Impact of phenylalanine on Hanseniaspora vineae aroma metabolism during wine fermentation.

Hanseniaspora vineae exhibits extraordinary positive oenological characteristics contributing to the aroma and texture of wines, especially by its ability to produce great concentrations of benzenoid and phenylpropanoid compounds compared with conventional Saccharomyces yeasts. Consequently, in practice, sequential inoculation of H. vineae and Saccharomyces cerevisiae allows to improve the aromatic quality of wines. In this work, we evaluated the impact on wine aroma produced by increasing the concentration of phenylalanine, the main amino acid precursor of phenylpropanoids and benzenoids. Fermentations were carried out using a Chardonnay grape juice containing 150 mg N/L yeast assimilable nitrogen. Fermentations were performed adding 60 mg/L of phenylalanine without any supplementary addition to the juice. Musts were inoculated sequentially using three different H. vineae strains isolated from Uruguayan vineyards and, after 96 h, S. cerevisiae was inoculated to complete the process. At the end of the fermentation, wine aromas were analysed by both gas chromatography-mass spectrometry and sensory evaluation through a panel of experts. Aromas derived from aromatic amino acids were differentially produced depending on the treatments. Sensory analysis revealed more floral character and greater aromatic complexity when compared with control fermentations without phenylalanine added. Moreover, fermentations performed in synthetic must with pure H. vineae revealed that even tyrosine can be used in absence of phenylalanine, and phenylalanine is not used by this yeast for the synthesis of tyrosine derivatives.

Supramolecular fluorescent probe based on a host-guest complex for amino acids recognition and detection in aqueous solution.

A supramolecular fluorescent probe based on a host-guest complex has been developed for amino acid recognition and detection in aqueous solution. Cucurbit[7]uril (Q[7]) with 4-(4-dimethylamino-styrene) quinoline (DSQ) formed a fluorescent probe (DSQ@Q[7]). The DSQ@Q[7] fluorescent probe nearly generated changes in fluorescence in response to four amino acids (arginine, histidine, phenylalanine and tryptophan). These changes were attributed to the host-guest interaction between DSQ@Q[7] and amino acids, which occurred as a consequence of the subtle cooperation of ionic dipole and hydrogen bonding. Linear discriminant analysis showed that the fluorescent probe could recognize and distinguish four amino acids, and a mixture with different concentration ratios could be well categorized in ultrapure water and tap water.

Selection of allosteric dnazymes that can sense phenylalanine by expression-SELEX.

Aptamers are ligand-binding RNA or DNA molecules and have been widely examined as biosensors, diagnostic tools, and therapeutic agents. The application of aptamers as biosensors commonly requires an expression platform to produce a signal to report the aptamer-ligand binding event. Traditionally, aptamer selection and expression platform integration are two independent steps and the aptamer selection requires the immobilization of either the aptamer or the ligand. These drawbacks can be easily overcome through the selection of allosteric DNAzymes (aptazymes). Herein, we used the technique of Expression-SELEX developed in our laboratory to select for aptazymes that can be specifically activated by low concentrations of l-phenylalanine. We chose a previous DNA-cleaving DNAzyme known as II-R1 as the expression platform for its low cleavage rate and used stringent selection conditions to drive the selection of high-performance aptazyme candidates. Three aptazymes were chosen for detailed characterization and these DNAzymes were found to exhibit a dissociation constant for l-phenylalanine as low as 4.8 µM, a catalytic rate constant improvement as high as 20 000-fold in the presence of l-phenylalanine, and the ability to discriminate against closely related l-phenylalanine analogs including d-phenylalanine. This work has established the Expression-SELEX as an effective SELEX method to enrich high-quality ligand-responsive aptazymes.

Comparison of Tandem Mass Spectrometry and the Fluorometric Method-Parallel Phenylalanine Measurement on a Large Fresh Sample Series and Implications for Newborn Screening for Phenylketonuria.

