Towards an interdisciplinary formalization of soundscapes.

Soundscapes have been studied by researchers from various disciplines, each with different perspectives, approaches, and terminologies. Consequently, the research field determines the actual concept of a specific soundscape with the associated components and also affects the definition itself. This complicates interdisciplinary communication and comparison of results, especially when research areas are involved which are not directly focused on soundscapes. For this reason, we present a formalization that aims to be independent of the concepts from the various disciplines, with the goal of being able to capture the heterogeneous data structure in one layered model. Our model consists of time-dependent sound sources and geodata that influence the acoustic composition of a soundscape represented by our sensor function. Using a case study, we present the application of our formalization by classifying land use types. For this we analyze soundscapes in the form of recordings from different devices at 23 different locations using three-dimensional convolutional neural networks and frequency correlation matrices. In our results, we present that soundscapes can be grouped into classes, but the given land use categories do not have to correspond to them.

The Evolving Trends in Infective Endocarditis and Determinants of Mortality: a 10-year Experience From a Tertiary Care Epicenter.

The epidemiology of infective endocarditis (IE) continues to evolve in areas affected by the opioid epidemic. Understanding the demographics of the disease allows us to better tailor therapy towards this at-risk population. This was an observational study of adults (age ≥ 18) admitted to the University of Kentucky hospital with IE between January 2009 and December 2018. 1,255 patients were included in the final analysis. The mean age was 42 years, 45% were female and injection drug use was seen in 66% of patients. On multivariable analysis, higher Charlson comorbidity indices, left sided, and multivalve involvement were associated with increased mortality, whereas surgical intervention demonstrated a trend towards lower mortality. Our results highlight the alarming increase in injection drug use related IE and the high mortality rates despite therapeutic advances. Patients with left sided IE, multivalve involvement and a higher Charlson comorbidity index had decreased survival.

A High-Throughput Assay for In Vitro Determination of Release Factor-Dependent Peptide Release from a Pretermination Complex by Fluorescence Anisotropy-Application to Nonsense Suppressor Screening and Mechanistic Studies.

Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren's mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren's binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren's low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity.

ATP citrate lyase links increases in glycolysis to diminished release of vesicular suppressor of cytokine signaling 3 by alveolar macrophages.

Extracellular vesicles (EVs) are important vectors for intercellular communication. Lung-resident alveolar macrophages (AMs) tonically secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis in the distal lung via its actions in recipient neighboring epithelial cells. AMs are metabolically distinct and exhibit low levels of glycolysis at steady state. To our knowledge, whether cellular metabolism influences the packaging and release of an EV cargo molecule has never been explored in any cellular context. Here, we report that increases in glycolysis following in vitro exposure of AMs to the growth and activating factor granulocyte-macrophage colony-stimulating factor inhibit the release of vesicular SOCS3 by primary AMs. Glycolytically diminished SOCS3 secretion requires export of citrate from the mitochondria to the cytosol and its subsequent conversion to acetyl-CoA by ATP citrate lyase. Our data for the first time implicate perturbations in intracellular metabolites in the regulation of vesicular cargo packaging and secretion.

Ataluren binds to multiple protein synthesis apparatus sites and competitively inhibits release factor-dependent termination.

Genetic diseases are often caused by nonsense mutations, but only one TRID (translation readthrough inducing drug), ataluren, has been approved for clinical use. Ataluren inhibits release factor complex (RFC) termination activity, while not affecting productive binding of near-cognate ternary complex (TC, aa-tRNA.eEF1A.GTP). Here we use photoaffinity labeling to identify two sites of ataluren binding within rRNA, proximal to the decoding center (DC) and the peptidyl transfer center (PTC) of the ribosome, which are directly responsible for ataluren inhibition of termination activity. A third site, within the RFC, has as yet unclear functional consequences. Using single molecule and ensemble fluorescence assays we also demonstrate that termination proceeds via rapid RFC-dependent hydrolysis of peptidyl-tRNA followed by slow release of peptide and tRNA from the ribosome. Ataluren is an apparent competitive inhibitor of productive RFC binding, acting at or before the hydrolysis step. We propose that designing more potent TRIDs which retain ataluren's low toxicity should target areas of the RFC binding site proximal to the DC and PTC which do not overlap the TC binding site.

Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms.

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through-inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell's protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs.

Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3.

