Event-responsive scanning transmission electron microscopy.

An ever-present limitation of transmission electron microscopy is the damage caused by high-energy electrons interacting with any sample. By reconsidering the fundamentals of imaging, we demonstrate an event-responsive approach to electron microscopy that delivers more information about the sample for a given beam current. Measuring the time to achieve an electron count threshold rather than waiting a predefined constant time improves the information obtained per electron. The microscope was made to respond to these events by blanking the beam, thus reducing the overall dose required. This approach automatically apportions dose to achieve a given signal-to-noise ratio in each pixel, eliminating excess dose that is associated with diminishing returns of information. We demonstrate the wide applicability of our approach to beam-sensitive materials by imaging biological tissue and zeolite.

Targeting Cyclophilin A in the Cardiac Microenvironment Preserves Heart Function and Structure in Failing Hearts.

BACKGROUND: Cardiac hypertrophy is characterized by remodeling of the myocardium, which involves alterations in the ECM (extracellular matrix) and cardiomyocyte structure. These alterations critically contribute to impaired contractility and relaxation, ultimately leading to heart failure. Emerging evidence implicates that extracellular signaling molecules are critically involved in the pathogenesis of cardiac hypertrophy and remodeling. The immunophilin CyPA (cyclophilin A) has been identified as a potential culprit. In this study, we aimed to unravel the interplay between eCyPA (extracellular CyPA) and myocardial dysfunction and evaluate the therapeutic potential of inhibiting its extracellular accumulation to improve heart function. METHODS: Employing a multidisciplinary approach encompassing in silico, in vitro, in vivo, and ex vivo experiments we studied a mouse model of cardiac hypertrophy and human heart specimen to decipher the interaction of CyPA and the cardiac microenvironment in highly relevant pre-/clinical settings. Myocardial expression of CyPA (immunohistology) and the inflammatory transcriptome (NanoString) was analyzed in human cardiac tissue derived from patients with nonischemic, noninflammatory congestive heart failure (n=187). These analyses were paralleled by a mouse model of Ang (angiotensin) II-induced heart failure, which was assessed by functional (echocardiography), structural (immunohistology, atomic force microscopy), and biomolecular (Raman spectroscopy) analyses. The effect of inhibiting eCyPA in the cardiac microenvironment was evaluated using a newly developed neutralizing anti-eCyPA monoclonal antibody. RESULTS: We observed a significant accumulation of eCyPA in both human and murine-failing hearts. Importantly, higher eCyPA expression was associated with poor clinical outcomes in patients (P=0.043) and contractile dysfunction in mice (Pearson correlation coefficient, -0.73). Further, myocardial expression of eCyPA was critically associated with an increase in myocardial hypertrophy, inflammation, fibrosis, stiffness, and cardiac dysfunction in vivo. Antibody-based inhibition of eCyPA prevented (Ang II)-induced myocardial remodeling and dysfunction in mice. CONCLUSIONS: Our study provides strong evidence of the pathogenic role of eCyPA in remodeling, myocardial stiffening, and dysfunction in heart failure. The findings suggest that antibody-based inhibition of eCyPA may offer a novel therapeutic strategy for nonischemic heart failure. Further research is needed to evaluate the translational potential of these interventions in human patients with cardiac hypertrophy.

Structural color in the bacterial domain: The ecogenomics of a 2-dimensional optical phenotype.

Structural color is an optical phenomenon resulting from light interacting with nanostructured materials. Although structural color (SC) is widespread in the tree of life, the underlying genetics and genomics are not well understood. Here, we collected and sequenced a set of 87 structurally colored bacterial isolates and 30 related strains lacking SC. Optical analysis of colonies indicated that diverse bacteria from at least two different phyla (Bacteroidetes and Proteobacteria) can create two-dimensional packing of cells capable of producing SC. A pan-genome-wide association approach was used to identify genes associated with SC. The biosynthesis of uroporphyrin and pterins, as well as carbohydrate utilization and metabolism, was found to be involved. Using this information, we constructed a classifier to predict SC directly from bacterial genome sequences and validated it by cultivating and scoring 100 strains that were not part of the training set. We predicted that SCr is widely distributed within gram-negative bacteria. Analysis of over 13,000 assembled metagenomes suggested that SC is nearly absent from most habitats associated with multicellular organisms except macroalgae and is abundant in marine waters and surface/air interfaces. This work provides a large-scale ecogenomics view of SC in bacteria and identifies microbial pathways and evolutionary relationships that underlie this optical phenomenon.

