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Atopic dermatitis is the most common inflammatory skin disease and is characterized by a deficient epidermal barrier and cutaneous inflammation. Genetic studies suggest a key role of keratinocytes in atopic dermatitis pathogenesis, but the alterations in the proteome that occur in the full epidermis have not been defined. Using a pressure-cycling technology and data-independent acquisition approach, we performed quantitative proteomics of epidermis from healthy volunteers and lesional and nonlesional patient skin. Results were validated by targeted proteomics using parallel reaction monitoring mass spectrometry and immunofluorescence staining. Proteins that were differentially abundant in the epidermis of patients with atopic dermatitis versus in healthy control reflect the strong inflammation in lesional skin and the defect in keratinocyte differentiation and epidermal stratification that already characterizes nonlesional skin. Most importantly, they reveal impaired activation of the NRF2-antioxidant pathway and reduced abundance of mitochondrial proteins involved in key metabolic pathways in the affected epidermis. Analysis of primary human keratinocytes with small interfering RNA‒mediated NRF2 knockdown revealed that the impaired NRF2 activation and mitochondrial abnormalities are partially interlinked. These results provide insight into the molecular alterations in the epidermis of patients with atopic dermatitis and identify potential targets for pharmaceutical intervention., Journal of Investigative Dermatology, 143 (2), ISSN:0022-202X, ISSN:1523-1747

Graphical abstract, There is a knowledge gap regarding the factors that impede the ruminal digestion of plant cell walls or if rumen microbiota possess the functional activities to overcome these constraints. Innovative experimental methods were adopted to provide a high-resolution understanding of plant cell wall chemistries, identify higher-order structures that resist microbial digestion, and determine how they interact with the functional activities of the rumen microbiota. We characterized the total tract indigestible residue (TTIR) from cattle fed a low-quality straw diet using two comparative glycomic approaches: ELISA-based glycome profiling and total cell wall glycosidic linkage analysis. We successfully detected numerous and diverse cell wall glycan epitopes in barley straw (BS) and TTIR and determined their relative abundance pre- and post-total tract digestion. Of these, xyloglucans and heteroxylans were of higher abundance in TTIR. To determine if the rumen microbiota can further saccharify the residual plant polysaccharides within TTIR, rumen microbiota from cattle fed a diet containing BS were incubated with BS and TTIR ex vivo in batch cultures. Transcripts coding for carbohydrate-active enzymes (CAZymes) were identified and characterized for their contribution to cell wall digestion based on glycomic analyses, comparative gene expression profiles, and associated CAZyme families. High-resolution phylogenetic fingerprinting of these sequences encoded CAZymes with activities predicted to cleave the primary linkages within heteroxylan and arabinan. This experimental platform provides unprecedented precision in the understanding of forage structure and digestibility, which can be extended to other feed-host systems and inform next-generation solutions to improve the performance of ruminants fed low-quality forages.

Ion mobility spectrometry (IMS) is an analytical technique where ions are separated in the gas phase based on their mobility through a buffer gas in the presence of an electric field. An ion passing through an IMS device has a characteristic collisional cross section (CCS) value that depends on the buffer gas used. IMS can be coupled with mass spectrometry (MS), which characterizes an ion based on a mass-to-charge ratio (m/z), to increase analytical specificity and provide further physicochemical information. In particular, IMS-MS is of ever-increasing interest for the analysis of lipids, which can be problematic to accurately identify and quantify in bodily fluids by liquid chromatography (LC) with MS alone due to the presence of isomers, isobars, and structurally similar analogs. IMS provides an additional layer of separation when combined with front-end LC approaches, thereby, enhancing peak capacity and analytical specificity. CCS (and also ion mobility drift time) can be plotted against m/z ion intensity and/or LC retention time in order to generate in-depth molecular profiles of a sample. Utilization of IMS-MS for routine clinical laboratory testing remains relatively unexplored, but areas do exist for potential implementation. A brief update is provided here on lipid analysis using IMS-MS with a perspective on some applications in the clinical laboratory.

Detailed knowledge on tissue-specific metabolic reprogramming in diabetic nephropathy (DN) is vital for more accurate understanding the molecular pathological signature and developing novel therapeutic strategies. In the present study, a spatial-resolved metabolomics approach based on air flow-assisted desorption electrospray ionization (AFADESI) and matrix-assisted laser desorption ionization (MALDI) integrated mass spectrometry imaging (MSI) was proposed to investigate tissue-specific metabolic alterations in the kidneys of high-fat diet-fed and streptozotocin (STZ)-treated DN rats and the therapeutic effect of astragaloside IV, a potential anti-diabetic drug, against DN. As a result, a wide range of functional metabolites including sugars, amino acids, nucleotides and their derivatives, fatty acids, phospholipids, sphingolipids, glycerides, carnitine and its derivatives, vitamins, peptides, and metal ions associated with DN were identified and their unique distribution patterns in the rat kidney were visualized with high chemical specificity and high spatial resolution. These region-specific metabolic disturbances were ameliorated by repeated oral administration of astragaloside IV (100 mg/kg) for 12 weeks. This study provided more comprehensive and detailed information about the tissue-specific metabolic reprogramming and molecular pathological signature in the kidney of diabetic rats. These findings highlighted the promising potential of AFADESI and MALDI integrated MSI based metabolomics approach for application in metabolic kidney diseases., Graphical abstract The present study revealed region-specific metabolic reprogramming during diabetic nephropathy (DN) by using air flow-assisted desorption electrospray ionization (AFADESI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI).Image 1

Highlights • LC-MS methods are used to identify intact OXA-48 like enzymes from bacterial lysates. • Individual protein variants of the OXA-48 like carbapenemases can be detected with tandem mass spectrometry. • OXA-48 like carbapenemases can be distinguished from extended spectrum β-lactamases like CTX-M-15. • The method for detection can be run in five minutes or less., Introduction Antibiotic-resistant Gram-negative bacteria are of a growing concern globally, especially those producing enzymes conferring resistance. OXA-48-like carbapenemases hydrolyze most β-lactam antibiotics, with typically low-level hydrolysis of carbapenems, but have limited effect on broad-spectrum cephalosporins. These are frequently co-expressed with extended spectrum β-lactamases, especially CTX-M-15, which typically shows high level resistance to broad-spectrum cephalosporins, yet is carbapenem susceptible. The combined resistance profile makes the need for successful detection of these specific resistance determinants imperative for effective antibiotic therapy. Objectives The objective of this study is to detect and identify OXA-48-like and CTX-M-15 enzymes using mass spectrometry, and to subsequently develop a method for detection of both enzyme types in combination with liquid chromatography. Methods Cells grown in either broth or on agar were harvested, lysed, and, in some cases buffer-exchanged. Lysates produced from bacterial cells were separated and analyzed via liquid chromatography with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). Results The intact proteins of OXA-48, OXA-181, and OXA-232 (collectively OXA-48-like herein) and CTX-M-15 were characterized and detected. Acceptance criteria based on sequence-informative fragments from each protein group were established as confirmatory markers for the presence of the protein(s). A total of 25 isolates were successfully tested for OXA-48 like (2), CTX-M-15 (3), or expression of both (7) enzymes. Thirteen isolates served as negative controls. Conclusions Here we present a method for the direct and independent detection of both OXA-48-like carbapenemases and CTX-M-15 β-lactamases using LC-MS/MS. The added sensitivity of MS/MS allows for simultaneous detection of at least two co-eluting, co-isolated and co-fragmented proteins from a single mass spectrum.

