The FUT2 Variant c.461G>A (p.Trp154*) Is Associated With Differentially Expressed Genes and Nasopharyngeal Microbiota Shifts in Patients With Otitis Media

Otitis media (OM) is a leading cause of childhood hearing loss. Variants in FUT2, which encodes alpha-(1,2)-fucosyltransferase, were identified to increase susceptibility to OM, potentially through shifts in the middle ear (ME) or nasopharyngeal (NP) microbiotas as mediated by transcriptional changes. Greater knowledge of differences in relative abundance of otopathogens in carriers of pathogenic variants can help determine risk for OM in patients. In order to determine the downstream effects of FUT2 variation, we examined gene expression in relation to carriage of a common pathogenic FUT2 c.461G>A (p.Trp154*) variant using RNA-sequence data from saliva samples from 28 patients with OM. Differential gene expression was also examined in bulk mRNA and single-cell RNA-sequence data from wildtype mouse ME mucosa after inoculation with non-typeable Haemophilus influenzae (NTHi). In addition, microbiotas were profiled from ME and NP samples of 65 OM patients using 16S rRNA gene sequencing. In human carriers of the FUT2 variant, FN1, KMT2D, MUC16 and NBPF20 were downregulated while MTAP was upregulated. Post-infectious expression in the mouse ME recapitulated these transcriptional differences, with the exception of Fn1 upregulation after NTHi-inoculation. In the NP, Candidate Division TM7 was associated with wildtype genotype (FDR-adj-p=0.009). Overall, the FUT2 c.461G>A variant was associated with transcriptional changes in processes related to response to infection and with increased load of potential otopathogens in the ME and decreased commensals in the NP. These findings provide increased understanding of how FUT2 variants influence gene transcription and the mucosal microbiota, and thus contribute to the pathology of OM.

Myofibril orientation as a metric for characterizing heart disease

Myocyte disarray is a hallmark of cardiomyopathy. However, the relationship between alterations in the orientation of individual myofibrils and myofilaments to disease progression has been largely underexplored. This oversight has predominantly been due to a paucity of methods for objective and quantitative analysis. Here we introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions and demonstrate its superiority as compared to conventional histological assessments. Using small-angle X-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wildtype mouse myocardium by assessing the angular spread of the 1,0 equatorial reflection (angle sigma). At a sarcomere length (SL) of 1.9 microns, the angle sigma was 0.23 +/- 0.01 rad, decreased to 0.19 +/- 0.01 rad at a SL of 2.1 microns, and further decreased to 0.15 +/- 0.01 rad at a SL of 2.3 microns (p<0.0001). Angle sigma was significantly larger in R403Q (a MYH7 HCM model) porcine myocardium (0.24 +/- 0.01 rad) compared to WT myocardium (0.14 +/- 0.005 rad, p<0.0001) as well as in human heart failure tissue (0.19 +/- 0.006 rad) when compared to non-failing samples (0.17 +/- 0.007 rad, p=0.01). These data indicate that diseased myocardium suffers from greater myofibrillar disorientation compared to healthy controls. Finally, we showed that conventional, histology-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Our method for directly assessing myofibrillar orientation avoids the artifacts introduced by conventional histological methods that directly assess myocyte orientation and only indirectly assess myofibrillar orientation, and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent X-ray diffraction patterns from frozen human myocardium provides a new tool for investigating the structural bases of cardiomyopathies.

Abstract P350: Myofibril Orientation In Cardiac Muscle And Its Implication For Heart Disease

Rationale: Myocyte disarray is a hallmark of cardiomyopathy. However, the orientation of individual myofibrils and myofilaments and how their alignment may be altered in disease progression have been largely underexplored. This oversight has been predominantly due to a paucity of methods for objective and quantitative analysis. Objective: To introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions and demonstrate its superiority versus traditional histological assessments. Methods and Results: Using small-angle X-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in skinned, relaxed, wildtype mouse myocardium by assessing the angular spread of the 1,0 equatorial reflection (angle σ). At a sarcomere length (SL) of 1.9 μm, the angle σ was 0.23±0.01 rad, decreased to 0.19±0.01 rad at a SL of 2.1 μm, and further decreased to 0.15±0.01 rad at a SL of 2.3 μm (p<0.0001). Angle σ was significantly larger in R403Q (a MYH7 HCM model) porcine myocardium (0.24±0.01 rad) compared to WT myocardium (0.14±0.005 rad, p<0.0001) as well as in biopsied human heart failure tissue (0.19±0.006 rad) when compared to non-failing samples (0.17±0.007 rad, p=0.01). These data indicate that diseased myocardium suffers from myofibrillar disorientation compared to healthy controls. Finally, using control samples, we showed that traditional, histological-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Conclusions: Our method for assessing myofibrillar orientation limits the artifacts introduced by traditional histological processing and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent X-ray diffraction patterns from frozen, biopsied human myocardium opens up new avenues of inquiry regarding the relation of myofibrillar structure to function in health and disease.