Machine learning predicts emergency physician specialties from treatment strategies for patients suspected of myocardial infarction.

BACKGROUND: Our investigation aimed to determine how the diverse backgrounds and medical specialties of emergency physicians (Eps) influence the accuracy of diagnoses and the subsequent treatment pathways for patients presenting preclinically with MI symptoms. By scrutinizing the relationships between EPs' specialties and their approaches to patient care, we aimed to unveil potential variances in diagnostic accuracy and treatment choices. METHODS: In this retrospective, monocenter cohort study, we leveraged machine learning techniques to analyze a comprehensive dataset of 2328 patients with suspected MI, encompassing preclinical diagnoses, electrocardiogram (ECG) interpretations, and subsequent treatment strategies by attending EPs. RESULTS: We demonstrated that diagnosis and treatment patterns of different specialties were distinct enough, that machine learning (ML) was able to differentiate between specialties (maximum area under the receiver operating characteristic = 0.80 for general medicine and 0.80 for surgery). In our study, internist demonstrated the highest accuracy for preclinical identification of STEMI (0.96) whereas surgeons showed the highest accuracy for identifying NSTEMI. Our findings highlight significant correlations between EP specialties and the accuracy of both preclinical diagnoses and subsequent treatment pathways for patients with suspected MI. CONCLUSIONS: Our results offer valuable insights into how the diverse backgrounds and specialties of EPs can influence the optimization of patient care in emergency settings. Understanding these patterns can help in the development of tailored training programs and protocols to enhance diagnostic accuracy and treatment efficacy in emergency cardiac care, ultimately optimizing patient treatment and improving outcomes.

Preclinical identification of acute coronary syndrome without high sensitivity troponin assays using machine learning algorithms.

Preclinical management of patients with acute chest pain and their identification as candidates for urgent coronary revascularization without the use of high sensitivity troponin essays remains a critical challenge in emergency medicine. We enrolled 2760 patients (average age 70 years, 58.6% male) with chest pain and suspected ACS, who were admitted to the Emergency Department of the University Hospital Tübingen, Germany, between August 2016 and October 2020. Using 26 features, eight Machine learning models (non-deep learning models) were trained with data from the preclinical rescue protocol and compared to the "TropOut" score (a modified version of the "preHEART" score which consists of history, ECG, age and cardiac risk but without troponin analysis) to predict major adverse cardiac event (MACE) and acute coronary artery occlusion (ACAO). In our study population MACE occurred in 823 (29.8%) patients and ACAO occurred in 480 patients (17.4%). Interestingly, we found that all machine learning models outperformed the "TropOut" score. The VC and the LR models showed the highest area under the receiver operating characteristic (AUROC) for predicting MACE (AUROC = 0.78) and the VC showed the highest AUROC for predicting ACAO (AUROC = 0.81). A SHapley Additive exPlanations (SHAP) analyses based on the XGB model showed that presence of ST-elevations in the electrocardiogram (ECG) were the most important features to predict both endpoints.

Machine learning insights into thrombo-ischemic risks and bleeding events through platelet lysophospholipids and acylcarnitine species.

Coronary artery disease (CAD) often leads to adverse events resulting in significant disease burdens. Underlying risk factors often remain inapparent prior to disease incidence and the cardiovascular (CV) risk is not exclusively explained by traditional risk factors. Platelets inherently promote atheroprogression and enhanced platelet functions and distinct platelet lipid species are associated with disease severity in patients with CAD. Lipidomics data were acquired using mass spectrometry and processed alongside clinical data applying machine learning to model estimates of an increased CV risk in a consecutive CAD cohort (n = 595). By training machine learning models on CV risk measurements, stratification of CAD patients resulted in a phenotyping of risk groups. We found that distinct platelet lipids are associated with an increased CV or bleeding risk and independently predict adverse events. Notably, the addition of platelet lipids to conventional risk factors resulted in an increased diagnostic accuracy of patients with adverse CV events. Thus, patients with aberrant platelet lipid signatures and platelet functions are at elevated risk to develop adverse CV events. Machine learning combining platelet lipidome data and common clinical parameters demonstrated an increased diagnostic value in patients with CAD and might improve early risk discrimination and classification for CV events.

