Proteomic Profile of Flaxseed (Linum usitatissimum L.) Products as Influenced by Protein Concentration Method and Cultivar.

The research is focused on the quantitative evaluation of the flaxseed (Linum usitatissimum L.) proteome at the level of seed cake (SC), fine flour-sieved a fraction below 250 µm (FF)-and protein concentrate (PC). The evaluation was performed on three oilseed flax cultivars (Agriol, Raciol, and Libra) with different levels of α-linolenic acid content using LC-MS/MS (shotgun proteomics) analysis, which was finalized by database searching using the NCBI protein database for Linum usitatissimum and related species. A total of 2560 protein groups (PGs) were identified, and their relative abundance was calculated. A set of 33 quantitatively most significant PGs was selected for further characterization. The selected PGs were divided into four classes-seed storage proteins (11S globulins and conlinins), oleosins, defense- and stress-related proteins, and other major proteins (mainly including enzymes). Seed storage proteins were found to be the most abundant proteins. Specifically, 11S globulins accounted for 41-44% of SC proteins, 40-46% of FF proteins, and 72-84% of PC proteins, depending on the cultivar. Conlinins (2S albumins) were the most abundant in FF, ranging from 10 to 13% (depending on cultivar). The second most important class from the point of relative abundance was oleosins, which were represented in SC and FF in the range of 2.1-3.8%, but only 0.36-1.20% in PC. Surprisingly, a relatively high abundance of chitinase was found in flax products as a protein related to defence and stress reactions.

Discriminatory power of MALDI-TOF MS protein profiling analysis of pork meat and meat products.

Forty different sample preparation methods were tested to obtain the most informative MALDI-TOF MS protein profiles of pork meat. Extraction by 25% formic acid with the assistance of zirconia-silica beads followed by defatting by methanol:chloroform mixture (1:1, v/v) and deposition by using the layer-by-layer method was determined as the optimum sample preparation protocol. The discriminatory power of the method was then examined on samples of pork meat and meat products. The method was able to discriminate between selected salami based on the production method and brand and was able to monitor the ripening process in salami. However, it was not able to differentiate between different brands of pork ham or closely located parts of pork meat. In the latter case, a more comprehensive analysis using LC-MS/MS was used to assess the differences in protein abundance and their relation to the outputs of MALDI - TOF MS profiling.

Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer.

High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of 'core ascitic EV-associated proteins' but also defined their subset unique to HGSC ascites. Using single-cell RNA sequencing data, we mapped the origin of HGSC-specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non-malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type-specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics.

N-glycan profiling of tissue samples to aid breast cancer subtyping.

Breast cancer is a highly heterogeneous disease. Its intrinsic subtype classification for diagnosis and choice of therapy traditionally relies on the presence of characteristic receptors. Unfortunately, this classification is often not sufficient for precise prediction of disease prognosis and treatment efficacy. The N-glycan profiles of 145 tumors and 10 healthy breast tissues were determined using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry. The tumor samples were classified into Mucinous, Lobular, No-Special-Type, Human Epidermal Growth Factor 2 + , and Triple-Negative Breast Cancer subtypes. Statistical analysis was conducted using the reproducibility-optimized test statistic software package in R, and the Wilcoxon rank sum test with continuity correction. In total, 92 N-glycans were detected and quantified, with 59 consistently observed in over half of the samples. Significant variations in N-glycan signals were found among subtypes. Mucinous tumor samples exhibited the most distinct changes, with 28 significantly altered N-glycan signals. Increased levels of tri- and tetra-antennary N-glycans were notably present in this subtype. Triple-Negative Breast Cancer showed more N-glycans with additional mannose units, a factor associated with cancer progression. Individual N-glycans differentiated Human Epidermal Growth Factor 2 + , No-Special-Type, and Lobular cancers, whereas lower fucosylation and branching levels were found in N-glycans significantly increased in Luminal subtypes (Lobular and No-Special-Type tumors). Clinically normal breast tissues featured a higher abundance of signals corresponding to N-glycans with bisecting moiety. This research confirms that histologically distinct breast cancer subtypes have a quantitatively unique set of N-glycans linked to clinical parameters like tumor size, proliferative rate, lymphovascular invasion, and metastases to lymph nodes. The presented results provide novel information that N-glycan profiling could accurately classify human breast cancer samples, offer stratification of patients, and ongoing disease monitoring.

