Engineered nanoparticles promote cardiac tropism of AAV vectors.

Cardiac muscle targeting is a notoriously difficult task. Although various nanoparticle (NP) and adeno-associated viral (AAV) strategies with heart tissue tropism have been developed, their performance remains suboptimal. Significant off-target accumulation of i.v.-delivered pharmacotherapies has thwarted development of disease-modifying cardiac treatments, such as gene transfer and gene editing, that may address both rare and highly prevalent cardiomyopathies and their complications. Here, we present an intriguing discovery: cargo-less, safe poly (lactic-co-glycolic acid) particles that drastically improve heart delivery of AAVs and NPs. Our lead formulation is referred to as ePL (enhancer polymer). We show that ePL increases selectivity of AAVs and virus-like NPs (VLNPs) to the heart and de-targets them from the liver. Serotypes known to have high (AAVrh.74) and low (AAV1) heart tissue tropisms were tested with and without ePL. We demonstrate up to an order of magnitude increase in heart-to-liver accumulation ratios in ePL-injected mice. We also show that ePL exhibits AAV/NP-independent mechanisms of action, increasing glucose uptake in the heart, increasing cardiac protein glycosylation, reducing AAV neutralizing antibodies, and delaying blood clearance of AAV/NPs. Current approaches utilizing AAVs or NPs are fraught with challenges related to the low transduction of cardiomyocytes and life-threatening immune responses; our study introduces an exciting possibility to direct these modalities to the heart at reduced i.v. doses and, thus, has an unprecedented impact on drug delivery and gene therapy. Based on our current data, the ePL system is potentially compatible with any therapeutic modality, opening a possibility of cardiac targeting with numerous pharmacological approaches.

The correlation between brain structure characteristics and emotion regulation ability in children at high risk of autism spectrum disorder.

As indicated by longitudinal observation, autism has difficulty controlling emotions to a certain extent in early childhood, and most children's emotional and behavioral problems are further aggravated with the growth of age. This study aimed at exploring the correlation between white matter and white matter fiber bundle connectivity characteristics and their emotional regulation ability in children with autism using machine learning methods, which can lay an empirical basis for early clinical intervention of autism. Fifty-five high risk of autism spectrum disorder (HR-ASD) children and 52 typical development (TD) children were selected to complete the skull 3D-T1 structure and diffusion tensor imaging (DTI). The emotional regulation ability of the two groups was compared using the still-face paradigm (SFP). The classification and regression models of white matter characteristics and white matter fiber bundle connections of emotion regulation ability in the HR-ASD group were built based on the machine learning method. The volume of the right amygdala (R2 = 0.245) and the volume of the right hippocampus (R2 = 0.197) affected constructive emotion regulation strategies. FA (R2 = 0.32) and MD (R2 = 0.34) had the predictive effect on self-stimulating behaviour. White matter fiber bundle connection predicted constructive regulation strategies (positive edging R2 = 0.333, negative edging R2 = 0.334) and mother-seeking behaviors (positive edging R2 = 0.667, negative edging R2 = 0.363). The emotional regulation ability of HR-ASD children is significantly correlated with the connections of multiple white matter fiber bundles, which is a potential neuro-biomarker of emotional regulation ability.

Transport pathways and kinetics of cerebrospinal fluid tracers in mouse brain observed by dynamic contrast-enhanced MRI.

Recent studies have suggested the glymphatic system as a key mechanism of waste removal in the brain. Dynamic contrast-enhanced MRI (DCE-MRI) using intracisternally administered contrast agents is a promising tool for assessing glymphatic function in the whole brain. In this study, we evaluated the transport kinetics and distribution of three MRI contrast agents with vastly different molecular sizes in mice. Our results demonstrate that oxygen-17 enriched water (H217O), which has direct access to parenchymal tissues via aquaporin-4 water channels, exhibited significantly faster and more extensive transport compared to the two gadolinium-based contrast agents (Gd-DTPA and GadoSpin). Time-lagged correlation and clustering analyses also revealed different transport pathways for Gd-DTPA and H217O. Furthermore, there were significant differences in transport kinetics of the three contrast agents to the lateral ventricles, reflecting the differences in forces that drive solute transport in the brain. These findings suggest the size-dependent transport pathways and kinetics of intracisternally administered contrast agents and the potential of DCE-MRI for assessing multiple aspects of solute transport in the glymphatic system.

