Disentangling hydroxynitrile glucoside biosynthesis in a barley (Hordeum vulgare) metabolon provides access to elite malting barleys for ethyl carbamate-free whisky production.

Barley produces several specialized metabolites, including five α-, ß-, and γ-hydroxynitrile glucosides (HNGs). In malting barley, presence of the α-HNG epiheterodendrin gives rise to undesired formation of ethyl carbamate in the beverage production, especially after distilling. Metabolite-GWAS identified QTLs and underlying gene candidates possibly involved in the control of the relative and absolute content of HNGs, including an undescribed MATE transporter. By screening 325 genetically diverse barley accessions, we discovered three H. vulgare ssp. spontaneum (wild barley) lines with drastic changes in the relative ratios of the five HNGs. Knock-out (KO)-lines, isolated from the barley FIND-IT resource and each lacking one of the functional HNG biosynthetic genes (CYP79A12, CYP71C103, CYP71C113, CYP71U5, UGT85F22 and UGT85F23) showed unprecedented changes in HNG ratios enabling assignment of specific and mutually dependent catalytic functions to the biosynthetic enzymes involved. The highly similar relative ratios between the five HNGs found across wild and domesticated barley accessions indicate assembly of the HNG biosynthetic enzymes in a metabolon, the functional output of which was reconfigured in the absence of a single protein component. The absence or altered ratios of the five HNGs in the KO-lines did not change susceptibility to the fungal phytopathogen Pyrenophora teres causing net blotch. The study provides a deeper understanding of the organization of HNG biosynthesis in barley and identifies a novel, single gene HNG-0 line in an elite spring barley background for direct use in breeding of malting barley, eliminating HNGs as a source of ethyl carbamate formation in whisky production.

Structure-guided engineered urethanase from Candida parapsilosis with pH and ethanol tolerance to efficiently degrade ethyl carbamate in Chinese rice wine.

Urethane hydrolase can degrade the carcinogen ethyl carbamate (EC) in fermented food, but its stability and activity limit its application. In this study, a mutant G246A and a double mutant N194V/G246A with improved cpUH activity and stability of Candida parapsilosis were obtained by site-directed mutagenesis. The catalytic efficiency (Kcat/Km) of mutant G246A and double mutant N194V/G246A are 1.95 times and 1.88 times higher than that of WT, respectively. In addition, compared with WT, the thermal stability and pH stability of mutant G246A and double mutant N194V/G246A were enhanced. The ability of mutant G246A and double mutant N194V/G246A to degrade EC in rice wine was also stronger than that of WT. The mutation increased the stability of the enzyme, as evidenced by decreased root mean square deviation (RMSD) and increased hydrogen bonds between the enzyme and substrate by molecular dynamics simulation and molecular docking analysis. The molecule modification of new cpUH promotes the industrial process of EC degradation.

Formation and hazard of ethyl carbamate and construction of genetically engineered Saccharomyces cerevisiae strains in Huangjiu (Chinese grain wine).

Huangjiu, a well-known conventional fermented Chinese grain wine, is widely consumed in Asia for its distinct flavor. Trace amounts of ethyl carbamate (EC) may be generated during the fermentation or storage process. The International Agency for Research on Cancer elevated EC to a Class 2A carcinogen, so it is necessary to regulate EC content in Huangjiu. The risk of intake of dietary EC is mainly assessed through the margin of exposure (MOE) recommended by the European Food Safety Authority, with a smaller MOE indicating a higher risk. Interventions are necessary to reduce EC formation. As urea, one of the main precursors of EC formation in Huangjiu, is primarily produced by Saccharomyces cerevisiae through the catabolism of arginine, the construction of dominant engineered fermentation strains is a favorable trend for the future production and application of Huangjiu. This review summarized the formation and carcinogenic mechanism of EC from the perspectives of precursor substances, metabolic pathways after ingestion, and risk assessment. The methods of constructing dominant S. cerevisiae strains in Huangjiu by genetic engineering technology were reviewed, which provided an important theoretical basis for reducing EC content and strengthening practical control of Huangjiu safety, and the future research direction was prospected.

TNF-α-dependent lung inflammation upregulates PD-L1 in monocyte-derived macrophages to contribute to lung tumorigenesis.

