A guide to machine learning for biologists.

The expanding scale and inherent complexity of biological data have encouraged a growing use of machine learning in biology to build informative and predictive models of the underlying biological processes. All machine learning techniques fit models to data; however, the specific methods are quite varied and can at first glance seem bewildering. In this Review, we aim to provide readers with a gentle introduction to a few key machine learning techniques, including the most recently developed and widely used techniques involving deep neural networks. We describe how different techniques may be suited to specific types of biological data, and also discuss some best practices and points to consider when one is embarking on experiments involving machine learning. Some emerging directions in machine learning methodology are also discussed.

A Razor's Edge: Vascular Responses to Acute Inflammatory Lung Injury/Acute Respiratory Distress Syndrome.

Historically considered a metabolically inert cellular layer separating the blood from the underlying tissue, the endothelium is now recognized as a highly dynamic, metabolically active tissue that is critical to organ homeostasis. Under homeostatic conditions, lung endothelial cells (ECs) in healthy subjects are quiescent, promoting vasodilation, platelet disaggregation, and anti-inflammatory mechanisms. In contrast, lung ECs are essential contributors to the pathobiology of acute respiratory distress syndrome (ARDS), as the quiescent endothelium is rapidly and radically altered upon exposure to environmental stressors, infectious pathogens, or endogenous danger signals into an effective and formidable regulator of innate and adaptive immunity. These dramatic perturbations, produced in a tsunami of inflammatory cascade activation, result in paracellular gap formation between lung ECs, sustained lung edema, and multi-organ dysfunction that drives ARDS mortality. The astonishing plasticity of the lung endothelium in negotiating this inflammatory environment and efforts to therapeutically target the aberrant ARDS endothelium are examined in further detail in this review.

Julia for biologists.

Major computational challenges exist in relation to the collection, curation, processing and analysis of large genomic and imaging datasets, as well as the simulation of larger and more realistic models in systems biology. Here we discuss how a relative newcomer among programming languages-Julia-is poised to meet the current and emerging demands in the computational biosciences and beyond. Speed, flexibility, a thriving package ecosystem and readability are major factors that make high-performance computing and data analysis available to an unprecedented degree. We highlight how Julia's design is already enabling new ways of analyzing biological data and systems, and we provide a list of resources that can facilitate the transition into Julian computing.

Deficiency of the Deubiquitinase UCHL1 Attenuates Pulmonary Arterial Hypertension.

BACKGROUND: The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. METHODS: Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats (Uchl1-/-), and conditional Uchl1 knockout mice (Tie2Cre-Uchl1fl/fl) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1-/- animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. RESULTS: Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice (Gadd45α-/-) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. CONCLUSIONS: Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.

Ultrafast end-to-end protein structure prediction enables high-throughput exploration of uncharacterized proteins.

Deep learning-based prediction of protein structure usually begins by constructing a multiple sequence alignment (MSA) containing homologs of the target protein. The most successful approaches combine large feature sets derived from MSAs, and considerable computational effort is spent deriving these input features. We present a method that greatly reduces the amount of preprocessing required for a target MSA, while producing main chain coordinates as a direct output of a deep neural network. The network makes use of just three recurrent networks and a stack of residual convolutional layers, making the predictor very fast to run, and easy to install and use. Our approach constructs a directly learned representation of the sequences in an MSA, starting from a one-hot encoding of the sequences. When supplemented with an approximate precision matrix, the learned representation can be used to produce structural models of comparable or greater accuracy as compared to our original DMPfold method, while requiring less than a second to produce a typical model. This level of accuracy and speed allows very large-scale three-dimensional modeling of proteins on minimal hardware, and we demonstrate this by producing models for over 1.3 million uncharacterized regions of proteins extracted from the BFD sequence clusters. After constructing an initial set of approximate models, we select a confident subset of over 30,000 models for further refinement and analysis, revealing putative novel protein folds. We also provide updated models for over 5,000 Pfam families studied in the original DMPfold paper.

Mitochondrial DNA copy number variation in asthma risk, severity, and exacerbations.

