Interleukin-11/IL-11 Receptor Promotes Glioblastoma Cell Proliferation, Epithelial-Mesenchymal Transition, and Invasion.

Glioblastoma is highly proliferative and invasive. However, the regulatory cytokine networks that promote glioblastoma cell proliferation and invasion into other areas of the brain are not fully defined. In the present study, we define a critical role for the IL-11/IL-11Rα signalling axis in glioblastoma proliferation, epithelial to mesenchymal transition, and invasion. We identified enhanced IL-11/IL-11Rα expression correlated with reduced overall survival in glioblastoma patients using TCGA datasets. Proteomic analysis of glioblastoma cell lines overexpressing IL-11Rα displayed a proteome that favoured enhanced proliferation and invasion. These cells also displayed greater proliferation and migration, while the knockdown of IL-11Rα reversed these tumourigenic characteristics. In addition, these IL-11Rα overexpressing cells displayed enhanced invasion in transwell invasion assays and in 3D spheroid invasion assays, while knockdown of IL-11Rα resulted in reduced invasion. Furthermore, IL-11Rα-overexpressing cells displayed a more mesenchymal-like phenotype compared to parental cells and expressed greater levels of the mesenchymal marker Vimentin. Overall, our study identified that the IL-11/IL-11Rα pathway promotes glioblastoma cell proliferation, EMT, and invasion.

Structure activity relationship and target prediction for ABX464 analogues in Caenorhabditis elegans.

Global challenges with treatment failures and/or widespread resistance in parasitic worms against commercially available anthelmintics lend impetus to the development of new anthelmintics with novel mechanism(s) of action. The free-living nematode Caenorhabditis elegans is an important model organism used for drug discovery, including the screening and structure-activity investigation of new compounds, and target deconvolution. Previously, we conducted a whole-organism phenotypic screen of the 'Pandemic Response Box' (from Medicines for Malaria Venture, MMV) and identified a hit compound, called ABX464, with activity against C. elegans and a related, parasitic nematode, Haemonchus contortus. Here, we tested a series of 44 synthesized analogues to explore the pharmacophore of activity on C. elegans and revealed five compounds whose potency was similar or greater than that of ABX464, but which were not toxic to human hepatoma (HepG2) cells. Subsequently, we employed thermal proteome profiling (TPP), protein structure prediction and an in silico-docking algorithm to predict ABX464-target candidates. Taken together, the findings from this study contribute significantly to the early-stage drug discovery of a new nematocide based on ABX464. Future work is aimed at validating the ABX464-protein interactions identified here, and at assessing ABX464 and associated analogues against a panel of parasitic nematodes, towards developing a new anthelmintic with a mechanism of action that is distinct from any of the compounds currently-available commercially.

Global lipid remodelling by hypoxia aggravates migratory potential in pancreatic cancer while maintaining plasma membrane homeostasis.

Hypoxia plays an important role in pancreatic cancer progression. It drives various metabolic reprogramming in cells including that of lipids, which in turn, can modify the structure and function of cell membranes. Homeostatic adaptation of membranes is well-recognized, but how and if it is regulated in hypoxic pancreatic cancer and its relation to aggressive phenotype and metastasis remains elusive. Here we show hypoxia-induced extensive global lipid remodelling spanning changes in lipid classes, unsaturation levels, glyceryl backbone and acyl chain lengths. No major modulation of plasma membrane biophysical properties revealed a decoupling of lipidome modulation from membrane properties under hypoxia. This was supported by observing minor changes in the lipidome of plasma membranes under hypoxia. Further, hypoxia increased migration and invasion underpinned by reduced actin volume, cell cortical stiffness and facile tether dynamics. In conclusion, we demonstrate buffering of the lipidome alterations leading to a homeostatic membrane response. These findings will help to understand the hypoxic regulation of pancreatic membrane homeostasis and identify tangible theranostic avenues.

Liver-Secreted Hexosaminidase A Regulates Insulin-Like Growth Factor Signaling and Glucose Transport in Skeletal Muscle.

