Anticitrullinated antibodies recognize rheumatoid arthritis associated T-cell epitopes modified by bacterial L-asparaginase.

Citrullinated proteins and anti-citrullinated protein antibodies (ACPAs) play an important role in the pathogenesis of rheumatoid arthritis (RA). It has been suggested that during inflammation or dysbiosis, bacteria could initiate production of ACPAs. Most patients with RA are seropositive for ACPAs, but these antibodies have overlapping reactivity to different posttranslational modifications (PTMs). For initiation and development of RA, T lymphocytes and T cell epitopes are still required. In this study, we evaluated the ability of bacterial L-asparaginase to modify RA-related T cell epitopes within type II collagen (CII259-273 and CII311-325), as well as whether these modified epitopes are recognized by ACPAs from RA patients. We included 12 patients with early RA and 11 healthy subjects selected according to predefined specific criteria. LC-MS/MS analyses revealed that the bacterial L-asparaginase can modify investigated T cell epitopes. ELISA tests showed cross-reactivity of ACPA positive sera from early RA patients towards the enzymatically modified immunodominant T cell epitopes within type II collagen (CII), but not to the modified irrelevant peptides. These data suggest that the cross-reactive ACPAs recognize the "carbonyl-Gly-Pro" motif in CII. Moreover, the T cell recognition of the modified major immunodominant T cell epitope Gal264-CII259-273 was not affected. This epitope was still able to activate autoreactive T cells from early RA patients. It is likely that such modifications are the missing link between the T cell priming and the development of anti-modified protein antibodies (AMPAs). Our results provide additional information on the etiology and pathogenesis of RA.

Polyphasic characterisation of Microcoleusautumnalis (Gomont, 1892) Strunecky, Komárek & J.R.Johansen, 2013 (Oscillatoriales, Cyanobacteria) using a metabolomic approach as a complementary tool.

As a result of the continuous revision of cyanobacterial taxonomy, Phormidiumautumnale (Agardh) Trevisan ex Gomont, 1892 has been transferred to the genus Microcoleus as Microcoleusautumnalis (Gomont, 1892) Strunecky, Komárek & J.R.Johansen, 2013. This transfer was based on a single strain and literature data. In the present study, we revise the taxonomic position of Microcoleusautumnalis by applying the classical approach of polyphasic taxonomy and additionally using metabolomics. Cyanobacterial strains identified as Phormidiumautumnale and Microcoleusvaginatus (type species of the genus Microcoleus) were used for comparative analyses. In addition, the taxonomic relationship between the species Phormidiumautumnale and Phormidiumuncinatum was determined on the basis of polyphasic characteristics. Monitoring of the morphological variability of Phormidiumautumnale and Microcoleusvaginatus strains showed a difference in the morphology concerning the ends of the trichomes, the shape of the apical cells, as well as the presence/absence of the calyptra and its shape. The performed TEM analysis of the thylakoid arrangement of the studied strains showed parietal arrangement of the thylakoids in the representatives of genus Phormidium and fascicular arrangement in genus Microcoleus. Molecular genetic analyses, based on 16S rDNA, revealed grouping of the investigated P.autumnale strains in a separate clade. This clade is far from the subtree, which is very clearly formed by the representatives of the type species of genus Microcoleus, namely M.vaginatus. The metabolomic analysis involving P.autumnale and M.vaginatus strains identified 39 compounds that could be used as potential biochemical markers to distinguish the two cyanobacterial species. Based on the data obtained, we suggest changing of the current status of Microcoleusautumnalis by restoring its previous appurtenance to the genus Phormidium under the name Phormidiumautumnale (Agardh) Trevisan ex Gomont, 1892 and distinguishing this species from genus Microcoleus.

