Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC-MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness. ...Here we show that micro-flow LC-MS/MS using a 1 × 150 mm column shows excellent reproducibility of chromatographic retention time (<0.3% coefficient of variation, CV) and protein quantification (<7.5% CV) using data from >2000 samples of human cell lines, tissues and body fluids. Deep proteome analysis identifies >9000 proteins and >120,000 peptides in 16 h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16 h. The system identifies >30,000 phosphopeptides in 12 h and protein-protein or protein-drug interaction experiments can be analyzed in 20 min per sample. We show that the same column can be used to analyze >7500 samples without apparent loss of performance. This study demonstrates that micro-flow LC-MS/MS is suitable for a broad range of proteomic applications.
Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, ...we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.
Laser decontamination of radioactive surfaces is an innovative technology. Our contribution to improving this technology includes studies on laser beam decontamination with a pulsed laser of an ...average power of 150 W, equipped with a hand guided working head. Our investigations are focused on metallic surfaces typical in nuclear power plants, such as stainless steel, bright and rusted mild steel, galvanized steel, and painted steel. As typical nuclides of contaminated surfaces we chose Co-60 and Cs-137, the most frequently occurring nuclides in many nuclear plant components; Sr-85 as a representative of Sr-90, the potentially most harmful fission nuclide; and Am-241 as a representative of the minor alpha-radiation emitting actinides. Here, we present our results of decontamination and recovery ratios. Decontamination ratios of 90–100% were achieved on different surfaces.
Targeted therapies against oncogenic receptor tyrosine kinases (RTK) show promising results in the clinic. Unfortunately, despite the initial positive response, most patients develop therapeutic ...resistance. Most research has focused on acquired resistance occurring after an extensive time of treatment; however, the question remains as to how cells can survive an initial treatment, as early resistance to apoptosis will enable cells to develop any growth-stimulating mechanism. Here, the non-small cell lung cancer (NSCLC) PC9 cell line was used to systematically profile, by mass spectrometry, changes in the proteome, kinome, and phosphoproteome during early treatment with the EGFR inhibitor afatinib. Regardless of the response, initial drug-sensitive cells rapidly adapt to targeted therapy, and within days, cells regained the capacity to proliferate, despite persisting target inhibition. These data reveal a rapid reactivation of mTOR and MAPK signaling pathways after initial inhibition and an increase in abundance and activity of cytoskeleton and calcium signaling-related proteins. Pharmacologic inhibition of reactivated pathways resulted in increased afatinib efficacy. However more strikingly, cells that were restricted from accessing extracellular calcium were extremely sensitive to afatinib treatment. These findings were validated using three additional inhibitors tested in four different NSCLC cell lines, and the data clearly indicated a role for Ca
signaling during the development of adaptive resistance. From a therapeutic point of view, the increased inhibitor efficacy could limit or even prevent further resistance development.
Combined targeting of calcium signaling and RTKs may limit drug resistance and improve treatment efficacy.
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The development of reliable joining techniques for ceramics and metals is crucial for energy applications, such as fuel cells, nuclear reactors, and high-temperature sensors, most especially for the ...sealing of hydrothermal sensors to study multiphase flows. However, during one-step active laser brazing it is a serious problem that a high thermal stress concentration can occur at the joint interfaces or on the ceramic side of the joint due to mismatches between the CTEs (coefficients of thermal expansion) and/or elastic constants. The uncontrolled thermal residual stress can lead to cracks and defects in the brazement. In the present work, an elastoplastic finite element method/numerical model was formulated to study the thermal residual stresses developed in the brazement between ceramics and austenitic stainless steel during cooling in active laser brazing. Calculations and comparison experiments were conducted to validate the simulated stress distribution in un-patterned ceramics. Stress analyses were conducted for planar and cylindrical specimen geometries (lab joints) relevant for miniaturized energy sensors. Laser interface patterning was employed to create micro-scale features on ceramic interfaces that reduce thermal stress concentrations. The optimization of the interface designing parameters including hatch size, structure width, pattern depth and metal/ceramic thickness ratio was performed using the Taguchi method with orthogonal arrays. The study suggests that laser interface structuring can modify thermal residual stresses in ceramic-to-metal brazements, thereby increasing the reliability of active brazing joints.
ABSTRACT
Objectives:
Recently, our group detected that polyvinyl chloride (PVC) perfusion lines leach large amounts of the toxic plasticizer diethylhexylphthalate (DEHP) under conditions typical of ...intensive care units. In the present study, we investigated the extraction of DEHP from PVC connecting tubes that are commonly used for total parenteral nutrition (TPN) solutions. The aim of the study was to estimate the amount of DEHP to which children receiving home TPN are exposed for months and years.