Nonalcoholic fatty liver disease (NAFLD) and impaired glycemic control are closely linked; however, the pathophysiological mechanisms underpinning this bidirectional relationship remain unresolved. The high secretory capacity of the liver and impairments in protein secretion in NAFLD suggest that endocrine changes in the liver are likely to contribute to glycemic defects. We identify hexosaminidase A (HEXA) as an NAFLD-induced hepatokine in both mice and humans. HEXA regulates sphingolipid metabolism, converting GM2 to GM3 gangliosides-sphingolipids that are primarily localized to cell-surface lipid rafts. Using recombinant murine HEXA protein, an enzymatically inactive HEXA(R178H) mutant, or adeno-associated virus vectors to induce hepatocyte-specific overexpression of HEXA, we show that HEXA improves blood glucose control by increasing skeletal muscle glucose uptake in mouse models of insulin resistance and type 2 diabetes, with these effects being dependent on HEXA's enzymatic action. Mechanistically, HEXA remodels muscle lipid raft ganglioside composition, thereby increasing IGF-1 signaling and GLUT4 localization to the cell surface. Disrupting lipid rafts reverses these HEXA-mediated effects. In this study, we identify a pathway for intertissue communication between liver and skeletal muscle in the regulation of systemic glycemic control.

Differential gene expression and potential regulatory network of fatty acid biosynthesis during fruit and leaf development in yellowhorn (Xanthoceras sorbifolium), an oil-producing tree with significant deployment values.

Xanthoceras sorbifolium (yellowhorn) is a woody oil plant with super stress resistance and excellent oil characteristics. The yellowhorn oil can be used as biofuel and edible oil with high nutritional and medicinal value. However, genetic studies on yellowhorn are just in the beginning, and fundamental biological questions regarding its very long-chain fatty acid (VLCFA) biosynthesis pathway remain largely unknown. In this study, we reconstructed the VLCFA biosynthesis pathway and annotated 137 genes encoding relevant enzymes. We identified four oleosin genes that package triacylglycerols (TAGs) and are specifically expressed in fruits, likely playing key roles in yellowhorn oil production. Especially, by examining time-ordered gene co-expression network (TO-GCN) constructed from fruit and leaf developments, we identified key enzymatic genes and potential regulatory transcription factors involved in VLCFA synthesis. In fruits, we further inferred a hierarchical regulatory network with MYB-related (XS03G0296800) and B3 (XS02G0057600) transcription factors as top-tier regulators, providing clues into factors controlling carbon flux into fatty acids. Our results offer new insights into key genes and transcriptional regulators governing fatty acid production in yellowhorn, laying the foundation for efforts to optimize oil content and fatty acid composition. Moreover, the gene expression patterns and putative regulatory relationships identified here will inform metabolic engineering and molecular breeding approaches tailored to meet biofuel and bioproduct demands.

Respiratory strategy at birth initiates distinct lung injury phenotypes in the preterm lamb lung.

BACKGROUND: A lack of clear trial evidence often hampers clinical decision-making during support of the preterm lung at birth. Protein biomarkers have been used to define acute lung injury phenotypes and improve patient selection for specific interventions in adult respiratory distress syndrome. The objective of the study was to use proteomics to provide a deeper biological understanding of acute lung injury phenotypes resulting from different aeration strategies at birth in the preterm lung. METHODS: Changes in protein abundance against an unventilated group (n = 7) were identified via mass spectrometry in a biobank of gravity dependent and non-dependent lung tissue from preterm lambs managed with either a Sustained Inflation (SI, n = 20), Dynamic PEEP (DynPEEP, n = 19) or static PEEP (StatPEEP, n = 11). Ventilation strategy-specific pathways and functions were identified (PANTHER and WebGestalt Tool) and phenotypes defined using integrated analysis of proteome, physiological and clinical datasets (MixOmics package). RESULTS: 2372 proteins were identified. More altered proteins were identified in the non-dependent lung, and in SI group than StatPEEP and DynPEEP. Different inflammation, immune system, apoptosis and cytokine pathway enrichment were identified for each strategy and lung region. Specific integration maps of clinical and physiological outcomes to specific proteins could be generated for each strategy. CONCLUSIONS: Proteomics mapped the molecular events initiating acute lung injury and identified detailed strategy-specific phenotypes. This study demonstrates the potential to characterise preterm lung injury by the direct aetiology and response to lung injury; the first step towards true precision medicine in neonatology.

Potential allopolyploid origin of Ericales revealed with gene-tree reconciliation.