Extracellular vesicles (EVs) contain a diverse array of molecular cargoes that alter cellular phenotype and function following internalization by recipient cells. In the lung, alveolar macrophages (AMs) secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis via vesicular transfer to neighboring alveolar epithelial cells. Although changes in the secretion of EV molecules-including but not limited to SOCS3-have been described in response to microenvironmental stimuli, the cellular and molecular machinery that control alterations in vesicular cargo packaging remain poorly understood. Furthermore, the use of quantitative methods to assess the sorting of cytosolic cargo molecules into EVs is lacking. Here, we utilized cigarette smoke extract (CSE) exposure of AMs as an in vitro model of oxidative stress to address these gaps in knowledge. We demonstrate that the accumulation of reactive oxygen species (ROS) in AMs was sufficient to augment vesicular SOCS3 release in this model. Using nanoparticle tracking analysis (NTA) in tandem with a new carboxyfluorescein succinimidyl ester (CFSE)-based intracellular protein packaging assay, we show that the stimulatory effects of CSE were at least in part attributable to elevated amounts of SOCS3 packaged per EV secreted by AMs. Furthermore, the use of a 20S proteasome activity assay alongside treatment of AMs with conventional proteasome inhibitors strongly suggest that ROS stimulated SOCS3 release via inactivation of the proteasome. These data demonstrate that tuning of AM proteasome function by microenvironmental oxidants is a critical determinant of the packaging and secretion of cytosolic SOCS3 protein within EVs.

Microenvironmental Influences on Extracellular Vesicle-Mediated Communication in the Lung.

Extracellular vesicles (EVs) are increasingly appreciated as important vectors of information transmission between cells. Most research on EVs has emphasized their roles in inflammatory and pathologic conditions, and in the airways and alveoli, EV secretion by various cell types is implicated in various forms of lung disease. However, recent evidence also demonstrates a homeostatic role for lung EVs by mediating transmission of anti-inflammatory signals between alveolar macrophages and lung epithelial cells. Nevertheless, our understanding of the mechanisms responsible for EV cargo packaging, stability in the extracellular milieu, and acquisition by recipient cells remains limited. Here, we review the current understanding of these mechanistic aspects of vesicular communication and their potential modulation by constituents of the unique microenvironment of the lung.

A multi-resolution HEALPix data structure for spherically mapped point data.

Data describing entities with locations that are points on a sphere are described as spherically mapped. Several data structures designed for spherically mapped data have been developed. One of them, known as Hierarchical Equal Area iso-Latitude Pixelization (HEALPix), partitions the sphere into twelve diamond-shaped equal-area base cells and then recursively subdivides each cell into four diamond-shaped subcells, continuing to the desired level of resolution. Twelve quadtrees, one associated with each base cell, store the data records associated with that cell and its subcells. HEALPix has been used successfully for numerous applications, notably including cosmic microwave background data analysis. However, for applications involving sparse point data HEALPix has possible drawbacks, including inefficient memory utilization, overwriting of proximate points, and return of spurious points for certain queries. A multi-resolution variant of HEALPix specifically optimized for sparse point data was developed. The new data structure allows different areas of the sphere to be subdivided at different levels of resolution. It combines HEALPix positive features with the advantages of multi-resolution, including reduced memory requirements and improved query performance. An implementation of the new Multi-Resolution HEALPix (MRH) data structure was tested using spherically mapped data from four different scientific applications (warhead fragmentation trajectories, weather station locations, galaxy locations, and synthetic locations). Four types of range queries were applied to each data structure for each dataset. Compared to HEALPix, MRH used two to four orders of magnitude less memory for the same data, and on average its queries executed 72% faster.

NKG2A-Expressing Natural Killer Cells Dominate the Response to Autologous Lymphoblastoid Cells Infected with Epstein-Barr Virus.

Epstein-Barr virus (EBV) is a human γ-herpesvirus that establishes latency and lifelong infection in host B cells while achieving a balance with the host immune response. When the immune system is perturbed through immunosuppression or immunodeficiency, however, these latently infected B cells can give rise to aggressive B cell lymphomas. Natural killer (NK) cells are regarded as critical in the early immune response to viral infection, but their role in controlling expansion of infected B cells is not understood. Here, we report that NK cells from healthy human donors display increased killing of autologous B lymphoblastoid cell lines (LCLs) harboring latent EBV compared to primary B cells. Coculture of NK cells with autologous EBV+ LCL identifies an NK cell population that produces IFNγ and mobilizes the cytotoxic granule protein CD107a. Multi-parameter flow cytometry and Boolean analysis reveal that these functional cells are enriched for expression of the NK cell receptor NKG2A. Further, NKG2A+ NK cells more efficiently lyse autologous LCL than do NKG2A- NK cells. More specifically, NKG2A+2B4+CD16-CD57-NKG2C-NKG2D+ cells constitute the predominant NK cell population that responds to latently infected autologous EBV+ B cells. Thus, a subset of NK cells is enhanced for the ability to recognize and eliminate autologous, EBV-infected transformed cells, laying the groundwork for harnessing this subset for therapeutic use in EBV+ malignancies.

Transcription factor Dlx3 induces aryl hydrocarbon receptor promoter activity.