Small Neuron-Derived Extracellular Vesicles from Individuals with Down Syndrome Propagate Tau Pathology in the Wildtype Mouse Brain

Individuals with Down syndrome (DS) exhibit Alzheimer’s disease (AD) pathology at a young age, including amyloid plaques and neurofibrillary tangles (NFTs). Tau pathology can spread via extracellular vesicles, such as exosomes. The cargo of neuron-derived small extracellular vesicles (NDEVs) from individuals with DS contains p-Tau at an early age. The goal of the study was to investigate whether NDEVs isolated from the blood of individuals with DS can spread Tau pathology in the brain of wildtype mice. We purified NDEVs from the plasma of patients with DS-AD and controls and injected small quantities using stereotaxic surgery into the dorsal hippocampus of adult wildtype mice. Seeding competent Tau conformers were amplified in vitro from DS-AD NDEVs but not NDEVs from controls. One month or 4 months post-injection, we examined Tau pathology in mouse brains. We found abundant p-Tau immunostaining in the hippocampus of the mice injected with DS-AD NDEVs compared to injections of age-matched control NDEVs. Double labeling with neuronal and glial markers showed that p-Tau staining was largely found in neurons and, to a lesser extent, in glial cells and that p-Tau immunostaining was spreading along the corpus callosum and the medio-lateral axis of the hippocampus. These studies demonstrate that NDEVs from DS-AD patients exhibit Tau seeding capacity and give rise to tangle-like intracellular inclusions.

DYT-TOR1A Subcellular Proteomics Reveals Selective Vulnerability of the Nuclear Proteome to Cell Stress

TorsinA is a AAA+ ATPase that shuttles between the ER lumen and outer nuclear envelope in an ATP-dependent manner and is functionally implicated in nucleocytoplasmic transport. We hypothesized that the DYT-TOR1A dystonia disease-causing variant, ΔE TorsinA, may therefore disrupt the normal subcellular distribution of proteins between the nuclear and cytosolic compartments. To test this hypothesis, we performed proteomic analysis on nuclear and cytosolic subcellular fractions from DYT-TOR1A and wildtype mouse embryonic fibroblasts (MEFs). We further examined the compartmental proteomes following exposure to thapsigargin (Tg), an endoplasmic reticulum (ER) stressor, because DYT-TOR1A dystonia models have previously shown abnormalities in cellular stress responses. Across both subcellular compartments, proteomes of DYT-TOR1A cells showed basal state disruptions consistent with an activated stress response, and in response to thapsigargin, a blunted stress response. However, the DYT-TOR1A nuclear proteome under Tg cell stress showed the most pronounced and disproportionate degree of protein disruptions - 3-fold greater than all other conditions. The affected proteins extended beyond those typically associated with stress responses, including enrichments for processes critical for neuronal synaptic function. These findings highlight the advantage of subcellular proteomics to reveal events that localize to discrete subcellular compartments and refine thinking about the mechanisms and significance of cell stress in DYT-TOR1A pathogenesis.

