There is another: H3K27me3-mediated genomic imprinting.

DNA methylation has long been considered the primary epigenetic mediator of genomic imprinting in mammals. Recent epigenetic profiling during early mouse development revealed the presence of domains of trimethylation of lysine 27 on histone H3 (H3K27me3) and chromatin compaction specifically at the maternally derived allele, independent of DNA methylation. Within these domains, genes are exclusively expressed from the paternally derived allele. This novel mechanism of noncanonical imprinting plays a key role in the development of mouse extraembryonic tissues and in the regulation of imprinted X-chromosome inactivation, highlighting the importance of parentally inherited epigenetic histone modifications. Here, we discuss the mechanisms underlying H3K27me3-mediated noncanonical imprinting in perspective of the dynamic chromatin landscape during early mouse development and explore evolutionary origins of noncanonical imprinting.

STPP-UP: An alternative method for drug target identification using protein thermal stability.

Thermal proteome profiling (TPP) has significantly advanced the field of drug discovery by facilitating proteome-wide identification of drug targets and off-targets. However, TPP has not been widely applied for high-throughput drug screenings, since the method is labor intensive and requires a lot of measurement time on a mass spectrometer. Here, we present Single-tube TPP with Uniform Progression (STPP-UP), which significantly reduces both the amount of required input material and measurement time, while retaining the ability to identify drug targets for compounds of interest. By using incremental heating of a single sample, changes in protein thermal stability across a range of temperatures can be assessed, while alleviating the need to measure multiple samples heated to different temperatures. We demonstrate that STPP-UP is able to identify the direct interactors for anticancer drugs in both human and mice cells. In summary, the STPP-UP methodology represents a useful tool to advance drug discovery and drug repurposing efforts.

Decreasing Phosphorus Loss in Tile-Drained Landscapes Using Flue Gas Desulfurization Gypsum.

Elevated phosphorus (P) loading from agricultural nonpoint-source pollution continues to impair inland waterbodies throughout the world. The application of flue gas desulfurization (FGD) gypsum to agricultural fields has been suggested to decrease P loading because of its high calcium content and P sorbing potential. A before-after control-impact paired field experiment was used to examine the water quality effects of successive FGD gypsum applications (2.24 Mg ha; 1 ton acre each) to an Ohio field with high soil test P levels (>480 ppm Mehlich-3 P). Analysis of covariance was used to compare event discharge, dissolved reactive P (DRP), and total P (TP) concentrations and loadings in surface runoff and tile discharge between the baseline period (86 precipitation events) and Treatment Period 1 (42 precipitation events) and Treatment Period 2 (84 precipitation events). Results showed that, after the first application of FGD gypsum, event mean DRP and TP concentrations in treatment field tile water were significantly reduced by 21 and 10%, respectively, and DRP concentrations in surface runoff were significantly reduced by 14%; however, no significant reductions were noted in DRP or TP loading. After the second application, DRP and TP loads were significantly reduced in surface runoff (DRP, 41%; TP 40%), tile discharge (DRP, 35%; TP, 15%), and combined (surface + tile) discharge (DRP, 36%; TP, 38%). These findings indicate that surface application of FGD gypsum can be used as a tool to address elevated P concentrations and loadings in drainage waters.

[History of internal fixators. The subsequent importance for spinal surgery]. / Geschichte des Fixateur interne. Seine spätere Bedeutung für die Wirbelsäulenchirurgie.

BACKGROUND: This article presents a retrospective look at spinal implants of the 1970s and 1980s. OBJECTIVE: The historical development of internal fixators as the successor to external fixators. MECHANICAL PRINCIPLE: Pedicled screws are stably anchored in vertebral bodies of the thoracic or lumbar spine or the sacrum using a dorsal approach. They are joined by a rod as a longitudinal support, separated by freely selectable distances and in any desirable and initially modifiable angle. After locking this results in an angular and rotationally stable completely sunken bilateral construction for fixing two or more vertebrae together and the position can be manually adjusted using long lever arms on the pedicled screws. RESULTS: The first in vivo application in humans was on 22 December 1982 in Basel. The initial indications were unstable spinal fractures. The expectations placed on the new working principle of internal fixation and its realization were confirmed and short stretch fixation exclusively of the neighboring vertebra and immediate mobilization of patients could be routinely achieved. The indications were extended to include instability of the spine for conditions outside the field of traumatology. Further developments of implants and other technical solutions in the coupling system using the same basic principle in the direction of multisegmental applications, ease of operation and titanium-based materials became internationally established and were developed into universal spinal stabilization systems for spinal degeneration, deformities, tumors and olisthesis. CONCLUSION: The basic principle of spinal fixators (internal and external) is contained in the complete product range of dorsal stabilizing implants from practically all manufacturers worldwide and has become taken for granted.

Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics.

Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

Before and after hip fracture, vitamin D deficiency may not be treated sufficiently.

UNLABELLED: Our findings show that only about 20% of seniors receive vitamin D supplementation prior to their index hip fracture or after the event. We further confirm the high prevalence of severe vitamin D deficiency in this population and show that those who receive supplementation have significantly higher 25-hydroxyvitamin D (25(OH)D) status. INTRODUCTION: The aim of this study is to assess current practice in pre- and post-hip fracture care practice with respect to vitamin D supplementation. METHODS: We surveyed 1,090 acute hip fracture patients age 65 and older admitted to acute care for hip fracture repair; 844 had serum 25-hydroxyvitamin D levels measured upon admission to acute care, and 362 agreed to be followed at 12 month after their hip fracture. Prevalence of vitamin D supplementation was assessed upon admission to acute care (at the time of hip fracture), upon discharge from acute care, and at 6 and 12 months follow-up. RESULTS: Of 1,090 acute hip fracture patients (mean age 85 years, 78% women, 59 % community-dwelling), 19% had received any dose of vitamin D prior to the index hip fracture, 27% (of 854 assessed) at discharge from acute care, 22 % (of 321 assessed) at 6 month, and 21% (of 285 assessed) at 12 month after their hip fracture. At the time of fracture, 45% had 25(OH)D levels below 10 ng/ml, 81% had levels below 20 ng/ml, and 96% had levels below 30 ng/ml. Notably, 25(OH)D levels did not differ by season or gender but were significantly higher among 164 hip fracture patients, with any vitamin D supplementation compared with 680 without supplementation (19.9 versus 10.8 ng/ml; p < 0.0001). CONCLUSION: Only about 20% of seniors receive vitamin D at the time of their fracture and after the event. This is despite the documented 81% prevalence of vitamin D deficiency. Interdisciplinary efforts may be warranted to improve vitamin D supplementation in seniors both before a hip fracture occurs and after.

A novel antifolate suppresses growth of FPGS-deficient cells and overcomes methotrexate resistance.

Cancer cells make extensive use of the folate cycle to sustain increased anabolic metabolism. Multiple chemotherapeutic drugs interfere with the folate cycle, including methotrexate and 5-fluorouracil that are commonly applied for the treatment of leukemia and colorectal cancer (CRC), respectively. Despite high success rates, therapy-induced resistance causes relapse at later disease stages. Depletion of folylpolyglutamate synthetase (FPGS), which normally promotes intracellular accumulation and activity of natural folates and methotrexate, is linked to methotrexate and 5-fluorouracil resistance and its association with relapse illustrates the need for improved intervention strategies. Here, we describe a novel antifolate (C1) that, like methotrexate, potently inhibits dihydrofolate reductase and downstream one-carbon metabolism. Contrary to methotrexate, C1 displays optimal efficacy in FPGS-deficient contexts, due to decreased competition with intracellular folates for interaction with dihydrofolate reductase. We show that FPGS-deficient patient-derived CRC organoids display enhanced sensitivity to C1, whereas FPGS-high CRC organoids are more sensitive to methotrexate. Our results argue that polyglutamylation-independent antifolates can be applied to exert selective pressure on FPGS-deficient cells during chemotherapy, using a vulnerability created by polyglutamylation deficiency.

Integrated multi-omics reveal polycomb repressive complex 2 restricts human trophoblast induction.

Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates.

An association between vasomotion and oxygen extraction.

