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The purpose of single-exposure HDR image reconstruction is to recover the missing information in the saturated region of the LDR image, and use neural network to reconstruct the HDR image. In recent years, single-exposure HDR imaging using deep learning (DL) has made significant progress. In this study, the latest development of deep single-exposure HDR imaging method was comprehensively and deeply investigated and analyzed. At present, there are five main methods for deep single-exposure HDR imaging: direct learning from a single LDR image, generating bracketed LDR image stacks, computationally efficient learning, learning camera imaging pipeline, and learning neural sensors. Importantly, this study reviewed the limitations and future work of each category. In addition, this study also discussed the potential and future trends of each category constructively.

An interval type-2 fuzzy set and fuzzy Petri net combined risk assessment model for chemical production was proposed to solve the problems of disorganized hierarchy and poorly targeted measures, as well as the requirement for complex equipment associated with chemical production risk assessment. First, four different types of risk databases were established according to the production process of cyclohexane. Considering the intrinsic relationship between the risk factors in the fault database, the interval type-2 fuzzy set was used to improve the semantic transformation accuracy and calculate the confidence in the risk factors. The fuzzy Petri net model was used to simulate the dynamic development of accidents, and the parallel relationship between risk factors was intuitively described. Thereafter, the external relationship between risk factors was analyzed, and the net structure of each layer was divided to build a multilevel model. Finally, the catalyst activation process during cyclohexane production was taken as an example for risk assessment calculation, and the accident risk probability was calculated by multilevel fuzzy reasoning. The results demonstrate that the model is an improvement over traditional methods and can be used for precise prevention and control. Moreover, it can accurately analyze risk probability during chemical production, determine the risk associated with the reaction process, effectively prevent accidents, and provide a reference for risk evaluation and risk classification.

So far, scientists have researched to explore the emotional connotation of words in different native languages and genders. This study investigates how connotations of words differ by cultures and genders by inspecting valence values, which indicates how happy people feel about specific words in datasets obtained from USA, Spain, and Portuguese. To fulfill the objective, we categorized people’s feelings evoked by the words based on a criterion created by adding or subtracting standard deviation to or from valence means for the upper and lower bound. Then, words with valence values outside of the upper and lower bounds were categorized as emotional words (positive or negative) and were analyzed. The results show that people of different cultures are more connected regarding the perception of negative connotation of words than positive connotation. Moreover, Portuguese and Spanish are more connected than either of them with the US as they have more emotion words in common. On the gender aspect, we notice that females tend to give more extreme ratings for words than males. Furthermore, the analysis shows that males have a more positive feeling towards sex-related words than females. Overall, this study outlines a way for people interested in anthropology to understand the differences in the emotional connotation of words between males and females and across cultures.

Cancers are defined by genetic defects, which underlines the prospect of using gene therapy in patient care. During the past decade, CRISPR technology has rapidly evolved into a powerful gene editing tool with high fidelity and precision. However, one of the impediments slowing down the clinical translation of CRISPR-based gene therapy concerns the lack of ideal delivery vectors. Extracellular vesicles (EVs) are nano-sized membrane sacs naturally released from nearly all types of cells. Although EVs are secreted for bio-information conveyance among cells or tissues, they have been recognized as superior vectors for drug or gene delivery. Recently, emerging evidence has spotlighted EVs in CRISPR delivery towards cancer treatment. In this review, we briefly introduce the biology and function of the CRISPR system and follow this with a summary of current delivery methods for CRISPR applications. We emphasize the recent progress in EV-mediated CRISPR editing for various cancer types and target genes. The reported strategies for constructing EV-CRISPR vectors, as well as their limitations, are discussed in detail. The review aims to throw light on the clinical potential of engineered EVs and encourage the expansion of our available toolkit to defeat cancer.

