Tigilanol Tiglate-Induced Changes in Secretome Profiles Alter C-Met Phosphorylation and Cell Surface Protein Expression in H357 Head and Neck Cancer Cells.

Tigilanol tiglate (TT, also known as EBC-46) is a novel, plant-derived diterpene ester possessing anticancer and wound-healing properties. Here, we show that TT-evoked PKC-dependent S985 phosphorylation of the tyrosine kinase MET leads to subsequent degradation of tyrosine phosphorylated p-Y1003 and p-Y1234/5 MET species. PKC inhibition with BIM-1 blocked S985 phosphorylation of MET and led to MET cell surface accumulation. Treatment with metalloproteinase inhibitors prevented MET-ECD release into cell culture media, which was also blocked by PKC inhibitors. Furthermore, unbiased secretome analysis, performed using TMT-technology, identified additional targets of TT-dependent release of cell surface proteins from H357 head and neck cancer cells. We confirm that the MET co-signalling receptor syndecan-1 was cleaved from the cell surface in response to TT treatment. This was accompanied by rapid cleavage of the cellular junction adhesion protein Nectin-1 and the nerve growth factor receptor NGFRp75/TNFR16. These findings, that TT is a novel negative regulator of protumorigenic c-MET and NGFRp75/TNFR16 signalling, as well as regulating Nectin-1-mediated cell adhesion, further contribute to our understanding of the mode of action and efficacy of TT in the treatment of solid tumours.

Alterations in platelet proteome signature and impaired platelet integrin αIIbß3 activation in patients with COVID-19.

BACKGROUND: Patients with COVID-19 are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality. OBJECTIVES: To characterize the mechanism of altered platelet function in COVID-19 patients. METHODS: The platelet proteome, platelet functional responses, and platelet-neutrophil aggregates were compared between patients hospitalized with COVID-19 and healthy control subjects using tandem mass tag proteomic analysis, Western blotting, and flow cytometry. RESULTS: COVID-19 patients showed a different profile of platelet protein expression (858 altered of the 5773 quantified). Levels of COVID-19 plasma markers were enhanced in the platelets of COVID-19 patients. Gene ontology pathway analysis demonstrated that the levels of granule secretory proteins were raised, whereas those of platelet activation proteins, such as the thrombopoietin receptor and protein kinase Cα, were lowered. Basally, platelets of COVID-19 patients showed enhanced phosphatidylserine exposure, with unaltered integrin αIIbß3 activation and P-selectin expression. Agonist-stimulated integrin αIIbß3 activation and phosphatidylserine exposure, but not P-selectin expression, were decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This association was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction. CONCLUSIONS: Overall, our data suggest the presence of 2 platelet populations in patients with COVID-19: one of circulating platelets with an altered proteome and reduced functional responses and another of P-selectin-expressing neutrophil-associated platelets. Platelet-driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients.

Intraperitoneal administration of high doses of polyethylene glycol (PEG) causes hepatic subcapsular necrosis and low-grade peritonitis with a rise in hepatic biomarkers.

Polyethylene glycols (PEGs) are commonly employed as excipients in preclinical studies and in vitro experiments to dissolve poorly hydrosoluble drugs. Their use is generally considered safe in both animals and humans; however, limited data is available concerning the safety of PEGs when administered parenterally. The results of our investigation demonstrate that PEG-400 can have an irritant effect on serosal surfaces and causes subcapsular hepatocellular necrosis in mice when administered intraperitoneally at a high dose (4 mL/kg). Accordingly, levels of serum biomarkers of liver injury need to be carefully interpreted in studies where PEG is administered intraperitoneally and always in association with the results of the histological assessment.

CE-based sample quality assessment prior to 2-D gel electrophoresis: towards the standardization of gel-based proteomics.

2-DE remains one of the most commonly used separation techniques for complex protein mixtures. This article describes a new approach to 2-DE sample assessment using SDS capillary gel electrophoresis (in Beckman Coulter sieving medium) combined with multi-pixel detection. The performance of this platform was investigated using protein samples prepared for 2-DE. The capability to characterize 2-DE sample was tested and the results show that the repeatability of peak migration time and intensity are better than 2% RSD. The system gives good resolution, accurate molecular mass assignment, as well as absolute and relative quantification of proteins. Notably, this study also demonstrates the use of this platform to screen the quality of simple and complex 2-DE samples. Implementation of this technique in the proteomics workflow will not only improve the success rate of 2-DE, but will also enable sample verification before 2-DE and allow the relative quantification of proteins. The validation of differential protein expression is also demonstrated using the combined information of relative molecular mass and relative quantification. It is the first time that a rapid and visual evaluation method is reported for the quality assessment of 2-DE samples.