Highlights • Analysis of 2-methylcitric acid, methylmalonic acid, and homocysteine in DBS. • A dual derivatization LC-MS/MS strategy developed for use in second-tier NBS. • Second-tier panel replaces multiple initial follow-up tests. • Improved workflows without the use of ion pairing reagents in the LC mobile phases. • Anticipated increase in diagnostic specificity and reduction of parental anxiety., Inborn errors of propionate, cobalamin and methionine metabolism are targets for Newborn Screening (NBS) in most programs world-wide, and are primarily screened by analyzing for propionyl carnitine (C3) and methionine in dried blood spot (DBS) cards using tandem mass spectrometry (MS/MS). Single-tier NBS approaches using C3 and methionine alone lack specificity, which can lead to an increased false-positive rate if conservative cut-offs are applied to minimize the risk of missing cases. Implementation of liquid chromatography tandem mass spectrometry (LC-MS/MS) second-tier testing for 2-methylcitric acid (MCA), methylmalonic acid (MMA), and homocysteine (HCY) from the same DBS card can improve disease screening performance by reducing the false-positive rate and eliminating the need for repeat specimen collection. However, DBS analysis of MCA, MMA, and HCY by LC-MS/MS is challenging due to limited specimen size and analyte characteristics leading to a combination of low MS/MS sensitivity and poor reverse-phase chromatographic retention. Sufficient MS response and analytical performance can be achieved for MCA by amidation using DAABD-AE and by butylation for MMA and HCY. Herein we describe the validation of a second-tier dual derivatization LC-MS/MS approach to detect elevated MCA, MMA, and HCY in DBS cards for NBS. Clinical utility was demonstrated by retrospective analysis of specimens, an interlaboratory method comparison, and assessment of external proficiency samples. Imprecision was

Visual Abstract, Highlights • Myocardial matrix hydrogel preserves LV volumes and apical wall thickening in a chronic MI model. • Myocardial matrix hydrogel trends toward reduced fibrosis. • In vivo differential gene expression analysis shows the matrix modulates cardiac muscle contraction, metabolism, fibrosis, and the inflammatory/immune response in a chronic MI model., Summary A first-in-man clinical study on a myocardial-derived decellularized extracellular matrix hydrogel suggested the potential for efficacy in chronic myocardial infarction (MI) patients. However, little is understood about the mechanism of action in chronic MI. In this study, the authors investigated the efficacy and mechanism by which the myocardial matrix hydrogel can mitigate negative left ventricular (LV) remodeling in a rat chronic MI model. Assessment of cardiac function via magnetic resonance imaging demonstrated preservation of LV volumes and apical wall thickening. Differential gene expression analyses showed the matrix is able to prevent further negative LV remodeling in the chronic MI model through modulation of the immune response, down-regulation of pathways involved in heart failure progression and fibrosis, and up-regulation of genes important for cardiac muscle contraction.

Highlight • The role of Amaryllidaceae species from Ecuador as source of new drugs. • High amount of lycorine-type alkaloids in Phaedranassa dubia by GC-MS. • In vitro potential of Phaedranassa brevifolia as cholinesterase inhibitor. • Theoretical inhibitory activity of alkaloids against cholinesterase enzymes., Alzheimer's disease is considered the most common cause of dementia and, in an increasingly aging population worldwide, the quest for treatment is a priority. Amaryllidaceae alkaloids are of main interest because of their cholinesterase inhibition potential, which is the main palliative treatment available for this disease. We evaluated the alkaloidal profile and the in vitro inhibitory activity on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) of bulb alkaloid extract of Phaedranassa dubia and Phaedranassa brevifolia collected in Ecuador. Using gas chromatography coupled to mass spectrometry (GC-MS), we identified typical Amaryllidaceae alkaloids in these species, highlighting the presence of lycorine-type alkaloids in P. dubia and haemanthamine/crinine-type in P. brevifolia. The species P. dubia and P. brevifolia showed inhibitory activities against AChE (IC50 values of 25.48 ± 0.39 and 3.45 ± 0.29 μg.mL−1, respectively) and BuChE (IC50 values of 114.96 ± 4.94 and 58.89 ± 0.55 μg.mL−1, respectively). Computational experiments allowed us to understand the interactions of the alkaloids identified in these samples toward the active sites of AChE and BuChE. In silico, some alkaloids detected in these Amaryllidaceae species presented higher estimated binding free energy toward BuChE than galanthamine. This is the first study about the alkaloid profile and biological potential of P. brevifolia species.

Highlights • Evidence of poisoning with organophosphorus (OP) nerve agents requires biomedical verification. • OP nerve agents undergo common biotransformation pathways producing valuable biomarkers. • Internationally accepted methods target remaining poison, hydrolysis products and protein-adducts. • Mass spectrometry-based methods provide optimum selectivity and sensitivity for identification. • Methods, strategies, current proceedings, quality criteria and real cases of poisoning are presented., Intoxication by organophosphorus (OP) poisons, like nerve agents and pesticides, is characterized by the life-threatening inhibition of acetylcholinesterase (AChE) caused by covalent reaction with the serine residue of the active site of the enzyme (phosphylation). Similar reactions occur with butyrylcholinesterase (BChE) and serum albumin present in blood as dissolved proteins. For forensic purposes, products (adducts) with the latter proteins are highly valuable long-lived biomarkers of exposure to OP agents that are accessible by diverse mass spectrometric procedures. In addition, the evidence of poison incorporation might also succeed by the detection of remaining traces of the agent itself, but more likely its hydrolysis and/or enzymatic degradation products. These relatively short-lived molecules are distributed in blood and tissue, and excreted via urine. This review presents the mass spectrometry-based methods targeting the different groups of biomarkers in biological samples, which are already internationally accepted by the Organisation for the Prohibition of Chemical Weapons (OPCW), introduces novel approaches in the field of biomedical verification, and outlines the strict quality criteria that must be fulfilled for unambiguous forensic analysis.

Graphical abstract, Highlights • High-resolution solution NMR structure of rhNGF has been determined. • Quinary interactions characterize ATP binding to rhNGF. • SPR, ITC and STD-NMR reveal ATP binding to rhNGF with mM affinity. • NMR and MD analysis pinpoint to the presence of two binding sites of ATP on rhNGF. • Stoichiometry of ATP-Mg2+ or Zn2+-rhNGF mixtures affects KD affinity to TrkA/p75NTR., The Nerve Growth Factor (NGF) neurotrophin acts in the maintenance and growth of neuronal populations. Despite the detailed knowledge of NGF’s role in neuron physiology, the structural and mechanistic determinants of NGF bioactivity modulated by essential endogenous ligands are still lacking. We present the results of an integrated structural and advanced computational approach to characterize the extracellular ATP-NGF interaction. We mapped by NMR the interacting surface and ATP orientation on NGF and revealed the functional role of this interaction in the binding to TrkA and p75NTR receptors by SPR. The role of divalent ions was explored in conjunction with ATP. Our results pinpoint ATP as a likely transient molecular modulator of NGF signaling, in health and disease states.

