Distinct functional neutrophil phenotypes in sepsis patients correlate with disease severity.

Purpose: Sepsis is a clinical syndrome defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis is a highly heterogeneous syndrome with distinct phenotypes that impact immune function and response to infection. To develop targeted therapeutics, immunophenotyping is needed to identify distinct functional phenotypes of immune cells. In this study, we utilized our Organ-on-Chip assay to categorize sepsis patients into distinct phenotypes using patient data, neutrophil functional analysis, and proteomics. Methods: Following informed consent, neutrophils and plasma were isolated from sepsis patients in the Temple University Hospital ICU (n=45) and healthy control donors (n=7). Human lung microvascular endothelial cells (HLMVEC) were cultured in the Organ-on-Chip and treated with buffer or cytomix ((TNF/IL-1ß/IFNγ). Neutrophil adhesion and migration across HLMVEC in the Organ-on-Chip were used to categorize functional neutrophil phenotypes. Quantitative label-free global proteomics was performed on neutrophils to identify differentially expressed proteins. Plasma levels of sepsis biomarkers and neutrophil extracellular traps (NETs) were determined by ELISA. Results: We identified three functional phenotypes in critically ill ICU sepsis patients based on ex vivo neutrophil adhesion and migration patterns. The phenotypes were classified as: Hyperimmune characterized by enhanced neutrophil adhesion and migration, Hypoimmune that was unresponsive to stimulation, and Hybrid with increased adhesion but blunted migration. These functional phenotypes were associated with distinct proteomic signatures and differentiated sepsis patients by important clinical parameters related to disease severity. The Hyperimmune group demonstrated higher oxygen requirements, increased mechanical ventilation, and longer ICU length of stay compared to the Hypoimmune and Hybrid groups. Patients with the Hyperimmune neutrophil phenotype had significantly increased circulating neutrophils and elevated plasma levels NETs. Conclusion: Neutrophils and NETs play a critical role in vascular barrier dysfunction in sepsis and elevated NETs may be a key biomarker identifying the Hyperimmune group. Our results establish significant associations between specific neutrophil functional phenotypes and disease severity and identify important functional parameters in sepsis pathophysiology that may provide a new approach to classify sepsis patients for specific therapeutic interventions.

Dysregulated Autophagy and Sarcomere Dysfunction in Patients With Heart Failure With Co-Occurrence of P63A and P380S BAG3 Variants.

BACKGROUND: Mutations to the co-chaperone protein BAG3 (B-cell lymphoma-2-associated athanogene-3) are a leading cause of dilated cardiomyopathy (DCM). These mutations often impact the C-terminal BAG domain (residues 420-499), which regulates heat shock protein 70-dependent protein turnover via autophagy. While mutations in other regions are less common, previous studies in patients with DCM found that co-occurrence of 2 BAG3 variants (P63A, P380S) led to worse prognosis. However, the underlying mechanism for dysfunction is not fully understood. METHODS AND RESULTS: In this study, we used proteomics, Western blots, and myofilament functional assays on left ventricular tissue from patients with nonfailing, DCM, and DCM with BAG363/380 to determine how these mutations impact protein quality control and cardiomyocyte contractile function. We found dysregulated autophagy and increased protein ubiquitination in patients with BAG363/380 compared with nonfailing and DCM, suggesting impaired protein turnover. Expression and myofilament localization of BAG3-binding proteins were also uniquely altered in the BAG3,63/380 including abolished localization of the small heat shock protein CRYAB (alpha-crystallin B chain) to the sarcomere. To determine whether these variants impacted sarcomere function, we used cardiomyocyte force-calcium assays and found reduced maximal calcium-activated force in DCM and BAG363/380. Interestingly, myofilament calcium sensitivity was increased in DCM but not with BAG363/380, which was not explained by differences in troponin I phosphorylation. CONCLUSIONS: Together, our data support that the disease-enhancing mechanism for BAG3 variants outside of the BAG domain is through disrupted protein turnover leading to compromised sarcomere function. These findings suggest a shared mechanism of disease among pathogenic BAG3 variants, regardless of location.

Secreted folate receptor γ drives fibrogenesis in metabolic dysfunction-associated steatohepatitis by amplifying TGFß signaling in hepatic stellate cells.

