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Cystathionine β-synthase (CBS)-deficient patients are prone to vascular thrombosis. In contrast, Cbs −/− mice show no abnormalities in blood coagulation. To identify molecular basis underlying these disparately different thrombotic phenotypes, we analyzed plasma proteomes of Cbs −/− vs. Cbs +/+ mice (8-month-old, 12/group, sex-matched) and CBS −/− vs. CBS +/+ humans (37 ± 7-year-old, 10–14/group, sex-matched) using label-free mass spectrometry. We identified 117 and 41 differentiating plasma proteins in Cbs −/− mice and CBS −/− humans, respectively. Twenty-one proteins were shared between CBS −/− humans and Cbs −/− mice, with sixteen changed in the opposite direction. Proteins involved in blood coagulation and complement/coagulation cascades represented a greater fraction of the differentiating proteins in CBS −/− patients (51%) than in Cbs −/− mice (21%). Top canonical pathways, identified by Ingenuity Pathways Analysis, such as LXR/RXR, FXR/RXR activation (− log[ P -value] = 30–31) and atherosclerosis signaling (− log[ P -value] = 10–11) were similarly affected in Cbs −/− mice and CBS −/− humans. The Coagulation System was affected stronger in CBS −/− humans than in Cbs −/− mice (− log[ P -value] = 15 vs. 10, respectively) while acute phase response and complement system were affected stronger in Cbs −/− mice (− log[ P -value] = 33 and 22, respectively) than in humans (− log[ P -value] = 22 and 6, respectively). Other pathways, including IL-7 signaling and B cell development were affected only in Cbs −/− mice. Taken together, our findings suggest that differences in these processes, in particular in the Coagulation System, could account for the thrombotic phenotype in CBS −/− patients and the absence of thrombosis in Cbs −/− mice. Overall, our findings suggest that Cbs −/− mice have a better adaptive response to protect from prothrombotic effects of hyperhomocysteinemia than CBS −/− humans.

The purpose of this study was to present a chemical analysis of the metabolome of cell aggregates of the tree fern (J.D. Hooker) cell suspension culture. The LC/MS and GC/MS techniques were used for identification of metabolites. The kinetics of fresh weight, dry weight, and ash content showed 3.5-fold increases during 15-day-long culture. The analysis demonstrated high metabolic activity of cultured cells. In total, 160 metabolites from primary and secondary metabolism and almost 2000 compounds of unknown identity were identified. Three flavonoids-the chalcone isookanin [(2S)-2-(3,4-dihydroxyphenyl)-7,8-dihydroxy-2,3-dihydrochromen-4-one], a methoxy derivative of the flavone gardenin B (5-Hydroxy-2-(4-methoxyphenyl)-6,7,8-trimethoxy-4H-1-benzopyran-4-one), and the isoflavone tectoridin (4',5-Dihydro-6-methoxy-7-(O-glucoside)isoflavone)-had not been previously detected in the cell culture of . Principal component analysis revealed five distinct groups of samples; groups 4 and 5 showed the greatest similarity and corresponded to cultures on days 12 and 15, respectively. The number of differentiating compounds was 75, indicated by a heatmap showing positive and negative correlations between the days of culture. The studies described in this paper are crucial for further identification of metabolites and establishing the relationship between the metabolic composition of tree fern cells in culture and their biological activity, assessed by physiological parameters. By determining the relationship between the chemical composition of cells and their growth from culture initiation to senescence, we will provide a more complete picture of the potential for environmental factors to regulate this relationship. Based on previous studies, environmental stimuli such as electromagnetic fields or light of different wavelengths can result in altered growth physiology and cell mass, as well as metabolite diversification and accumulation. The research results presented in this paper provide a foundation for further studies aimed at predicting and regulating the productivity of cells in suspension culture and elucidating the significance of tree fern-derived metabolic products in human cell biology, particularly in thyroid cells.

Background T cell-mediated rejection (TCMR) remains a challenge in kidney transplantation. Based on a histopathological biopsy examination, patients can be classified into groups such as no rejection (NR), borderline rejection (BR; Banff category 3), and acute rejection (AR; Banff category 4). Yet, this classification is not sufficient, since for the borderline cases a number of patients may require a clinical intervention. Thus, a robust classification by biopsy proteome profiling may provide a solution. Methods In this work, kidney tissue from patients classified into NR, BR, and AR were subjected to MS-based proteomic profiling. Subsequently, a panel of four proteins (GNB4, PDK1, AGXT, CD73) was selected for validation by immunohistochemistry (IHC). This retrospective study was approved by the Bioethics Committee of the Medical University of Gdańsk, no. NKBBN/201/2021. Results Proteomic analysis identified 2547 proteins whose abundance profiles demonstrated strong concordance between the BR and AR groups. In a quantitative comparison between the BR and AR groups, GNB4 and AGXT emerged as significantly differentiating. Moreover, AGXT was indicated as a potential biomarker following ROC analysis. PDK1 and CD73 were found to best classify the samples in a binary analysis. IHC confirmed only upregulation of GNB4 in immune cells and PDK1 in macrophages, with no significant changes in the tubular epithelium. Conclusions Thus, GNB4 and PDK1 in immune cells and macrophages have been identified as a potential target for further extensive studies. If their relevance were to be confirmed in a larger patient cohort, their IHC analysis could serve as an extension of established histopathological classification in the context of kidney transplant rejection.