Few incidents of ancient allopolyploidization (polyploidization by hybridization or merging diverged genomes) were previously revealed, although there is significant evidence for the accumulation of whole genome duplications (WGD) in plants. Here, we focused on Ericales, one of the largest and most diverse angiosperm orders with significant ornamental and economic value. Through integrating 24 high-quality whole genome data selected from ~ 200 Superasterids genomes/species and an algorithm of topology-based gene-tree reconciliation, we explored the evolutionary history of in Ericales with ancient complex. We unraveled the allopolyploid origin of Ericales and detected extensive lineage-specific gene loss following the polyploidization. Our study provided a new hypothesis regarding the origin of Ericales and revealed an instructive perspective of gene loss as a pervasive source of genetic variation and adaptive phenotypic diversity in Ericales.

Risk factors and long-term prognosis for postoperative hypoxemia in patients with acute type A aortic dissection: A retrospective observational study.

Hypoxemia is 1 of the most common complications in the patients with acute Type A aortic dissection (ATAAD). This study aimed to summarize the risk factors, management strategies and long-term prognosis for postoperative hypoxemia in ATAAD patients. Baseline characteristics and clinical data of all the patients were collected. Patients were divided into 2 groups according to the PaO2/FiO2 after surgery: Hypoxemia group (n = 142) and Non-hypoxemia group (n = 68). The differences in gender, age, body mass index, operation time, cardiopulmonary bypass (CPB) time, aortic cross-clamping time, deep hypothermic circulatory arrest time, preoperative PaO2/FiO2, postoperative PaO2/FiO2, PaO2/FiO2 before extubating, time of mechanical ventilation, length of intensive care unit stay, length of hospital stay, in-hospital mortality, and overall mortality were compared between the 2 groups. The incidence of postoperative hypoxemia in this study was 67.6% (142/210). body mass index (26.4 ±â€…3.8 vs 24.4 ±â€…3.3kg/m2, P < .001) in the hypoxemia group were markedly higher and CPB time (196.3 ±â€…41.0 vs 181.0 ±â€…37.3 minutes, P = .010) in the hypoxemia group were significantly longer than those in the non-hypoxemia group. While preoperative PaO2/FiO2 (229.7 ±â€…91.4 vs 299.7 ±â€…101.2mmHg, P < .001) was significantly lower than those in the non-hypoxemia group. In the hypoxemia group, PaO2/FiO2 before extubating was significantly higher than that after operation, and the difference was significant. Logistic regression analysis showed that overweight (odds ratio [OR]: 1.113, P = .030), CPB time (OR: 1.009, P = .043) and preoperative PaO2/FiO2 (OR: 0.994, P = .001) were independent risk factors for postoperative hypoxemia. Further follow-up results showed no significant difference in long-term mortality between the 2 groups. Logistic regression analysis revealed that PaO2/FiO2 before extubating (OR: 0.985, P < .001), paraplegia (OR: 10.994, P = .019), acute renal failure (OR: 12.590, P < .001), re-operation (OR: 4.721, P = .014) and re-admission to intensive care unit (OR: 13.727, P = .001) were independent risk factors for long-term mortality. Our results showed that overweight and prolonged CPB time were risk factors for postoperative hypoxemia in ATAAD patients. While PaO2/FiO2 before extubating were independent risk factors for long-term mortality, indicating that active correction of hypoxemia and maintain a higher PaO2/FiO2 before extubating may help to improve the prognosis of the ATAAD patients.

Reaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy.

Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.