Adeno-associated viruses (AAVs) are the vectors of choice for long-term delivery of genes to the heart and due to their safety in clinics. Despite its many favorable features, a notable drawback is the presence of preexisting neutralizing antibodies (NAb), which bind to free AAVs and impair their clinical effect. Here, we describe delivery of AAVs encapsulated in extracellular vesicles (eAAV), as a superior cardiac gene delivery system that offers higher NAb resistance. Methods: Ultracentrifuge-isolated eAAVs were characterized using TEM, DLS, qNano, nanoflow cytometry, ExoView, qPCR and Western blot. Gene delivery efficacy of free AAV and eAAVs were compared with and without NAbs using human cardiomyocytes in vitro, and in mice in vivo, using flow cytometry (with mCherry) and bioluminescence imaging (with luciferase). Therapeutic efficacy of gene delivery by free AAV and eAAV in presence and absence of NAb was determined using a cardiac contractile gene SERCA2a in a mouse model of myocardial infarction (MI). eAAV uptake and trafficking was studied both in vitro and in vivo using fluorescence dye-labeled eAAV. Results: eAAV outperformed free AAV in delivering genes even in the presence of NAbs, both in vitro and in vivo. SERCA2a delivered by eAAV significantly improved cardiac function (ejection fraction, %EF, ~60%) compared to when delivered by free AAV (~40%) or saline control (~20%), in intramyocardially injected MI mice with NAb. Fluorescence-tagged eAAVs injected to mouse hearts were internalized into all cell types (CMs and non-CMs), but, eAAV-mCherry was primarily expressed in CMs, indicating that eAAV-mediated gene delivery is cardiotropic. eAAVs were uptaken in to acidic endosomal (Rab5 & 7 positive) compartments of cultured cardiomyocytes and delivered significantly higher amounts of luciferase to the nucleus of human CMs compared to free AAVs. This uptake of eAAVs was inhibited by the proton pump inhibitor in endosomes, Bafilomycin A1. Conclusion: Our study demonstrated that eAAV-mediated gene delivery can circumvent neutralizing antibody (NAb) issue for gene therapy. eAAVs are cardiotropic and deliver genes to the nucleus via uptake into acidic endosomes. eAAV is a superior gene delivery vector and a new platform to treat heart failure.

Lung carcinoma is the most common type of cancer and the leading cause of cancer-related death worldwide. Among the numerous therapeutic strategies for the treatment of lung cancer, adeno-associated virus (AAV)-mediated gene transfer has been demonstrated to have the potential to effectively suppress tumor growth or reverse the progression of the disease in a number of preclinical studies. AAV vector has a safety profile; however, the relatively low delivery efficacy to particular subtypes of lung carcinoma has limited its prospective clinical translation. Exosomes are nanosized extracellular vesicles secreted from nearly all known cell types. Exosomes have a membrane-enclosed structure carrying a range of cargo molecules for efficient intercellular transfer of functional entities, thus are considered as a superior vector for drug delivery. In the present study, we developed a novel strategy to produce and purify AAV-containing exosomes (AAVExo) from AAV-packaging HEK 293T cells. The cellular uptake capacity of exosomes assisted and enhanced AAV entry into cells and protected AAV from antibody neutralization, which was a serious challenge for AAV in vivo application. We tested a list of lung cancer cell lines representing non-small-cell lung cancer and small-cell lung cancer and found that AAVExo apparently improved the gene transfer efficiency compared to conventional AAV vector. Our in vitro results were supported in vivo in a lung cancer xenograft rodent model. Additionally, we evaluated the gene delivery efficiency in the presence of neutralizing antibody on lung cancer cells. The results demonstrated that AAVExo-mediated gene transfer was not impacted, while the AAV vectors were significantly blocked by the neutralizing antibody. Taken together, we established an efficient methodology for AAVExo purification, and the purified AAVExo largely enhanced gene delivery to lung cancer cells with remarkable resistance to antibody neutralization.

Background: Despite its functional importance in various fundamental bioprocesses, studies of N 6 -methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration. Methods: We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes. Results: We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis. Conclusions: Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