Formalin-fixed paraffin-embedded (FFPE) tissue specimens constitute a highly valuable source of clinical material for retrospective molecular studies. However, metabolomic assessment of such archival material remains still in its infancy. Hence, there is an urgent need for efficient methods enabling extraction and profiling of metabolites present in FFPE tissue specimens. Here we demonstrate the methodology for isolation of primary metabolites from archival tissues; either fresh-frozen, formalin-fixed or formalin-fixed and paraffin-embedded specimens of mouse kidney were analysed and compared in this work. We used gas chromatography followed by mass spectrometry (GC/MS approach) to identify about 80 metabolites (including amino acids, saccharides, carboxylic acids, fatty acids) present in such archive material. Importantly, about 75% of identified compounds were detected in all three types of specimens. Moreover, we observed that fixation with formalin itself (and their duration) did not affect markedly the presence of particular metabolites in tissue-extracted material, yet fixation for 24h could be recommended as a practical standard. Paraffin embedding influenced efficiency of extraction, which resulted in reduced quantities of several compounds. Nevertheless, we proved applicability of FFPE specimens for non-targeted GS/MS-based profiling of tissue metabolome, which is of great importance for feasibility of metabolomics studies using retrospective clinical material.

In this study, proteomic and metabolomic changes in leaves and roots of two barley (Hordeum vulgare L.) genotypes, with contrasting drought tolerance, subjected to water deficit were investigated. Our two-dimensional electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF and MALDI-TOF/TOF) analyses revealed 121 drought-responsive proteins in leaves and 182 in roots of both genotypes. Many of the identified drought-responsive proteins were associated with processes that are typically severely affected during water deficit, including photosynthesis and carbon metabolism. However, the highest number of identified leaf and root proteins represented general defense mechanisms. In addition, changes in the accumulation of proteins that represent processes formerly unassociated with drought response, e.g., phenylpropanoid metabolism, were also identified. Our tandem gas chromatography - time of flight mass spectrometry (GC/MS TOF) analyses revealed approximately 100 drought-affected low molecular weight compounds representing various metabolite types with amino acids being the most affected metabolite class. We compared the results from proteomic and metabolomic analyses to search for existing relationship between these two levels of molecular organization. We also uncovered organ specificity of the observed changes and revealed differences in the response to water deficit of drought susceptible and tolerant barley lines. Particularly, our results indicated that several of identified proteins and metabolites whose accumulation levels were increased with drought in the analyzed susceptible barley variety revealed elevated constitutive accumulation levels in the drought-resistant line. This may suggest that constitutive biochemical predisposition represents a better drought tolerance mechanism than inducible responses.

Spinocerebellar ataxia type 3 (SCA3/MJD) is a neurodegenerative disease caused by CAG expansion in mutant ATXN3 gene. The resulting PolyQ tract in mutant ataxin-3 protein is toxic to neurons and currently no effective treatment exists. Function of both normal and mutant ataxin-3 is pleiotropic by their interactions and the influence on protein level. Our new preclinical Ki150 model with over 150 CAG/Q in ataxin-3 has robust aggregates indicating the presence of a process that enhances the interaction between proteins. Interactions in large complexes may resemble the real-life inclusion interactions and was never examined before for mutant and normal ataxin-3 and in homozygous mouse model with long polyQ tract. We fractionated ataxin-3-positive large complexes and independently we pulled-down ataxin-3 from brain lysates, and both were followed by proteomics. Among others, mutant ataxin-3 abnormally interacted with subunits of large complexes such as Cct5 and 6, Tcp1, and Camk2a and Camk2b. Surprisingly, the complexes exhibit circular molecular structure which may be linked to the process of aggregates formation where annular aggregates are intermediate stage to fibrils which may indicate novel ataxin-3 mode of interactions. The protein complexes were involved in transport of mitochondria in axons which was confirmed by altered motility of mitochondria along SCA3 Ki150 neurites.