Comparison of Yield Characteristics, Chemical Composition, Lignans Content and Antioxidant Potential of Experimentally Grown Six Linseed (Linum usitatissimum L.) Cultivars.

Linseed represents a rich source of nutritional, functional and health-beneficial compounds. Nevertheless, the chemical composition and content of bioactive compounds may be quite variable and potentially affected by various factors, including genotype and the environment. In this study, the proximate chemical composition, lignans content and antioxidant potential of six experimentally grown linseed cultivars were assessed and compared. A diagonal cultivation trial in the University of South Bohemia Experimental Station in Ceské Budejovice, Czech Republic, was established in three subsequent growing seasons (2018, 2019 and 2020). The results showed that the cultivar and growing conditions influenced most studied parameters. The lack of precipitation in May and June 2019 negatively affected the seed yield and the level of secoisolariciresinol diglucoside but did not decrease the crude protein content, which was negatively related to the oil content. The newly developed method for lignans analysis allowed the identification and quantification of secoisolariciresinol diglucoside and matairesinol. Their content correlated positively with the total polyphenol content and antioxidant assays (DPPH and ABTS radical scavenging activity), indicating the significant contribution to the biofunctional properties of linseed. On the other hand, we did not detect minor linseed lignans, pinoresinol and lariciresinol. The results of this study showed the importance of cultivar and growing conditions factors on the linseed chemical composition and the lignans content, determining its nutritional and medicinal properties.

Unraveling Epigenetic Changes in A. thaliana Calli: Impact of HDAC Inhibitors.

The ability for plant regeneration from dedifferentiated cells opens up the possibility for molecular bioengineering to produce crops with desirable traits. Developmental and environmental signals that control cell totipotency are regulated by gene expression via dynamic chromatin remodeling. Using a mass spectrometry-based approach, we investigated epigenetic changes to the histone proteins during callus formation from roots and shoots of Arabidopsis thaliana seedlings. Increased levels of the histone H3.3 variant were found to be the major and most prominent feature of 20-day calli, associated with chromatin relaxation. The methylation status in root- and shoot-derived calli reached the same level during long-term propagation, whereas differences in acetylation levels provided a long-lasting imprint of root and shoot origin. On the other hand, epigenetic signs of origin completely disappeared during 20 days of calli propagation in the presence of histone deacetylase inhibitors (HDACi), sodium butyrate, and trichostatin A. Each HDACi affected the state of post-translational histone modifications in a specific manner; NaB-treated calli were epigenetically more similar to root-derived calli, and TSA-treated calli resembled shoot-derived calli.

Benchmarking of Two Peptide Clean-Up Protocols: SP2 and Ethyl Acetate Extraction for Sodium Dodecyl Sulfate or Polyethylene Glycol Removal from Plant Samples before LC-MS/MS.

The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for 10-ng, 250-ng, and 10-µg peptide samples, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the most robust approach, even for the simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction.

Unraveling adipose tissue proteomic landscapes in severe obesity: insights into metabolic complications and potential biomarkers.

In this study, we aimed to comprehensively characterize the proteomic landscapes of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) in patients with severe obesity, to establish their associations with clinical characteristics, and to identify potential serum protein biomarkers indicative of tissue-specific alterations or metabolic states. We conducted a cross-sectional analysis of 32 patients with severe obesity (16 males and 16 females) of Central European descent who underwent bariatric surgery. Clinical parameters and body composition were assessed using dual-energy X-ray absorptiometry (DXA) and bioelectrical impedance, with 15 patients diagnosed with type 2 diabetes (T2D) and 17 with hypertension. Paired SAT and VAT samples, along with serum samples, were subjected to state-of-the-art proteomics liquid chromatography-mass spectrometry (LC-MS). Our analysis identified 7,284 proteins across SAT and VAT, with 1,249 differentially expressed proteins between the tissues and 1,206 proteins identified in serum. Correlation analyses between differential protein expression and clinical traits suggest a significant role of SAT in the pathogenesis of obesity and related metabolic complications. Specifically, the SAT proteomic profile revealed marked alterations in metabolic pathways and processes contributing to tissue fibrosis and inflammation. Although we do not establish a definitive causal relationship, it appears that VAT might respond to SAT metabolic dysfunction by potentially enhancing mitochondrial activity and expanding its capacity. However, when this adaptive response is exceeded, it could possibly contribute to insulin resistance (IR) and in some cases, it may be associated with the progression to T2D. Our findings provide critical insights into the molecular foundations of SAT and VAT in obesity and may inform the development of targeted therapeutic strategies.NEW & NOTEWORTHY This study provides insights into distinct proteomic profiles of subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and serum in patients with severe obesity and their associations with clinical traits and body composition. It underscores SAT's crucial role in obesity development and related complications, such as insulin resistance (IR) and type 2 diabetes (T2D). Our findings emphasize the importance of understanding the SAT and VAT balance in energy homeostasis, proteostasis, and the potential role of SAT capacity in the development of metabolic disorders.