Phytochemical characterization and biological activity of apricot kernels' extract in yeast-cell based tests and hepatocellular and colorectal carcinoma cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Bitter apricot kernels' extract contains a broad spectrum of biologically active substances with a lot of attention to amygdalin - cyanogenic glycoside. The extract has been used in the pharmaceutical industry for years as an ingredient of different pharmaceuticals with anti-inflammatory, antimicrobial, or regenerative properties. In traditional medicine, the bitter apricot kernels are known as a remedy for respiratory disorders and skin diseases. The apricot kernels and amygdalin are often prescribed by practitioners for the prevention and treatment of various medical conditions, including colorectal cancer. THE PRESENT STUDY AIMS: to evaluate the phytochemical composition and the potential antimutagenic, antirecombinogenic, and antitumor effect of apricot kernels' extract at very low concentrations in yeast cell-based tests and mammalian hepatocellular and colon carcinoma cell lines. MATERIALS AND METHODS: Phytochemical analysis was performed by LC-MS profiling. Reverse-phase HPLC and UV detection were applied for the determination of amygdalin quantity in the extract. Biological activity was evaluated by Zimmermann's mutagenicity and Ty1 retrotransposition test. Cytotoxic/antiproliferative activity of apricot kernels' extract was performed on four types of cell lines - HepG2, HT-29, BALB/3T3, clone A31, and BJ using the standard MTT-dye reduction assay. RESULTS: Data revealed the presence of more than 1000 compounds and 4 cyanogenic glycosides among them - Amygdalin, Deidaclin, Linamarin and Prulaurasin. The Amygdalin concentration was measured to be 57.8 µg/ml. All extract concentrations demonstrated a strong antigenotoxic, antirecombinogenic, antimutagenic, and anticarcinogenic effect in the yeast cell-based tests. High selectivity of the extract action is established among different mammalian cell lines. Normal cell line BJ is found to be resistant to the extract action. HepG2 was found to be the most sensitive to apricot kernels' action. CONCLUSION: The present study provides the first phytochemical analysis of Bulgarian bitter apricot kernels. Three new cyanogenic glycosides were reported. Evidence is obtained that the apricot kernels' extract at low concentrations is not able to induce some of the events related to the initial steps of tumorigenesis. Additionally, a high selectivity of the extract action is established among different cell lines. The most sensitive cell line was found to be HepG2.

In vivo toxicity assessment of Clinopodium vulgare L. water extract characterized by UHPLC-HRMS.

Clinopodium vulgare L. (Lamiaceae) was used in the traditional Bulgarian medicine for treatment of wounds, diabetes and gastric ulcers. In this study we aimed at safety assessment of C. vulgare lyophilized water extract (CVE) characterized by ultra high-performance liquid chromatography-Orbitrap high resolution mass spectrometry (UHPLC-HRMS). The acute and sub-acute toxicity of CVE was determined in two rodent species (mice and rats), and two routes of administration - intraperitoneal (i.p.) and oral (p.o.). LD50 (i.p.), were found to be 675 mg/kg (mice) and 500 mg/kg (rats). An acute i. p. administration resulted in central nervous system toxic effects. LD50 (p.o.) was higher than 2000 mg/kg for both species. In sub-acute oral administration, CVE did not exert any toxic effect on hematology, blood and urine biochemistry, and histomorphology in pancreas, liver, spleen and kidney. In addition, based on accurate masses, MS/MS and comparison with standards, a variety of flavonoids, caffeic acid oligomers and saponins were tentatively elucidated in CVE. Rosmarinic acid was the major compound. In conclusion, CVE did not cause hematological, biochemical and histopathological changes after oral administration and it is safe for internal use. The obtained UHPLC-HRMS profile revealed CVE as a new rich source of water soluble caffeic acid oligomers.

Assessment of the cytotoxicity, antioxidant activity and chemical composition of extracts from the cyanobacterium Fischerella major Gomont.