Gene therapy is a promising approach for the treatment of cardiovascular disease. Current strategies for myocardial gene transfer include the use of adeno-associated virus (AAV) vectors. However, AAVs may not be ideal for gene therapy vectors owing to pre-existing AAV capsid immunity in the human population that may reduce transduction efficacy and hinder preclinical-to-clinical translation. Interestingly, recent studies suggest that exosome-mediated encapsulation may protect viruses from neutralizing antibodies (NAbs) against the capsid and promote viral infectivity. Here, we describe the ability of exosome-enveloped AAVs, i.e. exosomal AAVs (eAAVs), to evade NAbs and serve as a highly efficient gene delivery tool for cardiovascular therapeutics. We have developed a method to purifiy eAAVs from AAV-producing HEK-293T cells, and used electron/confocal microscopy, qPCR, immunoblotting, dynamic light scattering and interferometric imaging measurements to characterize eAAV morphology, contents and mechanism of action. We confirmed eAAVs represent vesicular fractions that exhibit common exosome phenotype, along with the presence of virus particles, and demonstrated that eAAV infectious entry potentially involves trafficking via endocytic compartments. Using flow cytometry, Langendorff perfusion system and bioluminescence imaging, we then evaluated efficiency of heart targeting for eAAV9/eAAV6 and standard AAV9/AAV6 encoding for mCherry or firefly luciferase in human cardiomyocytes in vitro and in mouse model in vivo . Regardless of the presence or absence of NAbs, we showed that eAAVs are more efficient in transduction in the same titer ranges as compared to standard AAVs. To test therapeutic efficacy, we intramyocardially injected eAAV9 or AAV9 vectors encoding for SERCA2a in NAb+ post-myocardial infarction mice and further evaluated cardiac function using echocardiography. Remarkably, eAAV9-SERCA2a outperformed standard AAVs significantly improving cardiac function in the presence of NAbs (%EF 55.14 ± 3.50 compared to 27.31 ± 1.63 at 6 weeks, respectively). In summary, delivery of AAVs protected by carrier exosomes (i.e. eAAVs) may retain the clinical benefits of AAVs while addressing one of its major challenges.

Cranial radiotherapy, although beneficial for the treatment of brain tumors, inevitably leads to normal tissue damage that can induce unintended neurocognitive complications that are progressive and debilitating. Ionizing radiation exposure has also been shown to compromise the structural integrity of mature neurons throughout the brain, an effect believed to be at least in part responsible for the deterioration of cognitive health. Past work has shown that cranially transplanted human neural stem cells (hNSCs) or their extracellular vesicles (EVs) afforded long‐term beneficial effects on many of these cognitive decrements. To provide additional insight into the potential neuroprotective mechanisms of cell‐based regenerative strategies, we have analyzed hippocampal neurons for changes in structural integrity and synaptic remodeling after unilateral and bilateral transplantation of hNSCs or EVs derived from those same cells. Interestingly, hNSCs and EVs similarly afforded protection to host neurons, ameliorating the impact of irradiation on dendritic complexity and spine density for neurons present in both the ipsilateral and contralateral hippocampi 1 month following irradiation and transplantation. These morphometric improvements were accompanied by increased levels of glial cell‐derived growth factor and significant attenuation of radiation‐induced increases in postsynaptic density protein 95 and activated microglia were found ipsi‐ and contra‐lateral to the transplantation sites of the irradiated hippocampus treated with hNSCs or hNSC‐derived EVs. These findings document potent far‐reaching neuroprotective effects mediated by grafted stem cells or EVs adjacent and distal to the site of transplantation and support their potential as therapeutic agents to counteract the adverse effects of cranial irradiation., Regenerative medicine holds promise for restoring brain structure and function following cancer relevant radiotherapy. These studies document potent distal neuroprotective effects mediated by bilateral and unilateral grafted stem cells or extracellular vesicles to the site of transplantation and support their potential as therapeutic agents to counteract the adverse effects of cranial irradiation.

Rationale: Paracrine secretions seem to mediate therapeutic effects of human CD34 + stem cells locally transplanted in patients with myocardial and critical limb ischemia and in animal models. Earlier, we had discovered that paracrine secretion from human CD34 + cells contains proangiogenic, membrane-bound nanovesicles called exosomes (CD34Exo). Objective: Here, we investigated the mechanisms of CD34Exo-mediated ischemic tissue repair and therapeutic angiogenesis by studying their miRNA content and uptake. Methods and Results: When injected into mouse ischemic hindlimb tissue, CD34Exo, but not the CD34Exo-depleted conditioned media, mimicked the beneficial activity of their parent cells by improving ischemic limb perfusion, capillary density, motor function, and their amputation. CD34Exo were found to be enriched with proangiogenic miRNAs such as miR-126-3p. Knocking down miR-126-3p from CD34Exo abolished their angiogenic activity and beneficial function both in vitro and in vivo. Interestingly, injection of CD34Exo increased miR-126-3p levels in mouse ischemic limb but did not affect the endogenous synthesis of miR-126-3p, suggesting a direct transfer of stable and functional exosomal miR-126-3p. miR-126-3p enhanced angiogenesis by suppressing the expression of its known target, SPRED1 , simultaneously modulating the expression of genes involved in angiogenic pathways such as VEGF (vascular endothelial growth factor), ANG1 (angiopoietin 1), ANG2 (angiopoietin 2), MMP9 (matrix metallopeptidase 9), TSP1 (thrombospondin 1), etc. Interestingly, CD34Exo, when treated to ischemic hindlimbs, were most efficiently internalized by endothelial cells relative to smooth muscle cells and fibroblasts, demonstrating a direct role of stem cell–derived exosomes on mouse endothelium at the cellular level. Conclusions: Collectively, our results have demonstrated a novel mechanism by which cell-free CD34Exo mediates ischemic tissue repair via beneficial angiogenesis. Exosome-shuttled proangiogenic miRNAs may signify amplification of stem cell function and may explain the angiogenic and therapeutic benefits associated with CD34 + stem cell therapy.