BACKGROUND: Asthma pathophysiology is associated with mitochondrial dysfunction. Mitochondrial DNA copy number (mtDNA-CN) has been used as a proxy of mitochondrial function, with lower levels indicating mitochondrial dysfunction in population studies of cardiovascular diseases and cancers. OBJECTIVES: We investigated whether lower levels of mtDNA-CN are associated with asthma diagnosis, severity, and exacerbations. METHODS: mtDNA-CN is evaluated in blood from 2 cohorts: UK Biobank (UKB) (asthma, n = 39,147; no asthma, n = 302,302) and Severe Asthma Research Program (SARP) (asthma, n = 1283; nonsevere asthma, n = 703). RESULTS: Individuals with asthma have lower mtDNA-CN compared to individuals without asthma in UKB (beta, -0.006 [95% confidence interval, -0.008 to -0.003], P = 6.23 × 10-6). Lower mtDNA-CN is associated with asthma prevalence, but not severity in UKB or SARP. mtDNA-CN declines with age but is lower in individuals with asthma than in individuals without asthma at all ages. In a 1-year longitudinal study in SARP, mtDNA-CN was associated with risk of exacerbation; those with highest mtDNA-CN had the lowest risk of exacerbation (odds ratio 0.333 [95% confidence interval, 0.173 to 0.542], P = .001). Biomarkers of inflammation and oxidative stress are higher in individuals with asthma than without asthma, but the lower mtDNA-CN in asthma is independent of general inflammation or oxidative stress. Mendelian randomization studies suggest a potential causal relationship between asthma-associated genetic variants and mtDNA-CN. CONCLUSION: mtDNA-CN is lower in asthma than in no asthma and is associated with exacerbations. Low mtDNA-CN in asthma is not mediated through inflammation but is associated with a genetic predisposition to asthma.

Studying the Pulmonary Endothelium in Health and Disease: An Official American Thoracic Society Workshop Report.

Lung endothelium resides at the interface between the circulation and the underlying tissue, where it senses biochemical and mechanical properties of both the blood as it flows through the vascular circuit and the vessel wall. The endothelium performs the bidirectional signaling between the blood and tissue compartments that is necessary to maintain homeostasis while physically separating both, facilitating a tightly regulated exchange of water, solutes, cells, and signals. Disruption in endothelial function contributes to vascular disease, which can manifest in discrete vascular locations along the artery-to-capillary-to-vein axis. Although our understanding of mechanisms that contribute to endothelial cell injury and repair in acute and chronic vascular disease have advanced, pathophysiological mechanisms that underlie site-specific vascular disease remain incompletely understood. In an effort to improve the translatability of mechanistic studies of the endothelium, the American Thoracic Society convened a workshop to optimize rigor, reproducibility, and translation of discovery to advance our understanding of endothelial cell function in health and disease.

eNAMPT is a novel therapeutic target for mitigation of coronary microvascular disease in type 2 diabetes.

AIMS/HYPOTHESIS: Individuals with diabetes are at high risk of cardiovascular complications, which significantly increase morbidity/mortality. Coronary microvascular disease (CMD) is recognised as a critical contributor to the increased cardiac mortality observed in people with diabetes. Therefore, there is an urgent need for treatments that are specific to CMD. eNAMPT (extracellular nicotinamide phosphoribosyltransferase) is a damage-associated molecular pattern and TLR4 ligand, whose plasma levels are elevated in people with diabetes. This study was thus designed to investigate the pathogenic role of intracellular nicotinamide phosphoribosyltransferase (iNAMPT) and eNAMPT in promoting the development of CMD in a preclinical murine model of type 2 diabetes. METHODS: An inducible type 2 diabetic mouse model was generated by a single injection of low-dose streptozocin (75 mg/kg, i.p.) combined with a high-fat diet for 16 weeks. The in vivo effects of i/eNAMPT inhibition on cardiac endothelial cell (CEC) function were evaluated by using Nampt+/- heterozygous mice, chronic administration of eNAMPT-neutralising monoclonal antibody (mAb) or use of an NAMPT enzymatic inhibitor (FK866). RESULTS: As expected, diabetic wild-type mice exhibited significantly lower coronary flow velocity reserve (CFVR), a determinant of coronary microvascular function, compared with control wild-type mice. eNAMPT plasma levels or expression in CECs were significantly greater in diabetic mice than in control mice. Furthermore, in comparison with diabetic wild-type mice, diabetic Nampt+/- heterozygous mice showed markedly improved CFVR, accompanied by increased left ventricular capillary density and augmented endothelium-dependent relaxation (EDR) in the coronary artery. NAMPT inhibition by FK866 or an eNAMPT-neutralising mAb significantly increased CFVR in diabetic mice. Furthermore, administration of the eNAMPT mAb upregulated expression of angiogenesis- and EDR-related genes in CECs from diabetic mice. Treatment with either eNAMPT or NAD+ significantly decreased CEC migration and reduced EDR in coronary arteries, partly linked to increased production of mitochondrial reactive oxygen species. CONCLUSIONS/INTERPRETATION: These data indicate that increased i/eNAMPT expression contributes to the development of diabetic coronary microvascular dysfunction, and provide compelling support for eNAMPT inhibition as a novel and effective therapeutic strategy for CMD in diabetes.