Surface-applied insecticide treatments for quarantine control of Japanese beetle, Popillia japonica Newman (Coleoptera: Scarabaeidae), larvae in field-grown nursery plants.

BACKGROUND: Japanese beetles, Popillia japonica Newman, are a quarantine challenge for nursery shipments from infested to non-infested states. Marathon (imidacloprid) and Discus (imidacloprid + cyfluthrin) are approved preharvest nursery treatments (US Domestic Japanese Beetle Harmonization Plan; DJHP). This study evaluated approved and non-approved (acephate, carbaryl, clothianidin, dinotefuran, halofenozide, thiamethoxam, trichlorfon) preharvest treatments, optimal rates (labeled 1x, 0.3-0.75 x or 2-3 x) and optimal timings (June, July, August and September) to control early-instar (grubs) P. japonica in field nurseries. RESULTS: Most insecticides effectively reduced grub densities, except for acephate, carbaryl and trichlorfon. Clothianidin, thiamethoxam and halofenozide provided grub control equivalent to DJHP standards during most years. Across all test years and timings, percentage grub reductions were: Marathon (1x: 59.2-100; 3 x: 78.9-100), Discus (1x: 60.7-100), clothianidin (1x: 96.1-100; 3 x: 97.4-100), thiamethoxam (1x: 75.0-100; 3 x: 80.0-100), halofenozide (1x: 70.0-100; 3 x: 90.0-100) and dinotefuran (1x: 13.2-88.2; 3 x: 71.1-93.4). CONCLUSIONS: July application was more consistent and effective than other timings, and higher rates (2x and 3 x) did not generally improve grub control. Overall, clothianidin, thiamethoxam and halofenozide (and dinotefuran applied in August) were equivalent to current DJHP standards. These insecticides may be suitable for DJHP Category 2 states, potentially lowering grower costs.

Characterization of real-world activity, fuel use, and emissions for selected motor graders fueled with petroleum diesel and B20 biodiesel.

Motor graders are a common type of nonroad vehicle used in many road construction and maintenance applications. In-use activity, fuel use, and emissions were measured for six selected motor graders using a portable emission measurement system. Each motor grader was tested with petroleum diesel and B20 biodiesel. Duty cycles were quantified in terms of the empirical cumulative distribution function of manifold absolute pressure (MAP), which is an indicator of engine load. The motor graders were operated under normal duty cycles for road maintenance and repair at various locations in Wake and Nash Counties in North Carolina. Approximately 3 hr of quality-assured, second-by-second data were obtained during each test. An empirical modal-based model of vehicle fuel use and emissions was developed, based on stratifying the data with respect to ranges of normalized MAP, to enable comparisons between duty cycles, motor graders, and fuels. Time-based emission factors were found to increase monotonically with MAP. Fuel-based emission factors were mainly sensitive to differences between idle and non-idle engine operation. Cycle average emission factors were estimated for road "resurfacing," "roading," and "shouldering" activities. On average, the use of B20 instead of petroleum diesel leads to a negligible decrease of 1.6% in nitric oxide emission rate, and decreases of 19-22% in emission rates of carbon monoxide, hydrocarbons, and particulate matter. Emission rates decrease significantly when comparing newer engine tier vehicles to older ones. Significant reductions in tailpipe emissions accrue especially from the use of B20 and adoption of newer vehicles.

Real-world in-use activity, fuel use, and emissions for nonroad construction vehicles: a case study for excavators.

A study design was developed and demonstrated for deployment of a portable emission measurement system (PEMS) for excavators. Excavators are among the most commonly used vehicles in construction activities. The PEMS measured nitric oxide, carbon monoxide, hydrocarbons, carbon dioxide, and opacity-based particulate matter. Data collection, screening, processing, and analysis protocols were developed to assure data quality and to quantify variability in vehicle fuel consumption and emissions rates. The development of data collection procedures was based on securing the PEMS while avoiding disruption to normal vehicle operations. As a result of quality assurance, approximately 90% of the attempted measurements resulted in valid data. On the basis of field data collected for three excavators, an average of 50% of the total nitric oxide emissions was associated with 29% of the time of operation, during which the average engine speed and manifold absolute pressure were significantly higher than corresponding averages for all data. Mass per time emission rates during non-idle modes (i.e., moving and using bucket) were on average 7 times greater than for the idle mode. Differences in normalized average rates were influenced more by intercycle differences than intervehicle differences. This study demonstrates the importance of accounting for intercycle variability in real-world in-use emissions to develop more accurate emission inventories. The data collection and analysis methodology demonstrated here is recommended for application to more vehicles to better characterize real-world vehicle activity, fuel use, and emissions for nonroad construction equipment.