Hepatoprotective Activity of Lignin-Derived Polyphenols Dereplicated Using High-Resolution Mass Spectrometry, In Vivo Experiments, and Deep Learning

Chronic liver diseases affect more than 1 billion people worldwide and represent one of the main public health issues. Nonalcoholic fatty liver disease (NAFLD) accounts for the majority of mortal cases, while there is no currently approved therapeutics for its treatment. One of the prospective approaches to NAFLD therapy is to use a mixture of natural compounds. They showed effectiveness in alleviating NAFLD-related conditions including steatosis, fibrosis, etc. However, understanding the mechanism of action of such mixtures is important for their rational application. In this work, we propose a new dereplication workflow for deciphering the mechanism of action of the lignin-derived natural compound mixture. The workflow combines the analysis of molecular components with high-resolution mass spectrometry, selective chemical tagging and deuterium labeling, liver tissue penetration examination, assessment of biological activity in vitro, and computational chemistry tools used to generate putative structural candidates. Molecular docking was used to propose the potential mechanism of action of these structures, which was assessed by a proteomic experiment

Potential Urine Proteomic Biomarkers for Focal Segmental Glomerulosclerosis and Minimal Change Disease

Primary focal segmental glomerulosclerosis (FSGS), along with minimal change disease (MCD), are diseases with primary podocyte damage that are clinically manifested by the nephrotic syndrome. The pathogenesis of these podocytopathies is still unknown, and therefore, the search for biomarkers of these diseases is ongoing. Our aim was to determine of the proteomic profile of urine from patients with FSGS and MCD. Patients with a confirmed diagnosis of FSGS (n = 30) and MCD (n = 9) were recruited for the study. For a comprehensive assessment of the severity of FSGS a special index was introduced, which was calculated as follows: the first score was assigned depending on the level of eGFR, the second score—depending on the proteinuria level, the third score—resistance to steroid therapy. Patients with the sum of these scores of less than 3 were included in group 1, with 3 or more—in group 2. The urinary proteome was analyzed using liquid chromatography/mass spectrometry. The proteome profiles of patients with severe progressive FSGS from group 2, mild FSGS from group 1 and MCD were compared. Results of the label free analysis were validated using targeted LC-MS based on multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS) available for 47 of the 76 proteins identified as differentiating between at least one pair of groups. Quantitative MRM SIS validation measurements for these 47 proteins revealed 22 proteins with significant differences between at least one of the two group pairs and 14 proteins were validated for both comparisons. In addition, all of the 22 proteins validated by MRM SIS analysis showed the same direction of change as at the discovery stage with label-free LC-MS analysis, i.e., up or down regulation in MCD and FSGS1 against FSGS2. Patients from the FSGS group 2 showed a significantly different profile from both FSGS group 1 and MCD. Among the 47 significantly differentiating proteins, the most significant were apolipoprotein A-IV, hemopexin, vitronectin, gelsolin, components of the complement system (C4b, factors B and I), retinol- and vitamin D-binding proteins. Patients with mild form of FSGS and MCD showed lower levels of Cystatin C, gelsolin and complement factor I

Antitumor Effect of Bleomycin Nanoaerosol in Murine Carcinoma Model

Bleomycin, which is widely used as an antitumor agent, possesses serious adverse effects such as pulmonary toxicity. Local nanoaerosol deposition for lung cancer treatment is a promising alternative to drug delivery to lung lesions. The aim of this work is to test the hypothesis that bleomycin nanoaerosol can be effectively used to treat multiple lung metastases. To obtain bleomycin nanoaerosol, an aerosol generator based on electrospray of a solution of a nonvolatile substance with gas-phase neutralization of charged aerosol particles was used. Lung metastases in murine Lewis lung carcinoma and B16 melanoma animal models were counted. The effect of inhaled bleomycin nanoparticles on the number and volume of metastases, as well as pulmonary side effects, was investigated. Using a mouse exposure chamber, the dose-dependent effect of inhaled bleomycin on tumor volume was evaluated in comparison with intraperitoneal administration. Bleomycin nanoaerosol reduced the volume of metastases and produced a higher antitumor effect at much lower doses. It has been established that long-term exposure to nanoaerosol with a low dose of bleomycin is capable of suppressing cancer cell growth. The treatment was well tolerated. In the lungs, minor changes were found in the form of focal-diffuse infiltration of the lung parenchyma

Targeted MRM Quantification of Urinary Proteins in Chronic Kidney Disease Caused by Glomerulopathies