Doxorubicin Exposure and Breast Cancer Risk in Survivors of Adolescent and Adult Hodgkin Lymphoma.

PURPOSE: Female Hodgkin lymphoma (HL) survivors treated with chest radiotherapy (RT) at a young age have a strongly increased risk of breast cancer (BC). Studies in childhood cancer survivors have shown that doxorubicin exposure may also increase BC risk. Although doxorubicin is the cornerstone of HL chemotherapy, the association between doxorubicin and BC risk has not been examined in HL survivors treated at adult ages. METHODS: We assessed BC risk in a cohort of 1,964 female 5-year HL survivors, treated at age 15-50 years in 20 Dutch hospitals between 1975 and 2008. We calculated standardized incidence ratios, absolute excess risks, and cumulative incidences. Doxorubicin exposure was analyzed using multivariable Cox regression analyses. RESULTS: After a median follow-up of 21.6 years (IQR, 15.8-27.1 years), 252 women had developed invasive BC or ductal carcinoma in situ. The 30-year cumulative incidence was 20.8% (95% CI, 18.2 to 23.4). Survivors treated with a cumulative doxorubicin dose of >200 mg/m2 had a 1.5-fold increased BC risk (95% CI, 1.08 to 2.1), compared with survivors not treated with doxorubicin. BC risk increased 1.18-fold (95% CI, 1.05 to 1.32) per additional 100 mg/m2 doxorubicin (Ptrend = .004). The risk increase associated with doxorubicin (yes v no) was not modified by age at first treatment (hazard ratio [HR]age <21 years, 1.5 [95% CI, 0.9 to 2.6]; HRage ≥21 years, 1.3 [95% CI, 0.9 to 1.9) or chest RT (HRwithout mantle/axillary field RT, 1.9 [95% CI, 1.06 to 3.3]; HRwith mantle/axillary field RT, 1.2 [95% CI, 0.8 to 1.8]). CONCLUSION: This study shows that treatment with doxorubicin is associated with increased BC risk in both adolescent and adult HL survivors. Our results have implications for BC surveillance guidelines for HL survivors and treatment strategies for patients with newly diagnosed HL.

A recent gibbon ape leukemia virus germline integration in a rodent from New Guinea.

Germline colonization by retroviruses results in the formation of endogenous retroviruses (ERVs). Most colonization's occurred millions of years ago. However, in the Australo-Papuan region (Australia and New Guinea), several recent germline colonization events have been discovered. The Wallace Line separates much of Southeast Asia from the Australo-Papuan region restricting faunal and pathogen dispersion. West of the Wallace Line, gibbon ape leukemia viruses (GALVs) have been isolated from captive gibbons. Two microbat species from China appear to have been infected naturally. East of Wallace's Line, the woolly monkey virus (a GALV) and the closely related koala retrovirus (KoRV) have been detected in eutherians and marsupials in the Australo-Papuan region, often vertically transmitted. The detected vertically transmitted GALV-like viruses in Australo-Papuan fauna compared to sporadic horizontal transmission in Southeast Asia and China suggest the GALV-KoRV clade originates in the former region and further models of early-stage genome colonization may be found. We screened 278 samples, seven bat and one rodent family endemic to the Australo-Papuan region and bat and rodent species found on both sides of the Wallace Line. We identified two rodents (Melomys) from Australia and Papua New Guinea and no bat species harboring GALV-like retroviruses. Melomys leucogaster from New Guinea harbored a genomically complete replication-competent retrovirus with a shared integration site among individuals. The integration was only present in some individuals of the species indicating this retrovirus is at the earliest stages of germline colonization of the Melomys genome, providing a new small wild mammal model of early-stage genome colonization.

cGMP modulates hemin-mediated platelet death.