Cyanoprokaryotes (Cyanobacteria/Cyanophyta) are ancient photosynthetic prokaryotic organisms with cosmopolitan distribution. They are producers of a number of biologically active substances with antitumor and antifungal activity, vitamins, antibiotics, algaecides, insecticides, repellents, hormones, immunosuppressants and toxins. So far, the cyanobacterium Fischerella major Gomont has not been studied regarding its impact on the environment and human health. In this study, the cytotoxic, antioxidant and antitumor activities of four extracts prepared from Fischerella major were evaluated in vitro. In addition, the total phenolic content and the potential for production of cyanotoxins were also analyzed. The conducted GC/MS analysis identified 45 compounds with different chemical nature and biological activity. Presence of microcystins and saxitoxins was detected in all Fischerella major extracts. In vitro testing on cell cultures showed a significant concentration- and time-dependent cytotoxic effect on all cell lines (HeLa, SK-Hep-1 and FL) treated at three exposure times (24, 48 and 72 h) with four extracts. A selective antitumor effect was not observed. This is the first study demonstrating biological activity of extracts from Fischerella major, which makes it an interesting subject for further research, including environmental risk assessments (as producer of cyanotoxins) or as a potential source of pharmaceuticals.

A new liquid chromatography-high resolution Orbitrap mass spectrometry-based strategy to characterize Glucuronide Oleanane-type Triterpenoid Carboxylic Acid 3, 28-O-Bidesmosides (GOTCAB) saponins.A case study of Gypsophila glomerata Pall ex M. B. (Caryophyllaceae).

Glucuronide Oleanane-type Triterpenoid Carboxylic Acid 3, 28-Bidesmosides (GOTCAB) saponins are bioactive natural compounds spread in Caryophyllidae. The high complexity of GOTCAB occurring as closely related isobaric and positional isomers is a challenge in their separation and identification. A new liquid chromatography - high resolution Orbitrap mass spectrometry acquisition strategy would be important for the structural elucidation of GOTCAB in plant extracts. In this study, the fragmentation behaviors of GOTCAB from methanol-aqueous root extract of Gypsophila glomerata Pall ex M. B. (Caryophyllaceae) were investigated using ultra high-performance liquid chromatography (UHPLC) coupled with hybrid quadrupole-Orbitrap high resolution mass spectrometry (HRMS). A new saponin was isolated and its structure was established by 1D and 2D-NMR spectroscopic experiments as 3-O-ß-D-galactopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-ß-D-glucuronopyranosyl gypsogenin 28-O-α-L-arabinopyranosyl-(1→3)-[ß-D-xylopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→2)-ß-D-fucopyranosyl ester. On the basis of the accurate mass measurements, fragmentation patterns in MS/MS analyses and comparison with previously isolated authentic references, a total of 41 GOTCAB saponins were identified or tentatively elucidated in G. glomerata roots, including 14 pairs of isobars. Possible fragmentation pathways for three groups of GOTCAB are suggested. The group I appeared to be GOTCAB of gypsogenin with two carbohydrate chains: a branched trisaccharide at C-3 and tri- to hexa-saccharide attached to C-28 of the aglycone through a deoxyhexose residue. Saponins with monoacetylated (group II) or sulphated (group III) C-28 chain were evidenced, as well as quillaic and oleanolic acid GOTCAB. Sixteen GOTCAB were previously not described. The content of Gypsophila prosaponins, gypsogenin 3-O-glucuronide (7.4079 ±â€¯0.0723 mg/g dry weight, dw) and quillaic acid 3-O-glucuronide (4.4593 ±â€¯0.1207 mg/g dw), was determined by solid phase extraction - high-performance liquid chromatography (SPE-HPLC). In this study is presented the first systematic investigation on the fragmentation patterns and diagnostic fingerprints of the fragment ions in the MS/MS spectra of the gypsogenin -, quillaic acid - and oleanolic acid - bidesmosides. A LC-HRMS Orbitrap acquisition strategy could give an insight in the GOTCAB containing taxa.

Selective Profiling of Saponins from Gypsophila trichotoma Wend. by HILIC Separation and HRMS Detection.