Hepatic fibrosis is the primary determinant of mortality in patients with metabolic dysfunction-associated steatohepatitis (MASH). Transforming growth factor-ß (TGFß), a master profibrogenic cytokine, is a promising therapeutic target that has not yet been translated into an effective therapy in part because of liabilities associated with systemic TGFß antagonism. We have identified that soluble folate receptor γ (FOLR3), which is expressed in humans but not in rodents, is a secreted protein that is elevated in the livers of patients with MASH but not in those with metabolic dysfunction-associated steatotic liver disease, those with type II diabetes, or healthy individuals. Global proteomics showed that FOLR3 was the most highly significant MASH-specific protein and was positively correlated with increasing fibrosis stage, consistent with stimulation of activated hepatic stellate cells (HSCs), which are the key fibrogenic cells in the liver. Exposure of HSCs to exogenous FOLR3 led to elevated extracellular matrix (ECM) protein production, an effect synergistically potentiated by TGFß1. We found that FOLR3 interacts with the serine protease HTRA1, a known regulator of TGFBR, and activates TGFß signaling. Administration of human FOLR3 to mice induced severe bridging fibrosis and an ECM pattern resembling human MASH. Our study thus uncovers a role of FOLR3 in enhancing fibrosis.

Bag3 Regulates Mitochondrial Function and the Inflammasome Through Canonical and Noncanonical Pathways in the Heart.

B-cell lymphoma 2-associated athanogene-3 (Bag3) is expressed in all animal species, with Bag3 levels being most prominent in the heart, the skeletal muscle, the central nervous system, and in many cancers. Preclinical studies of Bag3 biology have focused on animals that have developed compromised cardiac function; however, the present studies were performed to identify the pathways perturbed in the heart even before the occurrence of clinical signs of dilatation and failure of the heart. These studies show that hearts carrying variants that knockout one allele of BAG3 have significant alterations in multiple cellular pathways including apoptosis, autophagy, mitochondrial homeostasis, and the inflammasome.

Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IRA Signal in Malignant Mesothelioma.

EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using targeted proteomics, protein-protein interaction methods, PCR cloning, and 3D modeling approaches, we identified a novel ubiquitin E3 ligase complex recruited by the EphB4 C tail upon autocrine IGF-II signal deprivation. We show this complex to contain a previously unknown N-Terminal isoform of Deltex3 E3-Ub ligase (referred as "DTX3c"), along with UBA1(E1) and UBE2N(E2) ubiquitin ligases and the ATPase/unfoldase Cdc48/p97. Upon autocrine IGF-II neutralization in cultured MSTO211H (a Malignant Mesothelioma cell line that is highly responsive to the EphB4 degradation rescue IGF-II signal), the inter-molecular interactions between these factors were enhanced and their association with the EphB4 C-tail increased consistently with the previously described EphB4 degradation pattern. The ATPase/unfoldase activity of Cdc48/p97 was required for EphB4 recruitment. As compared to the previously known isoforms DTX3a and DTX3b, a 3D modeling analysis of the DTX3c Nt domain showed a unique 3D folding supporting isoform-specific biological function(s). We shed light on the molecular machinery associated with autocrine IGF-II regulation of oncogenic EphB4 kinase expression in a previously characterized IGF-II+/EphB4+ Mesothelioma cell line. The study provides early evidence for DTX3 Ub-E3 ligase involvement beyond the Notch signaling pathway.

Incorporation of paclitaxel in mesenchymal stem cells using nanoengineering upregulates antioxidant response, CXCR4 expression and enhances tumor homing.