Chronic inflammation, which is dominated by macrophage-involved inflammatory responses, is an instigator of cancer initiation. Macrophages are the most abundant immune cells in healthy lungs, and associated with lung tumor development and promotion. PD-L1 is a negative molecule in macrophages and correlated with an immunosuppressive function in tumor environment. Macrophages expressing PD-L1, rather than tumor cells, exhibits a critical role in tumor growth and progression. However, whether and how PD-L1 in macrophages contributes to inflammation-induced lung tumorigenesis requires further elucidation. Here, we found that higher expression of PD-L1 in CD11b+ CD206+ macrophages was positively correlated with tumor progression and PD-1+ CD8+ T cells population in human adenocarcinoma patients. In the urethane-induced inflammation-driven lung adenocarcinoma (IDLA) mouse model, the infiltration of circulating CD11bhigh F4/80+ monocyte-derived macrophages (MoMs) was increased in pro-tumor inflamed lung tissues and lung adenocarcinoma. PD-L1 was mainly upregulated in MoMs associated with enhanced T cells exhaustion in lung tissues. Anti-PD-L1 treatment can reduce T cells exhaustion at pro-tumor inflammatory stage, and then inhibit tumorigenesis in IDLA. The pro-tumor lung inflammation depended on TNF-α to upregulate PD-L1 and CSN6 expression in MoMs, and induced cytokines production by alveolar type-II cells (AT-II). Furthermore, inflammatory AT-II cells could secret TNF-α to upregulate PD-L1 expression in bone-marrow driven macrophages (BM-M0). Inhibition of CSN6 decreased PD-L1 expression in TNF-α-activated macrophage in vitro, suggesting a critical role of CSN6 in PD-L1 upregulation. Thus, pro-tumor inflammation can depend on TNF-α to upregulate PD-L1 in recruited MoMs, which may be essential for lung tumorigenesis.

Features and application potential of microbial urethanases.

Urethanase (EC 3.5.1.75) can reduce ethyl carbamate (EC), a group 2A carcinogen found in foods and liquor. However, it is not yet commercially available. Urethanase has been detected as an intracellular enzyme from yeast, filamentous fungi, and bacteria. Based on the most recent progress in the sequence analysis of this enzyme, it was observed that amidase-type enzyme can degrade EC. All five enzymes had highly conserved sequences of amidase signature family, and their molecular masses were in the range of 52-62 kDa. The enzymes of Candida parapsilosis and Aspergillus oryzae formed a homotetramer, and that of Rhodococcus equi strain TB-60 existed as a monomer. Most urethanases exhibited amidase activity, and those of C. parapsilosis and A. oryzae also demonstrated high activity against acrylamide, which is a group 2A carcinogen. It was recently reported that urease and esterase also exhibited urethanase activity. Although research on the enzymatic degradation of EC has been very limited, recently some sequences of EC-degrading enzyme have been elucidated, and it is anticipated that new enzymes would be developed and applied into practical use. KEY POINTS: • Recently, some urethanase sequences have been elucidated • The amino acid residues that formed the catalytic triad were conserved • Urethanase shows amidase activity and can also degrade acrylamide.

Identification and expression of bifunctional acid urea-degrading enzyme/urethanase from Enterobacter sp. R-SYB082 and its application in degradation of ethyl carbamate in Chinese rice wine (Huangjiu).

BACKGROUND: Ethyl carbamate (EC) is a potential carcinogen existing in fermented foods such as Chinese rice wine (Huangjiu). Since urea is an important precursor of EC, the degradation of urea could be an effective way to reduce EC in foods. RESULTS: In this study, an Enterobacter sp. R-SYB082 with acid urea degradation characteristics was obtained through microbial screening. Further research isolated a new acid urea-degrading enzyme from R-SYB082 strain - ureidoglycolate amidohydrolase (UAH) - which could degrade EC directly. The cloning and expression of UAH in Escherichia coli BL21 (DE3) suggested that the activity of urea-degrading enzyme reached 3560 U L-1 , while urethanase activity reached 2883 U L-1 in the optimal fermentation condition. The enzyme had the dual ability of degrading substrate urea and product EC. The removal rate of EC in Chinese rice wine could reach 90.7%. CONCLUSION: This study provided a new method for the integrated control of EC in Chinese rice wine and other fermented foods. © 2022 Society of Chemical Industry.

Effects of the pre-fermentative addition of chitosan on the nitrogenous fraction and the secondary fermentation products of SO2 -free red wines.