Glomerulopathies with nephrotic syndrome that are resistant to therapy often progress to end-stage chronic kidney disease (CKD) and require timely and accurate diagnosis. Targeted quantitative urine proteome analysis by mass spectrometry (MS) with multiple-reaction monitoring (MRM) is a promising tool for early CKD diagnostics that could replace the invasive biopsy procedure. However, there are few studies regarding the development of highly multiplexed MRM assays for urine proteome analysis, and the two MRM assays for urine proteomics described so far demonstrate very low consistency. Thus, the further development of targeted urine proteome assays for CKD is actual task. Herein, a BAK270 MRM assay previously validated for blood plasma protein analysis was adapted for urine-targeted proteomics. Because proteinuria associated with renal impairment is usually associated with an increased diversity of plasma proteins being present in urine, the use of this panel was appropriate. Another advantage of the BAK270 MRM assay is that it includes 35 potential CKD markers described previously. Targeted LC-MRM MS analysis was performed for 69 urine samples from 46 CKD patients and 23 healthy controls, revealing 138 proteins that were found in ≥2/3 of the samples from at least one of the groups. The results obtained confirm 31 previously proposed CKD markers. Combination of MRM analysis with machine learning for data processing was performed. As a result, a highly accurate classifier was developed (AUC = 0.99) that enables distinguishing between mild and severe glomerulopathies based on the assessment of only three urine proteins (GPX3, PLMN, and A1AT or SHBG)

Prognosis of Alzheimer’s Disease Using Quantitative Mass Spectrometry of Human Blood Plasma Proteins and Machine Learning

Early recognition of the risk of Alzheimer’s disease (AD) onset is a global challenge that requires the development of reliable and affordable screening methods for wide-scale application. Proteomic studies of blood plasma are of particular relevance; however, the currently proposed differentiating markers are poorly consistent. The targeted quantitative multiple reaction monitoring (MRM) assay of the reported candidate biomarkers (CBs) can contribute to the creation of a consistent marker panel. An MRM-MS analysis of 149 nondepleted EDTA–plasma samples (MHRC, Russia) of patients with AD (n = 47), mild cognitive impairment (MCI, n = 36), vascular dementia (n = 8), frontotemporal dementia (n = 15), and an elderly control group (n = 43) was performed using the BAK 125 kit (MRM Proteomics Inc., Canada). Statistical analysis revealed a significant decrease in the levels of afamin, apolipoprotein E, biotinidase, and serum paraoxonase/arylesterase 1 associated with AD. Different training algorithms for machine learning were performed to identify the protein panels and build corresponding classifiers for the AD prognosis. Machine learning revealed 31 proteins that are important for AD differentiation and mostly include reported earlier CBs. The best-performing classifiers reached 80% accuracy, 79.4% sensitivity and 83.6% specificity and were able to assess the risk of developing AD over the next 3 years for patients with MCI. Overall, this study demonstrates the high potential of the MRM approach combined with machine learning to confirm the significance of previously identified CBs and to propose consistent protein marker panels

The Dynamics of β-Amyloid Proteoforms Accumulation in the Brain of a 5xFAD Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is the leading cause of dementia among the elderly. Neuropathologically, AD is characterized by the deposition of a 39- to 42-amino acid long β-amyloid (Aβ) peptide in the form of senile plaques. Several post-translational modifications (PTMs) in the N-terminal domain have been shown to increase the aggregation and cytotoxicity of Aβ, and specific Aβ proteoforms (e.g., Aβ with isomerized D7 (isoD7-Aβ)) are abundant in the senile plaques of AD patients. Animal models are indispensable tools for the study of disease pathogenesis, as well as preclinical testing. In the presented work, the accumulation dynamics of Aβ proteoforms in the brain of one of the most widely used amyloid-based mouse models (the 5xFAD line) was monitored. Mass spectrometry (MS) approaches, based on ion mobility separation and the characteristic fragment ion formation, were applied. The results indicated a gradual increase in the Aβ fraction of isoD7-Aβ, starting from approximately 8% at 7 months to approximately 30% by 23 months of age. Other specific PTMs, in particular, pyroglutamylation, deamidation, and oxidation, as well as phosphorylation, were also monitored. The results for mice of different ages demonstrated that the accumulation of Aβ proteoforms correlate with the formation of Aβ deposits. Although the mouse model cannot be a complete analogue of the processes occurring in the human brain in AD, and several of the observed parameters differ significantly from human values supposedly due to the limited lifespan of the model animals, this dynamic study provides evidence on at least one of the possible mechanisms that can trigger amyloidosis in AD, i.e., the hypothesis on the relationship between the accumulation of isoD7-Aβ and the progression of AD-like pathology

