The long path towards implementation of clinical proteomics: Exemplified based on CKD273

Clinical proteomics aims at the development and the implementation of novel biomarkers that demonstrate a clear clinical benefit in the management of diseases. However, though the attention in the field is increasing and multiple articles on biomarker research are published, clinical implementation of these biomarkers is scarce. In this paper, we aim towards identifying the hurdles on the path towards implementation, and present one successful approach, based on capillary electrophoresis coupled with mass spectrometry. A panel of biomarkers identified and assessed using this approach, termed CKD273, has recently received a Letter‐of‐Support from the US‐Food and Drug Administration (FDA), and is now implemented in the (early) management of chronic kidney disease. Based on this experience in the process towards implementation of CKD273, issues associated with implementation and suggestions how to meet these challenges are given.     

Polyphenolic profile,antioxidant properties and antimicrobial activity of grape skin extracts of 14 Vitis vinifera varieties grown in Dalmatia (Croatia)

The aim of the present study was to determine polyphenolic composition, related antioxidative and antimicrobial properties of grape skin extracts from 14 grape varieties (seven white and seven red grape) grown in Dalmatia (Croatia). The content of total phenols, flavonoids, catechins, flavanols and individual polyphenols ((+)-catechin, (−)-epicatechin, epicatechin gallate, procyanidin B1 and procyanidin B2, quercetin glucoside, resveratrol monomers, piceid and astringin) was variety dependent. Antioxidant properties were determined as DPPH radical-scavenging ability (IC₅₀), ferric reducing/antioxidant power (FRAP), Fe²⁺-chelating activity (IC₅₀), and using β-carotene bleaching assay. The high antioxidant capacity of all extracts, both red and white, has been observed and related to the relative amounts of polyphenolic compounds with good antioxidant properties. The antimicrobial activity was screened by broth microdilution test using Gram-positive (Staphylococcus aureus, Bacillus cereus) and Gram-negative bacteria (Escherichia coli O157:H7, Salmonella Infantis, Campylobacter coli). It was confirmed against all tested organisms. Minimum inhibitory concentrations (MICs), were found in the range 0.014–0.59 mg of gallic acid equivalents (GAE)/ml, with lower MICs of white cultivars, especially against Campylobacter and Salmonella.     

Use of copper shavings to remove mercury from contaminated groundwater or wastewater by amalgamation

The efficacy of copper shavings (Cu(0)) for the removal of Hg2+ from aqueous solution by amalgamation is demonstrated. Two kinds of copper shavings were investigated: (a) chemically processed shavings (Fluka) and (b) recycled shavings from scrap metal. Batch sorption experiments yielded very high retardation coefficients of 28 850-82 830 for the concentration range studied (1-10 000 microg/L Hg2+ dissolved in distilled water or in a 0.01 M CaCl2 matrix solution). Sorption data were well-described bythe Freundlich isotherm equation. Kinetic batch sorption experiments showed that 96-98% of Hg2+ was removed within 2 h. Column experiments were performed with a mercury solution containing 1000 microg/L Hg in a 0.01 M CaCl2 matrix with a flow rate of 0.5 m/d. No mercury breakthrough (c/c(0) = 0.5) could be detected after more than 2300 percolated pore volumes, and the high retardation coefficients determined in the batch studies could be confirmed. Copper was released from the shavings due to the amalgamation process and to copper corrosion by oxygen, resulting in concentrations of mobilized copper of 0.2-0.6 mg/L. Due to their high efficiency in removing Hg2+ from aqueous solution, the use of copper shavings for the removal of mercury from contaminated water is suggested, employing a sequential system of mercury amalgamation followed by the removal of mobilized copper by an ion exchanger such as zeolites. Possible applications could be in environmental technologies such as wastewater treatment or permeable reactive barriers for in situ groundwater remediation.     