Transport Pathways and Kinetics of Cerebrospinal Fluid Tracers in Mouse Brain Observed by Dynamic Contrast-Enhanced MRI.

Background: Recent studies have suggested the glymphatic system as a solute transport pathway and waste removal mechanism in the brain. Imaging intracisternally administered tracers provides the opportunity of assessing various aspects of the glymphatic function. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) allows the evaluation of both the kinetics and spatial distribution of tracer transport in the whole brain. However, assessing mouse glymphatic function by DCE-MRI has been challenged by the small size of a mouse brain and the limited volume of fluids that can be delivered intracisternally without significantly altering the intracranial pressure. Further, previous studies in rats suggest that assessment of glymphatic function by DCE-MRI is dependent on the molecular size of the contrast agents. Methods: We established and validated an intracisternal infusion protocol in mice that allowed the measurements of the entire time course of contrast agent transport for 2 hours. The transport kinetics and distribution of three MRI contrast agents with drastically different molecular weights (MWs): Gd-DTPA (MW=661.8 Da, n=7), GadoSpin-P (MW=200 kDa, n=6), and oxygen-17 enriched water (H 2 17 O, MW=19 Da, n=7), were investigated. Results: The transport of H 2 17 O was significantly faster and more extensive than the two gadolinium-based contrast agents. Time-lagged correlation analysis and clustering analysis comparing the kinetics of Gd-DTPA and H 2 17 O transport also showed different cluster patterns and lag time between different regions of the brain, suggesting different transport pathways for H 2 17 O because of its direct access to parenchymal tissues via the aquaporin-4 water channels. Further, there were also significant differences in the transport kinetics of the three tracers to the lateral ventricles, which reflects the differences in forces that drive tracer transport in the brain. Conclusions: Comparison of the transport kinetics and distribution of three MRI contrast agents with different molecular sizes showed drastically different transport profiles and clustering patterns, suggesting that the transport pathways and kinetics in the glymphatic system are size-dependent.

Plaque-targeted, proteolysis-resistant, activatable and MRI-visible nano-GLP-1 receptor agonist targets smooth muscle cell differentiation in atherosclerosis.

Background: Glucagon-like peptide-1 receptor (GLP-1R) agonists are powerful glycemia-lowering agents, which have systematically been shown to lower cardiovascular events and mortality. These beneficial effects were difficult to pinpoint within atherosclerotic plaque due to lack of particular specificity of such agonists to the vascular cells and an inadequate understanding of the GLP-1R expression in atherosclerosis. Here, we hypothesized that the direct engagement of the GLP-1R in atherosclerosis by targeted agonists will alleviate vascular inflammation and plaque burden, even at a very low dose. Methods: The expression of GLP-1 receptor (GLP-1R, Glp1r mRNA) in human lesions with pathologic intimal thickening, Apoe-/- mouse atheroma and cultured immune/non-immune cells was investigated using genetic lineage tracing, Southern blotting and validated antisera against human GLP-1R. Protease-resistant and "activatable" nanoparticles (NPs) carrying GLP-1R agonist liraglutide (GlpNP) were engineered and synthesized. Inclusion of gadolinium chelates into GlpNP allowed for imaging by MRI. Atherosclerotic Apoe-/- mice were treated intravenously with a single dose (30 µg/kg of liraglutide) or chronically (1 µg/kg, 6 weeks, 2x/week) with GlpNP, liraglutide or control NPs, followed by assessment of metabolic parameters, atheroma burden, inflammation and vascular function. Results: Humal plaque specimens expressed high levels of GLP-1R within the locus of de-differentiated smooth muscle cells that also expressed myeloid marker CD68. However, innate immune cells under a variety of conditions expressed very low levels of Glp1r, as seen in lineage tracing and Southern blotting experiments examining full-length open reading frame mRNA transcripts. Importantly, de-differentiated vascular smooth muscle cells demonstrated significant Glp1r expression levels, suggesting that these could represent the cells with predominant Glp1r-positivity in atherosclerosis. GlpNP resisted proteolysis and demonstrated biological activity including in vivo glycemia lowering at 30 µg/kg and in vitro cholesterol efflux. Activatable properties of GlpNP were confirmed in vitro by imaging cytometry and in vivo using whole organ imaging. GlpNP targeted CD11b+/CD11c+ cells in circulation and smooth muscle cells in aortic plaque in Apoe-/- mice when assessed by MRI and fluorescence imaging. At a very low dose of 1 µg/kg, previously known to have little effect on glycemia and weight loss, GlpNP delivered i.v. for six weeks reduced triglyceride-rich lipoproteins in plasma, plaque burden and plaque cholesterol without significant effects on weight, glycemia and plasma cholesterol levels. Conclusions: GlpNP improves atherosclerosis at weight-neutral doses as low as 1 µg/kg with the effects independent from the pancreas or the central nervous system. Our study underlines the importance of direct actions of GLP-1 analogs on atherosclerosis, involving cholesterol efflux and inflammation. Our findings are the first to suggest the therapeutic modulation of vascular targets by GlpNP, especially in the context of smooth muscle cell inflammation.