Rationale : Studies have demonstrated that exosomes can repair cardiac tissue post–myocardial infarction and recapitulate the benefits of cellular therapy. Objective : We evaluated the role of donor age and hypoxia of human pediatric cardiac progenitor cell (CPC)–derived exosomes in a rat model of ischemia–reperfusion injury. Methods and Results : Human CPCs from the right atrial appendages from children of different ages undergoing cardiac surgery for congenital heart defects were isolated and cultured under hypoxic or normoxic conditions. Exosomes were isolated from the culture-conditioned media and delivered to athymic rats after ischemia–reperfusion injury. Echocardiography at day 3 post–myocardial infarction suggested statistically improved function in neonatal hypoxic and neonatal normoxic groups compared with saline-treated controls. At 28 days post–myocardial infarction, exosomes derived from neonatal normoxia, neonatal hypoxia, infant hypoxia, and child hypoxia significantly improved cardiac function compared with those from saline-treated controls. Staining showed decreased fibrosis and improved angiogenesis in hypoxic groups compared with controls. Finally, using sequencing data, a computational model was generated to link microRNA levels to specific outcomes. Conclusions : CPC exosomes derived from neonates improved cardiac function independent of culture oxygen levels, whereas CPC exosomes from older children were not reparative unless subjected to hypoxic conditions. Cardiac functional improvements were associated with increased angiogenesis, reduced fibrosis, and improved hypertrophy, resulting in improved cardiac function; however, mechanisms for normoxic neonatal CPC exosomes improved function independent of those mechanisms. This is the first study of its kind demonstrating that donor age and oxygen content in the microenvironment significantly alter the efficacy of human CPC-derived exosomes.

Background: Exciting new discoveries in RNA biology underscore the importance of post-transcriptional chemical modifications to mRNAs (epitranscriptome) in regulating RNA stability, nuclear export, cellular compartmentalization, splicing, translation and degradation. The most abundant and functionally relevant modification in RNA, N6-methyladenosine (m6A) is reversibly demethylated by one of the m6A demethylases, fat mass and obesity-associated protein (FTO) whose function in the mammalian heart remains incompletely understood. Materials and Methods: We used clinical human samples, preclinical pig and mouse models and primary cardiomyocytes to study m6A and FTO in the heart and in cardiomyocytes. We modulated FTO expression using AAV9 (in vivo), adenovirus (in vivo and in vitro) and siRNAs (in vitro). We investigated m6A-induced changes to contractile protein expression using m6A RNA immunoprecipitation sequencing (MeRIP-seq) and stable isotope labeling of amino acids in cell culture (SILAC). Results: We discovered in human heart failure that reduced FTO expression is associated with aberrant increase in m6A mRNA methylation, which is conserved in swine and mouse models of myocardial ischemia (MI). AAV9-mediated FTO gene delivery in mouse MI attenuated m6A increase and improved cardiac function with enhanced contractility, angiogenesis and reduced fibrosis. At the molecular level, FTO-mediated mRNA demethylation serves to increase contractile protein expression in mouse hearts as well as in isolated primary cardiomyocytes. By comparing human and mouse transcriptome-wide m6A maps with SILAC proteomic profiling from cardiomyocytes, we identified FTO-mediated m6A demethylation is transcript-specific and leads to altered protein expression of several key contractile, angiogenic and regenerative proteins. Conclusion: Using new RNA-based investigations, we uncovered a novel regulatory layer beyond the genome working at the level of epitranscriptome governing cardiac function. Our findings on the dynamic nature of the cardiac m6A-epitranscriptome will lead to deeper understanding of the mechanism of cardiac remodeling on one hand and innovative therapeutic interventions on the other.