Highlights • Proteomics has aided HF biomarker discovery, which allows for greater disease insights. • Experiment design can be tailored to HF research to discover novel biomarkers. • Primary methods include MS, protein microarray, aptamer, and PEA-based technologies. • Proteomics can detect unique low abundance proteins and detect protein modifications., Summary Circulating protein biomarkers provide information regarding pathways in heart failure (HF) and can add important value to clinicians. Advancements in proteomics allow researchers to measure a multitude of proteins simultaneously with excellent sensitivity and selectivity to detect low abundance proteins. This helps identify previously unrecognized pathways in HF and discover biomarkers and potential targets for HF therapies. Although several proteomic methods exist, including mass spectrometry, protein microarray, aptamer, and proximity extension assay−based techniques, each have their unique advantages. This paper provides an overview of the various proteomic methods, with examples of how each has contributed to understanding the pathways in HF., Central Illustration

Natural products have provided numerous lead compounds for drug discovery. However, the traditional analytical methods cannot detect most of these active components, especially at their usual low concentrations, from complex natural products. Herein, we reviewed the recent technological advances (2015–2019) related to the separation and screening bioactive components from natural resources, especially the emerging screening methods based on the bioaffinity techniques, including biological chromatography, affinity electrophoresis, affinity mass spectroscopy, and the latest magnetic and optical methods. These screening methods are uniquely advanced compared to other traditional methods, and they can fish out the active components from complex natural products because of the affinity between target and components, without tedious separation works. Therefore, these new tools can reduce the time and cost of the drug discovery process and accelerate the development of more effective and better-targeted therapeutic agents., Graphical abstract This review summarizes the recent technological advances (2015–2019) related to the separation and screening bioactive components from natural resources, especially the emerging screening methods based on the bioaffinity techniques.Image 1

Summary: Background: Synaptic proteins are increasingly studied as biomarkers for synaptic dysfunction and loss, which are early and central events in Alzheimer's disease (AD) and strongly correlate with the degree of cognitive decline. In this study, we specifically investigated the synaptic binding partners neurexin (NRXN) and neuroligin (Nlgn) proteins, to assess their biomarker's potential. Methods: we developed a parallel reaction monitoring mass spectrometric method for the simultaneous quantification of NRXNs and Nlgns in cerebrospinal fluid (CSF) of neurodegenerative diseases, focusing on AD. Specifically, NRXN-1α, NRXN-1β, NRXN-2α, NRXN-3α and Nlgn1, Nlgn2, Nlgn3 and Nlgn4 proteins were targeted. Findings: The proteins were investigated in a clinical cohort including CSF from controls (n=22), mild cognitive impairment (MCI) due to AD (n=44), MCI due to other conditions (n=46), AD (n=77) and a group of non-AD dementia (n=28). No difference in levels of NRXNs and Nlgns was found between AD (both at dementia and MCI stages) or controls or the non-AD dementia group for any of the targeted proteins. NRXN and Nlgn proteins correlated strongly with each other, but only a weak correlation with the AD core biomarkers and the synaptic biomarkers neurogranin and growth-associated protein 43, was found, possibly reflecting different pathogenic processing at the synapse. Interpretation: we conclude that NRXN and Nlgn proteins do not represent suitable biomarkers for synaptic pathology in AD. The panel developed here could aid in future investigations of the potential involvement of NRXNs and Nlgns in synaptic dysfunction in other disorders of the central nervous system. Funding: a full list of funding can be found under the acknowledgments section.

The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as selectins, galectins, and sialic acid-binding immunoglobulin-like lectins are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human embryonic kidney 293 (HEK293) cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knockin of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galβ1-3GalNAcα1-O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3-O-sulfotransferase 2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3-O-sulfotransferase 4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step toward expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.

Graphical abstract, Genome mining is a computational method for the automatic detection and annotation of biosynthetic gene clusters (BGCs) from genomic data. This approach has been increasingly utilised in natural product (NP) discovery due to the large amount of sequencing data that is now available. Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a class of structurally complex NP with diverse bioactivities. RiPPs have recently been shown to occupy a much larger expanse of genomic and chemical space than previously appreciated, indicating that annotation of RiPP BGCs in genomes may have been overlooked in the past. This review provides an overview of the genome mining tools that have been specifically developed to aid in the discovery of RiPP BGCs, which have been built from an increasing knowledgebase of RiPP structures and biosynthesis. Given these recent advances, the application of targeted genome mining has great potential to accelerate the discovery of important molecules such as antimicrobial and anticancer agents whilst increasing our understanding about how these compounds are biosynthesised in nature.

Understanding of the nephrotoxicity induced by drug candidates is vital to drug discovery and development. Herein, an in situ metabolomics method based on air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) was established for direct analysis of metabolites in renal tissue sections. This method was subsequently applied to investigate spatially resolved metabolic profile changes in rat kidney after the administration of aristolochic acid I, a known nephrotoxic drug, aimed to discover metabolites associated with nephrotoxicity. As a result, 38 metabolites related to the arginine–creatinine metabolic pathway, the urea cycle, the serine synthesis pathway, metabolism of lipids, choline, histamine, lysine, and adenosine triphosphate were significantly changed in the group treated with aristolochic acid I. These metabolites exhibited a unique distribution in rat kidney and a good spatial match with histopathological renal lesions. This study provides new insights into the mechanisms underlying aristolochic acids nephrotoxicity and demonstrates that AFADESI-MSI-based in situ metabolomics is a promising technique for investigation of the molecular mechanism of drug toxicity., Graphical abstract This study investigated the spatially resolved metabolic profile changes in rat kidney after the administration of aristolochic acid I (AAI) by an in situ metabolomics method based on air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI), providing new insights into the mechanisms underlying aristolochic acids nephrotoxicity.Image 1

Graphical abstract, The Zika virus is a flavivirus that can cause fulminant outbreaks and lead to Guillain-Barré syndrome, microcephaly and fetal demise. Like other flaviviruses, the Zika virus is transmitted by mosquitoes and provokes neurological disorders. Despite its risk to public health, no antiviral nor vaccine are currently available. In the recent years, several studies have set to identify human host proteins interacting with Zika viral proteins to better understand its pathogenicity. Yet these studies used standard human protein sequence databases. Such databases rely on genome annotations, which enforce a minimal open reading frame (ORF) length criterion. An ever-increasing number of studies have demonstrated the shortcomings of such annotation, which overlooks thousands of functional ORFs. Here we show that the use of a customized database including currently non-annotated proteins led to the identification of 4 alternative proteins as interactors of the viral capsid and NS4A proteins. Furthermore, 12 alternative proteins were identified in the proteome profiling of Zika infected monocytes, one of which was significantly up-regulated. This study presents a computational framework for the re-analysis of proteomics datasets to better investigate the viral-host protein interplays upon infection with the Zika virus.

