8%), quetiapine (6.2%), chlorpheniramine (5.3%), hydroxyzine (4.9%), and ephedrine (3.5%). All have clinical significance as indicated by severity of possible side effects, interactions with other drugs, and/or misuse potential. Drugs were generally detected more frequently in oral fluid than urine, but gabapentin was more frequently detected in urine. The prevalences of the included drugs seem high enough, and their clinical significance important enough, to warrant consideration of expanding clinical drug test panels, either by direct-to-definitive testing or the addition of selected immunoassay screens when available. Oral fluid was usually more suitable than urine as the test matrix, given the higher rates of detection in oral fluid for most substances included in this study.

Background: The aims are to compare the results of presumptive drug testing with confirmation of positives vs. direct-to-definitive drug testing, combined with investigation of urine vs. oral fluid as test matrices., Methods: Paired oral fluid and urine specimens were collected voluntarily and anonymously from 1098 individuals applying for methadone treatment in 11 clinics across 7 U.S. states. All specimens were analyzed by immunoassay (IA) and liquid chromatography-tandem mass spectrometry (LC-MS-MS)., Results: Confirmed IA prevalences for urine were significantly higher than for oral fluid for 7 out of 10 drug classes - benzodiazepines, cannabis, cocaine, methadone, opiates, oxycodone and tramadol. Drug prevalences by direct-to-definitive LC-MS-MS were either the same or higher than prevalences by confirmed IA. Drug prevalences by LC-MS-MS were higher in urine for two drug classes (cocaine, methadone) and higher in oral fluid for two drug classes (buprenorphine, tramadol), but were equivalent in urine and oral fluid when averaged over all 10 drug classes. Certain drugs of special concern such as heroin and buprenorphine were more frequently detected in oral fluid than urine., Conclusions: Urine analysis showed some technical advantage over oral fluid in sensitivity to several drug classes within a confirmed IA testing protocol, but this may be outweighed if there is reason to believe that tampering with urine specimens is a significant problem. Overall drug detection by direct-to-definitive testing was similar for oral fluid and urine, but one matrix may be preferable if there is a particular drug of clinical or epidemiological interest., Competing Interests: Declaration of Competing Interest The authors have nothing to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

During thrombopoiesis, megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Phosphoinositide-dependent protein kinase-1 (PDK1) is required for Ca2+-dependent platelet activation, but its role in MK development and regulation of platelet production remained elusive. The present study explored the role of PDK1 in the regulation of MK maturation and polarization during thrombopoiesis using a MK/platelet-specific knockout approach. Pdk1-deficient mice (Pdk1-/-) developed a significant macrothrombocytopenia as compared with wild-type mice (Pdk1fl/fl). Pdk1 deficiency further dramatically increased the number of MKs without sinusoidal contact within the BM hematopoietic compartment, resulting in a pronounced MK hyperplasia and a significantly increased extramedullary thrombopoiesis. Cultured Pdk1-/- BM-MKs showed impaired spreading on collagen, associated with an altered actin cytoskeleton structure with less filamentous actin (F-actin) and diminished podosome formation, whereas the tubulin cytoskeleton remained unaffected. This phenotype was associated with abrogated phosphorylation of p21-activated kinase (PAK) as well as its substrates LIM domain kinase and cofilin, supporting the hypothesis that the defective F-actin assembly results from increased cofilin activity in Pdk1-deficient MKs. Pdk1-/- BM-MKs developed increased ploidy and exhibited an abnormal ultrastructure with disrupted demarcation membrane system (DMS). Strikingly, Pdk1-/- BM-MKs displayed a pronounced defect in DMS polarization and produced significantly less proplatelets, indicating that PDK1 is critically required for proplatelet formation. In human MKs, genetic PDK1 knockdown resulted in increased maturity but reduced platelet-like particles formation. The present observations reveal a pivotal role of PDK1 in the regulation of MK cytoskeletal dynamics and polarization, proplatelet formation, and thrombopoiesis., (© 2019 by The American Society of Hematology.)