Reliability, validity and developmental sensitivity of the Language Use Inventory (LUI) in the Chinese context.

BACKGROUND: Pragmatics has generally been defined as the ability to use language in social situations, it is commonly regarded as the third major component of language ability. To date, there is no tool for assessing early pragmatic development of Chinese-speaking children. AIMS: To describe the translation of the Language Use Inventory (LUI) from English to Mandarin Chinese and to report findings on the Chinese version's reliability, validity and developmental sensitivity. METHODS & PROCEDURES: The original English version of the LUI was translated into Mandarin Chinese. Parents of 177 typically developing (TD) toddlers and preschool children completed the inventory to examine its internal reliability and construct validity and how scores differed across ages and sexes. A total of 31 parents out of the 177 completed the LUI-Mandarin, again within 4 weeks, to assess test-retest reliability. To examine discriminative validity, 43 parents of age- and sex-matched TD children and children with autism spectrum disorder (ASD) recruited from Nanjing Brain Hospital affiliated with Nanjing Medical University completed the LUI-Mandarin. OUTCOMES & RESULTS: Cronbach's alpha values for the LUI-Mandarin's three parts and for 11 of 12 LUI-Mandarin subscales were 0.707-0.992, with most values in the 0.825-0.992 range. Test-retest reliability ranged from 0.66 to 0.95, indicating good to excellent reliability. Factor analysis of the LUI-Mandarin revealed two different factors, and the total variance explained was 74.38%. The LUI-Mandarin total scores and subscale scores increased with age for both boys and girls, providing evidence of the inventory's developmental sensitivity. Girls, however, had higher total scores than boys at earlier ages (18-23 months). The results of the discriminant validity study revealed that performance was significantly lower in the ASD group than in the TD group with respect to LUI total scores and subscale scores (except for subscale A). CONCLUSIONS & IMPLICATIONS: The LUI-Mandarin is the first and only questionnaire available in China that evaluates the pragmatic language skills of children aged between 18 and 47 months. The results of the study show that the LUI-Mandarin is a valid and reliable tool for Chinese toddlers and preschool children. WHAT THIS PAPER ADDS: What is already known on this subject The LUI is a parent-report questionnaire that can provide comprehensive information about very young children's communicative competence. It is widely used both for assessment and to guide intervention. Additionally, it has been translated into French, Italian, Polish, Arabic, Portuguese and Norwegian and it shows good reliability and validity. What this paper adds to existing knowledge In the present study we describe the translation of the LUI from English to Mandarin Chinese and report findings on the Chinese version's reliability, validity and developmental sensitivity. What are the potential or actual clinical implications of this work? The LUI-Mandarin is the first and only questionnaire available in China that can evaluate pragmatic language skills of children aged between 18 and 47 months. The results show that the LUI-Mandarin is a valid and reliable tool for use with Chinese toddlers and preschool children.

The characteristics and factors of the internalizing and externalizing behaviours of children at high risk for autism spectrum disorder.