INTRODUCTION: Roots of Gypsophila trichotoma Wend. (Caryophyllaceae) are rich sources of glucuronide oleanane-type triterpenoid carboxylic acid 3,28-O-bidesmosides (GOTCABs). These saponins have been reported to possess synergistic cytotoxicity in combination with type I ribosome-inactivating protein saporin. OBJECTIVE: To develop ultra high-performance liquid chromatography - electrospray ionisation/high resolution mass spectrometry (UHPLC-ESI/HRMS) acquisition strategy for the recognition of Gypsophila GOTCAB saponins. METHODOLOGY: A highly-selective hydrophilic interaction UHPLC method (Si-HILIC UHPLC) was developed for the separation of GOTCAB saponins from the methanol-aqueous root extract of G. trichotoma (GTR). UHPLC was coupled to an Orbitrap mass spectrometer equipped with heated electrospray ionisation (HESI) probe. ESI-HRMS and tandem mass spectrometry (MS/MS) data of the separated compounds was used for saponins structure assignment. RESULTS: Based on the conformity of the fragmentation of 11 previously identified GTR saponins, 21 GOTCAB forming between two and four isobaric and positional isomers are identified with proposals for their structures. Tables with assignment of characteristic fragment ions and more than 10 newly identified saponins in GTR were described. Fragmentation rules for tentative identification of three major types of saponins from GTR were summarised and possible fragmentation pathways were proposed. Type I and II consisted of acylated and sulphated GOTCABs, respectively, while type III included acylated and sulphated saponins. The type II sulphated GOTCAB saponins were all previously not described. CONCLUSIONS: The study demonstrates the potential of the coupling of highly-selective (Si)-HILIC UHPLC with HRMS and MS/MS detection for analysis and identification of triterpenoid saponins. Copyright © 2017 John Wiley & Sons, Ltd.

Antimutagenic, Antirecombinogenic, and Antitumor Effect of Amygdalin in a Yeast Cell-Based Test and Mammalian Cell Lines.

Amygdalin is a major component of the seeds of Rosaceae family of plants such as apricots, peaches, cherry, nectarines, apples, plums, and so on, as well as almonds. It is used in alternative medicine for cancer prevention, alleviation of fever, cough suppression, and quenching thirst. The aim of the present study is to determine the mutagenic and recombinogenic effects of amygdalin in a test system Saccharomyces cerevisiae and to evaluate its potential antitumor effect in a yeast cell-based test and colon cancer cell lines. Results obtained show that concentrations 25, 50, and 100 µg/mL did not have any cytotoxic, mutagenic, and carcinogenic effect in yeast cell-based tests. Pretreatment with amygdalin at concentration 100 µg/mL leads to around twofold of the cell survival and decrease of reverse mutation frequency, induced by the alkylating agent methyl methanesulfonate. The frequency of gene conversion and mitotic crossing-over is around threefold lower. The anticarcinogenic potential of amygdalin at the same concentration is presented as around fourfold reduction of Ty1 retrotransposition induced by hexavalent chromium. In summary, data presented in this study provide evidence concerning the inability of amygdalin itself to provoke events related to the initial steps of tumorigenesis. In addition, the observed antimutagenic/antirecombinogenic effect could be activation of error-free and error-prone recombination events. Based on the high selectivity toward normal or tumor cell lines, it could be speculated that amygdalin has higher cytotoxic effect in cell lines with higher proliferative and metabolic activity, which are the majority of fast developing tumors.

HPLC-HRMS method for fast phytochelatins determination in plants. Application to analysis of Clinopodium vulgare L.

An optimized analytical method based on C8 core-shell reverse phase chromatographic separation and high resolution mass spectral (HRMS) detection is developed for a fast analysis of unbound phytochelatins (PCs) in plants. Its application to analysis of Clinopodium vulgare L. is demonstrated where proper PCs liberating and preservation conditions were employed using dithiotreitol in the extraction step. A baseline separation of glutathione (GSH) and phytochelatins from 2 to 5 (PC2-PC5) for 3 min was achieved at conventional HPLC backpressure, with detection limits from 3 ppt (for GSH) to 2.5 ppb (for PC5). It is shown, that the use of HRMS with tandem mass spectral (MS/MS) capabilities permits additional wide range screening ability for iso-phytochelatins and PC similar compounds, based on exact mass and fragment spectra in a post acquisition manner.