Requirements engineering in health care: the example of chemotherapy planning in paediatric oncology

Health care is characterized by highly complex processes of patient care that require unusual amount of communication between different health care professionals of different institutions. Sub-optimal processes can significantly impact on the patient’s health, increase the consumption of services and resources and in severe cases can lead to the patient death. For these reasons, requirements engineering for the development of information technology in health care is a complex process as well: without constant and rigorous evaluation, the impact of new systems on the quality of care is unknown and it is possible that badly designed systems significantly harm patients. To overcome these limitations, we present and discuss an approach to requirements engineering that we applied for the development of applications for chemotherapy planning in paediatric oncology. Chemotherapy planning in paediatric oncology is complex and time-consuming and errors must be avoided by all means. In the multi-hospital/multi-trial-centre environment of paediatric oncology, it is especially difficult and time-consuming to analyse requirements. Our approach combines a grounded theory approach with evolutionary prototyping based on the constant development and refinement of a generic domain model, in this case a domain model for chemotherapy planning in paediatric oncology. The prototypes were introduced in medical centres and final results show that the developed generic domain model is adequate.     

Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS-CoV-2 Papain-Like Protease

The two SARS-CoV-2 proteases, i. e. the main protease (M^pro) and the papain-like protease (PL^pro), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PL^pro catalysis in vitro. The assay was applied to investigate the effect of reported small-molecule PL^pro inhibitors and selected M^pro inhibitors on PL^pro catalysis. The results reveal that some, but not all, PL^pro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing M^pro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PL^pro. Less selective M^pro inhibitors, e. g. auranofin, inhibit PL^pro, highlighting the potential for dual PL^pro/M^pro inhibition. MS-based PL^pro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.     

Autotaxin-LPA-LPP3 Axis in Energy Metabolism and Metabolic Disease

Besides serving as a structural membrane component and intermediate of the glycerolipid metabolism, lysophosphatidic acid (LPA) has a prominent role as a signaling molecule through its binding to LPA receptors at the cell surface. Extracellular LPA is primarily produced from lysophosphatidylcholine (LPC) through the activity of secreted lysophospholipase D, autotaxin (ATX). The degradation of extracellular LPA to monoacylglycerol is mediated by lipid phosphate phosphatases (LPPs) at the cell membrane. This review summarizes and interprets current literature on the role of the ATX-LPA-LPP3 axis in the regulation of energy homeostasis, insulin function, and adiposity at baseline and under conditions of obesity. We also discuss how the ATX-LPA-LPP3 axis influences obesity-related metabolic complications, including insulin resistance, fatty liver disease, and cardiomyopathy.     

MicroRNA-16 Restores Sensitivity to Tyrosine Kinase Inhibitors and Outperforms MEK Inhibitors in KRAS-Mutated Non-Small Cell Lung Cancer

Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Chemotherapy, the treatment of choice in non-operable cases, achieves a dismal success rate, raising the need for new therapeutic options. In about 25% of NSCLC, the activating mutations of the KRAS oncogene define a subclass that cannot benefit from tyrosine kinase inhibitors (TKIs). The tumor suppressor miR-16 is downregulated in many human cancers, including NSCLC. The main objectives of this study were to evaluate miR-16 treatment to restore the TKI sensitivity and compare its efficacy to MEK inhibitors in KRAS-mutated NSCLC. Methods: We performed in vitro and in vivo studies to investigate whether miR-16 could be exploited to overcome TKI resistance in KRAS-mutated NSCLC. We had three goals: first, to identify the KRAS downstream effectors targeted by mir-16, second, to study the effects of miR-16 restoration on TKI resistance in KRAS-mutated NSCLC both in vitro and in vivo, and finally, to compare miR-16 and the MEK inhibitor selumetinib in reducing KRAS-mutated NSCLC growth in vitro and in vivo. Results: We demonstrated that miR-16 directly targets the three KRAS downstream effectors MAPK3, MAP2K1, and CRAF in NSCLC, restoring the sensitivity to erlotinib in KRAS-mutated NSCLC both in vitro and in vivo. We also provided evidence that the miR-16–erlotinib regimen is more effective than the selumetinib–erlotinib combination in KRAS-mutated NSCLC. Conclusions: Our findings support the biological preclinical rationale for using miR-16 in combination with erlotinib in the treatment of NSCLC with KRAS-activating mutations.     