Background Recently, the distinction between left- and right-sided colon cancer (LCC and RCC) has been brought into focus. RCC is associated with an inferior overall survival and progression-free survival. We aimed to perform a detailed analysis of the diversity of extracellular vesicles (EV) between LCC and RCC using quantitative proteomics and to identify for new diagnostic and prognostic biomarkers. Methods We isolated EVs from patients with LCC, RCC and healthy volunteers, and treated colorectal cancer cell line with serum-derived EVs. We then performed a quantitative proteomics analysis of the serum-derived EVs and cell line treated with EVs. Proteomic data are available via ProteomeXchange with the identifiers PXD012283 and PXD012304. In addition, we assessed the performance of EV SPARC and LRG1 as diagnosis and prognosis biomarkers in colon cancer. Findings The expression profile of the serum EV proteome in patients with RCC was different from that of patients with LCC. Serum-derived EVs in RCC promoted cellular mobility more significantly than EVs derived from LCC. EV SPARC and LRG1 expression levels demonstrated area under the receiver-operating characteristic curve values of 0.95 and 0.93 for discriminating patients with colon cancer from healthy controls. Moreover, the expression levels of SPARC and LRG1 correlated with tumour sidedness and were predictive of tumour recurrence. Interpretation We identified differences in EV protein profiles between LCC and RCC. Serum-derived EVs of RCC may promote metastasis via upregulation of extracellular matrix (ECM)-related proteins, especially SPARC and LRG1, which may serve as diagnosis and prognosis biomarkers in colon cancer.

Graphical abstract, Highlights • Byrsonima sericea DC. (Malpighiaceae) was studied for phenolic contents. • Byrsonima sericea (BSEE) ethanolic extracts explored Indomethacin induced gastric lesion. • BSEE lowers rates of AST, ALT, animal weight and relative organ weight. • Anti-diarrheal and antioxidant activity from leaf extracts were observed in the current investigation., Byrsonima sericea DC. (Malpighiaceae) leaves are popularly folk medicine in Brazil used to treat gastro-intestinal disorders including diarrhea and gastric diseases. Ethanol extract (BSEE), ethyl acetate extract (BSEAE) and hexane extract (BSHE) of the leaf part of Byrsonima sericea DC were characterized for their total phenolics, proanthocyanidins and flavonoids content. The total antioxidant capacity of extracts was determined. The ethnopharmacological use of B. sericea leaves was evaluated by assaying BSEE for gastroprotective activity in stomach ulcer induced by indomethacin, intestinal motility and toxicity. Abundance of phenols mainly tannins was found in BSEE. Total phenolics, flavonoids and proanthocyanidins content in BSEE were found to be 0.371, 0.172 and 1.3 × 10-4 (mg/g) respectively. BSEE showed concentration dependent significant scavenging of DPPH values 90.0 (%) respectively. Moreover, oral doses of 500 and 1000 mg/kg did not cause mortality, and there was no difference in animals weight, organs relative weight and alanine transaminase (ALT) and aspartate transaminase (AST), as compared to the control group. Doses of 250, 500 and 1000 mg/kg inhibited the gastric lesions induced by indomethacin in 52, 60 and 62 % respectively. The dose of 1000 mg/kg decreased intestinal motility in animals. The presence of phenolic compounds, including tannins could be associated with the anti-diarrheal action and the antioxidant properties could collaborate to the gastroprotective and anti- diarrheal activities, confirming its popular use of the plant.

Well-orchestrated maternal-fetal crosstalk occurs via secreted ligands, interacting receptors, and coupled intracellular pathways between the conceptus and endometrium, and is essential for successful embryo implantation. However, previous studies mostly focus on either the conceptus or the endometrium in isolation. The lack of integrated analysis impedes our understanding of early maternal-fetal crosstalk. Herein, focusing on ligand–receptor complexes and coupled pathways at the maternal-fetal interface in sheep, we provide the first comprehensive proteomic map of ligand-receptor pathway cascades essential for embryo implantation. We demonstrate that these cascades are associated with cell adhesion and invasion, redox homeostasis, and the immune response. Candidate interactions and their physiological roles were further validated by functional experiments. We reveal the physical interaction of albumin and claudin 4 and their roles in facilitating embryo attachment to endometrium. We also demonstrate a novel function of enhanced conceptus glycolysis in remodeling uterine receptivity by inducing endometrial histone lactylation, a newly identified histone modification. Results from in vitro and in vivo models supported the essential role of lactate in inducing endometrial H3K18 lactylation and in regulating redox homeostasis and apoptotic balance to ensure successful implantation. By reconstructing a map of potential ligand-receptor pathway cascades at the maternal-fetal interface, our study presents new concepts for understanding molecular and cellular mechanisms that fine-tune conceptus-endometrium crosstalk during implantation. This provides more direct and accurate insights for developing potential clinical intervention strategies to improve pregnancy outcomes following both natural and assisted conception.

Highlights • Tendon and ligament collagens differ in their post-translational lysine and cross-linking chemistry. • In ligament collagen, hydroxylysyl aldehyde, permanent cross-linking dominates. • Tendon collagen has a mix of cross-links based on lysyl and hydroxylysyl aldehydes. • The profile in tendon appears more adapted to facilitating growth, structural remodeling and repair of the fibrillar matrix., Tendons and ligaments tend to be pooled into a single category as dense elastic bands of collagenous connective tissue. They do have many similar properties, for example both tissues are flexible cords of fibrous tissue that join bone to either muscle or bone. Tendons and ligaments are both prone to degenerate and rupture with only limited capacity to heal, although tendons tend to heal faster than ligaments. Type I collagen constitutes about 80% of the dry weight of tendons and ligaments and is principally responsible for the core strength of each tissue. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including proline and lysine hydroxylation, hydroxylysine glycosylation and covalent cross-linking. The chemistry, placement and quantity of intramolecular and intermolecular cross-links are believed to be key contributors to the tissue-specific variations in material strength and biological properties of collagens. As tendons and ligaments grow and develop, the collagen cross-links are known to chemically mature, strengthen and change in profile. Accordingly, changes in cross-linking and other post-translational modifications are likely associated with tissue development and degeneration. Using mass spectrometry, we have compared tendon and ligaments from fetal and adult bovine knee joints to investigate changes in collagen post-translational properties. Although hydroxylation levels at the type I collagen helical cross-linking lysine residues were similar in all adult tissues, ligaments had significantly higher levels of glycosylation at these sites compared to tendon. Differences in lysine hydroxylation were also found between the tissues at the telopeptide cross-linking sites. Total collagen cross-linking analysis, including mature trivalent cross-links and immature divalent cross-links, revealed unique cross-linking profiles between tendon and ligament tissues. Tendons were found to have a significantly higher frequency of smaller diameter collagen fibrils compared with ligament, which we suspect is functionally associated with the unique cross-linking profile of each tissue. Understanding the specific molecular characteristics that define and distinguish these specialized tissues will be important to improving the design of orthopedic treatment approaches.

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings., Graphical Abstract, Highlights • Integrating protein interaction data with metabolic models expands multiomic mapping. • Proteomic profiling of tumors and cell lines reveals altered metabolic-related signatures. • Metabolite measurements validate pathway alterations in cancer cell lines and tumors., In Brief Metabolism is recognized as an important driver of complex diseases, but global metabolite profiling remains a challenge. Protein expression is a poor proxy because pathway enrichment models provide an incomplete mapping between the proteome and metabolism. We developed MOMENTA, a multiomic network approach for interrogating metabolic pathways from proteomics data. Analysis of data from cancer cell lines and human tumors reveals metabolic network rewiring and oncogene connections. The metabolic networks altered in cancer are linked to clinical outcomes.