Proteomic analysis reveals exercise training induced remodelling of hepatokine secretion and uncovers syndecan-4 as a regulator of hepatic lipid metabolism.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is linked to impaired lipid metabolism and systemic insulin resistance, which is partly mediated by altered secretion of liver proteins known as hepatokines. Regular physical activity can resolve NAFLD and improve its metabolic comorbidities, however, the effects of exercise training on hepatokine secretion and the metabolic impact of exercise-regulated hepatokines in NAFLD remain unresolved. Herein, we examined the effect of endurance exercise training on hepatocyte secreted proteins with the aim of identifying proteins that regulate metabolism and reduce NAFLD severity. METHODS: C57BL/6 mice were fed a high-fat diet for six weeks to induce NAFLD. Mice were exercise trained for a further six weeks, while the control group remained sedentary. Hepatocytes were isolated two days after the last exercise bout, and intracellular and secreted proteins were detected using label-free mass spectrometry. Hepatocyte secreted factors were applied to skeletal muscle and liver ex vivo and insulin action and fatty acid metabolism were assessed. Syndecan-4 (SDC4), identified as an exercise-responsive hepatokine, was overexpressed in the livers of mice using adeno-associated virus. Whole-body energy homeostasis was assessed by indirect calorimetry and skeletal muscle and liver metabolism was assessed using radiometric techniques. RESULTS: Proteomics analysis detected 2657 intracellular and 1593 secreted proteins from mouse hepatocytes. Exercise training remodelled the hepatocyte proteome, with differences in 137 intracellular and 35 secreted proteins. Bioinformatic analysis of hepatocyte secreted proteins revealed enrichment of tumour suppressive proteins and proteins involved in lipid metabolism and mitochondrial function, and suppression of oncogenes and regulators of oxidative stress. Hepatocyte secreted factors from exercise trained mice improved insulin action in skeletal muscle and increased hepatic fatty acid oxidation. Hepatocyte-specific overexpression of SDC4 reduced hepatic steatosis, which was associated with reduced hepatic fatty acid uptake, and blunted pro-inflammatory and pro-fibrotic gene expression. Treating hepatocytes with recombinant ectodomain of SDC4 (secreted form) recapitulated these effects with reduced fatty acid uptake, lipid storage and lipid droplet accumulation. CONCLUSIONS: Remodelling of hepatokine secretion is an adaptation to regular exercise training that induces changes in metabolism in the liver and skeletal muscle. SDC4 is a novel exercise-responsive hepatokine that decreases fatty acid uptake and reduces steatosis in the liver. By understanding the proteomic changes in hepatocytes with exercise, these findings have potential for the discovery of new therapeutic targets for NAFLD.

UV-B and UV-C radiation trigger both common and distinctive signal perceptions and transmissions in Pinus tabuliformis Carr.

In plants, ultraviolet (UV)-light is an important driver for growth and natural distribution, and is also a valuable tool for manipulating productivity as well as biotic interactions. Understanding of plant responses to different UV radiation is sparse, especially from a systems biology perspective and particularly for conifers. Here, we evaluated the physiological and transcriptomic responses to the short-term application of high-irradiance UV-B and UV-C waves on Pinus tabuliformis Carr., a major conifer in Northern China. By undertaking time-ordered gene coexpression network analyses and network comparisons incorporating physiological traits and gene expression variation, we uncovered communalities but also differences in P. tabuliformis responses to UV-B and UV-C. Both types of spectral bands caused a significant inhibition of photosynthesis, and conversely, the improvement of antioxidant capacity, flavonoid production and signaling pathways related to stress resistance, indicating a clear switch from predominantly primary metabolism to enhanced defensive metabolism in pine. We isolated distinct subnetworks for photoreceptor-mediated signal transduction, maximum quantum efficiency of photosystem II (Fv/Fm) regulation and flavonoid biosynthesis in response to UV-B and UV-C radiation. From these subnetworks, we further identified phototropins as potentially important elements in both UV-B and UV-C signaling and, for the first time, suggesting peptide hormones to be involved in promoting flavonoid biosynthesis against UV-B, while these hormones seem not to be implicated in the defense against UV-C exposure. The present study employed an effective strategy for disentangling the complex physiological and genetic regulatory mechanisms in a nonmodel plant species, and thus, provides a suitable reference for future functional evaluations and artificial UV-light mediated growing strategies in plant production.

Transformation of hempseed (Cannabis sativa L.) oil cake proteome, structure and functionality after extrusion.

The entire protein fractions from hempseed, its oil cake (30-40% protein) and the extruded protein isolate (>90% protein) were investigated. The first semi-quantitative mass spectrometry-based proteomics on hempseed was performed, leading to a sum of 1879 differentially abundant proteins being identified from individual pairwise comparisons of each extruded group compared to unextruded hempseed cake. The 'free-form' amino acid content and total amino acid content of hempseed oil cake were enhanced by up to 315% and 18%, respectively, after extrusion. Changes in the structure and thermal properties of hempseed protein were confirmed through circular dichroism, Fourier transform infrared spectroscopy, scanning electron microscopy and differential scanning calorimetry. The proteomic and structural transformation in the extruded hempseed protein fractions contributed to greater values in majority of tested functionality parameters, such as protein solubility, water and oil binding capacity, emulsification properties, and in vitro digestibility, as compared to their unextruded counterparts.