BACKGROUND: Different red winemaking were carried out to evaluate the effects of the prefermentative addition of chitosan, as an alternative to the use of SO2 , on the secondary products of alcoholic fermentation, yeast available nitrogen (YAN), biogenic amines and ethyl carbamate. RESULTS: The wines made with chitosan presented higher total acidity and higher content of tartaric and succinic acids than those made only with SO2 . The use of chitosan in winemaking resulted in wines with higher glycerol and diacetyl content without increasing the concentration of ethanol, acetic acid, acetaldehyde or butanediol. YAN was lower in wines made with chitosan, which may mean an advantage for the microbial stability of the wines. Furthermore, the use of chitosan at the beginning of alcoholic fermentation did not increase the concentration of biogenic amines or the formation of ethyl carbamate in SO2 -free red wines. CONCLUSION: The total or partial substitution of SO2 for chitosan at the beginning of the alcoholic fermentation gives rise to quality red wines without negatively affecting their nitrogen fraction or their very important secondary fermentation products such as acetic acid or acetaldehyde. © 2020 Society of Chemical Industry.

UHPLC-QTOFMS-based metabolomic analysis of serum and urine in rats treated with musalais containing varying ethyl carbamate content.

The aim of this work is to investigate the effect of the ethyl carbamate (EC) content in musalais on the metabolism of rats. Electron beam irradiation was performed to decrease the content of EC in musalais, and Sprague Dawley rats were subjected to intragastric administration of musalais with varying EC content (high, medium, and low groups). Control rats were fed normally without any treatment. Serum and urine samples were analyzed using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. Principal component analysis and orthogonal projections to latent structures discriminant analysis (OPLS-DA) were performed to detect changes in the metabolite profile in the serum and urine in order to identify the differential metabolites and metabolic pathways. The results demonstrated clear differences in the serum and urine metabolic patterns between control and treatment groups. Ions in treatment groups with variable importance in the projection of >1 (selected from the OPLS-DA loading plots) and Ps < 0.05 (Student t test) compared to control group were identified as candidate metabolites. Analysis of the metabolic pathways relevant to the identified differential metabolites revealed that high EC content in musalais (10 mg/kg) mainly affected rats through valine, leucine, and isoleucine biosynthesis and nicotinate and nicotinamide metabolism, which were associated with energy metabolism. In addition, this work suggests that EC can induce oxidative stress via inhibition of glycine content.

Metabolic engineering of Saccharomyces cerevisiae using the CRISPR/Cas9 system to minimize ethyl carbamate accumulation during Chinese rice wine fermentation.

Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85DUR1,2/DUR3-c. Consequently, the DUR3 expression level was significantly enhanced in the modified strain, resulting in increased utilization of urea. The brewing test showed that N85DUR1,2/DUR3-c reduced urea and EC concentrations by 92.0% and 58.5%, respectively, compared with those of the original N85 strain. Moreover, the engineered strain showed good genetic stability in reducing urea content during the repeated brewing experiments. Importantly, the genetic manipulation had a negligible effect on the growth and fermentation characteristics of the yeast strain. Therefore, the constructed strain is potentially suitable for application to reduce urea and EC contents during production of Chinese rice wine. KEY POINTS: • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation. • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation.

Chinese Yellow Rice Wine Processing with Reduced Ethyl Carbamate Formation by Deleting Transcriptional Regulator Dal80p in Saccharomyces cerevisiae.

Ethyl carbamate (EC) is a potential carcinogen that forms spontaneously during Chinese rice wine fermentation. The primary precursor for EC formation is urea, which originates from both external sources and arginine degradation. Urea degradation is suppressed by nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. The regulation of NCR is mediated by two positive regulators (Gln3p, Gat1p/Nil1p) and two negative regulators (Dal80p/Uga43p, Deh1p/Nil2p/GZF3p). DAL80 revealed higher transcriptional level when yeast cells were cultivated under nitrogen-limited conditions. In this study, when DAL80-deleted yeast cells were compared to wild-type BY4741 cells, less urea was accumulated, and genes involved in urea utilization were up-regulated. Furthermore, Chinese rice wine fermentation was conducted using dal80Δ cells; the concentrations of urea and EC were both reduced when compared to the BY4741 and traditional fermentation starter. The findings of this work indicated Dal80p is involved in EC formation possibly through regulating urea metabolism and may be used as the potential target for EC reduction.

Metabolic engineering of arginine permeases to reduce the formation of urea in Saccharomyces cerevisiae.