BACKGROUND AND AIMS: Hemolysis is a known risk factor for thrombosis resulting in critical limb ischemia and microcirculatory disturbance and organ failure. Intravasal hemolysis may lead to life-threatening complications due to uncontrolled thrombo-inflammation. Until now, conventional antithrombotic therapies failed to control development and progression of these thrombotic events. Thus, the pathophysiology of these thrombotic events needs to be investigated to unravel underlying pathways and thereby identify targets for novel treatment strategies. METHODS: Here we used classical experimental set-ups as well as high-end flow cytometry, metabolomics and lipidomic analysis to in-depth analyze the effects of hemin on platelet physiology and morphology. RESULTS: Hemin does strongly and swiftly induce platelet activation and this process is modulated by the sGC-cGMP-cGKI signaling axis. cGMP modulation also reduced the pro-aggregatory potential of plasma derived from patients with hemolysis. Furthermore, hemin-induced platelet death evokes distinct platelet subpopulations. Typical cell death markers, such as ROS, were induced by hemin-stimulation and the platelet lipidome was specifically altered by high hemin concentration. Specifically, arachidonic acid derivates, such as PGE2, TXB2 or 12-HHT, were significantly increased. Balancing the cGMP levels by modulation of the sGC-cGMP-cGKI axis diminished the ferroptotic effect of hemin. CONCLUSION: We found that cGMP modulates hemin-induced platelet activation and thrombus formation in vitro and cGMP effects hemin-mediated platelet death and changes in the platelet lipidome. Thus, it is tempting to speculate that modulating platelet cGMP levels may be a novel strategy to control thrombosis and critical limb ischemia in patients with hemolytic crisis.

Plasmodesmal connectivity in C4 Gynandropsis gynandra is induced by light and dependent on photosynthesis.

In leaves of C4 plants, the reactions of photosynthesis become restricted between two compartments. Typically, this allows accumulation of C4 acids in mesophyll (M) cells and subsequent decarboxylation in the bundle sheath (BS). In C4 grasses, proliferation of plasmodesmata between these cell types is thought to increase cell-to-cell connectivity to allow efficient metabolite movement. However, it is not known whether C4 dicotyledons also show this enhanced plasmodesmal connectivity and so whether this is a general requirement for C4 photosynthesis is not clear. How M and BS cells in C4 leaves become highly connected is also not known. We investigated these questions using 3D- and 2D-electron microscopy on the C4 dicotyledon Gynandropsis gynandra as well as phylogenetically close C3 relatives. The M-BS interface of C4 G. gynandra showed higher plasmodesmal frequency compared with closely related C3 species. Formation of these plasmodesmata was induced by light. Pharmacological agents that perturbed photosynthesis reduced the number of plasmodesmata, but this inhibitory effect could be reversed by the provision of exogenous sucrose. We conclude that enhanced formation of plasmodesmata between M and BS cells is wired to the induction of photosynthesis in C4 G. gynandra.

A missense mutation in Ehd1 associated with defective spermatogenesis and male infertility.

Normal function of the C-terminal Eps15 homology domain-containing protein 1 (EHD1) has previously been associated with endocytic vesicle trafficking, shaping of intracellular membranes, and ciliogenesis. We recently identified an autosomal recessive missense mutation c.1192C>T (p.R398W) of EHD1 in patients who had low molecular weight proteinuria (0.7-2.1 g/d) and high-frequency hearing loss. It was already known from Ehd1 knockout mice that inactivation of Ehd1 can lead to male infertility. However, the exact role of the EHD1 protein and its p.R398W mutant during spermatogenesis remained still unclear. Here, we report the testicular phenotype of a knockin mouse model carrying the p.R398W mutation in the EHD1 protein. Male homozygous knockin mice were infertile, whereas the mutation had no effect on female fertility. Testes and epididymes were significantly reduced in size and weight. The testicular epithelium appeared profoundly damaged and had a disorganized architecture. The composition of developing cell types was altered. Malformed acrosomes covered underdeveloped and misshaped sperm heads. In the sperm tail, midpieces were largely missing indicating disturbed assembly of the sperm tail. Defective structures, i.e., nuclei, acrosomes, and sperm tail midpieces, were observed in large vacuoles scattered throughout the epithelium. Interestingly, cilia formation itself did not appear to be affected, as the axoneme and other parts of the sperm tails except the midpieces appeared to be intact. In wildtype mice, EHD1 co-localized with acrosomal granules on round spermatids, suggesting a role of the EHD1 protein during acrosomal development. Wildtype EHD1 also co-localized with the VPS35 component of the retromer complex, whereas the p.R398W mutant did not. The testicular pathologies appeared very early during the first spermatogenic wave in young mice (starting at 14 dpp) and tubular destruction worsened with age. Taken together, EHD1 plays an important and probably multifaceted role in spermatogenesis in mice. Therefore, EHD1 may also be a hitherto underestimated infertility gene in humans.