Role of endothelial microRNA 155 on capillary leakage in systemic inflammation

Background Capillary leakage is a key contributor to the pathological host response to infections. The underlying mechanisms remain incompletely understood, and the role of microRNAs (MIR) has not been investigated in detail. We hypothesized that specific MIRs might be regulated directly in the endothelium thereby contributing to vascular leakage. Methods SmallRNA sequencing of endotoxemic murine pulmonary endothelial cells (ECs) was done to detect regulated vascular MIRs. In vivo models: transgenic zebrafish (flk1:mCherry/l-fabp:eGFP-DPB), knockout/wildtype mouse (B6.Cg-Mir155tm1.1Rsky/J); disease models: LPS 17.5 mg/kgBW and cecal ligation and puncture (CLP); in vitro models: stimulated human umbilical vein EC (HUVECs), transendothelial electrical resistance. Results Endothelial MIR155 was identified as a promising candidate in endotoxemic murine pulmonary ECs (25 × upregulation). Experimental overexpression in a transgenic zebrafish line and in HUVECs was sufficient to induce spontaneous vascular leakage. To the contrary, genetic MIR155 reduction protects against permeability both in vitro and in endotoxemia in vivo in MIR155 heterozygote knockout mice thereby improving survival by 40%. A tight junction protein, Claudin-1, was down-regulated both in endotoxemia and by experimental MIR155 overexpression. Translationally, MIR155 was detectable at high levels in bronchoalveolar fluid of patients with ARDS compared to healthy human subjects. Conclusions We found that MIR155 is upregulated in the endothelium in mouse and men as part of a systemic inflammatory response and might contribute to the pathophysiology of vascular leakage in a Claudin-1-dependent manner. Future studies have to clarify whether MIR155 could be a potential therapeutic target.

The role of extracellular matrix phosphorylation on energy dissipation in bone

Protein phosphorylation, critical for cellular regulatory mechanisms, is implicated in various diseases. However, it remains unknown whether heterogeneity in phosphorylation of key structural proteins alters tissue integrity and organ function. Here, osteopontin phosphorylation level declined in hypo- and hyper- phosphatemia mouse models exhibiting skeletal deformities. Phosphorylation increased cohesion between osteopontin polymers, and adhesion of osteopontin to hydroxyapatite, enhancing energy dissipation. Fracture toughness, a measure of bone’s mechanical competence, increased with ex-vivo phosphorylation of wildtype mouse bones and declined with ex-vivo dephosphorylation. In osteopontin-deficient mice, global matrix phosphorylation level was not associated with toughness. Our findings suggest that phosphorylated osteopontin promotes fracture toughness in a dose-dependent manner through increased interfacial bond formation. In the absence of osteopontin, phosphorylation increases electrostatic repulsion, and likely protein alignment and interfilament distance leading to decreased fracture resistance. These mechanisms may be of importance in other connective tissues, and the key to unraveling cell–matrix interactions in diseases.

Transcriptome-(Phospho)proteome Characterization of the Brain of a Constitutional Model of Cytoplasmic-predominant Pten Expression With Autism-Like Phenotypes

BackgroundPTEN, a well-studied tumor suppressor, has one of the strongest Mendelian associations with autism spectrum disorder (ASD), representing a special case in autism’s complex genetic architecture. Animal modeling for constitutional Pten mutation creates an opportunity to study how disruption of Pten affects neurobiology, providing insights that may be generalizable or at least inform our understanding of ASD. Although the neural transcriptome has been well characterized in Pten models, little has been done concerning the proteome and phosphoproteome. This is a critical gap in knowledge given that these –omic landscapes are more proximal to the actively observed biology than the transcriptome.MethodsWe sought to comprehensively characterize the neural proteome and phosphoproteome of the Ptenm3m4/m3m4 mouse, which exhibits cytoplasmic-predominant Pten expression. Proteomic and phosphoproteomic scans of Ptenm3m4/m3m4 and wildtype mouse brain at two-weeks- (P14) and six-weeks-of-age (P40) were performed using liquid chromatography with tandem mass spectrometry technology. Following quantification of differentially expressed/phosphorylated proteins, we performed gene overlap, gene enrichment, pathway, and network analyses to identify the similarity across the various datasets and understand the affected biological landscape.ResultsWe identified numerous differentially expressed/phosphorylated proteins, finding that dysregulation was greater at P40, consistent with the prior neural transcriptome data. We found the affected biological pathways were largely related to PTEN function, neurological processes, or neuroinflammation. Although we found minimal overlap among differentially expressed transcriptome-proteome-phosphoproteome molecules between P14 and P40 brains, there was congruence amongst the affected pathways. Importantly, network analysis identified Pten and Psd-95 as predominant regulatory nodes in the proteome and phosphoproteome, respectively. Moreover, we found overlap between our differentially expressed/phosphorylated proteins and known ASD risk genes.ConclusionsDifferential expression/phosphorylation revealed by transcriptome-proteome/phosphoproteome analyses of a germline Pten mutation model point to ASD risk genes like Pten and Psd-95 as major hubs in the protein networks, highlighting their important regulatory influence. Our observations here suggest Pten and Psd-95, known interactors in biological networks in the brain, are critical to either initiation or maintenance of cellular and perhaps organismal phenotypes related to ASD. Future research should explore rescuing Pten and Psd-95 function in attempts to ameliorate neurological pathologies and behavioral abnormalities.