BACKGROUND: The behavioral characteristics of children with autism spectrum disorder (ASD) are not only affected by their disease, but also by their parenting environment. HR-ASD has the risk of developing internalization and externalization problems. How the early development of these behavioral problems is affected by parent-child interaction is worth exploring. We tested whether parent-child interactions and parenting characteristics were associated with behavioural problems during the infant periods. METHODS: This study collected data from 91 infants at high risk for ASD and 68 matched typically developing (TD) infants, about their internalizing and externalizing behavioural problems and engagement states (i.e. positive, negative, and parent-child interactions), using free play paradigm. Parent measures were assessed using the Broad Autism Phenotypic Questionnaire (BAPQ) and Parenting Stress Index Short Form (PSI-SF) questionnaire. The core symptoms of ASD were assessed using the the Autism Diagnostic Observational Schedule (ADOS). RESULTS: During free play, infants in the HR-ASD group showed more internalizing (P < 0.001) and externalizing (P < 0.05) behaviours and less positive engagement (P < 0.01) than the TD group. In the regression analysis, we found that parenting stress had an impact on the infants' externalizing behaviours (△R2 = 0.215). Parent negative engagement had an impact on the infants' internalizing behaviours (△R2 = 0.451). CONCLUSIONS: The present study revealed that children at high risk for ASD exhibited more severe internalizing and externalizing behavioural problems than TD group. The parent negative engagement is associated with behavioural problems. The findings on the contribution of parents' factors to behavioural problems suggests that the parenting stress and parent-child interactions are important factors for mitigating behavioural problems.

Designing S100A9-Targeted Plant Virus Nanoparticles to Target Deep Vein Thrombosis.

Thromboembolic conditions are a leading cause of death worldwide, and deep vein thrombosis (DVT), or occlusive venous clot formation, is a critical and rising problem that contributes to damage of vital organs, long-term complications, and life-threatening conditions such as pulmonary embolism. Early diagnosis and treatment are correlated to better prognosis. However, current technologies in these areas, such as ultrasonography for diagnostics and anticoagulants for treatment, are limited in terms of their accuracy and therapeutic windows. In this work, we investigated targeting myeloid related protein 14 (MRP-14, also known as S100A9) using plant virus-based nanoparticle carriers as a means to achieve tissue specificity aiding prognosis and therapeutic intervention. We used a combinatorial peptide library screen to identify peptide ligands that bind MRP-14. Candidates were selected and formulated as nanoparticles by using cowpea mosaic virus (CPMV) and tobacco mosaic virus (TMV). Intravascular delivery of our MRP-14-targeted nanoparticles in a murine model of DVT resulted in enhanced accumulation in the thrombi and reduced thrombus size, suggesting application of nanoparticles for molecular targeting of MRP-14 could be a promising direction for improving DVT diagnostics, therapeutics, and therefore prognosis.

A Hybrid Ensemble Algorithm Combining AdaBoost and Genetic Algorithm for Cancer Classification with Gene Expression Data.

The diversity of base classifiers and integration of multiple classifiers are two key issues in the field of ensemble learning. This paper puts forward a hybrid ensemble algorithm combining AdaBoost and genetic algorithm(GA) for cancer classification with gene expression data. The decision group is designed to increase the diversity of base classifier pool, and the GA is used to assign weight to each base classifier, thus to improve the classification performance by avoiding local extrema. The decision groups composed by using base classifiers, including K-nearest neighbor (KNN), Naïve Bayes (NB), and Decision Tree (C4.5). Experimental results show that the proposed algorithm is superior to those existing ensemble learning methods, such as Bagging, Random Forest (RF), Rotation Forest (RoF), AdaBoost, AdaBoost-BPNN, AdaBoost-SVM, and AdaBoost-RF, especially it has better performance on small samples and unbalanced gene expression data processing.

Dynamic oxygen-17 MRI with adaptive temporal resolution using golden-means-based 3D radial sampling.