Engineered mesenchymal stem cells (MSCs) have been investigated extensively for gene delivery and, more recently, for targeted small molecule delivery. While preclinical studies demonstrate the potential of MSCs for targeted delivery, clinical studies suggest that tumor homing of native MSCs may be inefficient. We report here a surprising finding that loading MSCs with the anticancer drug paclitaxel (PTX) by nanoengineering results in significantly improved tumor homing compared to naïve MSCs. Loading PTX in MSCs results in increased levels of mitochondrial reactive oxygen species (ROS). In response to this oxidative stress, MSCs upregulate two important set of proteins. First were critical antioxidant proteins, most importantly nuclear factor erythroid 2-like 2 (Nrf2), the master regulator of antioxidant responses; upregulation of antioxidant proteins may explain how MSCs protect themselves from drug-induced oxidative stress. The second was CXCR4, a direct target of Nrf2 and a key mediator of tumor homing; upregulation of CXCR4 suggested a mechanism that may underlie the improved tumor homing of nanoengineered MSCs. In addition to demonstrating the potential mechanism of improved tumor targeting of nanoengineered MSCs, our studies reveal that MSCs utilize a novel mechanism of resistance against drug-induced oxidative stress and cell death, explaining how MSCs can deliver therapeutic concentrations of cytotoxic payload while maintaining their viability.

Molecular Framework of Mouse Endothelial Cell Dysfunction during Inflammation: A Proteomics Approach.

A key aspect of cytokine-induced changes as observed in sepsis is the dysregulated activation of endothelial cells (ECs), initiating a cascade of inflammatory signaling leading to leukocyte adhesion/migration and organ damage. The therapeutic targeting of ECs has been hampered by concerns regarding organ-specific EC heterogeneity and their response to inflammation. Using in vitro and in silico analysis, we present a comprehensive analysis of the proteomic changes in mouse lung, liver and kidney ECs following exposure to a clinically relevant cocktail of proinflammatory cytokines. Mouse lung, liver and kidney ECs were incubated with TNF-α/IL-1ß/IFN-γ for 4 or 24 h to model the cytokine-induced changes. Quantitative label-free global proteomics and bioinformatic analysis performed on the ECs provide a molecular framework for the EC response to inflammatory stimuli over time and organ-specific differences. Gene Ontology and PANTHER analysis suggest why some organs are more susceptible to inflammation early on, and show that, as inflammation progresses, some protein expression patterns become more uniform while additional organ-specific proteins are expressed. These findings provide an in-depth understanding of the molecular changes involved in the EC response to inflammation and can support the development of drugs targeting ECs within different organs. Data are available via ProteomeXchange (identifier PXD031804).

HIV-1 gp120 Impairs Spatial Memory Through Cyclic AMP Response Element-Binding Protein.

HIV-associated neurocognitive disorders (HAND) remain an unsolved problem that persists despite using antiretroviral therapy. We have obtained data showing that HIV-gp120 protein contributes to neurodegeneration through metabolic reprogramming. This led to decreased ATP levels, lower mitochondrial DNA copy numbers, and loss of mitochondria cristae, all-important for mitochondrial biogenesis. gp120 protein also disrupted mitochondrial movement and synaptic plasticity. Searching for the mechanisms involved, we found that gp120 alters the cyclic AMP response element-binding protein (CREB) phosphorylation on serine residue 133 necessary for its function as a transcription factor. Since CREB regulates the promoters of PGC1α and BDNF genes, we found that CREB dephosphorylation causes PGC1α and BDNF loss of functions. The data was validated in vitro and in vivo. The negative effect of gp120 was alleviated in cells and animals in the presence of rolipram, an inhibitor of phosphodiesterase protein 4 (PDE4), restoring CREB phosphorylation. We concluded that HIV-gp120 protein contributes to HAND via inhibition of CREB protein function.

Dysregulation of S-adenosylmethionine Metabolism in Nonalcoholic Steatohepatitis Leads to Polyamine Flux and Oxidative Stress.

Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease worldwide, with 25% of these patients developing nonalcoholic steatohepatitis (NASH). NASH significantly increases the risk of cirrhosis and decompensated liver failure. Past studies in rodent models have shown that glycine-N-methyltransferase (GNMT) knockout results in rapid steatosis, fibrosis, and hepatocellular carcinoma progression. However, the attenuation of GNMT in subjects with NASH and the molecular basis for its impact on the disease process is still unclear. To address this knowledge gap, we show the reduction of GNMT protein levels in the liver of NASH subjects compared to healthy controls. To gain insight into the impact of decreased GNMT in the disease process, we performed global label-free proteome studies on the livers from a murine modified amylin diet-based model of NASH. Histological and molecular characterization of the animal model demonstrate a high resemblance to human disease. We found that a reduction of GNMT leads to a significant increase in S-adenosylmethionine (AdoMet), an essential metabolite for transmethylation reactions and a substrate for polyamine synthesis. Further targeted proteomic and metabolomic studies demonstrated a decrease in GNMT transmethylation, increased flux through the polyamine pathway, and increased oxidative stress production contributing to NASH pathogenesis.