Phenylketonuria (PKU) was the first disease to be identified by the newborn screening (NBS) program. Currently, there are various methods for determining phenylalanine (Phe) values, with tandem mass spectrometry (MS/MS) being the most widely used method worldwide. We aimed to compare the MS/MS method with the fluorometric method (FM) for measuring Phe in the dried blood spot (DBS) and the efficacy of both methods in the NBS program. The FM was performed using a neonatal phenylalanine kit and a VICTOR2TM D fluorometer. The MS/MS method was performed using a NeoBaseTM 2 kit and a Waters Xevo TQD mass spectrometer. The Phe values measured with the MS/MS method were compared to those determined by the FM. The cut-off value for the NBS program was set at 120 µmol/L for FM and 85 µmol/L for MS/MS. We analyzed 54,934 DBS. The measured Phe values varied from 12 to 664 µmol/L, with a median of 46 µmol/L for the MS/MS method and from 10 to 710 µmol/L, with a median of 70 µmol/L for the FM. The Bland-Altman analysis indicated a bias of -38.9% (-23.61 µmol/L) with an SD of 21.3% (13.89 µmol/L) when comparing the MS/MS method to the FM. The Phe value exceeded the cut-off in 187 samples measured with FM and 112 samples measured with MS/MS. The FM had 181 false positives, while the MS/MS method had 106 false positives. Our study showed that the MS/MS method gives lower results compared to the FM. Despite that, none of the true positives would be missed, and the number of false-positive results would be significantly lower compared to the FM.

Acibenzolar-S-methyl activates phenylpropanoid pathway to enhance resistance against Alternaria alternata in pear fruit.

BACKGROUND: Alternaria alternata is a causal agent of black spot rot of pear fruit after harvest. Acibenzolar-S-methyl (ASM) has been shown to be a potential elicitor of tolerance in several horticultural products. This work was performed to research the influence of ASM on black spot rot of Docteur Jules Guyot pears and vital enzyme activity and gene expression in the phenylpropanoid pathway. RESULTS: ASM remarkably decreased the lesion diameter of A. alternata-inoculated pears. ASM also increased phenylalanine ammonialyase, cinnamate 4-hydroxylase, cinnamyl alcohol dehydrogenase, peroxidase, polyphenol oxidase activities and gene expression, and enhanced 4-coumarate/coenzyme A ligase activity in pears. Moreover, ASM improved the content of phenylalanine, total phenolic compounds, caffeic acid, flavonoids, anthocyanin and lignin in pears. CONCLUSION: ASM could modulate vital enzyme activity and gene expression in the phenylpropanoid pathway to accelerate metabolite synthesis, thereby enhancing resistance against A. alternata in pears. © 2022 Society of Chemical Industry.

Impact of dietary supplementation with N-carbamoyl-aspartic acid on serum metabolites and intestinal microflora of sows.

BACKGROUND: N-Carbamoyl-aspartic acid (NCA) is a critical precursor for de novo biosynthesis of pyrimidine nucleotides. To investigate the cumulative effects of maternal supplementation with NCA on the productive performance, serum metabolites and intestinal microbiota of sows, 40 pregnant sows (∼day 80) were assigned into two groups: (1) the control (CON) and (2) treatment (NCA, 50 g t-1 NCA). RESULTS: Results showed that piglets from the NCA group had heavier birth weight than those in the CON group (P < 0.05). In addition, maternal supplementation with NCA decreased the backfat loss of sows during lactation (P < 0.05). Furthermore,16S-rRNA sequencing results revealed that maternal NCA supplementation decreased the abundance of Cellulosilyticum, Fournierella, Anaerovibrio, and Oribacterium genera of sows during late pregnancy (P < 0.05). Similarly, on the 14th day of lactation, maternal supplementation with NCA reduced the diversity of fecal microbes of sows as evidenced by significantly lower observed species, Chao1, and Ace indexes, and decreased the abundance of Lachnospire, Faecalibacterium, and Anaerovorax genera, while enriched the abundance of Catenisphaera (P < 0.05). Untargeted metabolomics showed that a total of 48 differentially abundant biomarkers were identified, which were mainly involved in metabolic pathways of arginine/proline metabolism, phenylalanine/tyrosine metabolism, and fatty acid biosynthesis, etc. CONCLUSION: Overall, the results indicated that NCA supplementation regulated intestinal microbial composition of sows and serum differential metabolites related to arginine, proline, phenylalanine, tyrosine, and fatty acids metabolism that may contribute to regulating the backfat loss of sows, and the birth weight and diarrhea rate of piglets. © 2022 Society of Chemical Industry.