PURPOSE: The aim of this study was to develop a high-resolution 3D oxygen-17 (17 O) MRI method to delineate the kinetics of 17 O-enriched water (H217 O) across the entire mouse brain after a bolus injection via the tail vein. METHODS: The dynamic 17 O signal was acquired with a golden-means-based 3D radial sampling scheme. To achieve adequate temporal resolution with preserved spatial resolution, a k-space-weighted view sharing strategy was used in image reconstruction with an adaptive window size tailored to the kinetics of the 17 O signal. Simulation studies were performed to determine the adequate image reconstruction parameters. The established method was applied to delineating the kinetics of intravenously injected H217 O in vivo in the post-stroke mouse brain. RESULTS: The proposed dynamic 17 O-MRI method achieved an isotropic resolution of 1.21 mm (0.77 mm nominal) in mouse brain at 9.4T, with the temporal resolution increased gradually from 3 s at the initial phase of rapid signal increase to 15 s at the steady-state. The high spatial resolution enabled the delineation of the heterogeneous H217 O uptake and washout kinetics in stroke-affected mouse brain. CONCLUSION: The current study demonstrated a 3D 17 O-MRI method for dynamic monitoring of 17 O signal changes with high spatial and temporal resolution. The method can be utilized to quantify physiological parameters such as cerebral blood flow and blood-brain barrier permeability by tracking injected H217 O. It can also be used to measure oxygen consumption rate in 17 O-oxygen inhalation studies.

Publisher Correction: Systemically-delivered biodegradable PLGA alters gut microbiota and induces transcriptomic reprogramming in the liver in an obesity mouse model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Systemically-delivered biodegradable PLGA alters gut microbiota and induces transcriptomic reprogramming in the liver in an obesity mouse model.

Biodegradable materials, including the widely used poly (lactic-co-glycolic acid) (PLGA) nanoparticles contained in slow-release drug formulations, scaffolds and implants, are ubiquitous in modern biomedicine and are considered inert or capable of being metabolized through intermediates such as lactate. However, in the presence of metabolic stress, such as in obesity, the resulting degradation products may play a detrimental role, which is still not well understood. We evaluated the effect of intravenously-administered PLGA nanoparticles on the gut-liver axis under conditions of caloric excess in C57BL/6 mice. Our results show that PLGA nanoparticles accumulate and cause gut acidification in the cecum, accompanied by significant changes in the microbiome, with a marked decrease of Firmicutes and Bacteroidetes. This was associated with transcriptomic reprogramming in the liver, with a downregulation of mitochondrial function, and an increase in key enzymatic, inflammation and cell activation pathways. No changes were observed in systemic inflammation. Metagenome analysis coupled with publicly available microarray data suggested a mechanism of impaired PLGA degradation and intestinal acidification confirming an important enterohepatic axis of metabolite-microbiome interaction resulting in maintenance of metabolic homeostasis. Thus, our results have important implications for the investigation of PLGA use in metabolically-compromised clinical and experimental settings.

Nano-Antagonist Alleviates Inflammation and Allows for MRI of Atherosclerosis.

Specific targeting of inflammation remains a challenge in many pathologies, because of the necessary balance between host tolerance and efficacious inflammation resolution. Here, we discovered a short, 4-mer peptide which possesses antagonist properties towards CC chemokine receptor 2 (CCR2), but only when displayed on the surface of lipid nanoparticles. According to BLAST analysis, this peptide motif is a common repeating fragment in a number of proteins of the CC chemokine family, which are key players in the inflammatory response. In this study, self-assembled, peptide-conjugated nanoparticles (CCTV) exhibited typical properties of CCR2 antagonism, including affinity to the CCR2 receptor, inhibition of chemotactic migration of primary monocytes, and prevention from CC chemokine ligand 2 (CCL2)-induced actin polymerization. Furthermore, CCTV ameliorated NFkB activation and downregulated the secondary, but not the primary, inflammatory response in cultured macrophages. When conjugated with gadolinium or europium cryptates, CCTV enabled targeted imaging (via magnetic resonance imaging and time-resolved fluorescence) of atherosclerosis, a chronic inflammatory condition in which the CCL2/CCR2 axis is highly dysfunctional. CCTV targeted CCR2hiLy6Chi inflammatory monocytes in blood and the atherosclerotic plaque, resulting in cell-specific transcriptional downregulation of key inflammatory genes. Finally, CCTV generated pronounced inflammasome inactivation, likely mediated through reactive oxygen species scavenging and downregulation of NLRP3. In summary, our work demonstrates for the first time that a short peptide fragment presented on a nanoparticle surface exhibit potent receptor-targeted antagonist effects, which are not seen with the peptide alone. Unlike commonly used cargo-carrying, vector-directed drug delivery vehicles, CCTV nanoparticles may act as therapeutics/theranostics themselves, particularly in inflammatory conditions with CCL2/CCR2 pathogenesis, including cardiovascular disease and cancer.