Ectopic callose deposition into woody biomass modulates the nano-architecture of macrofibrils.

Plant biomass plays an increasingly important role in the circular bioeconomy, replacing non-renewable fossil resources. Genetic engineering of this lignocellulosic biomass could benefit biorefinery transformation chains by lowering economic and technological barriers to industrial processing. However, previous efforts have mostly targeted the major constituents of woody biomass: cellulose, hemicellulose and lignin. Here we report the engineering of wood structure through the introduction of callose, a polysaccharide novel to most secondary cell walls. Our multiscale analysis of genetically engineered poplar trees shows that callose deposition modulates cell wall porosity, water and lignin contents and increases the lignin-cellulose distance, ultimately resulting in substantially decreased biomass recalcitrance. We provide a model of the wood cell wall nano-architecture engineered to accommodate the hydrated callose inclusions. Ectopic polymer introduction into biomass manifests in new physico-chemical properties and offers new avenues when considering lignocellulose engineering.

Electron counting detectors in scanning transmission electron microscopy via hardware signal processing.

Transmission electron microscopy is a pivotal instrument in materials and biological sciences due to its ability to provide local structural and spectroscopic information on a wide range of materials. However, the electron detectors used in scanning transmission electron microscopy are often unable to provide quantified information, that is the number of electrons impacting the detector, without exhaustive calibration and processing. This results in arbitrary signal values with slow response times that cannot be used for quantification or comparison to simulations. Here we demonstrate and optimise a hardware signal processing approach to augment electron detectors to perform single electron counting.

Thrombus architecture is influenced by the antiplatelet loading treatment in patients with acute myocardial infarction.

BACKGROUND: Intracoronary thrombus formation is a main cause of acute myocardial infarction triggered by platelet activation. However, there are no data on the impact of different treatment strategies with antiplatelet agents before percutaneous coronary intervention (PCI) on histological characteristics of thrombus formation. OBJECTIVE: In this study, we investigate the impact of preinterventional administration of the P2Y12-inhibitors clopidogrel and prasugrel on thrombus composition, highlighting significant changes associated with the antiplatelet pre-treatment. METHODS: We prospectively enrolled 104 consecutive patients with ST-segment elevation myocardial infarction (STEMI) undergoing immediate PCI and thrombus aspiration by immunohistochemical staining along with RNA-sequencing employing Nanostring analysis. Fifty-two patients were treated with either prasugrel loading (60 mg) or clopidogrel loading (600 mg) prior to PCI, respectively. RESULTS: In Patients with STEMI, intracoronary thrombus architecture was significantly altered between patients pre-treated with prasugrel when compared to clopidogrel. Fibrin content of thrombi was significantly decreased (41.8 % versus 66.7 %, p = 0.009) after pre-treatment with prasugrel compared to clopidogrel. Furthermore, levels of MPO positive cells in intracoronary thrombi were significantly decreased in patients with prasugrel pre-treatment (90.5 versus 201.1, p = 0.014) indicating an association of antiplatelet pre-treatment and the inflammatory responses during thrombus formation. Most strikingly, we observed significant differences among both pre-treatment groups regarding altered RNA expression and signaling pathways of thrombo-inflammatory processes within the thrombotic material, which were independently associated with antiplatelet strategies. CONCLUSIONS: Our study elucidates the impact of antiplatelet pre-treatment on thrombus remodeling and architecture, thereby lowering the risk of recurrent adverse cardiovascular events in prasugrel-treated patients.