[Metabolomics study on the difference of tongue coating metabolites between patients with intra-oral halitosis and healthy individual].

PURPOSE: To analyze the difference of metabolites of tongue coating between patients with intra-oral halitosis and healthy individuals by untargeted metabolomics, and to explore significant differences in metabolites of intra-oral halitosis as biomarkers. METHODS: The untargeted metabolomics of tongue coating samples from 12 patients with intra-oral halitosis and 12 healthy individuals were studied by liquid chromatography and mass spectrometry combined with principal component analysis and orthogonal partial least squares discriminant analysis. The value of variable importance in projection ＞1 and P＜0.05 of Student's t test in the orthogonal partial least squares-discriminant analysis model were used as the criteria to screen and determine the differential metabolites. RESULTS: There were differences in the metabolites of tongue coating between patients with intra-oral halitosis and healthy individuals, and 11 different metabolites were identified. They were valyl-arginine, glycine-phenylalanine, tryptophyl-proline, deoxyadenosine, 4,5-dihydroniveusin A, N-acetyl-DL-tryptophan, paramethasone acetate, cyclopentanol, [(2-hexylcyclopentylidene) amino]thiourea, L-pipecolic acid and taurine. In the intra-oral halitosis group, the expressions of Glycine-phenylalanine and N-acetyl-DL-tryptophan were significantly up-regulated, while the expressions of taurine were significantly down-regulated. CONCLUSIONS: There are differences in the metabolites of tongue coating between patients with intra-oral halitosis and healthy individuals. The differential metabolites with diagnostic value may be used as diagnostic markers of intra-oral halitosis.

Endogenous benzoic acid interferes with the signatures of amino acids and thiol compounds through perturbing N-methyltransferase, glutamate-cysteine ligase, and glutathione S-transferase activity in dairy products.

Endogenous benzoic acid causes adverse effects on individual health, but the potential mechanisms often remain elusive. The positive rate of benzoic acid in seventy-two goat milk samples in triplicate was 93.6 %, verifying the presence of endogenous benzoic acid. In this study, we investigated the differences in protein expression and metabolites among goat milk with different final concentrations of benzoic acid via combined proteomics and metabolomics (LOQ 3.25 to 56.63 µg L-1) analysis based on UHPLC-Q-Orbitrap HRMS. Integrated analysis showed that benzoic acid reduced the content of l-histidine (from 1.27 to 0.49 mg/L) and 1-methylhistidine (from 1.40 to 0.68 mg/L), due to the increase of benzoic acid (0-30 mg/L) concentration significantly reduced the level and activity of N-methyltransferase. Protein-metabolite interactions suggested that benzoic acid enhanced glutamate-cysteine ligase and glutathione S-transferase expression and affected l-glutamate (from 1.22 to 0.49 mg/L) and glutathione contents, eventually leading to the formation of off-flavors and oxidation of goat milk. Meanwhile, the level of l-phenylalanine (from 4.17 to 1.94 mg/L) and l-tyrosine (from 1.05 to 0.26 mg/L) progressively decreased with the increase of benzoic acid concentration, which had a deleterious effect on the nutritional value and flavor formation of goat milk. These findings clarified the mechanism by which low-dose benzoic acid negatively affects the nutritional quality and flavor formation of goat milk.

Alterations in the Gut Microbiota and Metabolomics of Seafarers after a Six-Month Sea Voyage.

