Search Results (14)

Background/Objectives: Identifying effective and safe treatment options for non-infectious uveitis remains challenging due to chronic and relapsing ocular inflammation. Previous studies have shown that artesunate (ART) plays an immunosuppressive role in several classic autoimmune diseases, including uveitis. However, its impact on the plasma metabolic profile of recurrent autoimmune uveitis remains unclear. This study aims to explore the effect of ART on the plasma metabolic features of recurrent experimental autoimmune uveitis (EAU) in a Lewis rat. Methods: Rats were clinically and pathologically evaluated for the development of recurrent EAU induced by inter-photoreceptor retinoid-binding protein (IRBP) R16 peptide-specific T-cells (tEAU). The disruptive effects of ART on tEAU were investigated to evaluate the potential role of rat recurrent EAU. Differentially expressed metabolites were identified in the plasma of rats by untargeted metabolomics analysis after ART treatment. The differential metabolites were applied to subsequent pathway analysis and biomarker analysis by MetaboAnalyst. Results: ART can significantly alleviate the severity of clinical signs and pathological injuries of eyeballs with tEAU. Both non-supervised principal component analysis and orthogonal partial least-squares discriminant analysis showed 84 differential metabolites enriched in 16 metabolic pathways in the tEAU group compared with heathy controls and 51 differential metabolites enriched in 17 metabolic pathways, including arginine and proline metabolism, alanine metabolism, and aminoacyl-tRNA biosynthesis, in the ART-treated group compared with the tEAU group. Particularly, upregulated L-alanine levels in both alanine metabolism and aminoacyl-tRNA biosynthesis were associated with T-cell activation, while elevated spermidine and N-acetyl putrescine levels in arginine and proline metabolism related to T-cell differentiation proved to be valuable biomarkers for ART treatment. Conclusions: Our study demonstrates that ART treatment can alleviate recurrent uveitis by altering the plasma metabolic characteristics associated with T-cell activation and differentiation, which might provide novel insights for potential therapeutic treatments. Full article

Enteral nutrition (EN) formulas are necessary for critically ill patients to meet their metabolic requirements. Polyamines (putrescine, spermidine, and spermine) are crucial dietary components, with spermidine being particularly interesting due to its multiple proposed benefits. The requirements for and intake of polyamines have yet to be investigated in adult patients hospitalised in intensive care units (ICUs) who are exclusively fed via commercial EN formulas. The aim of this study was to determine the polyamine content and other biogenic amines of EN formulas and the total intake and gastric residual volume (GRV) in adult ICU patients during their first seven days of hospitalisation. The amines were analysed in 16 EN formulas using high-performance liquid chromatography (HPLC). The clinical data of eight patients of both sexes aged 47 to 77 admitted to the ICU were analysed. Differences existed among the analysed EN formulas. The N-acetyl putrescine content was higher than that of the remaining amines. The daily intake of polyamines in the ICU was less than 100 μmol (the dietary intake is above 400 μmol). An inverse correlation existed between total daily polyamine intake and daily GRV, without effects from other biogenic amines being analysed. Polyamine intake in critically ill patients receiving EN is low and could impact these patients’ feeding tolerance. These findings underscore the need for further research to explore the clinical implications of increasing the polyamine content of EN formulas. Full article

Escherichia coli and Shigella flexneri are challenging to differentiate using methods such as phenotyping, 16S rRNA sequencing, or protein profiling through matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) due to their close relatedness. This study explores the potential for identifying E. coli and S. flexneri by incorporating reference spectra of metabolite profiles, obtained via surface-assisted laser desorption/ionization mass spectrometry (SALDI MS) employing gold nanoparticles (AuNPs), into the Bruker Biotyper database. Metabolite extracts from E. coli and S. flexneri cells were prepared using liquid–liquid extraction in a chloroform–methanol–water system. The extracts were analyzed using Au-SALDI MS in positive ion mode, and reference spectra, compiled from 30 spectra for each bacterium, were added to the database. Identification of bacteria based on metabolite fingerprints in the Biotyper database produced correct results with scores exceeding 2.75. The results of Partial Least Squares-Discriminant Analysis (PLS-DA) demonstrated that the metabolomic approach could accurately differentiate the microorganisms under study. A panel of nine m/z values was also identified, each with an area under the ROC curve of above 0.8, enabling accurate identification of E. coli and S. flexneri. A search of metabolite databases allowed the following compounds to be assigned to the selected m/z values: N-acetylputrescine, arginine, 2-maleylacetate, benzoyl phosphate, N8-acetylspermidine, alanyl-glutamate, 4-hydroxy-2,3,4,5-tetrahydrodipicolinate, and sucrose. The analyses showed that identification of bacteria based on metabolite profiles obtained by the Au-SALDI MS method is feasible and can be useful for distinguishing closely related microorganisms that are difficult to differentiate by other techniques. Full article