KLF15 regulates endobiotic and xenobiotic metabolism.

Hepatic metabolism and elimination of endobiotics (for example, steroids, bile acids) and xenobiotics (for example, drugs, toxins) is essential for health. While the enzymatic (termed phase I-II) and transport machinery (termed phase III) controlling endobiotic and xenobiotic metabolism (EXM) is known, understanding of molecular nodal points that coordinate EXM function in physiology and disease remains incomplete. Here we show that the transcription factor Kruppel-like factor 15 (KLF15) regulates all three phases of the EXM system by direct and indirect pathways. Unbiased transcriptomic analyses coupled with validation studies in cells, human tissues, and animals, support direct transcriptional control of the EXM machinery by KLF15. Liver-specific deficiency of KLF15 (Li-KO) results in altered expression of numerous phase I-III targets, and renders animals resistant to the pathologic effects of bile acid and acetaminophen toxicity. Furthermore, Li-KO mice demonstrate enhanced degradation and elimination of endogenous steroid hormones, such as testosterone and glucocorticoid, resulting in reduced male fertility and blood glucose levels, respectively. Viral reconstitution of hepatic KLF15 expression in Li-KO mice reverses these phenotypes. Our observations identify a previously unappreciated transcriptional pathway regulating metabolism and elimination of endobiotics and xenobiotics.

S100A9-targeted tobacco mosaic virus nanoparticles exhibit high specificity toward atherosclerotic lesions in ApoE-/- mice.

We integrate a biocompatible plant virus-based nanotechnology (tobacco mosaic virus, TMV) with S100A9-targeting peptides for its application in imaging and diagnosis of atherosclerosis. S100A9-targeted TMV nanoparticles exhibit remarkable specificity to S100A9 and targeting of atherosclerosis lesions in ApoE-/- mice.

Delivery of thrombolytic therapy using rod-shaped plant viral nanoparticles decreases the risk of hemorrhage.

Cardiovascular thrombotic disease is an underlying cause of stroke, myocardial infarction and pulmonary embolism - some of the leading causes of death worldwide. Reperfusion therapy with anticoagulant, antiplatelet, and fibrinolytic agents has significantly reduced early mortality and morbidity from acute myocardial infarction and stroke. Nevertheless, bleeding side effects (e.g., intracranial hemorrhage) associated with the anti-thrombotic therapy can offset its benefits and limit its applicability to strictly defined short therapeutic windows. We have previously shown that elongated plant virus based nanoparticles can target cardiovascular thrombi and exhibited their utility for the delivery of streptokinase in an ex vivo model of thrombosis. Herein, we build upon our previous findings and demonstrate plant viral delivery of the current standard-of-care tissue plasminogen activator (tPA). Studies on a pre-clinical mouse model of arterial thrombosis indicate that while the therapeutic efficacy of free tPA and tPA-conjugated TMV are similar, the safety profile of the tPA-TMV formulation is improved, i.e. administration of the latter has less impact on hemostasis as demonstrated by decreased bleeding time. Thus, our data suggest that TMV-based delivery of thrombolytic therapies could be a promising and safer alternative to reperfusion therapy with the tPA.

S-Nitrosylation of ß-Arrestins Biases Receptor Signaling and Confers Ligand Independence.