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR., Graphical Abstract, Highlights • Temporal proteomic dynamics in the imatinib or adriamycin-induced drug resistance. • Comparison of four DIA software tools (OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA). • Sirtuin signaling pathway was significantly regulated in resistant K562 cells. • IDH2 was identified as a potential drug target correlated for resistant K562 cells., In Brief To understand the underlying resistance mechanisms in response to imatinib (IMA) and adriamycin (ADR), we explored two unique drug resistance models of K562 cells. We applied an optimized DIA–MS method to quantify 98,232 peptides from 7082 proteotypic proteins from these samples using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. The sirtuin signaling pathway was found significantly regulated in both models, and IDH2 was identified as a druggable regulator of acquired drug resistance.

To explore the recovery of renal function in severely ill coronavirus disease (COVID-19) survivors and determine the plasma metabolomic profile of patients with different renal outcomes 3 months after discharge, we included 89 severe COVID-19 survivors who had been discharged from Wuhan Union Hospital for 3 months. All patients had no underlying kidney disease before admission. At patient recruitment, renal function assessment, laboratory examination, chest computed tomography (CT) were performed. Liquid chromatography–mass spectrometry was used to detect metabolites in the plasma. We analyzed the longitudinally change in the estimated glomerular filtration rate (eGFR) based on serum creatinine and cystatin-c levels using the CKD-EPI equation and explored the metabolomic differences in patients with different eGFR change patterns from hospitalization to 3 months after discharge. Lung CT showed good recovery; however, the median eGFR significantly decreased at the 3-month follow-up. Among the 89 severely ill COVID-19 patients, 69 (77.5%) showed abnormal eGFR (, Graphical abstract Unlabelled Image

Highlights • BSP increased production of SCFAs and CO2 while reduced the ammonia concentrations. • BSP fermentation increased the diversity of the bacterial communities. • BSP fermented by intestinal microbiota to produce beneficial organic acid metabolites. • BSP with potential prebiotic properties were beneficial to human health., The effects of Moso bamboo (Phyllostachys edulis) shoot polysaccharide (BSP) on the human gut microbiota composition and volatile metabolite components were investigated by in vitro fermentation. After fermentation for 48 h, BSP utilization reached 40.29% and the pH of the fermentation solution decreased from 6.89 to 4.57. Moreover, the total short-chain fatty acid concentration significantly (P

Human apoptosis-linked gene-2 interacting protein X (ALIX), a versatile adapter protein, regulates essential cellular processes by shuttling between late endosomal membranes and the cytosol, determined by its interactions with Src kinase. Here, we investigate the molecular basis of these transitions and the effects of tyrosine phosphorylation on the interplay between structure, assembly, and intramolecular and intermolecular interactions of ALIX. As evidenced by transmission electron microscopy, fluorescence and circular dichroism spectroscopy, the proline-rich domain of ALIX, which encodes binding epitopes of multiple cellular partners, formed rope-like β-sheet-rich reversible amyloid fibrils that dissolved upon Src-mediated phosphorylation and were restored on protein-tyrosine phosphatase 1B-mediated dephosphorylation of its conserved tyrosine residues. Analyses of the Bro1 domain of ALIX by solution NMR spectroscopy elucidated the conformational changes originating from its phosphorylation by Src and established that Bro1 binds to hyperphosphorylated proline-rich domain and to analogs of late endosomal membranes via its highly basic surface. These results uncover the autoinhibition mechanism that relocates ALIX to the cytosol and the diverse roles played by tyrosine phosphorylation in cellular and membrane functions of ALIX.

Analysis of nucleotide variants is a cornerstone of cancer medicine. Although only 2% of the genomic sequence is protein coding, mutations occurring in these regions have the potential to influence protein structure or modification status and may have severe impact on disease aetiology. Proteogenomics enables the analysis of sample-specific nonsynonymous nucleotide variants with regard to their effect at the proteome and phosphoproteome levels. Here, we developed a proof-of-concept proteogenomics workflow and applied it to the malignant melanoma cell line A375. Initially, we studied the resistance to serine/threonine-protein kinase B-raf (BRAF) inhibitor (BRAFi) vemurafenib in A375 cells. This allowed identification of several oncogenic nonsynonymous nucleotide variants, including a gain-of-function variant on aurora kinase A (AURKA) at F31I. We also detected significant changes in abundance among (phospho)proteins, which led to reactivation of the MAPK signaling pathway in BRAFi-resistant A375 cells. Upon reconstruction of the multiomic integrated signaling networks, we predicted drug therapies with the potential to disrupt BRAFi resistance mechanism in A375 cells. Notably, we showed that AURKA inhibition is effective and specific against BRAFi-resistant A375 cells. Subsequently, we investigated amino acid variants that interfere with protein posttranslational modification (PTM) status and potentially influence A375 cell signaling irrespective of BRAFi resistance. Mass spectrometry (MS) measurements confirmed variant-driven PTM changes in 12 proteins. Among them was the runt-related transcription factor 1 (RUNX1) displaying a variant on a known phosphorylation site S(Ph)276L. We confirmed the loss of phosphorylation site by MS and demonstrated the impact of this variant on RUNX1 interactome., Graphical Abstract, Highlights • Proteogenomics workflow to study the malignant melanoma cell line A375. • Key signaling pathways are perturbed in BRAFi-sensitive and -resistant A375. • The signaling network in BRAFi-resistant cells can be targeted by several drugs. • Multiple amino acid variants directly affect protein phosphorylation status. • Loss of a phosphorylation site on RUNX1 alters its interactome., In Brief Proteogenomics is a powerful tool to study the mode of action of disease-associated mutations at the genome, transcriptome, proteome, and PTM level. Here, we applied a proteogenomics workflow to study the malignant melanoma cell line A375. Such workflow, used here as a proof of concept on A375 cells, may be applicable to other cancer types, cell lines, or even patient-derived samples.

International audience; The rigid core of intracellular tau filaments from Alzheimer’s disease (AD), Pick’s disease (PiD), and Corticobasal disease (CBD) brains has been shown to differ in their cryo-EM atomic structure. Despite providing critical information on the intimate arrangement of a fraction of htau molecule within the fibrillar scaffold, the cryo-EM studies neither yield a complete picture of tau fibrillar assemblies structure nor contribute insights into the surfaces that define their interactions with numerous cellular components. Here, using proteomic approaches such as proteolysis and molecular covalent painting, we mapped the exposed amino acid stretches at the surface and those constituting the fibrillar core of in vitro-assembled fibrils of human htau containing one N-terminal domain and three (1N3R) or four (1N4R) C-terminal microtubule-binding repeat domains as a result of alternative splicing. Using limited proteolysis, we identified the proteolytic fragments composing the molecular “bar-code” for each type of fibril. Our results are in agreement with structural data reported for filamentous tau from AD, PiD, and CBD cases predigested with the protease pronase. Finally, we report two amino acid stretches, exposed to the solvent in 1N4R not in 1N3R htau, which distinguish the surfaces of these two kinds of fibrils. Our findings open new perspectives for the design of highly specific ligands with diagnostic and therapeutic potential.