Adeno-associated viruses (AAVs) are promising therapeutic tools for gene delivery to the heart. However, pre-existing antibodies (NAbs) to many cardiotropic AAV serotypes naturally present in humans pose a critical challenge for the translation of gene therapies to clinical applications. Here, we describe the use of exosomal AAVs (eAAV) as a robust heart gene delivery system that improves transduction efficiency while protecting from pre-existing immunity to the viral capsid. To obtain eAAV specimens from conditioned medium from AAV-producing HEK-293T cells, we have developed a state-of-the-art multi-step ultracentrifugation strategy. We demonstrated through electron microscopy-based visualization, size distribution measurements and distribution of AAV genomes in post-centrifugation iodixanol gradients, that our purification process enables isolation of eAAVs with high purity and minimal contamination with standard AAVs. Efficiency of heart targeting was then evaluated for eAAV9 or eAAV6 and standard AAV9 or AAV6 in human cardiomyocytes (hCMs) in vitro and in passive immunity nude mouse model in vivo . Regardless of the presence or absence of NAbs, we demonstrated that eAAVs are more efficient in transduction of cells in the same titer ranges as standard AAVs. To test the therapeutic efficacy, eAAV9-SERCA2a or AAV9-SERCA2a were injected intramyocardially in post-myocardial infarction (MI) mice preinjected with NAbs. Remarkably, eAAV9-SERCA2a outperformed standard AAVs 6 weeks post-MI, significantly improving cardiac function in the presence of NAbs (%EF 55.14 ± 3.50 vs. 27.31 ± 1.63, respectively). Additionally, we demonstrated in vivo that eAAV9-mediated gene delivery is more specific to CMs than to other cell types present in the heart, which suggests that eAAVs preserve cardiotropic properties of AAV9 serotype. With examination of colocalization of eAAVs and markers specific for endosomes (Rab5 and Rab7) in hCMs in vitro , our preliminary data indicated that eAAV infectious entry potentially involves trafficking via endocytic compartments. In conclusion, these results underline the therapeutic potential of eAAVs to evade NAbs, and to facilitate the clinical translation of AAV-based gene therapies to a larger human population.

Rationale: Abnormal SUMOylation has emerged as a characteristic of heart failure (HF) pathology. Previously, we found reduced SUMO1 (small ubiquitin-like modifier 1) expression and SERCA2a (sarcoplasmic reticulum Ca 2+ -ATPase) SUMOylation in human and animal HF models. SUMO1 gene delivery or small molecule activation of SUMOylation restored SERCA2a SUMOylation and cardiac function in HF models. Despite the critical role of SUMO1 in HF, the regulatory mechanisms underlying SUMO1 expression are largely unknown. Objective: To examine miR-146a–mediated SUMO1 regulation and its consequent effects on cardiac morphology and function. Methods and Results: In this study, miR-146a was identified as a SUMO1-targeting microRNA in the heart. A strong correlation was observed between miR-146a and SUMO1 expression in failing mouse and human hearts. miR-146a was manipulated in cardiomyocytes through AAV9 (adeno-associated virus serotype 9)-mediated gene delivery, and cardiac morphology and function were analyzed by echocardiography and hemodynamics. Overexpression of miR-146a reduced SUMO1 expression, SERCA2a SUMOylation, and cardiac contractility in vitro and in vivo. The effects of miR-146a inhibition on HF pathophysiology were examined by transducing a tough decoy of miR-146a into mice subjected to transverse aortic constriction. miR-146a inhibition improved cardiac contractile function and normalized SUMO1 expression. The regulatory mechanisms of miR-146a upregulation were elucidated by examining the major miR-146a–producing cell types and transfer mechanisms. Notably, transdifferentiation of fibroblasts triggered miR-146a overexpression and secretion through extracellular vesicles, and the extracellular vesicle–associated miR-146a transfer was identified as the causative mechanism of miR-146a upregulation in failing cardiomyocytes. Finally, extracellular vesicles isolated from failing hearts were shown to contain high levels of miR-146a and exerted negative effects on the SUMO1/SERCA2a signaling axis and hence cardiomyocyte contractility. Conclusions: Taken together, our results show that miR-146a is a novel regulator of the SUMOylation machinery in the heart, which can be targeted for therapeutic intervention.