Vasomotion is defined as a spontaneous local oscillation in vascular tone whose function is unclear but may have a beneficial effect on tissue oxygenation. Optical reflectance spectroscopy and laser Doppler fluximetry provide unique insights into the possible mechanisms of vasomotion in the cutaneous microcirculation through the simultaneous measurement of changes in concentration of oxyhemoglobin ([HbO(2)]), deoxyhemoglobin ([Hb]), and mean blood saturation (S(mb)O(2)) along with blood volume and flux. The effect of vasomotion at frequencies <0.02 Hz attributed to endothelial activity was studied in the dorsal forearm skin of 24 healthy males. Fourier analysis identified periodic fluctuations in S(mb)O(2) in 19 out of 24 subjects, predominantly where skin temperatures were >29.3°C (X(2) = 6.19, P < 0.02). A consistent minimum threshold in S(mb)O(2) (mean: 39.4%, range: 24.0-50.6%) was seen to precede a sudden transient surge in flux, inducing a fast rise in S(mb)O(2). The integral increase in flux correlated with the integral increase in [HbO(2)] (Pearson's correlation r(2) = 0.50, P < 0.001) and with little change in blood volume suggests vasodilation upstream, responding to a low S(mb)O(2) downstream. This transient surge in flux was followed by a sustained period where blood volume and flux remained relatively constant and a steady decrease in [HbO(2)] and equal and opposite increase in [Hb] was considered to provide a measure of oxygen extraction. A measure of this oxygen extraction has been approximated by the mean half-life of the decay in S(mb)O(2) during this period. A comparison of the mean half-life in the 8 normal subjects [body mass index (BMI) <26.0 kg/m(2)] of 12.2 s and the 11 obese subjects (BMI >29.5 kg/m(2)) of 18.8 s was statistically significant (Mann Whitney, P < 0.004). The S(mb)O(2) fluctuated spontaneously in this saw tooth manner by an average of 9.0% (range 4.0-16.2%) from mean S(mb)O(2) values ranging from 30 to 52%. These observations support the hypothesis that red blood cells may act as sensors of local tissue hypoxia, through the oxygenation status of the hemoglobin, and initiate improved local perfusion to the tissue through hypoxic vasodilation.

Endothelial glycocalyx structure in the intact carotid artery: a two-photon laser scanning microscopy study.

BACKGROUND: The endothelial glycocalyx (EG) is the carbohydrate-rich luminal lining of endothelial cells that mediates permeability and blood cell-vessel wall interactions. To establish an atheroprotective role of the EG, adequate imaging and quantification of its properties in intact, viable, atherogenesis-prone arteries is needed. METHODS: Carotid arteries of C57Bl6/J mice (n=22) were isolated including the bifurcation, mounted in a perfusion chamber, and perfused with fluorescent lectin wheat germ agglutinin-fluorescein isothiocyanate. The EG was visualized through the vessel wall using two-photon laser scanning microscopy. An image quantification protocol was developed to assess EG thickness, which was sensitive to hyaluronidase-induced changes. RESULTS: In the lesion-protected common carotid artery, EG thickness was found to be 2.3 ± 0.1 µm (mean ± SEM), while the surface area devoid of (wheat germ agglutinin-sensitive) EG was 8.9 ± 4.2%. Data from the external carotid artery were similar (2.5 ± 0.1 µm; 9.1 ± 5.0%). In the atherogenesis-prone internal carotid artery the EG-devoid surface area was significantly higher (27.4 ± 5.5%, p<0.05); thickness at the remaining areas was 2.5 ± 0.1 µm. CONCLUSION: The EG can be adequately imaged and quantified using two-photon laser scanning microscopy in intact, viable mounted carotid arteries. Spatial EG differences could underlie atherogenesis.

Evaluation of magnetic resonance vessel size imaging by two-photon laser scanning microscopy.