Urea is an important precursor of the harmful carcinogenic product ethyl carbamate in fermented wines. To decipher more fully the contributions of three arginine permeases, Can1p, Gap1p and Alp1p in urea formation, various engineered strains were examined for their ability to form urea. This included seven mutants with different combinations of permease deficiency and grown in both simple and more complex media, and the wild-type strain modified to overexpress the three arginine permeases. A truncated GATA transcription factor, Gln3p1-653, was also overexpressed in the arginine permease deficient mutants to determine whether the permeases have a synergistic effect on urea formation with other urea reducing modules. Additionally, in this study, transcriptional changes of four genes related to arginine metabolism and urea formation were investigated. We found that the three amino acids permeases affect urea formation mainly through the utilization of arginine in YNB medium containing the 20 common amino acids. The deletion mutant Δgap1Δcan1 showed a significant reduction (68%) in extracellular urea compared to the wild-type strain grown in YPD medium. Overexpression of a truncated Gln3p in Δgap1Δcan1 reduced the extracellular urea concentration even further (by 67%) than that in the wild-type strain and showed a synergistic effect with Δgap1Δcan1 and Δalp1Δgap1Δcan1 for extracellular reduction. Moreover, the results of this study provide a promising way to reduce urea accumulation during wine fermentation using S. cerevisiae, and present an approach to control metabolism and product formation through the regulation of amino acid permeases.

CAR1 deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by Saccharomyces cerevisiae.

Enormous advances in genome editing technology have been achieved in recent decades. Among newly born genome editing technologies, CRISPR/Cas9 is considered revolutionary because it is easy to use and highly precise for editing genes in target organisms. CRISPR/Cas9 technology has also been applied for removing unfavorable target genes. In this study, we used CRISPR/Cas9 technology to reduce ethyl carbamate (EC), a potential carcinogen, which was formed during the ethanol fermentation process by yeast. Because the yeast CAR1 gene encoding arginase is the key gene to form ethyl carbamate, we inactivated the yeast CAR1 gene by the complete deletion of the gene or the introduction of a nonsense mutation in the CAR1 locus using CRISPR/Cas9 technology. The engineered yeast strain showed a 98 % decrease in specific activity of arginase while displaying a comparable ethanol fermentation performance. In addition, the CAR1-inactivated mutants showed reduced formation of EC and urea, as compared to the parental yeast strain. Importantly, CRISPR/Cas9 technology enabled generation of a CAR1-inactivated yeast strains without leaving remnants of heterologous genes from a vector, suggesting that the engineered yeast by CRISPR/Cas9 technology might sidestep GMO regulation.

Reduced production of ethyl carbamate for wine fermentation by deleting CAR1 in Saccharomyces cerevisiae.

Ethyl carbamate (EC), a pluripotent carcinogen, is mainly formed by a spontaneous chemical reaction of ethanol with urea in wine. The arginine, one of the major amino acids in grape musts, is metabolized by arginase (encoded by CAR1) to ornithine and urea. To reduce the production of urea and EC, an arginase-deficient recombinant strain YZ22 (Δcarl/Δcarl) was constructed from a diploid wine yeast, WY1, by successive deletion of two CAR1 alleles to block the pathway of urea production. The RT-qPCR results indicated that the YZ22 almost did not express CAR1 gene and the specific arginase activity of strain YZ22 was 12.64 times lower than that of parent strain WY1. The fermentation results showed that the content of urea and EC in wine decreased by 77.89 and 73.78 %, respectively. Furthermore, EC was forming in a much lower speed with the lower urea during wine storage. Moreover, the two CAR1 allele deletion strain YZ22 was substantially equivalent to parental strain in terms of growth and fermentation characteristics. Our research also suggested that EC in wine originates mainly from urea that is produced by the arginine.

[Characterization of arginine utilization strains from fermented grains and evaluation of their contribution to citrulline accumulation in Chinese Luzhou-flavor spirits].

Objective: This study aimed to characterize microorganisms responsible for accumulation of ethyl carbamate precursor citrulline in fermented grains during Luzhou-flavor spirits fermentation process, to provide theoretical basis for clarifying mechanisms of ethyl carbamate formation. Methods: High-throughput strain screening approach was used to isolate strains with high arginine utilization and high citrulline accumulation capability. Essential genes that comprise the arginine deiminase pathway of these isolates were verified and a modified Chinese liquor fermentation process was conducted by adding these strains. Results: A total of 20 strains with high arginine utilizing ability were obtained. Among them, Lactococcus garvieae LD3, Bacillus amyloliquefaciens BG5, Pediococcus acidilactici PH7 and Staphylococcus pasteuri SH11 exhibited high efficacy to produce citrulline from arginine. These strains could also increase citrulline in fermented grains. Conclusion: The accumulation of citrulline in fermented grains was confirmed to be corresponding to arginine utilization by Lactococcus garvieae, Bacillus amyloliquefaciens, Pediococcus acidilactici and Staphylococcus pasteuri through the arginine deiminase pathway.