Maintaining the health of seafarers is a difficult task during long-term voyages. Little is known about the corresponding changes in the gut microbiome-host interaction. This study recruited 30 seafarers undertaking a 6-month voyage and analyzed their gut microbiota using 16S rRNA gene sequencing. Fecal untargeted metabolomics analysis was performed using liquid chromatography-mass spectrometry. Significant changes in the composition of the gut microbiota and an increased ratio of Firmicutes/Bacteroidetes at the end (day 180) of the 6-month voyage, relative to the start (day 0), were observed. At the genus level, the abundances of Holdemanella and Plesiomonas were significantly increased, while the abundance of Bacteroides was decreased. Predicted microbial functional analysis revealed significant decreases in folate biosynthesis and biotin metabolism. Furthermore, 20 differential metabolites within six differentially enriched human metabolic pathways (including arginine biosynthesis, lysine degradation, phenylalanine metabolism, sphingolipid metabolism, pentose and glucuronate interconversions, and glycine, serine, and threonine metabolism) were identified by comparing the fecal metabolites at day 0 and day 180. Spearman correlation analysis revealed close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might affect specific human metabolic pathways. This study adopted a multi-omics approach and provides potential targets for maintaining the health of seafarers during long-term voyages. These findings are worthy of more in-depth exploration in future studies. IMPORTANCE Maintaining the health of seafarers undertaking long-term voyages is a difficult task. Apart from the alterations in the gut microbiome and fecal metabolites after a long-term voyage, our study also revealed that 20 differential metabolites within six differentially enriched human metabolic pathways are worthy of attention. Moreover, we found close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might impact specific human metabolic pathways. Accordingly, preventative measures, such as adjusting the gut microbiota by decreasing potential pathobionts or increasing potential probiotics as well as offsetting the decrease in B vitamins and beneficial metabolites (e.g., d-glucuronic acid and citrulline) via dietary adjustment or nutritional supplements, might improve the health of seafarers during long-term sea voyages. These findings provide valuable clues about gut microbiome-host interactions and propose potential targets for maintaining the health of seafarers engaged in long-term sea voyages.

Chemical Composition Assessment of Structural Parts (Seeds, Peel, Pulp) of Physalis alkekengi L. Fruits.

In recent years there has been an extensive search for nature-based products with functional potential. All structural parts of Physalis alkekengi (bladder cherry), including fruits, pulp, and less-explored parts, such as seeds and peel, can be considered sources of functional macro- and micronutrients, bioactive compounds, such as vitamins, minerals, polyphenols, and polyunsaturated fatty acids, and dietetic fiber. The chemical composition of all fruit structural parts (seeds, peel, and pulp) of two phenotypes of P. alkekengi were studied. The seeds were found to be a rich source of oil, yielding 14-17%, with abundant amounts of unsaturated fatty acids (over 88%) and tocopherols, or vitamin E (up to 5378 mg/kg dw; dry weight). The predominant fatty acid in the seed oils was linoleic acid, followed by oleic acid. The seeds contained most of the fruit's protein (16-19% dw) and fiber (6-8% dw). The peel oil differed significantly from the seed oil in fatty acid and tocopherol composition. Seed cakes, the waste after oil extraction, contained arginine and aspartic acid as the main amino acids; valine, phenylalanine, threonine, and isoleucine were present in slightly higher amounts than the other essential amino acids. They were also rich in key minerals, such as K, Mg, Fe, and Zn. From the peel and pulp fractions were extracted fruit concretes, aromatic products with specific fragrance profiles, of which volatile compositions (GC-MS) were identified. The major volatiles in peel and pulp concretes were ß-linalool, α-pinene, and γ-terpinene. The results from the investigation substantiated the potential of all the studied fruit structures as new sources of bioactive compounds that could be used as prospective sources in human and animal nutrition, while the aroma-active compounds in the concretes supported the plant's potential in perfumery and cosmetics.

Defining the HIV Capsid Binding Site of Nucleoporin 153.

The interaction between the HIV-1 capsid and human nucleoporin 153 (NUP153) is vital for delivering the HIV-1 preintegration complex into the nucleus via the nuclear pore complex. The interaction with the capsid requires a phenylalanine/glycine-containing motif in the C-terminus of NUP153 (NUP153C). This study used molecular modeling and biochemical assays to comprehensively determine the amino acids in NUP153 that are important for capsid interaction. Molecular dynamics, FoldX, and PyRosetta simulations delineated the minimal capsid binding motif of NUP153 based on the known structure of NUP153 bound to the HIV-1 capsid hexamer. Computational predictions were experimentally validated by testing the interaction of NUP153 with capsid using an in vitro binding assay and a cell-based TRIM-NUP153C restriction assay. This work identified eight amino acids from P1411 to G1418 that stably engage with capsid, with significant correlations between the interactions predicted by molecular models and empirical experiments. This validated the usefulness of this multidisciplinary approach to rapidly characterize the interaction between human proteins and the HIV-1 capsid. IMPORTANCE The human immunodeficiency virus (HIV) can infect nondividing cells by interacting with the host nuclear pore complex. The host nuclear pore protein NUP153 directly interacts with the HIV capsid to promote viral nuclear entry. This study used a multidisciplinary approach combining computational and experimental techniques to comprehensively map the effect of mutating the amino acids of NUP153 on HIV capsid interaction. This work showed a significant correlation between computational and empirical data sets, revealing that the HIV capsid interacted specifically with only six amino acids of NUP153. The simplicity of the interaction motif suggested other FG-containing motifs could also interact with the HIV-1 capsid. Furthermore, it was predicted that naturally occurring polymorphisms in human and nonhuman primates would disrupt NUP153 interaction with capsid, potentially protecting certain populations from HIV-1 infection.