MR vessel size imaging (MR-VSI) is increasingly applied to noninvasively assess microvascular properties of tumors and to evaluate tumor response to antiangiogenic treatment. MR-VSI provides measures for the microvessel radius and fractional blood volume of tumor tissue. However, data have not yet been evaluated with three-dimensional microscopy techniques. Therefore, three-dimensional two-photon laser scanning microscopy (TPLSM) was performed to assess microvascular radius and fractional vessel volume in tumor and muscle tissue. TPLSM data displayed a mazelike architecture of the tumor microvasculature and mainly parallel oriented muscle microvessels. For both MR-VSI and TPLSM, a larger vessel radius and fractional blood volume were found in the tumor rim than in the core. The microvessel radius was approximately six times larger in tumor and muscle for MR-VSI than for TPLSM. The tumor blood volume was 4-fold lower with MR-VSI than with TPLSM, whereas muscle blood volume was comparable for both techniques. Differences between the tumor rim, core, and muscle tissue showed similar trends for both MR-VSI and TPLSM parameters. These results indicate that MR-VSI does not provide absolute measures of microvascular morphology; however, it does reflect heterogeneity in microvascular morphology. Hence, MR-VSI may be used to assess differences in microvascular morphology.

Frontal and lateral characteristics of the osseous configuration in chronic ankle instability.

OBJECTIVE: The osseous ankle configuration (tibiotalar sector, talar radius and height) has been discovered as intrinsic risk factor for chronic ankle instability (CAI). These measurements were done on lateral radiographs only. In this study, the osseous characteristics in the frontal plane and further lateral values were measured. DESIGN: Level III case-control study. SETTING: Radiological measurement of frontal and lateral radiographs by one independent, blinded radiologist using a digital DICOM/PACS system. PATIENTS: A group of 52 patients with CAI was compared with an age- and sex-matched control group of 52 healthy subjects. MAIN OUTCOME MEASUREMENTS: In the frontal plane, the depth of the talar curvature (frontal curvature (froCu)) and the lateral and medial malleolar lengths were measured. In the lateral plane, the position of the centre of rotation to the tibial axis (talar centre of rotation to the anatomical axis of the tibia (TibCOR)) and the tibial lateral surface angle (TLS) were also measured. RESULTS: The froCu was deeper in patients with CAI (1.8 (0.5) mm) than in healthy subjects (1.0 (0.5) mm, p<0.05). The TibCOR was more anterior in patients with CAI (2.5 (1.9) mm) than in healthy subjects (1.6 (2.2) mm, p<0.05). The distance from the fibular tip to the centre of rotation was smaller in patients with CAI (3.5 (3.4) mm) than in healthy subjects (6.5 (3.3) mm, p<0.05). The TLS and the length of the lateral and medial ankle were not significantly different. CONCLUSIONS: This study supports that the osseous joint configuration is an intrinsic risk factor for CAI. It could be shown that CAI is characterised by a deeper frontal curvature of the talus and a more anterior position of the talus to the tibia.

Two Functional Axes of Feedback-Enforced PRC2 Recruitment in Mouse Embryonic Stem Cells.

Polycomb Repressive Complex 2 (PRC2) plays an essential role in gene repression during development, catalyzing H3 lysine 27 trimethylation (H3K27me3). MTF2 in the PRC2.1 sub-complex, and JARID2 in PRC2.2, are central in core PRC2 recruitment to target genes in mouse embryonic stem cells (mESCs). To investigate how PRC2.1 and PRC2.2 cooperate, we combined Polycomb mutant mESCs with chemical inhibition of binding to H3K27me3. We find that PRC2.1 and PRC2.2 mediate two distinct paths for recruitment, which are mutually reinforced. Whereas PRC2.1 recruitment is mediated by MTF2 binding to DNA, JARID2-containing PRC2.2 recruitment is more dependent on PRC1. Both recruitment axes are supported by core subunit EED binding to H3K27me3, but EED inhibition exhibits a more pronounced effect in Jarid2 null cells. Finally, we show that PRC1 and PRC2 enhance reciprocal binding. Together, these data disentangle the interdependent interactions that are important for PRC2 recruitment.

Nanoparticles for optical molecular imaging of atherosclerosis.