The purpose of this study was to investigate the suitability of the microDiamond detector (mDD) type 60019 (PTW-Freiburg, Germany) for radial dose function measurements with High Dose Rate (HDR) 192 Ir brachytherapy sources. An HDR 192 Ir source model mHDR v2r (Nucletron BV, an Elekta company, The Netherlands) was placed at the centre of a MP3 water phantom (PTW-Freiburg, Germany) within a 4F needle. Three mDDs were employed to measure the radial dose function of the source by acquiring profiles along the source transverse axis. Meanwhile, the experimental setup was simulated using the Monte Carlo (MC) code MCNP6.1™ (Los Alamos National Laboratory, USA) to calculate phantom-size, absorbed-dose energy dependence and volume averaging correction factors. After applying the correction factors, the radial dose function g L (r) for the line source approximation was calculated as defined in the TG-43 formalism at radial distances from 0.5cm to 10cm and compared to the consensus g L (r) (ESTRO and AAPM). The percentage differences to the consensus g L (r) for all the three mDDs were from -2.3% to +1.4% for distances r≤5cm and -6.2% to +2.6% for larger distances. These results indicate the suitability of the mDD for HDR brachytherapy measurements when all required corrections are applied., (Copyright © 2018. Published by Elsevier GmbH.)

Platelet aggregation, dense granule secretion and thrombus formation are dependent on sphingolipids like ceramide and sphingosine as well as sphingosine-1 phosphate. Sphingosine/ceramide metabolism involves ceramide synthases and ceramidases. However, the role of ceramide synthase and ceramidase in the regulation of platelet function remained ill-defined. The present study determined transmission light aggregometry, employed luciferase based ATP release measurements and studied in vitro thrombus formation under high arterial shear rates in order to define the impact of pharmacological inhibition of serine palmitoyltransferase, ceramide synthase and ceramidase on platelet function. As a result, inhibition of ceramidase significantly blunted collagen related peptide (CRP) induced glyocoprotein VI (GPVI)-dependent platelet aggregation, ATP release and thrombus formation on a collagen-coated surface under shear rates of 1700 -sec . Defective platelet aggregation after ceramidase inhibition could partially be overcome by exogenous sphingosine treatment reflecting a pivotal role of ceramidase-derived sphingosine in platelet function. Inhibition of serine palmitoyltransferase and ceramide synthase did not significantly modify GPVI-dependent platelet activation. In conclusion, the present study unraveled ceramidase as a crucial player in sphingosine-induced platelet activation following GPVI-dependent signaling., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory β-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2β ( ck2β -/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2 β -/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2β -/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca 2+ ) release. Presumably due to a blunted increase in the concentration of cytosolic Ca 2+ , activation-dependent increases of α and dense-granule secretion and integrin α IIb β 3 activation, and aggregation were abrogated in ck2β -/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2β -/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2β -/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2β fl/fl mice. The present observations reveal CK2β as a novel powerful regulator of thrombopoiesis, Ca 2+ -dependent platelet activation, and arterial thrombosis in vivo., (© 2017 by The American Society of Hematology.)

Cyclophilin A (CyPA) is involved in the pathophysiology of several inflammatory and cardiovascular diseases. To our knowledge, there is no specific inhibitor targeting extracellular CyPA without affecting other extracellular cyclophilins or intracellular CyPA functions. In this study, we developed an antibody-based inhibitor of extracellular CyPA and analysed its effects in vitro and in vivo . To generate a specific antibody, mice and rats were immunized with a peptide containing the extracellular matrix metalloproteinase inducer binding site and various antibody clones were selected and purified. At first, antibodies were tested for their binding capacity to recombinant CyPA and their functional activity. The clone 8H7-mAb was chosen for further experiments. 8H7-mAb reduced the CyPA-induced migration of inflammatory cells in vitro and in vivo . Furthermore, 8H7-mAb revealed strong antithrombotic effects by inhibiting CyPA-dependent activation of platelets and thrombus formation in vitro and in vivo . Surprisingly, 8H7-mAb did not influence in vivo tail bleeding time or in vitro whole blood coagulation parameters. Our study provides first evidence that antibody-based inhibition of extracellular CyPA inhibits thrombosis and thromboinflammation without affecting blood homeostasis. Thus, 8H7-mAb may be a promising compound for thrombi modulation in inflammatory diseases to prevent organ dysfunction., Competing Interests: Disclosure: None., (Schattauer GmbH Stuttgart.)

BackgroundThe preterm infant gut microbiota is vulnerable to different biotic and abiotic factors. Although the development of this microbiota has been extensively studied, the mobilome-i.e. the mobile genetic elements (MGEs) in the gut microbiota-has not been considered. Therefore, the aim of this study was to investigate the association of the mobilome with birth weight and hospital location in the preterm infant gut microbiota.MethodsThe data set consists of fecal samples from 62 preterm infants with and without necrotizing enterocolitis (NEC) from three different hospitals. We analyzed the gut microbiome by using 16S rRNA amplicon sequencing, shot-gun metagenome sequencing, and quantitative PCR. Predictive models and other data analyses were performed using MATLAB and QIIME.ResultSThe microbiota composition was significantly different between NEC-positive and NEC-negative infants and significantly different between hospitals. An operational taxanomic unit (OTU) showed strong positive and negative correlation with NEC and birth weight, respectively, whereas none showed significance for mode of delivery. Metagenome analyses revealed high levels of conjugative plasmids with MGEs and virulence genes. Results from quantitative PCR showed that the plasmid signature genes were significantly different between hospitals and in NEC-positive infants.ConclusionOur results point toward an association of the mobilome with hospital location in preterm infants.