The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases., Graphical Abstract, Highlights • Trichomonas vaginalis phagolysosome consist of over 460 proteins. • Lysosomes are involved in secretion of virulence factors such as TvCP2. • N-glycosylation is required for lysosomal protein targeting. • T. vaginalis possesses homologs of mannose 6-phosphate receptor., In Brief Lysosomes represent a central degradative compartment of eukaryotes, yet little is known about biogenesis and function of this organelle in the parasitic protist Trichomonas vaginalis. We analyzed the phagolysosomal proteome that consists of over 460 proteins including important virulence factors. We demonstrated that glycosylation is involved in lysosomal protein targeting in T. vaginalis, which is unprecedented in parasitic protists. In addition to the classical secretory pathway, lysosomes are involved in unconventional protein secretion.

DNA-protein cross-links are formed when proteins become covalently trapped with DNA in the presence of exogenous or endogenous alkylating agents. If left unrepaired, they inhibit transcription as well as DNA unwinding during replication and may result in genome instability or even cell death. The DNA repair protein O6-alkylguanine DNA-alkyltransferase (AGT) is known to form DNA cross-links in the presence of the carcinogen 1,2-dibromoethane, resulting in G:C to T:A transversions and other mutations in both bacterial and mammalian cells. We hypothesized that AGT-DNA cross-links would be processed by nuclear proteases to yield peptides small enough to be bypassed by translesion (TLS) polymerases. Here, a 15-mer and a 36-mer peptide from the active site of AGT were cross-linked to the N2 position of guanine via conjugate addition of a thiol containing a peptide dehydroalanine moiety. Bypass studies with DNA polymerases (pols) η and κ indicated that both can accurately bypass the cross-linked DNA peptides. The specificity constant (kcat/Km) for steady-state incorporation of the correct nucleotide dCTP increased by 6-fold with human (h) pol κ and 3-fold with hpol η, with hpol η preferentially inserting nucleotides in the order dC > dG > dA > dT. LC-MS/MS analysis of the extension product also revealed error-free bypass of the cross-linked 15-mer peptide by hpol η. We conclude that a bulky 15-mer AGT peptide cross-linked to the N2 position of guanine can retard polymerization, but that overall fidelity is not compromised because only correct bases are inserted and extended.

As the demand for laboratory testing by mass spectrometry increases, so does the need for automated methods for data analysis. Clinical mass spectrometry (MS) data is particularly well-suited for machine learning (ML) methods, which deal nicely with structured and discrete data elements. The alignment of these two fields offers a promising synergy that can be used to optimize workflows, improve result quality, and enhance our understanding of high-dimensional datasets and their inherent relationship with disease. In recent years, there has been an increasing number of publications that examine the capabilities of ML-based software in the context of chromatography and MS. However, given the historically distant nature between the fields of clinical chemistry and computer science, there is an opportunity to improve technological literacy of ML-based software within the clinical laboratory scientist community. To this end, we present a basic overview of ML and a tutorial of an ML-based experiment using a previously published MS dataset. The purpose of this paper is to describe the fundamental principles of supervised ML, outline the steps that are classically involved in an ML-based experiment, and discuss the purpose of good ML practice in the context of a binary MS classification problem.

Background & Aims Increased serum bile acids (BAs) have been observed in patients with non-alcoholic steatohepatitis (NASH). Pegbelfermin (PGBF), a polyethylene glycol-modified (PEGylated) analogue of human fibroblast growth factor 21 (FGF21), significantly decreased hepatic steatosis and improved fibrosis biomarkers and metabolic parameters in patients with NASH in a phase IIa trial. This exploratory analysis evaluated the effect of PGBF on serum BAs and explored potential underlying mechanisms. Methods Serum BAs and 7α-hydroxy-4-cholesten-3-one (C4) were measured by HPLC-mass spectrometry (MS) using serum collected in studies of patients with NASH (NCT02413372) and in overweight/obese adults (NCT03198182) who received PGBF. Stool samples were collected in NCT03198182 to evaluate faecal BAs by liquid chromatography (LC)-MS and the faecal microbiome by metagenetic and metatranscriptomic analyses. Results Significant reductions from baseline in serum concentrations of the secondary BA, deoxycholic acid (DCA), and conjugates, were observed with PGBF, but not placebo, in patients with NASH; primary BA concentrations did not significantly change in any arm. Similar effects of PGBF on BAs were observed in overweight/obese adults, allowing for an evaluation of the effects of PGBF on the faecal microbiome and BAs. Faecal transcriptomic analysis showed that the relative abundance of the gene encoding choloylglycine hydrolase, a critical enzyme for secondary BA synthesis, was reduced after PGBF, but not placebo, administration. Furthermore, a trend of reduction in faecal secondary BAs was observed. Conclusions PGBF selectively reduced serum concentrations of DCA and conjugates in patients with NASH and in healthy overweight/obese adults. Reduced choloylglycine hydrolase gene expression and decreased faecal secondary BA levels suggest a potential role for PGBF in modulating secondary BA synthesis by gut microbiome. The clinical significance of DCA reduction post-PGBF treatment warrants further investigation. Lay summary Pegbelfermin (PGBF) is a hormone that is currently being studied in clinical trials for the treatment of non-alcoholic fatty liver disease. In this study, we show that PGBF treatment can reduce bile acids that have previously been shown to have toxic effects on the liver. Additional studies to understand how PGBF regulates bile acids may provide additional information about its potential use as a treatment for fatty liver., Graphical abstract, Highlights • Bile acids are elevated in patients with non-alcoholic steatohepatitis. • Pegbelfermin, a PEGylated human FGF21 analogue, is in phase II trials for non-alcoholic steatohepatitis. • Pegbelfermin treatment was associated with secondary, but not primary, bile acid reductions. • Pegbelfermin reduced expression of a gene responsible for secondary bile acid synthesis. • Further study is needed to assess the clinical significance of these observations.

The extracellular matrix is a key component of tissues, yet it is underrepresented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested, and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared with a standard in-solution digest, the optimized method yielded a fourfold improvement in unique ECM peptide identifications., Graphical abstract, Highlights • Decellularization drastically improves ECM coverage. • ECM coverage is the highest with a dual chemical/enzymatic digestion method. • SPEED is the best tested single-shot method for ECM identification. • A fourfold improvement in ECM peptide IDs was achieved compared with standard methods., Due to their insolubility, many extracellular matrix (ECM) proteins are resistant to typical extraction and require optimized extraction methods for proteomic analysis. A wide variety of decellularization and ECM extraction methods have been published, but it remains unclear how these methods perform compared with one another on a complex, ECM-rich sample. A direct comparison of both cell and ECM extraction methods on a whole organism sample and four additional organs serves as a reference for future ECM proteomics.