Materials and Methods:
1000 mL of TPN, identical in constitution and amount to the home TPN of 1 of our patients, were perfused through 5 different connecting tube systems and collected in hexane‐rinsed glass bottles. The concentration of DEHP in the TPN was analyzed before and after perfusion.
Results:
Before perfusion of the lines, the solution had a DEHP concentration of 0.05 to 0.69 μg/mL (baseline value). After perfusion of the lines, the load of DEHP in the solution varied between 1.41 and 2.07 μg/mL. This TPN was established for children weighing 20 kg. The daily dosage is between 71 and 104 μg · kg−1 · day−1. TPN is administered at home for many months and years. The monthly charge of DEHP is between 42.3 and 62.1 mg. Children weighing 20 kg therefore receive a dosage between 2.1 and 3.1 mg · kg−1 · month−1.
Conclusions:
Diethylhexylphthalate and its metabolite monoethylhexylphthalate have been demonstrated to be carcinogenic, embryotoxic, hepatotoxic, pneumotoxic, and cardiotoxic and are known to disrupt endocrine pathways and liver detoxifying capacity in animals. They are suspected of having multiple effects in humans as well. The doses presented above should therefore be avoided in children receiving home TPN by the use of tubing systems that are completely free of DEHP. Such systems are available.
The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. ...Several individual omics studies have extended our knowledge of COVID-19 pathophysiology
. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-β pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.
Abstract
ProteomicsDB (https://www.ProteomicsDB.org) started as a protein-centric in-memory database for the exploration of large collections of quantitative mass spectrometry-based proteomics data. ...The data types and contents grew over time to include RNA-Seq expression data, drug-target interactions and cell line viability data. In this manuscript, we summarize new developments since the previous update that was published in Nucleic Acids Research in 2017. Over the past two years, we have enriched the data content by additional datasets and extended the platform to support protein turnover data. Another important new addition is that ProteomicsDB now supports the storage and visualization of data collected from other organisms, exemplified by Arabidopsis thaliana. Due to the generic design of ProteomicsDB, all analytical features available for the original human resource seamlessly transfer to other organisms. Furthermore, we introduce a new service in ProteomicsDB which allows users to upload their own expression datasets and analyze them alongside with data stored in ProteomicsDB. Initially, users will be able to make use of this feature in the interactive heat map functionality as well as the drug sensitivity prediction, but ultimately will be able to use all analytical features of ProteomicsDB in this way.
Looking for a candidate substance inducing hepatobiliary dysfunction under parenteral nutrition (PN) in newborns, we recently discovered that newborn infusions extract large amounts of the ...plasticizer diethylhexylphthalate (DEHP) from commonly used polyvinylchloride (PVC) infusion lines. This plasticizer is well known to be genotoxic and teratogenic in animals and to cause changes in various organs and enzyme systems even in humans. The aim of this study was to examine the effect of DEHP, extracted in the same way and in the same amount as in newborns, on livers of young rabbits.
Prepubertal rabbits received lipid emulsion through central IV lines continuously for 3 weeks either via PVC or polyethylene (PE) infusion systems. Livers were examined after 1 and 3 weeks by light and electron microscopy.
By light microscopy, hydropic degeneration, single-cell necrosis, fibrosis, and bile duct proliferation were observed more in the PVC group. Electron microscopy revealed multiple nuclear changes, clusters and atypical forms of peroxisomes, proliferation of smooth endoplasmic reticulum, increased deposition of lipofuscin, and a mild perisinusoidal fibrosis only in the PVC group. These changes, which are generally regarded as reaction upon a toxic stimulus, could be exclusively attributed to DEHP.
This investigation proved that DEHP produces toxin-like changes in livers of young rabbits in the same dose, duration, and method of administration as in newborn infants. For this reason, it is likely that DEHP is the substance that causes hepatobiliary dysfunction in newborns under PN. Possible modes of action of DEHP are proposed.
New drugs are desperately needed to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report screening commercial kinase inhibitors for antibacterial activity and found ...the anticancer drug sorafenib as major hit that effectively kills MRSA strains. Varying the key structural features led to the identification of a potent analogue, PK150, that showed antibacterial activity against several pathogenic strains at submicromolar concentrations. Furthermore, this antibiotic eliminated challenging persisters as well as established biofilms. PK150 holds promising therapeutic potential as it did not induce in vitro resistance, and shows oral bioavailability and in vivo efficacy. Analysis of the mode of action using chemical proteomics revealed several targets, which included interference with menaquinone biosynthesis by inhibiting demethylmenaquinone methyltransferase and the stimulation of protein secretion by altering the activity of signal peptidase IB. Reduced endogenous menaquinone levels along with enhanced levels of extracellular proteins of PK150-treated bacteria support this target hypothesis. The associated antibiotic effects, especially the lack of resistance development, probably stem from the compound's polypharmacology.
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