The Distal-less (Dlx) homeobox transcription factors (TFs) play a prominent role in regulating multiple facets of vertebrate biology. Though widely studied as mediators of tissue development, recent work has uncovered a role for this TF family in modulating the vertebrate hematopoietic compartment. Pertinent to our study, murine Dlx1-3 are expressed in an innate lymphocyte population known as natural killer (NK) cells, and they are implicated to assume a functional role in the NK cell maturation pathway. However, Dlx target genes are poorly understood. In Drosophila, the invertebrate Dlx ortholog Distal-less (Dll) regulates another transcription factor called Spineless (ss), which is critical for specifying distal antennal segments. Importantly, the vertebrate ortholog of ss is the aryl hydrocarbon receptor (AhR), a transcription factor recently shown to be important in the regulation of a number of immune cell subsets, including NK cells. Given these findings, we investigated whether Dlx TF family members might analogously regulate AhR in an NK cell context. Our results demonstrate that Dlx3 is constitutively co-expressed with AhR in murine and human CD127+ NK cells. Critically, we show that Dlx3 induces AhR promoter activity by binding to a regulatory region that resides ~5.5 kb upstream of the transcriptional start site. This mechanism is functionally relevant, as Dlx3 expression in human NK cells significantly enhances TF activity at AhR DNA-binding elements (Xenobiotic Responsive Elements, XREs). Thus, our study defines Dlx3 as a positive regulator of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor is required for the maintenance of liver-resident natural killer cells.

A tissue-resident population of natural killer cells (NK cells) in the liver has recently been described to have the unique capacity to confer immunological memory in the form of hapten-specific contact hypersensitivity independent of T and B cells. Factors regulating the development and maintenance of these liver-resident NK cells are poorly understood. The aryl hydrocarbon receptor (AhR) is a transcription factor modulated by exogenous and endogenous ligands that is important in the homeostasis of immune cells at barrier sites, such as the skin and gut. In this study, we show that liver-resident NK (NK1.1+CD3-) cells, defined as CD49a+TRAIL+CXCR6+DX5- cells in the mouse liver, constitutively express AhR. In AhR-/- mice, there is a significant reduction in the proportion and absolute number of these cells, which results from a cell-intrinsic dependence on AhR. This deficiency in liver-resident NK cells appears to be the result of higher turnover and increased susceptibility to cytokine-induced cell death. Finally, we show that this deficiency has functional implications in vivo. Upon hapten exposure, AhR-/- mice are not able to mount an NK cell memory response to hapten rechallenge. Together, these data demonstrate the requirement of AhR for the maintenance of CD49a+TRAIL+CXCR6+DX5- liver-resident NK cells and their hapten memory function.

Bidirectional Crosstalk between C5a Receptors and the NLRP3 Inflammasome in Macrophages and Monocytes.

C5a is an inflammatory mediator generated by complement activation that positively regulates various arms of immune defense, including Toll-like receptor 4 (TLR4) signaling. The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is activated by pathogen products and cellular/tissue damage products and is a major contributor of IL-1ß. In this study, we investigate whether C5a modulates lipopolysaccharide- (LPS-) induced NLRP3 inflammasome activation in myeloid cells. Appearance of plasma IL-1ß during endotoxemia was reduced in C5aR1(-/-) mice when compared to wild-type mice. In vitro, C5a significantly enhanced LPS-induced production of IL-1ß in bone marrow Ly6C-high inflammatory monocytes, accompanied by augmented intracellular pro-IL-1ß expression. This effect was abolished during p38 blockade by SB 203580 and in the absence of C5aR1. Conversely, C5a suppressed LPS-induced macrophage production of IL-1ß, which was accompanied by attenuated levels of pro-IL-1ß, NLRP3, and caspase-1 expression. C5a's suppressive effects were negated during phosphoinositide 3-kinase (PI3K) inhibition by wortmannin but were largely preserved in the absence of C5aR1. Thus, C5a bidirectionally amplifies TLR4-mediated NLRP3 inflammasome activation in monocytes while suppressing this pathway in macrophages. However, as C5aR1 deficiency attenuates the IL-1ß response to LPS challenge in vivo, our results suggest overall that C5a augments physiologic inflammasome responses.

Infective Endocarditis Complicated by Mitral Valve Aneurysm: Pathologic and Echocardiographic Correlations.

Infective endocarditis is a well-described cardiovascular disease that causes significant morbidity and mortality despite medical and surgical advances. Complications of endocarditis include heart failure, systemic embolization, and valvular destruction including valve aneurysms which increase morbidity and mortality. Mitral valve aneurysms are rarely encountered in the clinical setting. We present eight mitral valve aneurysm cases and discuss a new potential pathogenesis of this deadly endocarditis complication. Pathologic evaluation suggests that neovascularization of the anterior mitral valve leaflet predisposes this territory to abscess and aneurysm formation. In conclusion, mitral valve aneurysms appear to be another form of intravalvular abscess which has expanded and should be approached aggressively with surgical intervention if indicated.