Adiponectin is secreted via caveolin 1-dependent mechanisms in white adipocytes

Here we have investigated the role of the protein caveolin 1 (Cav1) and caveolae in the secretion of the white adipocyte hormone adiponectin. Using mouse primary subcutaneous adipocytes genetically depleted of Cav1, we show that the adiponectin secretion, stimulated either adrenergically or by insulin, is abrogated while basal (unstimulated) release of adiponectin is elevated. Adiponectin secretion is similarly affected in wildtype mouse and human adipocytes where the caveolae structure was chemically disrupted. The altered ex vivo secretion in adipocytes isolated from Cav1 null mice is accompanied by lowered serum levels of the high-molecular weight (HMW) form of adiponectin, whereas the total concentration of adiponectin is unaltered. Interestingly, levels of HMW adiponectin are maintained in adipose tissue from Cav1-depleted mice, signifying that a secretory defect is present. The gene expression of key regulatory proteins known to be involved in cAMP/adrenergically triggered adiponectin exocytosis (the beta-3-adrenergic receptor and exchange protein directly activated by cAMP) remains intact in Cav1 null adipocytes. Microscopy and fractionation studies indicate that adiponectin vesicles do not co-localise with Cav1 but that some vesicles are associated with a specific fraction of caveolae. Our studies propose that Cav1 has an important role in secretion of HMW adiponectin, even though adiponectin-containing vesicles are not obviously associated with this protein. We suggest that Cav1, and/or the caveolae domain, is essential for the organisation of signalling pathways involved in the regulation of HMW adiponectin exocytosis, a function that is disrupted in Cav1/caveolae-depleted adipocytes.

Ryanodine Receptor Type 2: A Molecular Target for Dichlorodiphenyltrichloroethane (DDT)- and Dichlorodiphenyldichloroethylene (DDE)-Mediated Cardiotoxicity

Dichlorodiphenyltrichloroethane (DDT) and its metabolite dichlorodiphenyl-dichloroethylene (DDE) are ubiquitously found in the environment and linked to cardiovascular diseases (CVDs) – with a majority of the work focused on hypertension. Studies investigating whether DDx can interact with molecular targets on cardiac tissue to directly affect cardiac function are lacking. Therefore, we investigated whether o,p’-DDT, p,p’-DDT, o,p’-DDE, or p,p’-DDE (DDx, collectively) can directly alter the function of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) by assessing their effect(s) on hiPSC-CMs Ca2+ dynamics. DDx (0.1-10 µM) affected hiPSC-CMs synchronous Ca2+ oscillation (SCO) frequency in a concentration-dependent manner, with p,p’-DDT and p,p’-DDE also decreasing Ca2+ stores. HEK-RyR2 cells cultured under antibiotic selection to induce expression of wildtype mouse ryanodine receptor type 2 are used to further investigate whether DDx alters hiPSC-CMs Ca2+ dynamics through engagement with ryanodine receptor type 2 (RyR2), a protein critical for cardiac muscle excitation-contraction coupling (ECC). Acute treatment with 10 µM DDx failed to induce Ca2+ release in HEK293-RyR2, whereas pre-treatment with DDx (0.1-10 µM) for 12- or 24-h significantly decreased SR Ca2+ stores in HEK-RyR2 cells challenged with caffeine (1 mM), an RyR agonist. [3H]ryanodine binding analysis using murine cardiac RyR2 homogenates further confirmed that all DDx isomers (10 µM) can directly engage with RyR2 to favor an open (leaky) confirmation, whereas only the DDT isomers (10 µM) modestly (≤10%) inhibited SERCA2a activity. The data demonstrate that DDx increases heart rate and depletes Ca2+ stores in human cardiomyocytes through a mechanism that impairs RyR2 function and Ca2+ dynamics.