Involvement of eNAMPT/TLR4 inflammatory signaling in progression of non-alcoholic fatty liver disease, steatohepatitis, and fibrosis.

Although the progression of non-alcoholic fatty liver disease (NAFLD) from steatosis to steatohepatitis (NASH) and cirrhosis remains poorly understood, a critical role for dysregulated innate immunity has emerged. We examined the utility of ALT-100, a monoclonal antibody (mAb), in reducing NAFLD severity and progression to NASH/hepatic fibrosis. ALT-100 neutralizes eNAMPT (extracellular nicotinamide phosphoribosyltransferase), a novel damage-associated molecular pattern protein (DAMP) and Toll-like receptor 4 (TLR4) ligand. Histologic and biochemical markers were measured in liver tissues and plasma from human NAFLD subjects and NAFLD mice (streptozotocin/high-fat diet-STZ/HFD, 12 weeks). Human NAFLD subjects (n = 5) exhibited significantly increased NAMPT hepatic expression and significantly elevated plasma levels of eNAMPT, IL-6, Ang-2, and IL-1RA compared to healthy controls, with IL-6 and Ang-2 levels significantly increased in NASH non-survivors. Untreated STZ/HFD-exposed mice displayed significant increases in NAFLD activity scores, liver triglycerides, NAMPT hepatic expression, plasma cytokine levels (eNAMPT, IL-6, and TNFα), and histologic evidence of hepatocyte ballooning and hepatic fibrosis. Mice receiving the eNAMPT-neutralizing ALT-100 mAb (0.4 mg/kg/week, IP, weeks 9 to 12) exhibited marked attenuation of each index of NASH progression/severity. Thus, activation of the eNAMPT/TLR4 inflammatory pathway contributes to NAFLD severity and NASH/hepatic fibrosis. ALT-100 is potentially an effective therapeutic approach to address this unmet NAFLD need.

Lamellipodia dynamics and microrheology in endothelial cell paracellular gap closure.

Vascular endothelial cells (ECs) form a semipermeable barrier separating vascular contents from the interstitium, thereby regulating the movement of water and molecular solutes across small intercellular gaps, which are continuously forming and closing. Under inflammatory conditions, however, larger EC gaps form resulting in increased vascular leakiness to circulating fluid, proteins, and cells, which results in organ edema and dysfunction responsible for key pathophysiologic findings in numerous inflammatory disorders. In this study, we extend our earlier work examining the biophysical properties of EC gap formation and now address the role of lamellipodia, thin sheet-like membrane projections from the leading edge, in modulating EC spatial-specific contractile properties and gap closure. Micropillars, fabricated by soft lithography, were utilized to form reproducible paracellular gaps in human lung ECs. Using time-lapse imaging via optical microscopy, rates of EC gap closure and motility were measured with and without EC stimulation with the barrier-enhancing sphingolipid, sphingosine-1-phosphate. Peripheral ruffle formation was ubiquitous during gap closure. Kymographs were generated to quantitatively compare the lamellipodia dynamics of sphingosine-1-phosphate-stimulated and -unstimulated ECs. Utilizing atomic force microscopy, we characterized the viscoelastic behavior of EC lamellipodia. Our results indicate decreased stiffness and increased liquid-like behavior of expanding lamellipodia compared with regions away from the cellular edge (lamella and cell body) during EC gap closure, results in sync with the rapid kinetics of protrusion/retraction motion. We hypothesize this dissipative EC behavior during gap closure is linked to actomyosin cytoskeletal rearrangement and decreased cross-linking during lamellipodia expansion. In summary, these studies of the kinetic and mechanical properties of EC lamellipodia and ruffles at gap boundaries yield insights into the mechanisms of vascular barrier restoration and potentially a model system for examining the druggability of lamellipodial protein targets to enhance vascular barrier integrity.