Critical role for the NLRP3 inflammasome during acute lung injury.

The inflammasome is a key factor in innate immunity and senses soluble pathogen and danger-associated molecular patterns as well as biological crystals (urate, cholesterol, etc.), resulting in expression of IL-1ß and IL-18. Using a standard model of acute lung injury (ALI) in mice featuring airway instillation of LPS, ALI was dependent on availability of NLRP3 as well as caspase-1, which are known features of the NLRP3 inflammasome. The appearance of IL-1ß, a product of NLRP3 inflammasome activation, was detected in bronchoalveolar lavage fluids (BALF) in a macrophage- and neutrophil-dependent manner. Neutrophil-derived extracellular histones appeared in the BALF during ALI and directly activated the NLRP3 inflammasome. Ab-mediated neutralization of histones significantly reduced IL-1ß levels in BALF during ALI. Inflammasome activation by extracellular histones in LPS-primed macrophages required NLRP3 and caspase-1 as well as extrusion of K(+), increased intracellular Ca(2+) concentration, and generation of reactive oxygen species. NLRP3 and caspase-1 were also required for full extracellular histone presence during ALI, suggesting a positive feedback mechanism. Extracellular histone and IL-1ß levels in BALF were also elevated in C5a-induced and IgG immune complex ALI models, suggesting a common inflammatory mechanism. These data indicate an interaction between extracellular histones and the NLRP3 inflammasome, resulting in ALI. Such findings suggest novel targets for treatment of ALI, for which there is currently no known efficacious drug.

The changing 'face' of endocarditis in Kentucky: an increase in tricuspid cases.

BACKGROUND: Advancements in medical technology and increased life expectancy have been described as contributing to the evolution of endocarditis. We sought to determine whether there has been a change in the incidence, demographics, microbiology, complications, and outcomes of infective endocarditis over a 10-year time span. METHODS: We screened 28,420 transthoracic and transesophageal echocardiogram reports performed at the Gill Heart Institute for the following indications: fever, masses, emboli (including stroke), sepsis, bacteremia, and endocarditis in 2 time periods: 1999 to 2000 and 2009 to 2010. Data were collected from diagnosed endocarditis cases. RESULTS: Overall, 143 cases of infective endocarditis were analyzed (48 in 1999-2000 and 95 in 2009-2010). The endocarditis incidence per number of admissions remained nearly constant at 0.113% for 1999-2000 and 0.148% for 2009-2010 (P = .153). However, tricuspid valve involvement increased markedly from 6% to 36% (P < .001). Also, reported history of intravenous drug use increased from 15% to 40% (P = .002). Valvular complications doubled from 17% to 35% (P = .031). Septic pulmonary emboli increased from 10% to 25% (P = .047). Despite these noted differences, inpatient mortality remained unchanged at 25% and 28% (P = .696) for the 2 time periods, respectively. CONCLUSIONS: The incidence of endocarditis at the University of Kentucky Medical Center has not changed and mortality remains high, but the "face of endocarditis" in Kentucky has evolved with an increased incidence of tricuspid valve involvement, valvular complications, and embolic events.

Induction of M2 regulatory macrophages through the ß2-adrenergic receptor with protection during endotoxemia and acute lung injury.

The main drivers of acute inflammation are macrophages, which are known to have receptors for catecholamines. Based on their function, macrophages are broadly categorized as having either M1 (proinflammatory) or M2 phenotypes (anti-inflammatory). In this study, we investigated catecholamine-induced alterations in the phenotype of activated macrophages. In the presence of lipopolysaccharide (LPS), mouse peritoneal macrophages acquired an M1 phenotype. However, the copresence of LPS and either epinephrine or norepinephrine resulted in a strong M2 phenotype including high levels of arginase-1 and interleukin-10, and a reduced expression of M1 markers. Furthermore, epinephrine enhanced macrophage phagocytosis and promoted type 2 T-cell responses in vitro, which are known features of M2 macrophages. Analysis of M2 subtype-specific markers indicated that LPS and catecholamine-cotreated macrophages were not alternatively activated but were rather of the regulatory macrophage subtype. Interestingly, catecholamines signaled through the ß2-adrenergic receptor but not the canonical cAMP/protein kinase A signaling pathway. Instead, the M2 pathway required an intact phosphoinositol 3-kinase pathway. Blockade of the ß2-adrenergic receptor reduced survival and enhanced injury in mouse models of endotoxemia and LPS-induced acute lung injury, respectively. These results demonstrate a role for the ß2-adrenergic receptor in promoting the M2 macrophage phenotype.