Hexosaminidase A (HEXA) regulates hepatic sphingolipid and lipoprotein metabolism in mice.

Hexosaminidase A (HexA), a heterodimer consisting of HEXA and HEXB, converts the ganglioside sphingolipid GM2 to GM3 by removing a terminal N-acetyl-d-galactosamine. HexA enzyme deficiency in humans leads to GM2 accumulation in cells, particularly in neurons, and is associated with neurodegeneration. While HexA and sphingolipid metabolism have been extensively investigated in the context of neuronal lipid metabolism, little is known about the metabolic impact of HexA and ganglioside degradation in other tissues. Here, we focussed on the role of HexA in the liver, which is a major regulator of systemic lipid metabolism. We find that hepatic Hexa expression is induced by lipid availability and increased in the presence of hepatic steatosis, which is associated with increased hepatic GM3 content. To assess the impact of HEXA on hepatic lipid metabolism, we used an adeno-associated virus to overexpress HEXA in the livers of high-fat diet fed mice. HEXA overexpression was associated with increased hepatic GM3 content and increased expression of enzymes involved in the degradation of glycated sphingolipids, ultimately driving sphingomyelin accumulation in the liver. In addition, HEXA overexpression led to substantial proteome remodeling in cell surface lipid rafts, which was associated with increased VLDL processing and secretion, hypertriglyceridemia and ectopic lipid accumulation in peripheral tissues. This study established an important role of HEXA in modulating hepatic sphingolipid and lipoprotein metabolism.

Chromosome-scale assembly and evolution of the tetraploid Salvia splendens (Lamiaceae) genome.

Polyploidization plays a key role in plant evolution, but the forces driving the fate of homoeologs in polyploid genomes, i.e., paralogs resulting from a whole-genome duplication (WGD) event, remain to be elucidated. Here, we present a chromosome-scale genome assembly of tetraploid scarlet sage (Salvia splendens), one of the most diverse ornamental plants. We found evidence for three WGD events following an older WGD event shared by most eudicots (the γ event). A comprehensive, spatiotemporal, genome-wide analysis of homoeologs from the most recent WGD unveiled expression asymmetries, which could be associated with genomic rearrangements, transposable element proximity discrepancies, coding sequence variation, selection pressure, and transcription factor binding site differences. The observed differences between homoeologs may reflect the first step toward sub- and/or neofunctionalization. This assembly provides a powerful tool for understanding WGD and gene and genome evolution and is useful in developing functional genomics and genetic engineering strategies for scarlet sage and other Lamiaceae species.

Occupational Allergic Sensitization Among Workers Processing King Crab (Paralithodes camtschaticus) and Edible Crab (Cancer pagurus) in Norway and Identification of Novel Putative Allergenic Proteins.

Introduction: Asthma and allergy occur frequently among seafood processing workers, with the highest prevalence seen in the crustacean processing industry. In this study we established for the first time the prevalence of allergic sensitization in the Norwegian king- and edible crab processing industry and characterized the IgE-reactive proteins. Materials and Methods: Two populations of crab processing workers participated; 119 king crab and 65 edible crab workers. The investigation included information on work tasks and health through a detailed questionnaire. Allergic sensitization was investigated by crab-specific IgE quantification and skin prick tests (SPT) to four in-house prepared crab extracts; raw meat, cooked meat, raw intestines and raw shell. Allergen-specific IgE binding patterns were analyzed by IgE immunoblotting to the four allergen extracts using worker serum samples. Total proteins in crab SPT extracts and immunoblot-based IgE binding proteins were identified by mass spectrometric analysis. Results: Positive SPTs were established in 17.5% of king- and 18.1% of edible crab workers, while elevated IgE to crab were demonstrated in 8.9% of king- and 12.2% of edible crab processing workers. There was no significant difference between the king and edible crab workers with respect to self-reported respiratory symptoms, elevated specific IgE to crab or SPT results. Individual workers exhibited differential IgE binding patterns to different crab extracts, with most frequent binding to tropomyosin and arginine kinase and two novel IgE binding proteins, hemocyanin and enolase, identified as king- and edible crab allergens. Conclusions: Occupational exposure to king- and edible crabs may frequently cause IgE mediated allergic sensitization. Future investigations addressing the diagnostic value of crab allergens including tropomyosin and arginine kinase and the less well-known IgE-binding proteins hemocyanin and enolase in a component-resolved diagnostic approach to crab allergy should be encouraged.