Extraction Protocol for Parallel Analysis of Proteins and DNA from Ancient Teeth and Dental Calculus.

Dental calculus is becoming a crucial material in the study of past populations with increasing interest in its proteomic and genomic content. Here, we suggest further development of a protocol for analysis of ancient proteins and a combined approach for subsequent ancient DNA extraction. We tested the protocol on recent teeth, and the optimized protocol was applied to ancient tooth to limit the destruction of calculus as it is a precious and irreplaceable source of dietary, microbiological, and ecological information in the archeological context. Finally, the applicability of the protocol was demonstrated on samples of the ancient calculus.

Spatial positioning of preimplantation mouse embryo cells is regulated by mTORC1 and m7G-cap-dependent translation at the 8- to 16-cell transition.

Preimplantation mouse embryo development involves temporal-spatial specification and segregation of three blastocyst cell lineages: trophectoderm, primitive endoderm and epiblast. Spatial separation of the outer-trophectoderm lineage from the two other inner-cell-mass (ICM) lineages starts with the 8- to 16-cell transition and concludes at the 32-cell stages. Accordingly, the ICM is derived from primary and secondary contributed cells; with debated relative EPI versus PrE potencies. We report generation of primary but not secondary ICM populations is highly dependent on temporal activation of mammalian target of Rapamycin (mTOR) during 8-cell stage M-phase entry, mediated via regulation of the 7-methylguanosine-cap (m7G-cap)-binding initiation complex (EIF4F) and linked to translation of mRNAs containing 5' UTR terminal oligopyrimidine (TOP-) sequence motifs, as knockdown of identified TOP-like motif transcripts impairs generation of primary ICM founders. However, mTOR inhibition-induced ICM cell number deficits in early blastocysts can be compensated by the late blastocyst stage, after inhibitor withdrawal; compensation likely initiated at the 32-cell stage when supernumerary outer cells exhibit molecular characteristics of inner cells. These data identify a novel mechanism specifically governing initial spatial segregation of mouse embryo blastomeres, that is distinct from those directing subsequent inner cell formation, contributing to germane segregation of late blastocyst lineages.

BABA-induced pathogen resistance: a multi-omics analysis of the tomato response reveals a hyper-receptive status involving ethylene.

Prior exposure to microbial-associated molecular patterns or specific chemical compounds can promote plants into a primed state with stronger defence responses. ß-aminobutyric acid (BABA) is an endogenous stress metabolite that induces resistance protecting various plants towards diverse stresses. In this study, by integrating BABA-induced changes in selected metabolites with transcriptome and proteome data, we generated a global map of the molecular processes operating in BABA-induced resistance (BABA-IR) in tomato. BABA significantly restricts the growth of the pathogens Oidium neolycopersici and Phytophthora parasitica but not Botrytis cinerea. A cluster analysis of the upregulated processes showed that BABA acts mainly as a stress factor in tomato. The main factor distinguishing BABA-IR from other stress conditions was the extensive induction of signaling and perception machinery playing a key role in effective resistance against pathogens. Interestingly, the signalling processes and immune response activated during BABA-IR in tomato differed from those in Arabidopsis with substantial enrichment of genes associated with jasmonic acid (JA) and ethylene (ET) signalling and no change in Asp levels. Our results revealed key differences between the effect of BABA on tomato and other model plants studied until now. Surprisingly, salicylic acid (SA) is not involved in BABA downstream signalization whereas ET and JA play a crucial role.

Characteristics of N-Glycosylation and Its Impact on the Molecular Behavior of Lupinus angustifolius γ-Conglutin.