The modification of protein lysine residues by the thioester homocysteine (Hcy)-thiolactone has been implicated in cardiovascular and neurodegenerative diseases. However, only a handful of proteins carrying Hcy on specific lysine residues have been identified and quantified in humans or animals. In the present work, we developed a liquid chromatography/mass spectrometry targeted assay, based on multiple reaction monitoring, for quantification of N -Hcy-Lys212 (K212Hcy) and N -Hcy-Lys525 (K525Hcy) sites in serum albumin in mice. Using this assay, we found that female (n = 20) and male (n = 13) Cbs −/− mice had significantly elevated levels of K212Hcy and K525Hcy modifications in serum albumin relative to their female (n = 19) and male (n = 17) Cbs +/− littermates. There was significantly more K212Hcy modification in Cbs −/− males than in Cbs −/− females (5.78 ± 4.21 vs . 3.15 ± 1.38 units, P  = 0.023). Higher K212Hcy levels in males than in females were observed also in Cbs +/− mice (2.72 ± 0.81 vs . 1.89 ± 1.07 units, P  = 0.008). In contrast, levels of the K525Hcy albumin modification were similar between males and females, both in Cbs −/− and Cbs +/− mice. These findings suggest that the sex-specific K212Hcy modification in albumin might have an important biological function in mice that is not affected by the Cbs genotype.

The aim of this study was to investigate the effect of an abiotic factor, i.e., lead at various concentrations (low causing a hormesis effect and causing high toxicity effects), on the generation of signalling molecules in pea (Pisum sativum L. cv. Cysterski) seedlings and then during infestation by the pea aphid (Acyrthosiphon pisum Harris). The second objective was to verify whether the presence of lead in pea seedling organs and induction of signalling pathways dependent on the concentration of this metal trigger defense responses to A. pisum. Therefore, the profile of flavonoids and expression levels of genes encoding enzymes of the flavonoid biosynthesis pathway (phenylalanine ammonialyase and chalcone synthase) were determined. A significant accumulation of total salicylic acid (TSA) and abscisic acid (ABA) was recorded in the roots and leaves of pea seedlings growing on lead-supplemented medium and next during infestation by aphids. Increased generation of these phytohormones strongly enhanced the biosynthesis of flavonoids, including a phytoalexin, pisatin. This research provides insights into the cross-talk between the abiotic (lead) and biotic factor (aphid infestation) on the level of the generation of signalling molecules and their role in the induction of flavonoid biosynthesis.

A wide range of compounds that occur in the genus Hypericum are listed as effective drugs of natural origin. The main biological activities of several Hypericum representatives are due to the presence of naphthodianthrones, phloroglucinols, and other diverse groups of secondary metabolites that synergistically contribute to their therapeutic effects. The regulation of biosynthesis of hypericin as the key bioactive naphthodianthrone remains uncertain. Here, we present liquid chromatography mass spectrometry-based phenotyping of 17 Hypericum species, the results of which suggest an important role for skyrin and its derivatives in the polyketide pathway that leads to hypericin formation. Moreover, we report for the first time the presence of new metabolites in the genus Hypericum that are related to classes of anthraquinones, their derivatives, and phloroglucinols. As skyrin and other species of anthraquinones are rarely found in higher plants but frequently occur in fungal microorganisms, the obtained results suggest that further research on the synthesis pathways of hypericin and the role of anthraquinone derivatives in plant metabolism should be carried out. The fact that these compounds are commonly synthesized in endophytic fungi and perhaps there is some similarity in the metabolic pathways between these organisms should also be investigated.

Spinocerebellar ataxia type 3 (SCA3/MJD) is caused by CAG expansion mutation resulting in a long polyQ domain in mutant ataxin-3. The mutant protein is a special type of protease, deubiquitinase, which may indicate its prominent impact on the regulation of cellular proteins levels and activity. Yet, the global model picture of SCA3 disease progression on the protein level, molecular pathways in the brain, and neurons, is largely unknown. Here, we investigated the molecular SCA3 mechanism using an interdisciplinary research paradigm combining behavioral and molecular aspects of SCA3 in the knock-in ki91 model. We used the behavior, brain magnetic resonance imaging (MRI) and brain tissue examination to correlate the disease stages with brain proteomics, precise axonal proteomics, neuronal energy recordings, and labeling of vesicles. We have demonstrated that altered metabolic and mitochondrial proteins in the brain and the lack of weight gain in Ki91 SCA3/MJD mice is reflected by the failure of energy metabolism recorded in neonatal SCA3 cerebellar neurons. We have determined that further, during disease progression, proteins responsible for metabolism, cytoskeletal architecture, vesicular, and axonal transport are disturbed, revealing axons as one of the essential cell compartments in SCA3 pathogenesis. Therefore we focus on SCA3 pathogenesis in axonal and somatodendritic compartments revealing highly increased axonal localization of protein synthesis machinery, including ribosomes, translation factors, and RNA binding proteins, while the level of proteins responsible for cellular transport and mitochondria was decreased. We demonstrate the accumulation of axonal vesicles in neonatal SCA3 cerebellar neurons and increased phosphorylation of SMI-312 positive adult cerebellar axons, which indicate axonal dysfunction in SCA3. In summary, the SCA3 disease mechanism is based on the broad influence of mutant ataxin-3 on the neuronal proteome. Processes central in our SCA3 model include disturbed localization of proteins between axonal and somatodendritic compartment, early neuronal energy deficit, altered neuronal cytoskeletal structure, an overabundance of various components of protein synthesis machinery in axons.