Proteomics reveals region-specific hemostatic alterations in response to mechanical ventilation in a preterm lamb model of lung injury.

INTRODUCTION: Preterm infants often require assisted ventilation, however ventilation when applied to the immature lung can initiate ventilator-induced lung injury (VILI). The biotrauma which underscores VILI is largely undefined, and is likely to involve vascular injury responses, including hemostasis. We aimed to use a ventilated, preterm lamb model to: (1) characterize regional alterations in hemostatic mediators within the lung and (2) assess the functional impact of protein alterations on hemostasis by analyzing temporal thrombin generation. MATERIALS AND METHODS: Preterm lambs delivered at 124 to 127 days gestation received 90 min of mechanical ventilation (positive end-expiratory pressure = 8 cm H2O, VT = 6-8 ml/kg) and were compared with unventilated control lambs. At study completion, lung tissue was taken from standardized nondependent and gravity-dependent regions, and Orbitrap-mass spectrometry and KEGG were used to identify and map regional alterations in hemostasis pathway members. Temporal alterations in plasma thrombin generation were assessed. RESULTS: Ventilation was distributed towards the nondependent lung. Significant changes in hemostatic protein abundance, were detected at a two-fold higher rate in the nondependent lung when compared with the gravity-dependent lung. Seven proteins were uniquely altered in non-dependent lung (SERPINA1, MYL12A, RAP1B, RHOA, ITGB1, A2M, GNAI2), compared with a single proteins in gravity-dependent lung (COL1A2). Four proteins were altered in both regions (VTN, FGG, FGA, and ACTB). Tissue protein alterations were mirrored by plasma hypocoagulability at 90-minutes of ventilation. CONCLUSIONS: We observed regionally specific, hemostatic alterations within the preterm lung together with disturbed fibrinolysis following a short period of mechanical ventilation.

Widespread remodeling of proteome solubility in response to different protein homeostasis stresses.

The accumulation of protein deposits in neurodegenerative diseases has been hypothesized to depend on a metastable subproteome vulnerable to aggregation. To investigate this phenomenon and the mechanisms that regulate it, we measured the solubility of the proteome in the mouse Neuro2a cell line under six different protein homeostasis stresses: 1) Huntington's disease proteotoxicity, 2) Hsp70, 3) Hsp90, 4) proteasome, 5) endoplasmic reticulum (ER)-mediated folding inhibition, and 6) oxidative stress. Overall, we found that about one-fifth of the proteome changed solubility with almost all of the increases in insolubility were counteracted by increases in solubility of other proteins. Each stress directed a highly specific pattern of change, which reflected the remodeling of protein complexes involved in adaptation to perturbation, most notably, stress granule (SG) proteins, which responded differently to different stresses. These results indicate that the protein homeostasis system is organized in a modular manner and aggregation patterns were not correlated with protein folding stability (ΔG). Instead, distinct cellular mechanisms regulate assembly patterns of multiple classes of protein complexes under different stress conditions.

Sensitivity and binding kinetics of an ultra-sensitive chemiluminescent enzyme-linked immunosorbent assay at arrays of antibodies.