γ-Conglutin, a lupin seed protein, is an intriguing protein both in terms of the complexity of its molecular structure and a broad spectrum of unique health-promoting properties manifested in animal and human trials. Moreover, this protein is an evolutionary cornerstone whose physiological significance for the plant has not been determined yet. Herein, a comprehensive characterization of γ-conglutin glycosylation is presented and includes (i) the identification of the N-glycan-bearing site, (ii) the qualitative and quantitative composition of glycan-building saccharides, as well as (iii) the effect of oligosaccharide removal on structural and thermal stability. The obtained results indicate the presence of glycans belonging to different classes attached to the Asn98 residue. In addition, the detachment of the oligosaccharide significantly affects secondary structure composition, which disturbs the oligomerization process. The structural changes were also reflected in biophysical parameters, i.e., at a pH value of 4.5, an increase in γ-conglutin thermal stability was observed for the deglycosylated monomeric form. Collectively, the presented results provide evidence of the high complexity of the post-translational maturation and suggest the possibility of a functional effect that glycosylation might have on γ-conglutin structure integrity.

New findings on the action of hypericin in hypoxic cancer cells with a focus on the modulation of side population cells.

The presence of key hypoxia regulators, namely, hypoxia-inducible factor (HIF)-1α or HIF-2α, in tumors is associated with poor patient prognosis. Hypoxia massively activates several genes, including the one encoding the BCRP transporter that proffers multidrug resistance to cancer cells through the xenobiotic efflux and is a determinant of the side population (SP) associated with cancer stem-like phenotypes. As natural medicine comes to the fore, it is instinctive to look for natural agents possessing powerful features against cancer resistance. Hypericin, a pleiotropic agent found in Hypericum plants, is a good example as it is a BCRP substrate and potential inhibitor, and an SP and HIF modulator. Here, we showed that hypericin efficiently accumulated in hypoxic cancer cells, degraded HIF-1/2α, and decreased BCRP efflux together with hypoxia, thus diminishing the SP population. On the contrary, this seemingly favorable result was accompanied by the stimulated migration of this minor population that preserved the SP phenotype. Because hypoxia unexpectedly decreased the BCRP level and SP fraction, we compared the SP and non-SP proteomes and their changes under hypoxia in the A549 cell line. We identified differences among protein groups connected to the epithelial-mesenchymal transition, although major changes were related to hypoxia, as the upregulation of many proteins, including serpin E1, PLOD2 and LOXL2, that ultimately contribute to the initiation of the metastatic cascade was detected. Altogether, this study helps in clarifying the innate and hypoxia-triggered resistance of cancer cells and highlights the ambivalent role of natural agents in the biology of these cells.

A previously uncharacterized Factor Associated with Metabolism and Energy (FAME/C14orf105/CCDC198/1700011H14Rik) is related to evolutionary adaptation, energy balance, and kidney physiology.

In this study we use comparative genomics to uncover a gene with uncharacterized function (1700011H14Rik/C14orf105/CCDC198), which we hereby name FAME (Factor Associated with Metabolism and Energy). We observe that FAME shows an unusually high evolutionary divergence in birds and mammals. Through the comparison of single nucleotide polymorphisms, we identify gene flow of FAME from Neandertals into modern humans. We conduct knockout experiments on animals and observe altered body weight and decreased energy expenditure in Fame knockout animals, corresponding to genome-wide association studies linking FAME with higher body mass index in humans. Gene expression and subcellular localization analyses reveal that FAME is a membrane-bound protein enriched in the kidneys. Although the gene knockout results in structurally normal kidneys, we detect higher albumin in urine and lowered ferritin in the blood. Through experimental validation, we confirm interactions between FAME and ferritin and show co-localization in vesicular and plasma membranes.

A complex role of Arabidopsis CDKD;3 in meiotic progression and cytokinesis.

Meiosis is a specialized cell division that halves the number of chromosomes in two consecutive rounds of chromosome segregation. In angiosperm plants is meiosis followed by mitotic divisions to form rudimentary haploid gametophytes. In Arabidopsis, termination of meiosis and transition to gametophytic development are governed by TDM1 and SMG7 that mediate inhibition of translation. Mutants deficient in this mechanism do not form tetrads but instead undergo multiple cycles of aberrant nuclear divisions that are likely caused by the failure to downregulate cyclin dependent kinases during meiotic exit. A suppressor screen to identify genes that contribute to meiotic exit uncovered a mutation in cyclin-dependent kinase D;3 (CDKD;3) that alleviates meiotic defects in smg7 deficient plants. The CDKD;3 deficiency prevents aberrant meiotic divisions observed in smg7 mutants or delays their onset after initiation of cytokinesis, which permits formation of functional microspores. Although CDKD;3 acts as an activator of cyclin-dependent kinase A;1 (CDKA;1), the main cyclin dependent kinase that regulates meiosis, cdkd;3 mutation appears to promote meiotic exit independently of CDKA;1. Furthermore, analysis of CDKD;3 interactome revealed enrichment for proteins implicated in cytokinesis, suggesting a more complex function of CDKD;3 in cell cycle regulation.