Sex-controlled sperm combined with artificial insemination allows animals to reproduce offspring according to the desired sex, accelerates the process of animal genetics and breeding and promotes the development of animal husbandry. However, the molecular markers for sexual sperm sorting are unusual. To identify the molecular markers of boar sperm sorting, proteomics and metabolomics techniques were applied to analyze the differences in proteins and metabolism between X and Y sperm. Label-free quantitative proteomics identified 254 differentially expressed proteins (DEPs) in the X and Y sperm of boars, including 106 proteins that were highly expressed in X sperm and 148 proteins that were highly expressed in Y sperm. Among the differential proteins, COX6A1, COX1, CYTB, FUT8, GSTK1 and PFK1 were selected as potential biological markers for X and Y sperm sorting. Moreover, 760 metabolites from X and Y sperm were detected. There were 439 positive ion mode metabolites and 321 negative ion mode metabolites identified. The various metabolites were phosphoenolpyruvate, phytosphingosine, L-arginine, N-acetylputrescine, cytidine-5′-diphosphate and deoxyuridine. These metabolites were mainly involved in the TCA cycle, oxidative phosphorylation pathway, glycolysis pathway, lipid metabolism pathway, amino acid metabolism pathway, pentose phosphate pathway and nucleic acid metabolism pathway. The differential proteins and differential metabolites obtained by the combined proteomics and metabolomics analysis were projected simultaneously to the KEGG pathway, and a total of five pathways were enriched, namely oxidative phosphorylation pathway, purine metabolism, unsaturated fatty acid biosynthesis, ABC transporters and peroxisomes. In summary, COX6A1 and CYTB were identified as potential biomarkers for boar X and Y sperm sorting. Full article

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. Early detection and the modification of risk factors, such as diet, can reduce its incidence. Among food components, polyamines are important for maintaining gastrointestinal health and are metabolites of gut microbiota. Their disruption is linked to CRC, making polyamines a potential marker of the disease. This study analyzed the relationship between dietary components, including polyamines, and the presence of polyamines in feces to determine whether their presence could contribute to predicting the occurrence of colorectal lesions in patients. In total, 59 participants of both sexes (aged 50 to 70 years) who had undergone colonoscopy screening for CRC (18 without and 41 with colorectal lesions) participated in the study. A nutritional survey and determination of fecal polyamine content were performed. Specific dietary components and putrescine levels were higher in patients with colorectal lesions. The diet ratio of putrescine–spermidine and the fecal content of N-acetyl putrescine and cadaverine were elevated in patients with precancerous lesions and adenocarcinomas, showing a potential predictive value for the presence of colorectal lesions. These findings suggest that N-acetyl putrescine and cadaverine could be complementary markers for the diagnosis of suspected colorectal lesions. Full article

D-arginine (D-Arg) can promote embryogenic callus (EC) proliferation and increase the rate of somatic embryo induction of litchi (Litchi chinensis Sonn.), yet the mechanism underlying the processes is incompletely understood. To investigate the mechanism, physiological responses of polyamines (PAs) [putrescine (Put), spermidine (Spd), and spermine (Spm)] were investigated for D-Arg-treated litchi EC and enzyme activity related to polyamine metabolism, plant endogenous hormones, and polyamine- and embryogenic-related genes were explored. Results showed that the exogenous addition of D-Arg reduces the activity of diamine oxidase (DAO) and polyamine oxidase (PAO) in EC, reduces the production of H₂O₂, promotes EC proliferation, and increases the (Spd + Spm)/Put ratio to promote somatic embryo induction. Exogenous D-Arg application promoted somatic embryogenesis (SE) by increasing indole-3-acetyl glycine (IAA-Gly), kinetin-9-glucoside (K9G), and dihydrozeatin-7-glucoside (DHZ7G) levels and decreasing trans-zeatin riboside (tZR), N-[(-)-jasmonoyl]-(L)-valine (JA-Val), jasmonic acid (JA), and jasmonoyl-L-isoleucine (Ja-ILE) levels on 18 d, as well as promoting cell division and differentiation. The application of exogenous D-Arg regulated EC proliferation and somatic embryo induction by altering gene expression levels of the WRKY family, AP2/ERF family, C3H family, and C2H2 family. These results indicate that exogenous D-Arg could regulate the proliferation of EC and the SE induction of litchi by changing the biosynthesis of PAs through the alteration of gene expression pattern and endogenous hormone metabolism. Full article