We have characterized the sensitivity and kinetics of a multiplex immunoassay system based on detection of chemiluminescence (CL) at arrays of antibodies. This enzyme-linked immunosorbent assay (ELISA) was based on the spotting of different antibodies in a circular pattern at the bottom of a well of a microtiter plate. Sandwich immunocomplexes within each spot were labeled with horse radish peroxidase, and CL was generated locally to each spot in the array from turnover of luminol substrate. CL from the arrays across the plate was collected in single images; long exposure times were used to maximize sensitivity, and short exposure times were used to extend the dynamic range at higher signals. Image analysis was used to determine the intensity of light from each spot in the array, and intensity was converted to concentration of protein via comparison to a calibration curve. To determine the intrinsic sensitivity of the CL ELISA array, streptavidin horseradish peroxidase (SA-HRP) was captured on an array spotted with biotinylated detection antibodies. The limit of detection (LOD) of SA-HRP was 105 aM, or 3200 enzymes per 50 µL. A single-plex assay for prostate specific antigen (PSA) was developed that had an LOD of 79 aM when the microtiter plate was shaken orbitally, comparable to the most sensitive immunoassays reported to date. Normalization of CL signals in the PSA assay to signal per molecule of SA-HRP showed that the efficiency of the shaken assay was ~40%. When the plates were not shaken, the efficiency was ~4.5%, i.e., ~9-fold lower than when shaken. To better understand the theoretical basis of the sensitivity of these assays, we developed COMSOL numerical models of the binding kinetics at the array for plates that were shaken orbitally and those not shaken. Experimental data from the orbitally shaken PSA assay were best modeled by inertial mixing in a three-layer system that included a 8-µm-thick concentration boundary layer. Experimental data from the unshaken PSA assay were well modeled by diffusion-limited kinetics. A single-plex assay for IL-10 was developed with an LOD of 69 aM or 1.5 fg/mL, and used to measure this cytokine in plasma and serum of 10 healthy individuals. A 5-plex assay for IL-5, IL-6, IL-10, IL-22, and TNF-α was developed with LODs of 56 aM, 237 aM, 69 aM, 88 aM, and 373 aM, respectively. The assay was used to measure these 5 cytokines in the plasma and serum of the same individuals. The correlation in concentration of IL-10 measured in single-plex and multiplex assays was good (r2 = 0.89; bias = 14.5%). The factors that result in the high sensitivity of CL ELISA arrays-mostly high signal to noise ratio of extended chemiluminescent imaging-are discussed. This multiplex CL ELISA could be used for sensitive profiling of multiple proteins for in vitro diagnostics and biomarker detection in the development of therapeutics.

Preterm Lung Exhibits Distinct Spatiotemporal Proteome Expression at Initiation of Lung Injury.

The development of regional lung injury in the preterm lung is not well understood. This study aimed to characterize time-dependent and regionally specific injury patterns associated with early ventilation of the preterm lung using a mass spectrometry-based proteomic approach. Preterm lambs delivered at 124-127 days gestation received 15 or 90 minutes of mechanical ventilation (positive end-expiratory pressure = 8 cm H2O, Vt = 6-8 ml/kg) and were compared with unventilated control lambs. At study completion, lung tissue was taken from standardized nondependent and dependent regions, and assessed for lung injury via histology, quantitative PCR, and proteomic analysis using Orbitrap-mass spectrometry. Ingenuity pathway analysis software was used to identify temporal and region-specific enrichments in pathways and functions. Apoptotic cell numbers were ninefold higher in nondependent lung at 15 and 90 minutes compared with controls, whereas proliferative cells were increased fourfold in the dependent lung at 90 minutes. The relative gene expression of lung injury markers was increased at 90 minutes in nondependent lung and unchanged in gravity-dependent lung. Within the proteome, the number of differentially expressed proteins was fourfold higher in the nondependent lung than the dependent lung. The number of differential proteins increased over time in both lung regions. A total of 95% of enriched canonical pathways and 94% of enriched cellular and molecular functions were identified only in nondependent lung tissue from the 90-minute ventilation group. In conclusion, complex injury pathways are initiated within the preterm lung after 15 minutes of ventilation and amplified by continuing ventilation. Injury development is region specific, with greater alterations within the proteome of nondependent lung.

The Effect of Ultraviolet Radiation on Bitumen Aging Depth.

The aging effect of ultraviolet (UV) radiation on bitumen has gained increasing attention from researchers, resulting in the emergence of a new method to simulate the UV aging that occurs during the service life of bitumen. However, the UV aging degree is closely related to bitumen thickness and the effect of UV radiation on aging depth is not clear. The relationship between ultraviolet (UV) radiation and bitumen UV aging depth was investigated in this paper. Three groups of samples were UV aged using different aging procedures to investigate the bitumen aging mechanism of UV radiation. The results from the first group showed that UV aging depth increased along with aging time. After aging for five hours, the complex modulus of the second and third layers increased. The second group’s results indicated that the aging effect of ozone was small and that the increase in aging depth was uncorrelated with ozone. The results from the third group showed that the transmittance of bitumen increased after UV aging and that the real reason why aging depth increased was permeation.