Development of a peripheral blood transcriptomic gene signature to predict bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is the most common lung disease of extreme prematurity, yet mechanisms that associate with or identify neonates with increased susceptibility for BPD are largely unknown. Combining artificial intelligence with gene expression data is a novel approach that may assist in better understanding mechanisms underpinning chronic lung disease and in stratifying patients at greater risk for BPD. The objective of this study is to develop an early peripheral blood transcriptomic signature that can predict preterm neonates at risk for developing BPD. Secondary analysis of whole blood microarray data from 97 very low birth weight neonates on day of life 5 was performed. BPD was defined as positive pressure ventilation or oxygen requirement at 28 days of age. Participants were randomly assigned to a training (70%) and testing cohort (30%). Four gene-centric machine learning models were built, and their discriminatory abilities were compared with gestational age or birth weight. This study adheres to the transparent reporting of a multivariable prediction model for individual prognosis or diagnosis (TRIPOD) statement. Neonates with BPD (n = 62 subjects) exhibited a lower median gestational age (26.0 wk vs. 30.0 wk, P < 0.01) and birth weight (800 g vs. 1,280 g, P < 0.01) compared with non-BPD neonates. From an initial pool (33,252 genes/patient), 4,523 genes exhibited a false discovery rate (FDR) <1%. The area under the receiver operating characteristic curve (AUC) for predicting BPD utilizing gestational age or birth weight was 87.8% and 87.2%, respectively. The machine learning models, using a combination of five genes, revealed AUCs ranging between 85.8% and 96.1%. Pathways integral to T cell development and differentiation were associated with BPD. A derived five-gene whole blood signature can accurately predict BPD in the first week of life.

The Differing Roles of Flavins and Quinones in Extracellular Electron Transfer in Lactiplantibacillus plantarum.

Lactiplantibacillus plantarum is a lactic acid bacterium that is commonly found in the human gut and fermented food products. Despite its overwhelmingly fermentative metabolism, this microbe can perform extracellular electron transfer (EET) when provided with an exogenous quinone, 1,4-dihydroxy-2-naphthoic acid (DHNA), and riboflavin. However, the separate roles of DHNA and riboflavin in EET in L. plantarum have remained unclear. Here, we seek to understand the role of quinones and flavins in EET by monitoring iron and anode reduction in the presence and absence of these small molecules. We found that addition of either DHNA or riboflavin can support robust iron reduction, indicating electron transfer to extracellular iron occurs through both flavin-dependent and DHNA-dependent routes. Using genetic mutants of L. plantarum, we found that flavin-dependent iron reduction requires Ndh2 and EetA, while DHNA-dependent iron reduction largely relies on Ndh2 and PplA. In contrast to iron reduction, DHNA-containing medium supported more robust anode reduction than riboflavin-containing medium, suggesting electron transfer to an anode proceeds most efficiently through the DHNA-dependent pathway. Furthermore, we found that flavin-dependent anode reduction requires EetA, Ndh2, and PplA, while DHNA-dependent anode reduction requires Ndh2 and PplA. Taken together, we identify multiple EET routes utilized by L. plantarum and show that the EET route depends on access to environmental biomolecules and on the electron acceptor. This work expands our molecular-level understanding of EET in Gram-positive microbes and provides additional opportunities to manipulate EET for biotechnology. IMPORTANCE Lactic acid bacteria are named because of their nearly exclusive fermentative metabolism. Thus, the recent observation of EET activity-typically associated with anaerobic respiration-in this class of organisms has forced researchers to rethink the rules governing microbial metabolic strategies. Our identification of multiple routes for EET in L. plantarum that depend on two different redox active small molecules expands our understanding of how microbes metabolically adapt to different environments to gain an energetic edge and how these processes can be manipulated for biotechnological uses. Understanding the role of EET in lactic acid bacteria is of great importance due to the significance of lactic acid bacteria in agriculture, bioremediation, food production, and gut health. Furthermore, the maintenance of multiple EET routes speaks to the importance of this process to function under a variety of environmental conditions.