The influence of polysialic acid (PSA) and sialic acid (SA) on the gut microbial community composition and metabolites in healthy humans was investigated using a bionic gastrointestinal reactor. The results indicated that PSA and SA significantly changed the gut microbiota and metabolites to different degrees. PSA can increase the relative abundances of Faecalibacterium and Allisonella, whereas SA can increase those of Bifidobacterium and Megamonas. Both can significantly increase the content of short-chain fatty acids. The results of metabolome analysis showed that PSA can upregulate ergosterol peroxide and gallic acid and downregulate the harmful metabolite N-acetylputrescine. SA can upregulate 4-pyridoxic acid and lipoic acid. PSA and SA affect gut microbiota and metabolites in different ways and have positive effects on human health. These results will provide a reference for the further development of PSA- and SA-related functional foods and health products. Full article

The typical Korean diet contains a significant quantity of doenjang owing to its unique taste and health benefits. However, the presence of anti-nutritional and toxic substances, such as biogenic amines and microbial pathogens, in doenjang has resulted in a loss of revenue and poor consumer health. The present study focused on the identification and quantification of different biogenic amines, pathogenic Bacillus cereus, and yeast counts in 36 doenjang products (designated as De-1 to De-36, 500 g each) procured from the different cottage industries situated in different parts of the Republic of Korea. The results indicated, only three samples were contaminated with B. cereus, exceeding the recommended limit (4 log CFU/g) suggested by the national standards of Korea. A total of six distinct yeasts were identified in different doenjang samples, whose comprehensive enzymatic profiling suggested the absence of harmful enzymes such as N-acetyl-β-glucosaminidase, α-chymotrypsin, and β-glucuronidase. The biogenic amines were detected in the range of 67.68 mg/kg to 2556.68 mg/kg and classified into six major groups based on hierarchical cluster analysis. All doenjang samples contained tryptamine, putrescine, cadaverine, histamine, and tyramine, while 94.44% were positive for spermidine and spermine. The results documented the analysis of traditional cottage industry doenjang and suggest the need for constant monitoring to ensure the safety of food for the consumer. Full article

Most forms of life, including the archaea, bacteria, and eukaryotes synthesize the polyamine putrescine. Although putrescine is widely distributed, several Gram-positive bacteria, including Staphylococcus aureus (S. aureus), appear to be the exceptions. We report here that strains of S. aureus can produce the polyamine putrescine, as well as the derivative N-acetyl-putrescine. Three strains of S. aureus from the American Type Culture Collection (ATCC), one strain listed in the National Center for Biotechnology Information (NCBI) database, whose genomic sequence is well defined, and well as eight strains from S. aureus-induced brain abscesses of individual patients from multiple geographic locations were evaluated. Each strain was grown in complete chemically defined medium (CDM) under stringent conditions, after which the partially purified conditioned medium (CM) was analyzed by mass spectroscopy (MS), and the data were reported as the ratio of experimental results to controls. We confirmed the synthesis of putrescine by S. aureus by using ¹³C/¹⁵N-labeled arginine as a tracer. We found that agmatine, N-acetyl-putrescine, ornithine, citrulline, proline, and NH₃ were all labeled with heavy isotope derived from ¹³C/¹⁵N-labeled arginine. None of the strains examined produced spermine or spermidine, but strains from either ATCC or human brain abscesses produced putrescine and/or its derivative N-acetyl-putrescine. Full article

A simultaneous quantitative profiling method for polyamines and steroids using liquid chromatography–tandem mass spectrometry was developed and validated. We applied this method to human serum samples to simultaneously evaluate polyamine and steroid levels. Chemical derivatization was performed using isobutyl chloroformate to increase the sensitivity of polyamines. The method was validated, and the matrix effects were in the range of 78.7–126.3% and recoveries were in the range of 87.8–123.6%. Moreover, the intra-day accuracy and precision were in the ranges of 86.5–116.2% and 0.6–21.8%, respectively, whereas the inter-day accuracy and precision were in the ranges of 82.0–119.3% and 0.3–20.2%, respectively. The linearity was greater than 0.99. The validated method was used to investigate the differences in polyamine and steroid levels between treated breast cancer patients and normal controls. In our results, N-acetyl putrescine, N-acetyl spermidine, cadaverine, 1,3-diaminopropane, and epitestosterone were significantly higher in the breast cancer patient group. Through receiver operating characteristic curve analysis, all metabolites that were significantly increased in patient groups with areas under the curve >0.8 were shown. This mass spectrometry-based quantitative profiling method, used for the investigation of breast cancer, is also applicable to androgen-dependent diseases and polyamine-related diseases. Full article