Aims: Hyperlipidaemia enhances susceptibility to thrombosis, while platelet oxidixed LDL (oxLDL) binding in acute coronary syndrome (ACS) correlates with activation status. This study explores the platelet lipidome in symptomatic coronary artery disease (CAD) patients and the functional consequences of the chemokine CXCL12 and its receptors CXCR-4/-7 on lipid uptake in platelets., Methods and Results: Platelet-oxLDL detected by flow cytometry was enhanced (P = 0.04) in CAD patients, moderately correlated with platelet CXCR7 surface expression (ρ = 0.39; P < 0.001), while inversely with CXCR4 (ρ = 0.35; P < 0.001). Platelet-oxLDL was elevated (P = 0.01) in ACS patients with angiographic evidence of intracoronary thrombi. Ex vivo analysis of intracoronary thrombi sections revealed oxLDL deposition in platelet-enriched areas verified by immunofluorescence confocal microscopy. LDL-oxLDL uptake enhanced reactive oxygen species, mitochondrial superoxide generation, intraplatelet LDL to oxLDL conversion, and lipid peroxidation, counteracted by SOD2-mimetic MnTMPyP. Lipidomic analysis revealed enhanced intraplatelet-oxidized phospholipids, cholesteryl esters, sphingomyelin, ceramides, di- and triacylglycerols, acylcarnitines in CAD patients compared with age-matched controls as ascertained by liquid chromatography hyphenated to high-resolution mass spectrometry. LDL-oxLDL induced degranulation, αIIbβ3-integrin activation, apoptosis, thrombin generation estimated by calibrated automated thrombinoscopy, and shape change verified by live imaging using scanning ion conductance microscopy. Further, LDL-oxLDL enhanced thrombus formation ex vivo and in vivo in mice (ferric chloride-induced carotid artery injury). LDL-oxLDL enhanced platelet CXCL12 release, differentially regulated CXCR4-CXCR7 surface exposure, while CXCL12 prompted LDL-oxLDL uptake and synergistically augmented the LDL-oxLDL-induced pro-oxidative, thrombogenic impact on platelet function., Conclusion: An altered platelet lipidome might be associated with thrombotic disposition in CAD, a mechanism potentially regulated by CXCL12-CXCR4-CXCR7 axis., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Platelet adhesion, activation, and aggregation are essential for primary hemostasis, but are also critically involved in the development of acute arterial thrombotic occlusion. Stimulation of the collagen receptor glycoprotein VI (GPVI) leads to phospholipase Cγ2-dependent inositol triphosphate (IP 3 ) production with subsequent platelet activation, due to increased intracellular Ca 2+ concentration ([Ca 2+ ] i ). Although tricyclic antidepressants have been shown to potentially impair platelet activation, nothing is hitherto known about potential effects of the tricyclic antidepressant doxepin on platelet Ca 2+ signaling and thrombus formation. As shown in the present study, doxepin significantly diminished the stimulatory effect of GPVI agonist collagen-related peptide (CRP) on intracellular Ca 2+ release as well as subsequent extracellular Ca 2+ influx. Doxepin was partially effective by impairment of CRP-dependent IP 3 production. Moreover, doxepin abrogated CRP-induced platelet degranulation and integrin α IIb β 3 activation and aggregation. Finally, doxepin markedly blunted in vitro platelet adhesion to collagen and thrombus formation under high arterial shear rates (1,700 -s ). In conclusion, doxepin is a powerful inhibitor of GPVI-dependent platelet Ca 2+ signaling, platelet activation, and thrombus formation., (Copyright © 2017 the American Physiological Society.)