Most G protein-coupled receptors (GPCRs) signal through both heterotrimeric G proteins and ß-arrestins (ßarr1 and ßarr2). Although synthetic ligands can elicit biased signaling by G protein- vis-à-vis ßarr-mediated transduction, endogenous mechanisms for biasing signaling remain elusive. Here we report that S-nitrosylation of a novel site within ßarr1/2 provides a general mechanism to bias ligand-induced signaling through GPCRs by selectively inhibiting ßarr-mediated transduction. Concomitantly, S-nitrosylation endows cytosolic ßarrs with receptor-independent function. Enhanced ßarr S-nitrosylation characterizes inflammation and aging as well as human and murine heart failure. In genetically engineered mice lacking ßarr2-Cys253 S-nitrosylation, heart failure is exacerbated in association with greatly compromised ß-adrenergic chronotropy and inotropy, reflecting ßarr-biased transduction and ß-adrenergic receptor downregulation. Thus, S-nitrosylation regulates ßarr function and, thereby, biases transduction through GPCRs, demonstrating a novel role for nitric oxide in cellular signaling with potentially broad implications for patho/physiological GPCR function, including a previously unrecognized role in heart failure.

Elongated Plant Virus-Based Nanoparticles for Enhanced Delivery of Thrombolytic Therapies.

Thrombotic cardiovascular disease, including acute myocardial infarction, ischemic stroke, and venous thromboembolic disease, is the leading cause of morbidity and mortality worldwide. While reperfusion therapy with thrombolytic agents reduces mortality from acute myocardial infarction and disability from stroke, thrombolysis is generally less effective than mechanical reperfusion and is associated with fatal intracerebral hemorrhage in up to 2-5% of patients. To address these limitations, we propose the tobacco mosaic virus (TMV)-based platform technology for targeted delivery of thrombolytic therapies. TMV is a plant virus-based nanoparticle with a high aspect ratio shape measuring 300 × 18 nm. These soft matter nanorods have favorable flow and margination properties allowing the targeting of the diseased vessel wall. We have previously shown that TMV homes to thrombi in a photochemical mouse model of arterial thrombosis. Here we report the synthesis of TMV conjugates loaded with streptokinase (STK). Various TMV-STK formulations were produced through bioconjugation of STK to TMV via intervening PEG linkers. TMV-STK was characterized using SDS-PAGE and Western blot, transmission electron microscopy, cryo-electron microscopy, and cryo-electron tomography. We investigated the thrombolytic activity of TMV-STK in vitro using static phantom clots, and in a physiologically relevant hydrodynamic model of shear-induced thrombosis. Our findings demonstrate that conjugation of STK to the TMV surface does not compromise the activity of STK. Moreover, the nanoparticle conjugate significantly enhances thrombolysis under flow conditions, which can likely be attributed to TMV's shape-mediated flow properties resulting in enhanced thrombus accumulation and dissolution. Together, these data suggest TMV to be a promising platform for the delivery of thrombolytics to enhance clot localization and potentially minimize bleeding risk.

Corrigendum: Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα.

This corrects the article DOI: 10.1038/ncomms15559.

Myeloid-related protein-14 regulates deep vein thrombosis.

Using transcriptional profiling of platelets from patients presenting with acute myocardial infarction, we identified myeloid-related protein-14 (MRP-14, also known as S100A9) as an acute myocardial infarction gene and reported that platelet MRP-14 binding to platelet CD36 regulates arterial thrombosis. However, whether MRP-14 plays a role in venous thrombosis is unknown. We subjected WT and Mrp-14-deficient (Mrp-14-/-) mice to experimental models of deep vein thrombosis (DVT) by stasis ligation or partial flow restriction (stenosis) of the inferior vena cava. Thrombus weight in response to stasis ligation or stenosis was reduced significantly in Mrp-14-/- mice compared with WT mice. The adoptive transfer of WT neutrophils or platelets, or the infusion of recombinant MRP-8/14, into Mrp-14-/- mice rescued the venous thrombosis defect in Mrp-14-/- mice, indicating that neutrophil- and platelet-derived MRP-14 directly regulate venous thrombogenesis. Stimulation of neutrophils with MRP-14 induced neutrophil extracellular trap (NET) formation, and NETs were reduced in venous thrombi harvested from Mrp-14-/- mice and in Mrp-14-/- neutrophils stimulated with ionomycin. Given prior evidence that MRP-14 also regulates arterial thrombosis, but not hemostasis (i.e., reduced bleeding risk), MRP-14 appears to be a particularly attractive molecular target for treating thrombotic cardiovascular diseases, including myocardial infarction, stroke, and venous thromboembolism.