It is known that culture media (CM) promotes cellular growth, adhesion, and protects explanted primary brain cells from in vitro stresses. The fetal bovine serum (FBS) supplement used in most CM, however, contains significant quantities of extracellular vesicles (EVs) that confound quantitative and qualitative analyses from the EVs produced by the cultured cells. We quantitatively tested the ability of common FBS EV-depletion protocols to remove exogenous EVs from FBS-supplemented CM and evaluated the influence such methods have on primary astrocyte culture growth and viability. We assessed two methodologies utilized for FBS EV removal prior to adding to CM: (1) an 18-h ultracentrifugation (UC); and (2) a commercial EV-depleted FBS (Exo-FBS™). Our analysis demonstrated that Exo-FBS™ CM provided the largest depletion (75%) of total FBS EVs, while still providing 6.92 × 109 ± 1.39 × 108 EVs/mL. In addition, both UC and Exo-FBS™ CM resulted in poor primary astrocyte cell growth and viability in culture. The two common FBS EV-depletion methods investigated, therefore, not only contaminate in vitro primary cell-derived EV analyses, but also provide a suboptimal environment for primary astrocyte cell growth and viability. It appears likely that future CM optimization, using a serum-free alternative, might be required to advance analyses of cell-specific EVs isolated in vitro.

Background: Despite its functional importance in various fundamental bioprocesses, the studies of N6-methyladenosine (m6A) in the heart are lacking. In this study, we investigated the role of fat mass and obesity-associated (FTO), an m6A demethylase, in cardiac contractile function during homeostasis and remodeling. Methods: We used clinical human samples, preclinical pig and mouse models and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO using AAV9 (in vivo), adenovirus (both in vivo and in vitro) and siRNAs (in vitro). We performed methylated (m6A) RNA immunoprecipitation sequencing (MeRIP-seq) to map transcriptome-wide m6A, and MeRIP qPCR assays to map and validate m6A in individual transcripts, in healthy and failing hearts and myocytes. We performed proteomics analysis using stable isotope labeling with amino acids in cell culture to study m6A role in mRNA to protein translation. Results: We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is carried out by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. Conclusion: Collectively, our study demonstrates the functional importance of FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

Background: Post-transcriptional modifications in the form of m6A (N6-methyladenosine) regulate mRNA fate and translation, miRNA biogenesis, lncRNA function and several cellular processes. However, m6A mechanisms in the mature post-mitotic tissues such as the mammalian heart remain unexplored. Here, we investigated the role of transcriptome-wide m6A in cardiac protein expression as well as lncRNA function. Methods: Using biochemical assays, we investigated the spatiotemporal gene expression patterns of m6A regulators in non-failing, failing (ischemic) human hearts, in mouse and pig MI models. In mouse heart, we mapped transcriptome-wide m6A by developing state-of-the-art MeRIP-seq coupled with novel bioinformatics analysis. To investigate the functional relevance of m6A to cardiac proteome, we used SILAC-LC-MS. By silencing (siRNA) m6A-reader proteins, we investigated the role of m6A in cardiomyocyte mRNA stability, decay and nuclear export. For in vivo myocardial gene delivery, we used AAV9 and adenovirus vectors. Finally, we performed immunohistology in cardiomyocytes and mouse heart tissues to study nuclei size, fibrosis and angiogenesis. Results: We discovered that the expression of m6A demethylase, FTO is decreased in ischemic myocardium and cardiomyocytes, thus in vivo FTO gene delivery resulted in attenuation of ischemia-induced increase in m6A and decrease in cardiac contractile function post-MI. FTO overexpression in mouse heart and human cardiomyocytes revealed selective demethylation (MeRIP-seq) of cardiac contractile transcripts resulting in induced contractile protein expression (SILAC-LC-MS) thus rescuing heart function post-MI. Mechanistically, we demonstrate that the cardioprotective mechanism of FTO is mediated by selective demethylation of cardiac contractile transcripts under ischemia, which prevents mRNA degradation as well as enhanced nuclear compaction. Finally, we demonstrate that FTO overexpression in mouse models of MI resulted in decreased fibrosis and enhanced angiogenesis. Conclusion: Our study provides the first description of a cardiac active m6A demethylase working at post-transcriptional level as a critical regulator of cardiac contractile function, fibrosis and angiogenesis.