Posiphen Reduces the Levels of Huntingtin Protein through Translation Suppression.

Posiphen tartrate (Posiphen) is an orally available small molecule that targets a conserved regulatory element in the mRNAs of amyloid precursor protein (APP) and α-synuclein (αSYN) and inhibits their translation. APP and αSYN can cause neurodegeneration when their aggregates induce neurotoxicity. Therefore, Posiphen is a promising drug candidate for neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Posiphen's safety has been demonstrated in three independent phase I clinical trials. Moreover, in a proof of concept study, Posiphen lowered neurotoxic proteins and inflammatory markers in cerebrospinal fluid of mild cognitive impaired patients. Herein we investigated whether Posiphen reduced the expression of other proteins, as assessed by stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry (MS)-based proteomics. Neuroblastoma SH-SY5Y cells, an in vitro model of neuronal function, were used for the SILAC protein profiling response. Proteins whose expression was altered by Posiphen treatment were characterized for biological functions, pathways and networks analysis. The most significantly affected pathway was the Huntington's disease signaling pathway, which, along with huntingtin (HTT) protein, was down-regulated by Posiphen in the SH-SY5Y cells. The downregulation of HTT protein by Posiphen was confirmed by quantitative Western blotting and immunofluorescence. Unchanged mRNA levels of HTT and a comparable decay rate of HTT proteins after Posiphen treatment supported the coclusion that Posiphen reduced HTT via downregulation of the translation of HTT mRNA. Meanwhile, the downregulation of APP and αSYN proteins by Posiphen was also confirmed. The mRNAs encoding HTT, APP and αSYN contain an atypical iron response element (IRE) in their 5'-untranslated regions (5'-UTRs) that bind iron regulatory protein 1 (IRP1), and Posiphen specifically bound this complex. Conversely, Posiphen did not bind the IRP1/IRE complex of mRNAs with canonical IREs, and the translation of these mRNAs was not affected by Posiphen. Taken together, Posiphen shows high affinity binding to the IRE/IRP1 complex of mRNAs with an atypical IRE stem loop, inducing their translation suppression, including the mRNAs of neurotoxic proteins APP, αSYN and HTT.

A Novel Benzopyrane Derivative Targeting Cancer Cell Metabolic and Survival Pathways.

(1) Background: Today, the discovery of novel anticancer agents with multitarget effects and high safety margins represents a high challenge. Drug discovery efforts indicated that benzopyrane scaffolds possess a wide range of pharmacological activities. This spurs on building a skeletally diverse library of benzopyranes to identify an anticancer lead drug candidate. Here, we aim to characterize the anticancer effect of a novel benzopyrane derivative, aiming to develop a promising clinical anticancer candidate. (2) Methods: The anticancer effect of SIMR1281 against a panel of cancer cell lines was tested. In vitro assays were performed to determine the effect of SIMR1281 on GSHR, TrxR, mitochondrial metabolism, DNA damage, cell cycle progression, and the induction of apoptosis. Additionally, SIMR1281 was evaluated in vivo for its safety and in a xenograft mice model. (3) Results: SIMR1281 strongly inhibits GSHR while it moderately inhibits TrxR and modulates the mitochondrial metabolism. SIMR1281 inhibits the cell proliferation of various cancers. The antiproliferative activity of SIMR1281 was mediated through the induction of DNA damage, perturbations in the cell cycle, and the inactivation of Ras/ERK and PI3K/Akt pathways. Furthermore, SIMR1281 induced apoptosis and attenuated cell survival machinery. In addition, SIMR1281 reduced the tumor volume in a xenograft model while maintaining a high in vivo safety profile at a high dose. (4) Conclusions: Our findings demonstrate the anticancer multitarget effect of SIMR1281, including the dual inhibition of glutathione and thioredoxin reductases. These findings support the development of SIMR1281 in preclinical and clinical settings, as it represents a potential lead compound for the treatment of cancer.