Downregulation of extraembryonic tension controls body axis formation in avian embryos.

Embryonic tissues undergoing shape change draw mechanical input from extraembryonic substrates. In avian eggs, the early blastoderm disk is under the tension of the vitelline membrane (VM). Here we report that the chicken VM characteristically downregulates tension and stiffness to facilitate stage-specific embryo morphogenesis. Experimental relaxation of the VM early in development impairs blastoderm expansion, while maintaining VM tension in later stages resists the convergence of the posterior body causing stalled elongation, failure of neural tube closure, and axis rupture. Biochemical and structural analysis shows that VM weakening is associated with the reduction of outer-layer glycoprotein fibers, which is caused by an increasing albumen pH due to CO2 release from the egg. Our results identify a previously unrecognized potential cause of body axis defects through mis-regulation of extraembryonic tissue tension.

Platelet versus plasma CXCL14, coronary artery disease, and clinical outcomes.

Background: Platelets express CXCL14, while platelet-derived CXCL14 induces monocyte chemotaxis and exerts an angiostatic effect on endothelial cells. Objectives: This study investigated both platelet surface-associated and circulating levels of CXCL14 in patients with heart disease and associations of this chemokine with myocardial function and outcomes in patients with coronary artery disease (CAD). Methods: This prospective study enrolled 450 patients with symptomatic heart disease. Platelet surface-associated and plasma CXCL14 levels were analyzed. All patients were followed up for 360 days for a primary composite outcome consisting of all-cause mortality, myocardial infarction, and/or ischemic stroke. Secondary outcomes consisted of the single events of all-cause mortality or myocardial infarction. Results: Baseline platelet-associated but not circulating CXCL14 levels were significantly lower in patients with chronic coronary syndrome (mean fluorescence intensity logarithmized, 1.35 ± 0.35) when compared to those with acute coronary syndrome (1.47 ± 0.38) and without CAD (1.51 ± 0.40). Platelet CXCL14 levels were significantly lower (1.37 ± 0.37 vs 1.48 ± 0.39) and circulating CXCL14 levels were significantly higher (lg, 2.88 ± 0.20 pg/mL vs 2.82 ± 0.26 pg/mL) in patients with normal baseline left ventricular ejection fraction (LVEF) when compared to those with impaired LVEF. Low baseline circulating CXCL14 (hazard ratio, 2.33; 1.00-5.46) but not platelet CXCL14 was associated with worse outcome in patients with CAD. Conclusion: Platelet-associated and circulating CXCL14 levels show differential regulation in patients with and without CAD. Although platelet-associated CXCL14 increased and circulating CXCL14 decreased with impairment of LVEF, only lower circulating CXCL14 upon admission was associated with worse prognosis in patients with CAD.

Porcine spermadhesin AQN-3 binds to negatively charged phospholipids.

The spermadhesin AQN-3 is a major component of porcine seminal plasma. While various studies suggest that this protein binds to boar sperm cells, its attachment to the cells is poorly understood. Therefore, the capacity of AQN-3 to interact with lipids was investigated. For that purpose, AQN-3 was recombinantly expressed in E. coli and purified via the included His-tag. Characterizing the quaternary structure by size exclusion chromatography revealed that recombinant AQN-3 (recAQN-3) is largely present as multimer and/or aggregate. To determine the lipid specificity of recAQN-3, a lipid stripe method and a multilamellar vesicle (MLV)-based binding assay were used. Both assays show that recAQN-3 selectively interacts with negatively charged lipids, like phosphatidic acid, phosphatidylinositol phosphates, and cardiolipin. No interaction was observed with phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or cholesterol. The affinity to negatively charged lipids can be explained by electrostatic interactions because binding is partly reversed under high-salt condition. However, more factors have to be assumed like hydrogen bonds and/or hydrophobic forces because the majority of bound molecules was not released by high salt. To confirm the observed binding behavior for the native protein, porcine seminal plasma was incubated with MLVs comprising phosphatidic acid or phosphatidyl-4,5-bisphosphate. Attached proteins were isolated, digested, and analyzed by mass spectrometry. Native AQN-3 was detected in all samples analyzed and was - besides AWN - the most abundant protein. It remains to be investigated whether AQN-3, together with other sperm associated seminal plasma proteins, acts as decapacitation factor by targeting negative lipids with signaling or other functional roles in fertilization.