Background: Tachycardiomyopathy or tachycardia-induced cardiomyopathy (TCM) has been known for decades as a reversible form of nonischemic cardiomyopathy. However, its mechanism and properties remain poorly understood., Objectives: The current study investigated endomyocardial biopsy samples from patients with TCM and compared them with samples from patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (ICM)., Methods: The study included 189 patients with new-onset heart failure and severely reduced ejection fraction not caused by valvular or ischemic heart disease. Nineteen patients retrospectively fulfilled common criteria of TCM, 79 patients had a diagnosis of DCM, and 91 had a diagnosis of ICM., Results: Patients with TCM, on the basis of clinical criteria, had stronger myocardial expression of major histocompatibility complex class II molecule and enhanced infiltration of CD68 + macrophages compared with patients with DCM. Furthermore, when compared with patients with ICM, the presence of T cells and macrophages was significantly reduced in TCM. Myocardial fibrosis was detected to a significantly lower degree in patients with TCM compared with patients with DCM and ICM. Electron microscopic examination revealed severe structural changes in patients with TCM. A disturbed distribution pattern of mitochondria was predominantly present in TCM. Quantitative assessment of myocyte morphology revealed significantly enhanced myocyte size compared with patients with ICM. Ribonucleic acid expression analysis identified changes in metabolic pathways among the patient groups., Conclusions: TCM is characterized by changes in cardiomyocyte and mitochondrial morphology accompanied by a macrophage-dominated cardiac inflammation. Thus, further prospective studies are warranted to characterize patients with TCM by endomyocardial biopsy more clearly., (Copyright © 2017 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objective: Activation of platelets by subendothelial collagen results in an increase of cytosolic Ca(2+) concentration ([Ca(2+)]i) and is followed by platelet activation and thrombus formation that may lead to vascular occlusion. The present study determined the role of phosphoinositide-dependent protein kinase 1 (PDK1) in collagen-dependent platelet Ca(2+) signaling and ischemic stroke in vivo., Approach and Results: Platelet activation with collagen receptor glycoprotein VI agonists collagen-related peptide or convulxin resulted in a significant increase in PDK1 activity independent of second-wave signaling. PDK1 deficiency was associated with reduced platelet phospholipase Cγ2-dependent inositol-1,4,5-trisphosphate production and intracellular [Ca(2+)]i in response to stimulation with collagen-related peptide or convulxin. The defective increase of [Ca(2+)]i resulted in a substantial defect in activation-dependent platelet secretion and aggregation on collagen-related peptide stimulation. Furthermore, Rac1 activation and spreading, adhesion to collagen, and thrombus formation under high arterial shear rates were significantly diminished in PDK1-deficient platelets. Mice with PDK1-deficient platelets were protected against arterial thrombotic occlusion after FeCl3-induced mesenteric arterioles injury and ischemic stroke in vivo. These mice had significantly reduced brain infarct volumes, with a significantly increased survival of 7 days after transient middle cerebral artery occlusion without increase of intracerebral hemorrhage. Tail bleeding time was prolonged in pdk1(-/-) mice, reflecting an important role of PDK1 in primary hemostasis., Conclusions: PDK1 is required for Ca(2+)-dependent platelet activation on stimulation of collagen receptor glycoprotein VI, arterial thrombotic occlusion, and ischemic stroke in vivo., (© 2016 American Heart Association, Inc.)

Apoptosis mediates neutrophil (PMN) phagocytosis and is influenced by cytokines and the Fas/Fas ligand pathway. To determine whether apoptosis of cord blood PMN differs from those of adults, cultured PMN were evaluated by morphological analysis, flow cytometry (TUNEL assay), and DNA gel electrophoresis. In addition, we studied the effect of anti-Fas IgM or cycloheximide on induction of PMN apoptosis. Spontaneous apoptosis (24 h) was less in cord blood PMN (mean +/- SD; 29 +/- 9 vs. adults, 56 +/- 14%, P < 0.001). Treatment (6 h) with anti-Fas IgM induced less apoptosis in cord blood PMN (24 +/- 6 vs. adults, 63 +/- 7%, P < 0.001), as did treatment with cycloheximide (13 +/- 10 vs. adults, 55 +/- 16%, P < 0.01). These data suggest the pre-existence of proteins that inhibit apoptosis or the absence of those that promote apoptosis in cord blood PMN.

After administration of high-dose codeine we found that in two cases 20-40% of the total morphine-equivalents in the 24-hour urine sample were free morphine. After another 24 hours the proportion of free morphine was up to 70% and after roughly 96 hours even went up to 96%. Although 10% of the administered codeine was eliminated as morphine. In contrast to the values reported in literature we found that even with codeine/morphine ratios greater than 0.5 and morphine concentrations of up to 2000 ng/ml urine one cannot naturally conclude that morphine/diamorphine has been consumed. The short half-life of codeine in serum was 2-4 hours, the final half-life was 9-11 hours. In urine we found a short half-life of 1-6 hours as well as a long half-life of 7-12 hours. When diazepam was administered simultaneously with codeine the expected half-life in serum was up to 1.5 times and in urine 2.5 times. The codeine/morphine ratios were hardly affected so far as evidence goes which they give of preceding drug-intake.