Molecular imaging contributes to future personalized medicine dedicated to the treatment of cardiovascular disease, the leading cause of mortality in industrialized countries. Endoscope-compatible optical imaging techniques would offer a stand-alone alternative and high spatial resolution validation technique to clinically accepted imaging techniques in the (intravascular) assessment of vulnerable atherosclerotic lesions, which are predisposed to initiate acute clinical events. Efficient optical visualization of molecular epitopes specific for vulnerable atherosclerotic lesions requires targeting of high-quality optical-contrast-enhancing particles. In this review, we provide an overview of both current optical nanoparticles and targeting ligands for optical molecular imaging of atherosclerotic lesions and speculate on their applicability in the clinical setting.

Design and hydraulic characteristics of a field-scale bi-phasic bioretention rain garden system for storm water management.

A field-scale bioretention rain garden system was constructed using a novel bi-phasic (i.e. sequence of anaerobic to aerobic) concept for improving retention and removal of storm water runoff pollutants. Hydraulic tests with bromide tracer and simulated runoff pollutants (nitrate-N, phosphate-P, Cu, Pb, and Zn) were performed in the system under a simulated continuous rainfall. The objectives of the tests were (1) to determine hydraulic characteristics of the system, and (2) to evaluate the movement of runoff pollutants through the system. For the 180 mm/24 h rainfall, the bi-phasic bioretention system effectively reduced both peak flow (approximately 70%) and runoff volume (approximately 42%). The breakthrough curves (BTCs) of bromide tracer suggest that the transport pattern of the system is similar to dispersed plug flow under this large runoff event. The BTCs of bromide showed mean 10% and 90% breakthrough times of 5.7 h and 12.5 h, respectively. Under the continuous rainfall, a significantly different transport pattern was found between each runoff pollutant. Nitrate-N was easily transported through the system with potential leaching risk from the initial soil medium, whereas phosphate-P and metals were significantly retained indicating sorption-mediated transport. These findings support the importance of hydraulics, in combination with the soil medium, when creating bioretention systems for bioremediation that are effective for various rainfall sizes and intervals.

Two-photon microscopy on vital carotid arteries: imaging the relationship between collagen and inflammatory cells in atherosclerotic plaques.

We used two-photon laser scanning microscopy (TPLSM) to demonstrate for the first time its potential in studying relational details at the cellular level of atherogenesis in intact, viable mouse carotid arteries. Isolated and mounted arteries of ApoE-/-mice, aged 15 or 21 weeks (7 and 13 weeks on western diet), were imaged after labeling with specific fluorescent markers for cell nuclei, inflammatory cells, collagen, and lipids. Data were compared with C57BL6/J mice fed a chow diet. Control vessels had intact endothelium without adhering blood cells or significant intimal collagen labeling. In ApoE-/-mice already at 15 weeks, inflammatory cells adhered to the endothelium and increased labeling of collagen was observed in tunica intima at both lesion-prone and non-lesion-prone sites, indicating endothelium activation. In plaques, internalized inflammatory cell density increased with age and plaque progression in tunicae adventitia and intima, but not media. In the whole plaque, aging or plaque progression did not alter the direct relationship between inflammatory cells and collagen. However, within the fibrous caps specifically, direct contact between inflammatory cells and collagen increased with age. This study demonstrates the potential of TPLSM in determining detailed information regarding the complex relationship between inflammatory cells and collagen during atherogenesis.

Two-photon lifetime imaging of fluorescent probes in intact blood vessels: a window to sub-cellular structural information and binding status.