Objectives: Despite reports highlighting citrate association with different diseases, serum citrate is scarcely used for diagnosis. Existing methods to quantify citrate are limited by their complexity and practicality of implementation. A simple and rapid NMR-based method to measure circulating citrate is described here, and its analytical performance evaluated. Design: and Methods: Citrate was quantified from NMR spectra using a non-negative linear least squares deconvolution algorithm. The analytical characteristics of the assay were evaluated using CLSI guidelines. To determine if the assay has adequate sensitivity to measure clinically relevant concentrations of citrate, the assay was used to quantify citrate in apparently healthy adults (n ​= ​553), and in the general population (n ​= ​133,576). Results: The LOQ for the assay was determined to be 1.48 ​mg/dL. Linearity was demonstrated over a wide range of concentrations (1.40–4.46 ​mg/dL). Coefficients of variation (%CV) for intra- and inter-assay precision ranged from 5.8–9.3 and 5.2–9.6%, respectively. Substances tested did not elicit interference with assay results. Specimen type comparison revealed

A20 is a potent anti-inflammatory protein that mediates both inflammation and ubiquitination in mammals, but the related mechanisms are not clear. In this study, we performed mass spectrometry (MS) screening, gene ontology (GO) analysis, and coimmunoprecipitation (co-IP) in a lipopolysaccharide (LPS)-induced inflammatory cell model to identify novel A20-interacting proteins. We confirmed that the E3 ubiquitin ligase Nrdp1, also known as ring finger protein 41 (RNF41), interacted with A20 in LPS-stimulated cells. Further co-IP analysis demonstrated that when A20 was knocked out, degradation-inducing K48-linked ubiquitination of inflammatory effector MyD88 was decreased, but protein interaction-mediating K63-linked ubiquitination of another inflammatory effector TBK1 was increased. Moreover, western blot experiments showed that A20 inhibition induced an increase in levels of MyD88 and phosphorylation of downstream effector proteins as well as of TBK1 and a downstream effector, while Nrdp1 inhibition induced an increase in MyD88 but a decrease in TBK1 levels. When A20 and Nrdp1 were coinhibited, no further change in MyD88 was observed, but TBK1 levels were significantly decreased compared with those upon A20 inhibition alone. Gain- and loss-of-function analyses revealed that the ZnF4 domain of A20 is required for Nrdp1 polyubiquitination. Upon LPS stimulation, the inhibition of Nrdp1 alone increased the secretion of IL-6 and TNF-α but decreased IFN-β secretion, as observed in other studies, suggesting that Nrdp1 preferentially promotes the production of IFN-β. Taken together, these results demonstrated that A20/Nrdp1 interaction is important for A20 anti-inflammation, thus revealing a novel mechanism for the anti-inflammatory effects of A20.

The analysis of ethanol and of its congeners in blood plays an important role in forensic cases, especially when allegations are made that alcohol has been consumed after an accident. In alcoholic beverages, congener alcohols are by-products and are generated during fermentation. The assay of these compounds in serum samples and beverages has been previously performed using headspace-gas chromatography-flame ionization detection methods (HS-GC-FID). As an alternative, a robust headspace-gas chromatography-mass spectrometry (HS-GC-MS) procedure was developed and validated, which has the following advantages:•Simultaneous determination of ethanol, congener alcohols and other endogenous substances.•Reduction of matrix interference by increasing selectivity and specificity.•Clear separation of the positional isomers 3-methyl-1-butanol and 2-methyl-1-butanol., Graphical abstract Image, graphical abstract

Visual Abstract, Highlights • MDA conjugated with HSA activates T cells from human atherosclerotic plaques through a direct mechanism. • MDA-HSA also induces maturation and activation of human dendritic cells, which in turn promote activation of autologous T cells from the same donor's plaques, although this effect appears somewhat weaker than the direct activation. • M1 polarization of macrophages, potentially an atherogenic effect, were also induced by MDA-HSA. • Heat shock protein 60 was induced in T cells by MDA-HSA, is atherogenic, and could promote dendritic cell/T cell activation. • Two peptide modifications of serum albumin in atherosclerotic patients’ HSA were similar to those generated by treatment of HSA with MDA in vitro., Summary Human dendritic cells were differentiated from blood monocytes and treated with malondialdehyde (MDA) conjugated with human serum albumin (HSA). Autologous T cells from human plaques or blood were co-cultured with the pre-treated dendritic cells or treated directly. MDA modifications were studied by mass spectrometry. MDA-HSA induced a pro-inflammatory DC-mediated T-cell activation and also a strong direct effect on T cells, inhibited by an inhibitor of oxidative stress and antibodies against MDA. Atherogenic heat shock protein-60 was strongly induced in T cells activated by MDA-HSA. Two peptide modifications in atherosclerotic patients' HSA were similar to those present in in vitro MDA-modified HSA.

Interferon-induced transmembrane proteins IFITM1 and IFITM3 (IFITM1/3) play a role in both RNA viral restriction and in human cancer progression. Using immunohistochemical staining of FFPE tissue, we identified subgroups of cervical cancer patients where IFITM1/3 protein expression is inversely related to metastasis. Guide RNA-CAS9 methods were used to develop an isogenic IFITM1/IFITM3 double null cervical cancer model in order to define dominant pathways triggered by presence or absence of IFITM1/3 signalling. A pulse SILAC methodology identified IRF1, HLA-B, and ISG15 as the most dominating IFNγ inducible proteins whose synthesis was attenuated in the IFITM1/IFITM3 double-null cells. Conversely, SWATH-IP mass spectrometry of ectopically expressed SBP-tagged IFITM1 identified ISG15 and HLA-B as dominant co-associated proteins. ISG15ylation was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. Proximity ligation assays indicated that HLA-B can interact with IFITM1/3 proteins in parental SiHa cells. Cell surface expression of HLA-B was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. SWATH-MS proteomic screens in cells treated with IFITM1-targeted siRNA cells resulted in the attenuation of an interferon regulated protein subpopulation including MHC Class I molecules as well as IFITM3, STAT1, B2M, and ISG15. These data have implications for the function of IFITM1/3 in mediating IFNγ stimulated protein synthesis including ISG15ylation and MHC Class I production in cancer cells. The data together suggest that pro-metastatic growth associated with IFITM1/3 negative cervical cancers relates to attenuated expression of MHC Class I molecules that would support tumor immune escape., Graphical abstract Unlabelled Image, Highlights • IFITM1/3 expression in cervical cancers inversely correlates with metastases. • Isogenic IFITM1 and IFITM3 null cervical cancer cells were developed. • Pulse SILAC approaches were used to define IFITM1/3 dependent signalling pathways. • The major IFITM1/3-interferon-γ dependent effectors are HLA-B and ISG15. • IFITM1/3 loss would be predicted to reduce HLA expression and ISG15ylation in vivo.

Background & Aims In Wilson disease, ATP7B mutations impair copper excretion into bile. Hepatic copper accumulation may induce mild to moderate chronic liver damage or even acute liver failure. Etiologic factors for this heterogeneous phenotype remain enigmatic. Liver steatosis is a frequent finding in Wilson disease patients, suggesting that impaired copper homeostasis is linked with liver steatosis. Hepatic mitochondrial function is affected negatively both by copper overload and steatosis. Therefore, we addressed the question of whether a steatosis-promoting high-calorie diet aggravates liver damage in Wilson disease via amplified mitochondrial damage. Methods Control Atp7b+/- and Wilson disease Atp7b-/- rats were fed either a high-calorie diet (HCD) or a normal diet. Copper chelation using the high-affinity peptide methanobactin was used in HCD-fed Atp7b-/- rats to test for therapeutic reversal of mitochondrial copper damage. Results In comparison with a normal diet, HCD feeding of Atp7b-/- rats resulted in a markedly earlier onset of clinically apparent hepatic injury. Strongly increased mitochondrial copper accumulation was observed in HCD-fed Atp7b-/- rats, correlating with severe liver injury. Mitochondria presented with massive structural damage, increased H2O2 emergence, and dysfunctional adenosine triphosphate production. Hepatocellular injury presumably was augmented as a result of oxidative stress. Reduction of mitochondrial copper by methanobactin significantly reduced mitochondrial impairment and ameliorated liver damage. Conclusions A high-calorie diet severely aggravates hepatic mitochondrial and hepatocellular damage in Wilson disease rats, causing an earlier onset of the disease and enhanced disease progression., Graphical abstract