Aryl Bis‐Sulfonamide Inhibitors of IspF from Arabidopsis thaliana and Plasmodium falciparum

2‐Methylerythritol 2,4‐cyclodiphosphate synthase (IspF) is an essential enzyme for the biosynthesis of isoprenoid precursors in plants and many human pathogens. The protein is an attractive target for the development of anti‐infectives and herbicides. Using a photometric assay, a screen of 40 000 compounds on IspF from Arabidopsis thaliana afforded symmetrical aryl bis‐sulfonamides that inhibit IspF from A. thaliana (AtIspF) and Plasmodium falciparum (PfIspF) with IC₅₀ values in the micromolar range. The ortho‐bis‐sulfonamide structural motif is essential for inhibitory activity. The best derivatives obtained by parallel synthesis showed IC₅₀ values of 1.4 μm against PfIspF and 240 nm against AtIspF. Substantial herbicidal activity was observed at a dose of 2 kg ha^?1. Molecular modeling studies served as the basis for an in silico search targeted at the discovery of novel, non‐symmetrical sulfonamide IspF inhibitors. The designed compounds were found to exhibit inhibitory activities in the double‐digit micromolar IC₅₀ range.     

The simultaneous addition of styrene maleic anhydride copolymer and multiwall carbon nanotubes during melt‐mixing on the morphology of binary blends of polyamide6 and acrylonitrile butadiene styrene copolymer

The effect of simultaneous addition of multiwall carbon nanotubes (MWNTs) and a reactive compatibilizer (styrene maleic anhydride copolymer, SMA) during melt‐mixing on the phase morphology of 80/20 (wt/wt) PA6/ABS blend has been investigated. Morphological analysis through scanning and transmission electron microscopic analysis revealed finer morphology of the blends in presence of SMA + MWNTs. Fourier transform infrared spectroscopic analysis indicated the formation of imide bonds during melt‐mixing. Non‐isothermal crystallization studies exhibited the presence of a majority faction of MWNTs in the PA6 phase of 80/20 (wt/wt) PA6/ABS blend in presence of SMA + MWNTs. Rheological analysis, dynamic mechanical thermal analysis, and thermogravimetric analysis have demonstrated the compatibilization action of simultaneous addition of a reactive compatibilizer (SMA copolymer) and MWNTs in PA6/ABS blends. An attempt has been made to investigate the role of simultaneous addition of SMA copolymer and MWNTs on the morphology of 80/20 (wt/wt) PA6/ABS blend through various characterization techniques. POLYM. ENG. SCI., 55:457–465, 2015. © 2014 Society of Plastics Engineers     

Synthesis and magnetorheological characteristics of ribbon‐like,polypyrrole‐coated carbonyl iron suspensions under oscillatory shear

One of the crucial problems of classical magnetorheological (MR) fluids is their high rate of sedimentation. This disadvantage may be substantially eliminated using core‐shell particles. The aim of this study is to prepare spherical carbonyl iron (CI) particles coated with conducing polymer polypyrrole (PPy) with ribbon‐like morphology. Scanning electron microscopy proved the formation of the ribbon‐like layer onto CI particles while Fourier transform infrared spectroscopy confirmed the chemical structure of PPy. The magnetic properties observed via vibrating sample magnetometer showed decreased magnetization saturation of core‐shell‐structured particles due to the existence of non‐magnetic surface layer. MR measurements performed under oscillatory shear flow as a function of the applied magnetic flux density, temperature, and particle concentration showed that core‐shell particle‐based MR suspension exhibits sufficient MR performance for practical applications. Moreover, the suspension stability is promoted significantly when core‐shell particles are used as a dispersed phase. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013