Polyamines are involved in the regulation of many cellular functions and are promising biomarkers of numerous physiological conditions. Since the concentrations of these compounds in biological fluids are low, sample extraction is one of the most critical steps of their analysis. Here, we developed a comprehensive, sensitive, robust, and high-throughput LC-MS/MS stable-isotope dilution method for the simultaneous determination of 19 metabolites related to polyamine metabolism, including polyamines, acetylated and diacetylated polyamines, precursors, and catabolites from liquid biopsies. The sample extraction was optimized to remove interfering compounds and to reduce matrix effects, thus being useful for large clinical studies. The method consists of two-step liquid-liquid extraction with a Folch extraction and ethyl acetate partitioning combined with dansyl chloride derivatization. The developed method was applied to a small gender-related trial concerning human serum and urine samples from 40 obese subjects. Sex differences were found for cadaverine, putrescine, 1,3-diaminopropane, γ-aminobutyric acid, N8-acetylspermidine, and N-acetylcadaverine in urine; N1-acetylspermine in serum; and spermine in both serum and urine. The results demonstrate that the developed method can be used to analyze biological samples for the study of polyamine metabolism and its association with human diseases. Full article

Enzymes in the transcarbamylase family catalyze the transfer of a carbamyl group from carbamyl phosphate (CP) to an amino group of a second substrate. The two best-characterized members, aspartate transcarbamylase (ATCase) and ornithine transcarbamylase (OTCase), are present in most organisms from bacteria to humans. Recently, structures of four new transcarbamylase members, N-acetyl-l-ornithine transcarbamylase (AOTCase), N-succinyl-l-ornithine transcarbamylase (SOTCase), ygeW encoded transcarbamylase (YTCase) and putrescine transcarbamylase (PTCase) have also been determined. Crystal structures of these enzymes have shown that they have a common overall fold with a trimer as their basic biological unit. The monomer structures share a common CP binding site in their N-terminal domain, but have different second substrate binding sites in their C-terminal domain. The discovery of three new transcarbamylases, l-2,3-diaminopropionate transcarbamylase (DPTCase), l-2,4-diaminobutyrate transcarbamylase (DBTCase) and ureidoglycine transcarbamylase (UGTCase), demonstrates that our knowledge and understanding of the spectrum of the transcarbamylase family is still incomplete. In this review, we summarize studies on the structures and function of transcarbamylases demonstrating how structural information helps to define biological function and how small structural differences govern enzyme specificity. Such information is important for correctly annotating transcarbamylase sequences in the genome databases and for identifying new members of the transcarbamylase family. Full article

Corynebacterium glutamicum shows great potential for the production of the glutamate-derived diamine putrescine, a monomeric compound of polyamides. A genome-scale stoichiometric model of a C. glutamicum strain with reduced ornithine transcarbamoylase activity, derepressed arginine biosynthesis, and an anabolic plasmid-addiction system for heterologous expression of E. coli ornithine decarboxylase gene speC was investigated by flux balance analysis with respect to its putrescine production potential. Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering. Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%. Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene snaA and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant. The resulting C. glutamicum strain NA6 obtained by systems metabolic engineering accumulated two fold more putrescine than the base strain, i.e., 58.1 ± 0.2 mM, and showed a specific productivity of 0.045 g·g⁻¹·h⁻¹ and a yield on glucose of 0.26 g·g⁻¹. Full article

A simple and highly sensitive procedure is described enabling the simultaneous determination of biogenic polyamines (PAs) and their related monoacetyl derivatives in stomach tissue. The method is based on HPLC using octane sulfonate as an ion-pairing agent employed in acetate buffers at pH 4.5. The application is accompanied with fluorescence detection followed by post-column derivatization with o-phthaldialdehyde at room temperature (20±0.5°C). N1- and N8-acetylspermidines (ASPD) can be determined with this method in the same run without performing any special procedures or pre-purification in concentrations exceeding 8.5 pmoles. The variability in reproducibility of the day-today precision and duplicate determination, and simultaneous determination of standard mixture and biological samples were found < 2%. The mean (± s.e.mean) retention times (n=12) for putrescine (Put), N1-ASPD, N8-ASPD, spermidine (Spd) and spermine (Spm) are 8.97±0.025; 17.64±0.063; 18.99±0.133; 28.20±0.070 and 39.81±0.098 min, respectively. The method was applied to determine PAs and specifically N1- and N8-ASPD in glandular part of stomach tissue of fasting rats (STFR) without any interference with endogenous aminoacids, histamine, and other reactive moieties. PAs and both mono-ASPD have been successfully determined in the STFR and the values are as follows: Put 37.2±10.1; N1-ASPD 5.88±0.48; N8-ASPD 4.43±0.94; Spd 750.7±22.7 and Spm 618.2±37.4 nmole/g of wet tissue. Information on gastric tissue polyamines and their acetylated derivatives may be useful in understanding the mechanism of drugs or agents that play some part in gastric ulcer production or its repair mechanisms. Full article