[Metabolism of ethyl carbamate precursors in soy sauce by Zygosaccharomyces rouxii ZQ02Metabolism of ethyl carbamate precursors in soy sauce by Zygosaccharomyces rouxii ZQ02].

Objective: To study nitrogen metabolism of Zygosaccharomyces rouxii and its relationship with the formation of soy sauce ethyl carbamate precursors. Methods: Z. rouxii ZQ02 was cultivated with single source of nitrogen, preferred nitrogen sources or under salt stress, to investigate its ability of using arginine, citrulline and urea. Results: Alanine, glycine and asparaginate were confirmed to be the preferred nitrogen sources of Z. rouxii ZQ02. Addition of preferred nitrogen sources did not inhibit the use of urea and citrulline, on the contrary, the consumption of urea and citrulline by Z. rouxii ZQ02 was stimulated with the addition of alanine and glycine. Z. rouxii ZQ02 did not accumulate any citrulline and urea from degradation of arginine. Urea and citrulline were used by Z. rouxii ZQ02 in the medium with single source of nitrogen. However, use of citrulline and urea by Z. rouxii ZQ02 was strongly inhibited under saline stress, resulting in the incomplete use of ethyl carbamate precursors. Conclusion: Use of citrulline and urea by Z. rouxii ZQ02 was strongly inhibited under high salt stress, resulting in the accumulation of ethyl carbamate precursors produced by other microorganisms during soy sauce fermentation.

Function of the loading module in CorI and of the O-methyltransferase CorH in vinyl carbamate biosynthesis of the antibiotic corallopyronin A.

Corallopyronin A is a promising in vivo active antibiotic, currently undergoing preclinical evaluation. This myxobacterial compound interferes with a newly identified drug target site, i.e., the switch region of the bacterial DNA-dependent RNA-polymerase (RNAP). Since this target site differs from that of known RNAP inhibitors such as the rifamycins, corallopyronin A shows no cross-resistance with other antibacterial agents. Corallopyronin A is a polyketide synthase- and nonribosomal peptide synthetase-derived molecule whose structure and biosynthesis is distinguished by several peculiarities, such as the unusual vinyl carbamate functionality whose formation involves carbonic acid as an unprecedented C1-starter unit. Using in vitro experiments the nature of this starter molecule was revealed to be the methyl ester of carbonic acid. Biochemical investigations showed that methylation of carbonic acid is performed by the O-methyltransferase CorH. These experiments shed light on the biosynthesis of the Eastern chain of α-pyrone antibiotics such as corallopyronin A.

Metabolic engineering of the regulators in nitrogen catabolite repression to reduce the production of ethyl carbamate in a model rice wine system.

Rice wine has been one of the most popular traditional alcoholic drinks in China. However, the presence of potentially carcinogenic ethyl carbamate (EC) in rice wine has raised a series of food safety issues. During rice wine production, the key reason for EC formation is urea accumulation, which occurs because of nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. NCR represses urea utilization by retaining Gln3p in the cytoplasm when preferred nitrogen sources are present. In order to increase the nuclear localization of Gln3p, some possible phosphorylation sites on the nuclear localization signal were mutated and the nuclear localization regulation signal was truncated, and the disruption of URE2 provided an additional method of reducing urea accumulation. By combining these strategies, the genes involved in urea utilization (DUR1,2 and DUR3) could be significantly activated in the presence of glutamine. During shake flask fermentations of the genetically modified strains, very little urea accumulated in the medium. Furthermore, the concentrations of urea and EC were reduced by 63% and 72%, respectively, in a model rice wine system. Examination of the normal nutrients in rice wine indicated that there were few differences in fermentation characteristics between the wild-type strain and the genetically modified strain. These results show that metabolic engineering of the NCR regulators has great potential as a method for eliminating EC during rice wine production.

Reduced production of Ethyl Carbamate in wine by regulating the accumulation of arginine in Saccharomyces cerevisiae.