Fluorescence lifetime imaging (FLIM) provides a complementary contrast mechanism to fluorescence intensity and ratio imaging in intact tissue. With FLIM the time-resolved decay in fluorescence intensity of (interacting) fluorophores can be quantified by means of time correlated single photon counting (TCSPC). Here we focus on fluorescence lifetime imaging in intact blood vessels. Requisites for imaging in intact tissue are good penetration depth and limited tissue damage. Therefore, in this pilot-study, we performed TCSPC-FLIM using two-photon laser scanning microscopy to determine, with sub-cellular resolution, the fluorescence lifetime of two fluorescent probes. First, we focused on the nucleic acid dye SYTO41 in the various compartments of cells in vitro and in situ in the wall of intact mouse carotid arteries. Second, it was assessed whether the interaction of the lectin WGA-FITC with the endothelial glycocalyx affects its fluorescence lifetime. Results showed comparable mono-exponential fluorescence lifetimes of SYTO41 in the nuclei of cells in vitro and in situ. The slightly shorter fluorescence lifetime observed in the cytoplasm allowed discrimination of the nuclei. SYTO41 displayed strong mitochondrial staining, as was verified by the mitochondrion-specific probe CMXRos. In addition, mitochondrial staining by SYTO41 was accompanied by a green shift in emission. In the mitochondrial region, SYTO41 showed a highly bi-exponential and relatively fast decay, with two distinct lifetime components. It is hypothesized that the fitted bi-exponential decay can either be contributed to (1) the mathematical approximation of the fluorescence intensity decay or (2) the presence of free and DNA-bound SYTO41 in the mitochondrial compartment, leading to two lifetime components. The fluorescence lifetime of WGA-FITC decreased by approximately 25% upon binding to the endothelial glycocalyx. From this study, we conclude that FLIM offers an additional contrast mechanism in imaging intact tissue and provides information on binding status between a probe and its ligand.

Influence of soil pH and application rate on the oxidation of calcium sulfite derived from flue gas desulfurization.

Calcium sulfite hemihydrate (CaSO(3).0.5H2O), a common byproduct of coal-fired utilities, is fairly insoluble and can decompose to release toxic SO2 under highly acidic soil conditions; however, it can also oxidize to form gypsum. The objective of this study was to examine the effects of application rate and soil pH on the oxidation of calcium sulfite under laboratory conditions. Oxidation rates measured by release of SO4-S to solution decreased with increasing application rate. Leachate SO4-S from soils amended with 1.0 to 3.0 g kg-1 CaSO3 increased over a 21 to 28 d period before reaching a plateau. At 4 g kg-1, maximum SO4-S release was delayed until Week 7. Oxidation and release of SO4-S from soil amended with 3.0 g kg-1 calcium sulfite increased markedly with decreasing soil pH. After only 3 d incubation, the concentrations of SO4-S in aqueous leachates were 77, 122, 170, 220, and 229 mg L-1 for initial soil pH values of 7.8, 6.5, 5.5, 5.1, and 4.0, respectively. At an initial soil pH value of 4.0, oxidation/dissolution did not increase much after 3 d. At higher pH values, oxidation was maximized after 21 d. These results suggest that autumn surface applications of calcium sulfite in no-till systems should permit ample time for oxidation/dissolution reactions to occur without introducing biocidal effects related to oxygen scavenging. Soil and annual crops can thus benefit from additions of soluble Ca and SO4 if calcium sulfite is applied in advance of spring planting.

In vivo blockade of platelet ADP receptor P2Y12 reduces embolus and thrombus formation but not thrombus stability.

OBJECTIVE: ADP is a key platelet agonist in thromboembolism. One of the receptors involved in ADP-induced platelet activation is the P2Y12 receptor, which is a target for antithrombotic drugs. METHODS AND RESULTS: Here, we present first evidence for a differential role of this receptor in thrombus and embolus formation in vivo. Anesthetized rabbits were treated with the selective P2Y12 antagonists AR-C69931 MX (3 microg x kg x min(-1) IV) or clopidogrel (25 mg/kg orally). Efficacy of these treatments was monitored by aggregation and thrombin generation measurements in blood samples ex vivo. Mesenteric arterioles were mechanically injured; thrombus growth and subsequent embolus formation were visualized by real-time intravital microscopy. AR-C69931 MX and clopidogrel significantly (P<0.05) reduced the total duration of embolization (by 52% and 36%, respectively), and fewer and smaller emboli were produced. The size of the initial thrombus was significantly reduced (P<0.005), but its stability was unaffected: plug formation was still effective. CONCLUSIONS: These findings demonstrate that ADP and its P2Y12 receptor are involved in thrombus growth and especially in the formation of emboli on the downstream side of the initial thrombus. This may explain the beneficial effects of P2Y12 receptor antagonists in secondary prevention of ischemic events in patients with arterial thrombosis.