IL-18 mediates sickle cell cardiomyopathy and ventricular arrhythmias.

Previous reports indicate that IL18 is a novel candidate gene for diastolic dysfunction in sickle cell disease (SCD)-related cardiomyopathy. We hypothesize that interleukin-18 (IL-18) mediates the development of cardiomyopathy and ventricular tachycardia (VT) in SCD. Compared with control mice, a humanized mouse model of SCD exhibited increased cardiac fibrosis, prolonged duration of action potential, higher VT inducibility in vivo, higher cardiac NF-κB phosphorylation, and higher circulating IL-18 levels, as well as reduced voltage-gated potassium channel expression, which translates to reduced transient outward potassium current (Ito) in isolated cardiomyocytes. Administering IL-18 to isolated mouse hearts resulted in VT originating from the right ventricle and further reduced Ito in SCD mouse cardiomyocytes. Sustained IL-18 inhibition via IL-18-binding protein resulted in decreased cardiac fibrosis and NF-κB phosphorylation, improved diastolic function, normalized electrical remodeling, and attenuated IL-18-mediated VT in SCD mice. Patients with SCD and either myocardial fibrosis or increased QTc displayed greater IL18 gene expression in peripheral blood mononuclear cells (PBMCs), and QTc was strongly correlated with plasma IL-18 levels. PBMC-derived IL18 gene expression was increased in patients who did not survive compared with those who did. IL-18 is a mediator of sickle cell cardiomyopathy and VT in mice and a novel therapeutic target in patients at risk for sudden death.

Xanthine oxidoreductase gene polymorphisms are associated with high risk of sepsis and organ failure.

BACKGROUND: Sepsis and associated organ failures confer substantial morbidity and mortality. Xanthine oxidoreductase (XOR) is implicated in the development of tissue oxidative damage in a wide variety of respiratory and cardiovascular disorders including sepsis and sepsis-associated acute respiratory distress syndrome (ARDS). We examined whether single nucleotide polymorphisms (SNPs) in the XDH gene (encoding XOR) might influence susceptibility to and outcome in patients with sepsis. METHODS: We genotyped 28 tag SNPs in XDH gene in the CELEG cohort, including 621 European American (EA) and 353 African American (AA) sepsis patients. Serum XOR activity was measured in a subset of CELEG subjects. Additionally, we assessed the functional effects of XDH variants utilizing empirical data from different integrated software tools and datasets. RESULTS: Among AA patients, six intronic variants (rs206805, rs513311, rs185925, rs561525, rs2163059, rs13387204), in a region enriched with regulatory elements, were associated with risk of sepsis (P < 0.008-0.049). Two out of six SNPs (rs561525 and rs2163059) were associated with risk of sepsis-associated ARDS in an independent validation cohort (GEN-SEP) of 590 sepsis patients of European descent. Two common SNPs (rs1884725 and rs4952085) in tight linkage disequilibrium (LD) provided strong evidence for association with increased levels of serum creatinine (Padjusted<0.0005 and 0.0006, respectively), suggesting a role in increased risk of renal dysfunction. In contrast, among EA ARDS patients, the missense variant rs17011368 (I703V) was associated with enhanced mortality at 60-days (P < 0.038). We found higher serum XOR activity in 143 sepsis patients (54.5 ± 57.1 mU/mL) compared to 31 controls (20.9 ± 12.4 mU/mL, P = 1.96 × 10- 13). XOR activity was associated with the lead variant rs185925 among AA sepsis patients with ARDS (P < 0.005 and Padjusted<0.01). Multifaceted functions of prioritized XDH variants, as suggested by various functional annotation tools, support their potential causality in sepsis. CONCLUSIONS: Our findings suggest that XOR is a novel combined genetic and biochemical marker for risk and outcome in patients with sepsis and ARDS.

The Impact of Insulin Resistance on Loss of Lung Function and Response to Treatment in Asthma.