Short chain fatty acids delay the development of hepatocellular carcinoma in HBx transgenic mice.

Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The HBV encoded oncoprotein, HBx, alters the expression of host genes and the activity of multiple signal transduction pathways that contribute to the pathogenesis of HCC by multiple mechanisms independent of HBV replication. However, it is not clear which pathways are the most relevant therapeutic targets in hepatocarcinogenesis. Short chain fatty acids (SCFAs) have strong anti-inflammatory and anti-neoplastic properties, suggesting that they may block the progression of chronic liver disease (CLD) to HCC, thereby identifying the mechanisms relevant to HCC development. This hypothesis was tested in HBx transgenic (HBxTg) mice fed SCFAs. Groups of HBxTg mice were fed with SCFAs or vehicle from 6 to 9 months of age and then assessed for dysplasia, and from 9 to 12 months of age and then assessed for HCC. Livers from 12 month old mice were then analyzed for changes in gene expression by mass spectrometry-based proteomics. SCFA-fed mice had significantly fewer dysplastic and HCC nodules compared to controls at 9 and 12 months, respectively. Pathway analysis of SCFA-fed mice showed down-regulation of signaling pathways altered by HBx in human CLD and HCC, including those involved in inflammation, phosphatidylinositol 3-kinase, epidermal growth factor, and Ras. SCFA treatment promoted increased expression of the tumor suppressor, disabled homolog 2 (DAB2). DAB2 depresses Ras pathway activity, which is constitutively activated by HBx. SCFAs also reduced cell viability in HBx-transfected cell lines in a dose-dependent manner while the viability of primary human hepatocytes was unaffected. These unique findings demonstrate that SCFAs delay the pathogenesis of CLD and development of HCC, and provide insight into some of the underlying mechanisms that are relevant to pathogenesis in that they are responsive to treatment.

A novel platform for peptide-mediated affinity capture and LC-MS/MS identification of host receptors involved in Plasmodium invasion.

Successful Plasmodium falciparum invasion of red blood cells includes the orderly execution of highly specific receptor-ligand molecular interactions between the parasite's proteins and the red blood cell membrane proteins. There is a growing need for elucidating receptor-ligand pairings, which will help in understanding the parasite's biology and provide the fundamental basis for developing prophylactic or therapeutic alternatives leading to mitigating or eliminating this type of malaria. We have thus used Plasmodium falciparum RH5 - derived peptides and ghost red blood cell proteins in synthetic peptide affinity capture assays to identify important host receptors used by Plasmodium spp. in the invasion of red blood cells. LC-MS/MS analysis confirmed the extensively described interaction between PfRH5 and the basigin receptor on the red blood cell membrane. As shown here, tagged synthetic peptides displaying high binding ability to erythrocytes can be used to identify receptors present in protein extracts from ghost red blood cells via affinity capture and LC-MS/MS. SIGNIFICANCE: The article describes a novel approach for identifying red blood cell receptors based on the ability of synthetic peptides having high red blood cell binding capacity to capture Plasmodium spp. receptors on proteins extracted from ghost red blood cells. Specifically, novel methods to identify Plasmodium falciparum reticulocyte binding protein homolog 5 PfRH5 and basigin interaction using a combination of affinity capture and LC-MS/MS assays is described. Identification of these host RBC receptors interacting with malarial parasite proteins is of utmost importance in studying the disease's pathogenesis and will provide crucial information in understanding the parasite's biology. In addition, data from these studies can be used to identify potential therapeutic target(s) to mitigate or eliminate this debilitating disease.

Transient receptor potential ion channel TRPM2 promotes AML proliferation and survival through modulation of mitochondrial function, ROS, and autophagy.

Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in maintaining cell survival following oxidant injury. Here, we show that TRPM2 is highly expressed in acute myeloid leukemia (AML). The role of TRPM2 in AML was studied following depletion with CRISPR/Cas9 technology in U937 cells. In in vitro experiments and in xenografts, depletion of TRPM2 in AML inhibited leukemia proliferation, and doxorubicin sensitivity was increased. Mitochondrial function including oxygen consumption rate and ATP production was reduced, impairing cellular bioenergetics. Mitochondrial membrane potential and mitochondrial calcium uptake were significantly decreased in depleted cells. Mitochondrial reactive oxygen species (ROS) were significantly increased, and Nrf2 was decreased, reducing the antioxidant response. In TRPM2-depleted cells, ULK1, Atg7, and Atg5 protein levels were decreased, leading to autophagy inhibition. Consistently, ATF4 and CREB, two master transcription factors for autophagosome biogenesis, were reduced in TRPM2-depleted cells. In addition, Atg13 and FIP200, which are known to stabilize ULK1 protein, were decreased. Reconstitution with TRPM2 fully restored proliferation, viability, and autophagy; ATF4 and CREB fully restored proliferation and viability but only partially restored autophagy. TRPM2 expression reduced the elevated ROS found in depleted cells. These data show that TRPM2 has an important role in AML proliferation and survival through regulation of key transcription factors and target genes involved in mitochondrial function, bioenergetics, the antioxidant response, and autophagy. Targeting TRPM2 may represent a novel therapeutic approach to inhibit myeloid leukemia growth and enhance susceptibility to chemotherapeutic agents through multiple pathways.

The Central Role of Protein Kinase C Epsilon in Cyanide Cardiotoxicity and Its Treatment.

In adult mouse myocytes, brief exposure to sodium cyanide (CN) in the presence of glucose does not decrease ATP levels, yet produces profound reduction in contractility, intracellular Ca2+ concentration ([Ca2+]i) transient and L-type Ca2+ current (ICa) amplitudes. We analyzed proteomes from myocytes exposed to CN, focusing on ionic currents associated with excitation-contraction coupling. CN induced phosphorylation of α1c subunit of L-type Ca2+ channel and α2 subunit of Na+-K+-ATPase. Methylene blue (MB), a CN antidote that we previously reported to ameliorate CN-induced reduction in contraction, [Ca2+]i transient and ICa amplitudes, was able to reverse this phosphorylation. CN decreased Na+-K+-ATPase current contributed by α2 but not α1 subunit, an effect that was also counteracted by MB. Peptide consensus sequences suggested CN-induced phosphorylation was mediated by protein kinase C epsilon (PKCε). Indeed, CN stimulated PKC kinase activity and induced PKCε membrane translocation, effects that were prevented by MB. Pretreatment with myristoylated PKCε translocation activator or inhibitor peptides mimicked and inhibited the effects of CN on ICa and myocyte contraction, respectively. We conclude that CN activates PKCε, which phosphorylates L-type Ca2+ channel and Na+-K+-ATPase, resulting in depressed cardiac contractility. We hypothesize that this inhibition of ion fluxes represents a novel mechanism by which the cardiomyocyte reduces its ATP demand (decreased ion fluxes and contractility), diminishes ATP turnover and preserves cell viability. However, this cellular protective effect translates into life-threatening cardiogenic shock in vivo, thereby creating a profound disconnect between survival mechanisms at the cardiomyocyte level from those at the level of the whole organism.

Novel biomarkers to assess in utero effects of maternal opioid use: First steps toward understanding short- and long-term neurodevelopmental sequelae.

Maternal opioid use disorder is common, resulting in significant neonatal morbidity and cost. Currently, it is not possible to predict which opioid-exposed newborns will require pharmacotherapy for neonatal abstinence syndrome. Further, little is known regarding the effects of maternal opioid use disorder on the developing human brain. We hypothesized that novel methodologies utilizing fetal central nervous system-derived extracellular vesicles isolated from maternal blood can address these gaps in knowledge. Plasma from opioid users and controls between 9 and 21 weeks was precipitated and extracellular vesicles were isolated. Mu opioid and cannabinoid receptor levels were quantified. Label-free proteomics studies and unbiased small RNA next generation sequencing was performed in paired fetal brain tissue. Maternal opioid use disorder increased mu opioid receptor protein levels in extracellular vesicles independent of opioid equivalent dose. Moreover, cannabinoid receptor levels in extracellular vesicles were upregulated with opioid exposure indicating cross talk with endocannabinoids. Maternal opioid use disorder was associated with significant changes in extracellular vesicle protein cargo and fetal brain micro RNA expression, especially in male fetuses. Many of the altered cargo molecules and micro RNAs identified are associated with adverse clinical neurodevelopmental outcomes. Our data suggest that assays relying on extracellular vesicles isolated from maternal blood extracellular vesicles may provide information regarding fetal response to opioids in the setting of maternal opioid use disorder. Prospective clinical studies are needed to evaluate the association between extracellular vesicle biomarkers, risk of neonatal abstinence syndrome and neurodevelopmental outcomes.