Microcin B17 (MccB17) is an antibacterial peptide produced by strains of Escherichia coli harboring the plasmid-borne mccB17 operon. MccB17 possesses many notable features. It is able to stabilize the transient DNA gyrase–DNA cleavage complex, a very efficient mode of action shared with the highly successful fluoroquinolone drugs. MccB17 stabilizes this complex by a distinct mechanism making it potentially valuable in the fight against bacterial antibiotic resistance. MccB17 was the first compound discovered from the thiazole/oxazole-modified microcins family and the linear azole-containing peptides; these ribosomal peptides are post-translationally modified to convert serine and cysteine residues into oxazole and thiazole rings. These chemical moieties are found in many other bioactive compounds like the vitamin thiamine, the anti-cancer drug bleomycin, the antibacterial sulfathiazole and the antiviral nitazoxanide. Therefore, the biosynthetic machinery that produces these azole rings is noteworthy as a general method to create bioactive compounds. Our knowledge of MccB17 now extends to many aspects of antibacterial–bacteria interactions: production, transport, interaction with its target, and resistance mechanisms; this knowledge has wide potential applicability. After a long time with limited progress on MccB17, recent publications have addressed critical aspects of MccB17 biosynthesis as well as an explosion in the discovery of new related compounds in the thiazole/oxazole-modified microcins/linear azole-containing peptides family. It is therefore timely to summarize the evidence gathered over more than 40 years about this still enigmatic molecule and place it in the wider context of antibacterials., Graphical Abstract Unlabelled Image, Highlights • Microcin B17 (MccB17) is a microbial toxin with a unique mode of action. • MccB17 stabilizes the gyrase–DNA cleavage complex; it is a potential substitute for fluoroquinolones. • The structures of microcin synthase and TldD/E have given key insight into its biosynthesis. • A variety of modified McB17s have been generated and characterized. • MccB17 has been implicated in IBD.

Background & Aims Porphyrias are caused by porphyrin accumulation resulting from defects in the heme biosynthetic pathway that typically lead to photosensitivity and possible end-stage liver disease with an increased risk of hepatocellular carcinoma. Our aims were to study the mechanism of porphyrin-induced cell damage and protein aggregation, including liver injury, where light exposure is absent. Methods Porphyria was induced in vivo in mice using 3,5-diethoxycarbonyl-1,4-dihydrocollidine or in vitro by exposing human liver Huh7 cells and keratinocytes, or their lysates, to protoporphyrin-IX, other porphyrins, or to δ-aminolevulinic acid plus deferoxamine. The livers, cultured cells, or porphyrin exposed purified proteins were analyzed for protein aggregation and oxidation using immunoblotting, mass spectrometry, and electron paramagnetic resonance spectroscopy. Consequences on cell-cycle progression were assessed. Results Porphyrin-mediated protein aggregation required porphyrin-photosensitized singlet oxygen and porphyrin carboxylate side-chain deprotonation, and occurred with site-selective native protein methionine oxidation. Noncovalent interaction of protoporphyrin-IX with oxidized proteins led to protein aggregation that was reversed by incubation with acidified n-butanol or high-salt buffer. Phototoxicity and the ensuing proteotoxicity, mimicking porphyria photosensitivity conditions, were validated in cultured keratinocytes. Protoporphyrin-IX inhibited proteasome function by aggregating several proteasomal subunits, and caused cell growth arrest and aggregation of key cell proliferation proteins. Light-independent synergy of protein aggregation was observed when porphyrin was applied together with glucose oxidase as a secondary peroxide source. Conclusions Photo-excitable porphyrins with deprotonated carboxylates mediate protein aggregation. Porphyrin-mediated proteotoxicity in the absence of light, as in the liver, requires porphyrin accumulation coupled with a second tissue oxidative injury. These findings provide a potential mechanism for internal organ damage and photosensitivity in porphyrias., Graphical abstract

Background: There is an urgent need for the identification of new, clinically useful biomarkers of CRC to enhance diagnostic and prognostic capabilities. Methods: We performed proteomic profiling on serum samples from paired pre- and post-operative CRC patients, colorectal polyps patients and healthy controls using an approach combining magnetic bead-based weak cation exchange and matrix-assisted laser desorption ionization-time of flight mass spectrometry. We next performed liquid chromatography-electrospray ionization-tandem mass spectrometry to identify the proteins and selected potential biomarker based on bioinformatics analysis of the TCGA and GEO dataset. We examined SETD7 expression in serum and tissue samples by ELISA and immunohistochemistry respectively and explored the biological function of SETD7 in vitro. Findings: 85 differentially expressed peptides were identified. Five peptides showing the most significant changes in abundance across paired pre- and post-operation CRC patients, colorectal polyps patients and healthy controls were identified as peptide regions of FGA, MUC5AC and SETD7. Bioinformatics analysis suggested that the up-regulation of SETD7 in CRC is relatively specific. Validation studies showed that SETD7 expression increased from healthy controls to those with colorectal polyps and finally CRC patients, and decreased after surgery. The sensitivity and specificity of SETD7 were 92.17% and 81.08%, with a high diagnostic value (AUC = 0.9477). In addition, SETD7 expression was significantly correlated with tumor stage and microsatellite instability. Knockdown of SETD7 inhibited cancer cell proliferation, induced G1/S cell cycle arrest and increased apoptosis. Interpretation: Our data indicate that SETD7 could serve as a potential diagnostic and prognostic biomarker for CRC. Keywords: Colorectal cancer, Proteomics, Serum biomarker, SETD7

Many current anti-cancer therapies rely on increasing the intracellular reactive oxygen and nitrogen species (RONS) contents with the aim to induce irreparable damage, which subsequently results in tumor cell death. A novel tool in cancer therapy is the use of cold atmospheric plasma (CAP), which has been found to be very effective in the treatment of many different cancer cell types in vitro as well as in vivo, mainly through the vast generation of RONS. One of the key determinants of the cell's fate will be the interaction of RONS, generated by CAP, with important proteins, i.e. redox-regulatory proteins. One such protein is cytoglobin (CYGB), a recently discovered globin proposed to be involved in the protection of the cell against oxidative stress. In this study, the effect of plasma-produced RONS on CYGB was investigated through the treatment of CYGB with CAP for different treatment times. Spectroscopic analysis of CYGB showed that although chemical modifications occur, its secondary structure remains intact. Mass spectrometry experiments identified these modifications as oxidations of mainly sulfur-containing and aromatic amino acids. With longer treatment time, the treatment was also found to induce nitration of the heme. Furthermore, the two surface-exposed cysteine residues of CYGB were oxidized upon treatment, leading to the formation of intermolecular disulfide bridges, and potentially also intramolecular disulfide bridges. In addition, molecular dynamics and docking simulations confirmed, and further show, that the formation of an intramolecular disulfide bond, due to oxidative conditions, affects the CYGB 3D structure, thereby opening the access to the heme group, through gate functioning of His117. Altogether, the results obtained in this study (1) show that plasma-produced RONS can extensively oxidize proteins and (2) that the oxidation status of two redox-active cysteines lead to different conformations of CYGB., Graphical abstract fx1