Rationale: The role of obesity-associated insulin resistance (IR) in airflow limitation in asthma is uncertain. Objectives: Using data in the Severe Asthma Research Program 3 (SARP-3), we evaluated relationships between homeostatic measure of IR (HOMA-IR), lung function (cross-sectional and longitudinal analyses), and treatment responses to bronchodilators and corticosteroids. Methods: HOMA-IR values were categorized as without (<3.0), moderate (3.0-5.0), or severe (>5.0). Lung function included FEV1 and FVC measured before and after treatment with inhaled albuterol and intramuscular triamcinolone acetonide and yearly for 5 years. Measurements and Main Results: Among 307 participants in SARP-3, 170 (55%) were obese and 140 (46%) had IR. Compared with patients without IR, those with IR had significantly lower values for FEV1 and FVC, and these lower values were not attributable to obesity effects. Compared with patients without IR, those with IR had lower FEV1 responses to ß-adrenergic agonists and systemic corticosteroids. The annualized decline in FEV1 was significantly greater in patients with moderate IR (-41 ml/year) and severe IR (-32 ml/year,) than in patients without IR (-13 ml/year, P < 0.001 for both comparisons). Conclusions: IR is common in asthma and is associated with lower lung function, accelerated loss of lung function, and suboptimal lung function responses to bronchodilator and corticosteroid treatments. Clinical trials in patients with asthma and IR are needed to determine if improving IR might also improve lung function.

Epidemiology of Asthma: Prevalence and Burden of Disease.

Asthma, a common airway disease, results in a significant burden to both patients and society worldwide. Yet, despite global political commitment backed by the United Nations, progress to reduce the burden of asthma remains inadequate. This is particularly true in low-income countries. To date, progress has been delayed by the lack of uniform data collection, imperfect surveillance methods, inadequate resources, poor access to effective therapies, substandard asthma education, ineffective governmental policies, rapid urbanization, progressive increase in asthma prevalence, increased life expectancy and obesity rates worldwide, asthma heterogeneity and disease complexity, smoking, and environmental exposures to allergens and pollution. A thorough understanding of the challenges facing the international community is essential to define future strategies to improve the burden of asthma.

eNAMPT Is a Novel Damage-associated Molecular Pattern Protein That Contributes to the Severity of Radiation-induced Lung Fibrosis.

The paucity of therapeutic strategies to reduce the severity of radiation-induced lung fibrosis (RILF), a life-threatening complication of intended or accidental ionizing radiation exposure, is a serious unmet need. We evaluated the contribution of eNAMPT (extracellular nicotinamide phosphoribosyltransferase), a damage-associated molecular pattern (DAMP) protein and TLR4 (Toll-like receptor 4) ligand, to the severity of whole-thorax lung irradiation (WTLI)-induced RILF. Wild-type (WT) and Nampt+/- heterozygous C57BL6 mice and nonhuman primates (NHPs, Macaca mulatta) were exposed to a single WTLI dose (9.8 or 10.7 Gy for NHPs, 20 Gy for mice). WT mice received IgG1 (control) or an eNAMPT-neutralizing polyclonal or monoclonal antibody (mAb) intraperitoneally 4 hours after WTLI and weekly thereafter. At 8-12 weeks after WTLI, NAMPT expression was assessed by immunohistochemistry, biochemistry, and plasma biomarker studies. RILF severity was determined by BAL protein/cells, hematoxylin and eosin, and trichrome blue staining and soluble collagen assays. RNA sequencing and bioinformatic analyses identified differentially expressed lung tissue genes/pathways. NAMPT lung tissue expression was increased in both WTLI-exposed WT mice and NHPs. Nampt+/- mice and eNAMPT polyclonal antibody/mAb-treated mice exhibited significantly attenuated WTLI-mediated lung fibrosis with reduced: 1) NAMPT and trichrome blue staining; 2) dysregulated lung tissue expression of smooth muscle actin, p-SMAD2/p-SMAD1/5/9, TGF-ß, TSP1 (thrombospondin-1), NOX4, IL-1ß, and NRF2; 3) plasma eNAMPT and IL-1ß concentrations; and 4) soluble collagen. Multiple WTLI-induced dysregulated differentially expressed lung tissue genes/pathways with known tissue fibrosis involvement were each rectified in mice receiving eNAMPT mAbs.The eNAMPT/TLR4 inflammatory network is essentially involved in radiation pathobiology, with eNAMPT neutralization an effective therapeutic strategy to reduce RILF severity.

MRSA-induced endothelial permeability and acute lung injury are attenuated by FTY720 S-phosphonate.