Ethyl carbamate (EC), a multisite carcinogenic compound, is naturally produced from urea and ethanol in alcoholic beverages. In order to reduce the content of EC in wine, the accumulation of arginine in Saccharomyces cerevisiae was regulated by genetic modifying genes involved in arginine transport and synthesis pathways to reduce the production of urea. Knockout of genes encoding arginine permease (Can1p) and amino acid permease (Gap1p) on the cell membrane as well as argininosuccinate synthase (Arg1) respectively resulted in a maximum reduction of 66.88% (9.40 µg/L) in EC, while overexpressing the gene encoding amino acid transporter (Vba2) reduced EC by 52.94% (24.13 µg/L). Simultaneously overexpressing Vba2 and deleting Arg1 showed the lowest EC production with a decrease of 68% (7.72 µg/L). The yield of total higher alcohols of the mutants all decreased compared with that of the original strain. Comprehensive consideration of flavor compound contents and sensory evaluation results indicated that mutant YG21 obtained by deleting two allele coding Gap1p performed best in must fermentation of Cabernet Sauvignon with the EC content low to 9.40 µg/L and the contents of total higher alcohols and esters of 245.61 mg/L and 41.71 mg/L respectively. This study has provided an effective strategy for reducing the EC in wine.

Comparative analysis of hippocampal extracellular space uncovers widely altered peptidome upon epileptic seizure in urethane-anaesthetized rats.

BACKGROUND: The brain extracellular fluid (ECF), composed of secreted neurotransmitters, metabolites, peptides, and proteins, may reflect brain processes. Analysis of brain ECF may provide new potential markers for synaptic activity or brain damage and reveal additional information on pathological alterations. Epileptic seizure induction is an acute and harsh intervention in brain functions, and it can activate extra- and intracellular proteases, which implies an altered brain secretome. Thus, we applied a 4-aminopyridine (4-AP) epilepsy model to study the hippocampal ECF peptidome alterations upon treatment in rats. METHODS: We performed in vivo microdialysis in the hippocampus for 3-3 h of control and 4-AP treatment phase in parallel with electrophysiology measurement. Then, we analyzed the microdialysate peptidome of control and treated samples from the same subject by liquid chromatography-coupled tandem mass spectrometry. We analyzed electrophysiological and peptidomic alterations upon epileptic seizure induction by two-tailed, paired t-test. RESULTS: We detected 2540 peptides in microdialysate samples by mass spectrometry analysis; and 866 peptides-derived from 229 proteins-were found in more than half of the samples. In addition, the abundance of 322 peptides significantly altered upon epileptic seizure induction. Several proteins of significantly altered peptides are neuropeptides (Chgb) or have synapse- or brain-related functions such as the regulation of synaptic vesicle cycle (Atp6v1a, Napa), astrocyte morphology (Vim), and glutamate homeostasis (Slc3a2). CONCLUSIONS: We have detected several consequences of epileptic seizures at the peptidomic level, as altered peptide abundances of proteins that regulate epilepsy-related cellular processes. Thus, our results indicate that analyzing brain ECF by in vivo microdialysis and omics techniques is useful for monitoring brain processes, and it can be an alternative method in the discovery and analysis of CNS disease markers besides peripheral fluid analysis.

Wine composition plays an important role in the control of carcinogenic precursor formation by Lactobacillus hilgardii X1B.

BACKGROUND: Lactobacillus hilgardii, a wine lactic acid bacterium, is able to use arginine, through the arginine deiminase pathway with the formation of citrulline, a precursor of the carcinogen ethyl carbamate. The influence of different Argentine wine varieties (Merlot, Cabernet Sauvignon and Malbec), on bacterial growth and arginine metabolism was examined. Furthermore, the effect of different components normally present in wines on the enzyme activities of the arginine deiminase system was determined. RESULTS: Malbec wine under all conditions assayed (33, 50 and 100% supplemented wine:basal media) showed higher arginine consumption and citrulline production than the other wines, as well as the highest bacterial growth and survival of Lactobacillus hilgardii X1B. Glucose and L-malic inhibited both arginine deiminase enzymes while fructose and citric acid only inhibited arginine deiminase. The red wines assayed in this study had different composition, and this is an explanation for the different behavior of the bacterium. CONCLUSION: The highest citrulline production in Malbec wine could be correlated with its lower concentrations of glucose, fructose, citric and phenolic acid than the other wines. Therefore, a wine with lower concentration of these sugars and acids could be dangerous due to the formation of ethyl carbamate precursors.