Differential Diagnosis of Preeclampsia Based on Urine Peptidome Features Revealed by High Resolution Mass Spectrometry

Preeclampsia (PE) is a severe pregnancy complication, which may be considered as a systemic response in the second half of pregnancy to physiological failures in the first trimester, and can lead to very serious consequences for the health of the mother and fetus. Since PE is often associated with proteinuria, urine proteomic assays may represent a powerful tool for timely diagnostics and appropriate management. High resolution mass spectrometry was applied for peptidome analysis of 127 urine samples of pregnant women with various hypertensive complications: normotensive controls (n = 17), chronic hypertension (n = 16), gestational hypertension (n = 15), mild PE (n = 25), severe PE (n = 25), and 29 patients with complicated diagnoses. Analysis revealed 3869 peptides, which mostly belong to 116 groups with overlapping sequences. A panel of 22 marker peptide groups reliably differentiating PE was created by multivariate statistics, and included 15 collagen groups (from COL1A1, COL3A1, COL2A1, COL4A4, COL5A1, and COL8A1), and single loci from alpha-1-antitrypsin, fibrinogen, membrane-associated progesterone receptor component 1, insulin, EMI domain-containing protein 1, lysine-specific demethylase 6B, and alpha-2-HS-glycoprotein each. ROC analysis of the created model resulted in 88% sensitivity, 96.8% specificity, and receiver operating characteristic curve (AUC) = 0.947. Obtained results confirm the high diagnostic potential of urinary peptidome profiling for pregnancy hypertensive disorders diagnostics

Effect of MSCs and MSC-Derived Extracellular Vesicles on Human Blood Coagulation

Mesenchymal stem cells (MSCs) have emerged as a potent therapeutic tool for the treatment of a number of pathologies, including immune pathologies. However, unwelcome effects of MSCs on blood coagulation have been reported, motivating us to explore the thrombotic properties of human MSCs from the umbilical cord. We revealed strong procoagulant effects of MSCs on human blood and platelet-free plasma using rotational thromboelastometry and thrombodynamic tests. A similar potentiation of clotting was demonstrated for MSC-derived extracellular vesicles (EVs). To offer approaches to avoid unwanted effects, we studied the impact of a heparin supplement on MSC procoagulative properties. However, MSCs still retained procoagulant activity toward blood from children receiving a therapeutic dose of unfractionated heparin. An analysis of the mechanisms responsible for the procoagulant effect of MSCs/EVs revealed the presence of tissue factor and other proteins involved in coagulation-associated pathways. Also, we found that some MSCs and EVs were positive for annexin V, which implies the presence of phosphatidylserine on their surfaces, which can potentiate clot formation. Thus, we revealed procoagulant activity of MSCs/EVs associated with the presence of phosphatidylserine and tissue factor, which requires further analysis to avoid adverse effects of MSC therapy in patients with a risk of thrombosis

Combined Impact of Magnetic Force and Spaceflight Conditions on Escherichia coli Physiology

Changes in bacterial physiology caused by the combined action of the magnetic force and microgravity were studied in Escherichia coli grown using a specially developed device aboard the International Space Station. The morphology and metabolism of E. coli grown under spaceflight (SF) or combined spaceflight and magnetic force (SF + MF) conditions were compared with ground cultivated bacteria grown under standard (control) or magnetic force (MF) conditions. SF, SF + MF, and MF conditions provided the up-regulation of Ag43 auto-transporter and cell auto-aggregation. The magnetic force caused visible clustering of non-sedimenting bacteria that formed matrix-containing aggregates under SF + MF and MF conditions. Cell auto-aggregation was accompanied by up-regulation of glyoxylate shunt enzymes and Vitamin B12 transporter BtuB. Under SF and SF + MF but not MF conditions nutrition and oxygen limitations were manifested by the down-regulation of glycolysis and TCA enzymes and the up-regulation of methylglyoxal bypass. Bacteria grown under combined SF + MF conditions demonstrated superior up-regulation of enzymes of the methylglyoxal bypass and down-regulation of glycolysis and TCA enzymes compared to SF conditions, suggesting that the magnetic force strengthened the effects of microgravity on the bacterial metabolism. This strengthening appeared to be due to magnetic force-dependent bacterial clustering within a small volume that reinforced the effects of the microgravity-driven absence of convectional flows