Disruption of the lung endothelial barrier is a hallmark of acute respiratory distress syndrome (ARDS), for which no effective pharmacologic treatments exist. Prior work has demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine-1-phosphate (S1P) and FTY720, exhibits potent endothelial cell (EC) barrier protective properties. In this study, we investigated the in vitro and in vivo efficacy of Tys against methicillin-resistant Staphylococcus aureus (MRSA), a frequent bacterial cause of ARDS. Tys-protected human lung EC from barrier disruption induced by heat-killed MRSA (HK-MRSA) or staphylococcal α-toxin and attenuated MRSA-induced cytoskeletal changes associated with barrier disruption, including actin stress fiber formation and loss of peripheral VE-cadherin and cortactin. Tys-inhibited Rho and myosin light chain (MLC) activation after MRSA and blocked MRSA-induced NF-κB activation and release of the proinflammatory cytokines, IL-6 and IL-8. In vivo, intratracheal administration of live MRSA in mice caused significant vascular leakage and leukocyte infiltration into the alveolar space. Pre- or posttreatment with Tys attenuated MRSA-induced lung permeability and levels of alveolar neutrophils. Posttreatment with Tys significantly reduced levels of bronchoalveolar lavage (BAL) VCAM-1 and plasma IL-6 and KC induced by MRSA. Dynamic intravital imaging of mouse lungs demonstrated Tys attenuation of HK-MRSA-induced interstitial edema and neutrophil infiltration into lung tissue. Tys did not directly inhibit MRSA growth or viability in vitro. In conclusion, Tys inhibits lung EC barrier disruption and proinflammatory signaling induced by MRSA in vitro and attenuates acute lung injury induced by MRSA in vivo. These results support the potential utility of Tys as a novel ARDS therapeutic strategy.

Urinary total conjugated 3-bromotyrosine, asthma severity, and exacerbation risk.

Asthma is an inflammatory disease of the airways characterized by eosinophil recruitment, eosinophil peroxidase release, and protein oxidation through bromination, which following tissue remodeling results in excretion of 3-bromotyrosine. Predicting exacerbations and reducing their frequency is critical for the treatment of severe asthma. In this study, we aimed to investigate whether urinary total conjugated bromotyrosine can discriminate asthma severity and predict asthma exacerbations. We collected urine from participants with severe (n = 253) and nonsevere (n = 178) asthma, and the number of adjudicated exacerbations in 1-yr longitudinal follow-up was determined among subjects enrolled in the Severe Asthma Research Program, a large-scale National Institutes of Health (NIH)-funded consortium. Urine glucuronidated bromotyrosine and total conjugated forms were quantified by hydrolysis with either glucuronidase or methanesulfonic acid, respectively, followed by liquid chromatography-tandem mass spectrometry analyses of free 3-bromotyrosine. Blood and sputum eosinophils were also counted. The majority of 3-bromotyrosine in urine was found to exist in conjugated forms, with glucuronidated bromotyrosine representing approximately a third, and free bromotyrosine less than 1% of total conjugated bromotyrosine. Total conjugated bromotyrosine was poorly correlated with blood (r2 = 0.038) or sputum eosinophils (r2 = 0.0069). Compared with participants with nonsevere asthma, participants with severe asthma had significantly higher urinary total conjugated bromotyrosine levels. Urinary total conjugated bromotyrosine was independently associated with asthma severity, correlated with the number of asthma exacerbations, and served as a predictor of asthma exacerbation risk over 1-yr of follow-up.

Cohesive cancer invasion of the biophysical barrier of smooth muscle.

Smooth muscle is found around organs in the digestive, respiratory, and reproductive tracts. Cancers arising in the bladder, prostate, stomach, colon, and other sites progress from low-risk disease to high-risk, lethal metastatic disease characterized by tumor invasion into, within, and through the biophysical barrier of smooth muscle. We consider here the unique biophysical properties of smooth muscle and how cohesive clusters of tumor use mechanosensing cell-cell and cell-ECM (extracellular matrix) adhesion receptors to move through a structured muscle and withstand the biophysical forces to reach distant sites. Understanding integrated mechanosensing features within tumor cluster and smooth muscle and potential triggers within adjacent adipose tissue, such as the unique damage-associated molecular pattern protein (DAMP), eNAMPT (extracellular nicotinamide phosphoribosyltransferase), or visfatin, offers an opportunity to prevent the first steps of invasion and metastasis through the structured muscle.