The Subtilisin-Like Protease Furin Regulates Hemin-Dependent Ectodomain Shedding of Glycoprotein VI.

INTRODUCTION: Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis. METHODS: We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin. RESULTS: We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions. CONCLUSION: In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.

Coverage of Lateral Lymph Nodes in Rectal Cancer Patients with Routine Radiation Therapy Practice and Associated Locoregional Recurrence Rates.

PURPOSE: Involved internal iliac and obturator lateral lymph nodes (LLNs) are a known risk factor for the occurrence of ipsilateral local recurrences (LLR) in rectal cancer. This study examined coverage of LLNs with routine radiation therapy practice in the Netherlands and associated LLR rates. METHODS AND MATERIALS: Patients with a primary tumor ≤8 cm of the anorectal junction, cT3-4 stage, and at least 1 internal iliac or obturator LLN with short axis ≥5 mm who received neoadjuvant (chemo)radiation therapy, were selected from a national, cross-sectional study of patients with rectal cancer treated in the Netherlands in 2016. Magnetic resonance images and radiation therapy treatment plans were reviewed regarding segmented LLNs as gross tumor volume (GTV), location of LLNs within clinical target volume (CTV), and received proportion of the planned radiation therapy dose. RESULTS: A total of 223 out of 3057 patients with at least 1 LLN ≥5 mm were selected. Of those, 180 (80.7%) LLNs were inside the CTV, of which 60 (33.3%) were segmented as GTV. Overall, 202 LLNs (90.6%) received ≥95% of the planned dose. Four-year LLR rates were not significantly higher for LLNs situated outside the CTV compared with those inside (4.0% vs 12.5%, P = .092) or when receiving <95% versus ≥95% of the planned radiation therapy dose (7.1% vs 11.3%, P = .843), respectively. Two of 7 patients who received a dose escalation of 60 Gy developed an LLR (4-year LLR rate of 28.6%). CONCLUSIONS: This evaluation of routine radiation therapy practice showed that adequate coverage of LLNs was still associated with considerable 4-year LLR rates. Techniques resulting in better local control for patients with involved LLNs need to be explored further.

Statin Treatment Is Associated with Alterations in the Platelet Lipidome.

BACKGROUND: Platelets are key players in the pathophysiology of coronary artery disease (CAD) and platelet hyperreactivity leads to increased risk of developing adverse cardiovascular events. Further, significant changes in the platelet lipidome occur in patients with acute coronary syndrome (ACS) and critically regulated lipids lead to platelet hyperresponsiveness. Statin treatment is crucial in the treatment and prevention of patients with CAD by remodeling lipid metabolism. OBJECTIVE: In this study, we investigate the platelet lipidome of CAD patients by untargeted lipidomics, highlighting significant changes between statin-treated and naïve patients. METHODS: We characterized the platelet lipidome in a CAD cohort (n = 105) by an untargeted lipidomics approach using liquid chromatography coupled to mass spectrometry. RESULTS: Among the annotated lipids, 41 lipids were significantly upregulated in statin-treated patients, whereas 6 lipids were downregulated compared to naïve patients. The most prominent upregulated lipids in statin-treated patients belong to the class of triglycerides, cholesteryl esters, palmitic acid, and oxidized phospholipids, whereas mainly glycerophospholipids were downregulated compared to untreated patients. A more pronounced effect of statin treatment on the platelet lipidome was observed in ACS patients. We further highlight a dose-dependent influence on the platelet lipidome. CONCLUSION: Our results reveal that the platelet lipidome is altered in CAD patients with statin treatment and upregulated lipids embody mainly characteristic triglycerides, whereas downregulated lipids mostly compromise glycerophospholipids, which may play a role in the pathophysiology of CAD. Results of this study may contribute to the understanding of statin treatment softening the lipid phenotype.