Extracellularly secreted membrane vesicles (EMVs) including exosomes were first discovered 30 years ago and were considered to be garbage bags comprised of unwanted cellular components [(1)][1]. Exosomes are relatively homogenous in size (about 30 to 100 nm) and are different from other secreted

Background: Adenosine in RNA is a substrate for addition or removal of methyl group. Reported five decades ago, methylated adenosine (m6A), the most abundant and functionally relevant chemical modification in RNA, whose transcriptome-wide mapping became possible only recently due to next generation sequencing (NGS). Coupled with NGS, m6A-methylated RNA capture (MeRIP-seq) identified widespread m6A distribution in ~8000 mRNA and ~1000 lncRNA transcripts in human and mouse transcriptome. Methods and Results: In a first of its kind approach, we examined m6A RNA methylation in both failing and non-failing hearts. We discovered that global m6A RNA methylation is significantly higher in left ventricles (LV) of failing human, swine and mouse hearts as compared to non-failing controls. Increase in m6A was associated with significantly lower expression of one of the key m6A demethylases, FTO, in the ischemic heart. siRNA-mediated silencing of FTO resulted in significant arrhythmias, loss of Ca 2+ dynamics such as Ca 2+ transient decay (Tau) and cardiomyocyte relaxation time in isolated adult rat cardiomyocytes. Conversely, FTO gene transfer reduced m6A and improved Ca 2+ transients and contractile function in primary cardiomyocytes under hypoxia. In a mouse model of MI, AAV-mediated gene transfer of FTO significantly improved cardiac function post-MI. We identified transcriptome-wide m6A distribution signatures and conserved methylated sites of several mRNAs and lncRNAs using MeRIP-seq in both human and mouse failing and non-failing LV. Detailed MeRIP map of individual transcripts identified differentially methylated 3’-UTR, 5’-UTR and exon sites within several cardiac mRNAs that are important for cardiac function. Conclusion: Our data provide first evidence that m6A modification in RNA is a regulator of cardiomyocyte Ca 2+ dynamics and cardiac function. Our findings on the dynamic nature of the cardiac m6A-epitranscriptome will add another portfolio to mRNA and lncRNA regulation of cardiac remodeling.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines. We would like to thank Tejaswee P. Shah for her valuable input in the development of FunRich. We would like to thank Jan Lotvall for helping us in this effort by circulating the email within his group. We thanks many other users who requested for features/changes in FunRich though the e-mail and the online forum. Suresh Mathivanan is supported by Australian Research Council DECRA (DE150101777) and Award U54- DA036134 supported by the NIH Common Fund through the Office of Strategic Coordination/Office of the NIH Director.The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript. Sí

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs.

Exosomes are cell-derived nanovesicles that carry and shuttle microRNAs (miRNAs) to mediate cell-cell communication. Vast majority of cell types including cardiac myocytes and progenitors actively secrete exosomes, whose miRNA contents are altered after physiological or pathological changes such as myocardial ischemia (MI). In this new study, we have discovered that chemical modification to mRNAs is a novel regulator of ischemia-induced gene expression changes in the heart. We hypothesized that the benefits of human CD34 + stem cell-derived exosomes (CD34exo) are mediated by mRNA modifications in the target cells via miRNA delivery. MiRNA profiling and bioinformatic analysis identified that CD34exo is selectively enriched with a number of miRNAs that directly target genes implicated in regulation of mRNA modifications. Interestingly, under myocardial ischemia, there was a significant increase in mRNA modifications in the mouse heart, which was decreased by about 70% with CD34exo-treatment. In line with the in vivo MI data, in vitro hypoxic stimulation in neonatal / adult rodent myocytes and non-myocytes increased mRNA modifications and controls known regulators of those mRNA modifications. Loss-of-function studies for regulators of mRNA modifications attenuated hypoxia-induced changes to epitranscriptome indicating important roles for these molecules under stress conditions. Finally, using gain-of-function and loss-of-function studies, we demonstrate that miR-126, one of the most enriched miRNAs in CD34exo, plays a critical role in regulating the mRNA modifications. We conclude that miRNAs enriched in CD34exo mediate their cardioprotective effect at least in part, by regulating the mRNA epitranscriptome of the target cell. Our new data suggests hypoxia as a novel regulator of the mRNA epitranscriptome and provides novel insights to post-transcriptional gene regulation in the heart.