ELMO1 deficiency enhances platelet function.

Phosphatidylinositol 3-kinase is an important signaling molecule that, once activated, leads to the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). We performed a proteomic screen to identify PIP3-interacting proteins in human platelets. Among these proteins, we found engulfment and cell motility 1 (ELMO1), a scaffold protein with no catalytic activity. ELMO1 is expressed in platelets and interacts with active RhoG. However, the function of ELMO1 in platelets is not known. The focus of this study was to determine the function of ELMO1 in platelets utilizing ELMO1-/- mice. Platelet aggregation, granule secretion, integrin αIIbß3 activation, and thromboxane generation were enhanced in ELMO1-/- platelets in response to glycoprotein VI (GPVI) agonists but unaltered when a protease-activated receptor 4 agonist was used. The kinetics of spreading on immobilized fibrinogen was enhanced in ELMO1-/- platelets compared with wild-type (WT) littermate controls. This suggests that ELMO1 plays a role downstream of the GPVI and integrin αIIbß3 pathway. Furthermore, whole blood from ELMO1-/- mice perfused over collagen exhibited enhanced thrombus formation compared with WT littermate controls. ELMO1-/- mice showed reduced survival compared with control following pulmonary embolism. ELMO1-/- mice also exhibited a shorter time to occlusion using the ferric-chloride injury model and reduced bleeding times compared with WT littermate controls. These results indicate that ELMO1 plays an important role in hemostasis and thrombosis in vivo. RhoG activity was enhanced in ELMO1-/- murine platelets compared with WT littermate controls in response to GPVI agonist. Together, these data suggest that ELMO1 negatively regulates GPVI-mediated thrombus formation via RhoG.

Elevated levels of brain homocysteine directly modulate the pathological phenotype of a mouse model of tauopathy.

A high circulating level of homocysteine (Hcy), also known as hyperhomocysteinemia, is a risk factor for Alzheimer's disease (AD). Previous studies show that elevated Hcy promotes brain amyloidosis and behavioral deficits in mouse models of AD. However, whether it directly modulates the development of tau neuropathology independently of amyloid beta in vivo is unknown. Herein, we investigate the effect of diet-induced elevated levels of brain Hcy on the phenotype of a relevant mouse model of human tauopathy. Compared with controls, tau mice fed with low folate and B vitamins diet had a significant increase in brain Hcy levels and worsening of behavioral deficits. The same mice had a significant elevation of tau phosphorylation, synaptic pathology, and astrocytes activation. In vitro studies demonstrated that Hcy effect on tau phosphorylation was mediated by an upregulation of 5-lipoxygenase via cdk5 kinase pathway activation. Our findings support the novel concept that high Hcy level in the central nervous system is a metabolic risk factor for neurodegenerative diseases, specifically characterized by the progressive accumulation of tau pathology, namely tauopathies.

Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation.

Although many factors contribute to cellular differentiation, the role of mitochondria Ca2+ dynamics during development remains unexplored. Because mammalian embryonic epiblasts reside in a hypoxic environment, we intended to understand whether mCa2+ and its transport machineries are regulated during hypoxia. Tissues from multiple organs of developing mouse embryo evidenced a suppression of MICU1 expression with nominal changes on other MCU complex components. As surrogate models, we here utilized human embryonic stem cells (hESCs)/induced pluripotent stem cells (hiPSCs) and primary neonatal myocytes to delineate the mechanisms that control mCa2+ and bioenergetics during development. Analysis of MICU1 expression in hESCs/hiPSCs showed low abundance of MICU1 due to its direct repression by Foxd1. Experimentally, restoration of MICU1 established the periodic cCa2+ oscillations and promoted cellular differentiation and maturation. These findings establish a role of mCa2+ dynamics in regulation of cellular differentiation and reveal a molecular mechanism underlying this contribution through differential regulation of MICU1.