Regulatory dynamics of gene expression in the developing male gametophyte of Arabidopsis.

Sexual reproduction in angiosperms requires the production and delivery of two male gametes by a three-celled haploid male gametophyte. This demands synchronized gene expression in a short developmental window to ensure double fertilization and seed set. While transcriptomic changes in developing pollen are known for Arabidopsis, no studies have integrated RNA and proteomic data in this model. Further, the role of alternative splicing has not been fully addressed, yet post-transcriptional and post-translational regulation may have a key role in gene expression dynamics during microgametogenesis. We have refined and substantially updated global transcriptomic and proteomic changes in developing pollen for two Arabidopsis accessions. Despite the superiority of RNA-seq over microarray-based platforms, we demonstrate high reproducibility and comparability. We identify thousands of long non-coding RNAs as potential regulators of pollen development, hundreds of changes in alternative splicing and provide insight into mRNA translation rate and storage in developing pollen. Our analysis delivers an integrated perspective of gene expression dynamics in developing Arabidopsis pollen and a foundation for studying the role of alternative splicing in this model.

Proteomic Profiles of Whole Seeds, Hulls, and Dehulled Seeds of Two Industrial Hemp (Cannabis sativa L.) Cultivars.

As a source of nutritionally important components, hemp seeds are often dehulled for consumption and food applications by removing the hard hulls, which increases their nutritional value. The hulls thus become waste, although they may contain valuable protein items, about which there is a lack of information. The present work is therefore aimed at evaluating the proteome of hemp (Cannabis sativa L.) at the whole-seed, dehulled seed, and hull levels. The evaluation was performed on two cultivars, Santhica 27 and Uso-31, using LC-MS/MS analysis. In total, 2833 protein groups (PGs) were identified, and their relative abundances were determined. A set of 88 PGs whose abundance exceeded 1000 ppm (MP88 set) was considered for further evaluation. The PGs of the MP88 set were divided into ten protein classes. Seed storage proteins were found to be the most abundant protein class: the averages of the cultivars were 65.5%, 71.3%, and 57.5% for whole seeds, dehulled seeds, and hulls, respectively. In particular, 11S globulins representing edestin (three PGs) were found, followed by 7S vicilin-like proteins (four PGs) and 2S albumins (two PGs). The storage 11S globulins in Santhica 27 and Uso-31 were found to have a higher relative abundance in the dehulled seed proteome (summing to 58.6 and 63.2%) than in the hull proteome (50.5 and 54%), respectively. The second most abundant class of proteins was oleosins, which are part of oil-body membranes. PGs belonging to metabolic proteins (e.g., energy metabolism, nucleic acid metabolism, and protein synthesis) and proteins related to the defence and stress responses were more abundant in the hulls than in the dehulled seeds. The hulls can, therefore, be an essential source of proteins, especially for medical and biotechnological applications. Proteomic analysis has proven to be a valuable tool for studying differences in the relative abundance of proteins between dehulled hemp seeds and their hulls among different cultivars.

Composition and toxicity of venom produced by araneophagous white-tailed spiders (Lamponidae: Lampona sp.).

Prey-specialised spiders are adapted to capture specific prey items, including dangerous prey. The venoms of specialists are often prey-specific and less complex than those of generalists, but their venom composition has not been studied in detail. Here, we investigated the venom of the prey-specialised white-tailed spiders (Lamponidae: Lampona), which utilise specialised morphological and behavioural adaptations to capture spider prey. We analysed the venom composition using proteo-transcriptomics and taxon-specific toxicity using venom bioassays. Our analysis identified 208 putative toxin sequences, comprising 103 peptides < 10 kDa and 105 proteins > 10 kDa. Most peptides belonged to one of two families characterised by scaffolds containing eight or ten cysteine residues. Toxin-like proteins showed similarity to galectins, leucine-rich repeat proteins, trypsins and neprilysins. The venom of Lampona was shown to be more potent against the preferred spider prey than against alternative cricket prey. In contrast, the venom of a related generalist was similarly potent against both prey types. These data provide insights into the molecular adaptations of venoms produced by prey-specialised spiders.