Introduction: Clinical application of human CD34+ stem cells is associated with improved exercise tolerance and therapeutic angiogenesis in patients with myocardial ischemia. We reported the first description of independent therapeutic potential of CD34+ stem cell-derived exosomes (CD34Exo) to that of parent cell by mechanisms that still remain poorly understood. Hypothesis: Herein, we tested the hypothesis that CD34Exo may selectively carry non-coding RNA (ncRNA) cargo targeted for pro-angiogenic signaling and ischemic tissue repair. Methods and Results: Murine models of myocardial ischemia employed throughout the study. Cell-free CD34Exo replicated the therapeutic activity of their parent cells by significantly improving myocardial ischemia (ejection fraction, 42±4 v 22±6%; capillary density, 113±7 v 66±6/HPF; fibrosis, 27±2 v 48±7%; p Conclusion: Our results reveal specific CD34Exo-shuttled microRNAs and pathways regulated in the ischemic myocardium. Our work presents a molecular framework for CD34Exo mechanism and function in therapeutic angiogenesis. Precise understanding of CD34Exo mechanisms could significantly amplify the therapeutic benefits of CD34Exo in ischemic tissue regeneration and repair.

Previous studies in our lab have revealed a novel mechanism that therapeutically important human CD34+ stem cells secrete exosomes (Exo) to extracellular milieu that induce angiogenic activity independent of the cells. Further studies demonstrated that CD34+ exosomes (CD34Exo) carry and transfer of proangiogenic miRNAs, such as miR-126, which affect the therapeutic function of the exosomes. Herein we hypothesized that hypoxic treatment of CD34+ stem cells can modulate the miRNA content and regenerative efficacy of CD34Exo. Methods and Results: Exosomes from human CD34+ cells cultured under hypoxia (H-Exo) were more proliferative, anti-apoptotic and angiogenic in vitro, as compared to exosomes from cells under normoxia (N-Exo). In a mouse model of hind limb ischemia, H-Exo treatment significantly enhanced perfusion (ratio: 0.93±0.05 v 0.77±0.02), increased capillary density (1.6±0.2 v 1.1±0.1/HPF) and prevented ischemic limb amputation (0% v 37.5%) as compared to N-Exo (p Conclusion: Hypoxia induced miR-126 expression in CD34 cell-derived exosomes stimulating exosomes-mediated angiogenesis and therapeutic recovery via ETS-transcriptional pathway. Our work has important clinical implications for therapeutic angiogenesis, especially in diabetic and cardiovascular patients who have stem cells with diminished angiogenic potential.

Exosomes, also known as microvesicles (EMVs), are nano-sized membrane particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation and suppression, and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein cargo, which can dictate pathogenicity. While some mechanisms have been proposed for the regulation of EMV cargo selection, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from the overall cellular membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we explore further the role of glycosylation in EMV cargo selection. We identify specific glycosylated targets and associated proteins and demonstrate alteration of their recruitment as a functio...

Cell surface glycans form a critical interface with the biological milieu, informing diverse processes from the inflammatory cascade to cellular migration. Assembly of discrete carbohydrate structures requires the coordinated activity of a repertoire of proteins, including glycosyltransferases and glycosidases. Little is known about the regulatory networks controlling this complex biosynthetic process. Recent work points to a role for microRNA (miRNA) in the regulation of specific glycan biosynthetic enzymes. Herein we take a unique systems-based approach to identify connections between miRNA and the glycome. By using our glycomic analysis platform, lectin microarrays, we identify glycosylation signatures in the NCI-60 cell panel that point to the glycome as a direct output of genomic information flow. Integrating our glycomic dataset with miRNA data, we map miRNA regulators onto genes in glycan biosynthetic pathways (glycogenes) that generate the observed glycan structures. We validate three of these predicted miRNA/glycogene regulatory networks: high mannose, fucose, and terminal β-GalNAc, identifying miRNA regulation that would not have been observed by traditional bioinformatic methods. Overall, our work reveals critical nodes in the global glycosylation network accessible to miRNA regulation, providing a bridge between miRNA-mediated control of cell phenotype and the glycome.