Metallomic and Untargeted Metabolomic Signatures of Human Milk from SARS-CoV-2 Positive Mothers.

SCOPE: Lack of information about the impact of maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the elemental and metabolomic profile of human milk (HM). METHODS AND RESULTS: An observational study on HM from mothers with COVID-19 is conducted including a prepandemic control group. Maternal-infant clinical records and symptomatology are recorded. The absolute quantification of elements and untargeted relative metabolomic profiles are determined by inductively coupled plasma mass spectrometry and gas chromatography coupled to mass spectrometry, respectively. Associations of HM SARS-CoV-2 antibodies with elemental and metabolomic profiles are studied. COVID-19 has a significant impact on HM composition. COVID-19 reduces the concentrations of Fe, Cu, Se, Ni, V, and Aluminium (Al) and increases Zn compared to prepandemic control samples. A total of 18 individual metabolites including amino acids, peptides, fatty acids and conjugates, purines and derivatives, alcohols, and polyols are significantly different in HM from SARS-CoV-2 positive mothers. Aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine, and linoleic acid pathways are significantly altered. Differences are obtained depending on COVID-19 symptomatic and asymptomatic status. CONCLUSIONS: This study provides unique insights about the impact of maternal SARS-CoV-2 infection on the elemental and metabolomic profiles of HM that warrants further research due the potential implications for infant health.

Functional divergence in the proteins encoded by ARO80 from S. uvarum, S. kudriavzevii and S. cerevisiae explain differences in the aroma production during wine fermentation.

Phenylethanol (PE) and phenylethyl acetate (PEA) are commonly desired compounds in wine because of their rose-like aroma. The yeast S. cerevisiae produces the PE either through de novo biosynthesis by shikimate pathway followed by the Ehrlich pathway or the direct phenylalanine catabolism via Ehrlich pathway, and then converted into PEA. Previous work demonstrated that, compared to S. cerevisiae, other Saccharomyces species, such as S. kudriavzevii and S. uvarum, produce higher concentrations of PE and PEA from the precursor phenylalanine, which indicates differential activities of the biosynthetic-involved enzymes. A previous in-silico analysis suggested that the transcriptional activator Aro80p is one of the best candidates to explain these differences. An improved functional analysis identified significant radical amino acid changes in the S. uvarum and S. kudriavzevii Aro80p that could impact the expression of the catabolic genes ARO9 and ARO10, and hence, the production of PE from phenylalanine. Indeed, wine S. cerevisiae strains carrying the S. uvarum and S. kudriavzevii ARO80 alleles increased the production of both compounds in the presence of phenylalanine by increasing the expression of ARO9 and ARO10. This study provides novel insights of the unidentified Aro80p regulatory region and the potential usage of alternatives ARO80 alleles to enhance the PE and PEA concentration in wine.

Competitive Self-Assembly Interaction between Ferrocenyl Units and Amino Acids for Entry into the Cavity of ß-Cyclodextrin for Chiral Electroanalysis.

At present, chiral electroanalysis of nonelectroactive chiral compounds still remains a challenge because they cannot provide an electrochemical signal by themselves. Here, a strategy based on a competitive self-assembly interaction of a ferrocene (Fc) unit and the testing isomers entering into the cavity of ß-cyclodextrin (ß-CD) was carried out for chiral electroanalysis. First of all, the Fc derivative was directly bridged to silica microspheres, followed by inclusion into the cavity of ß-CD. As expected, once it was modified onto the surface of a carbon working electrode as an electrochemical sensor, SiO2@Fc-CD-WE, its differential pulse voltammetry signal would markedly decrease compared with the uncovered Fc. Next, when l- and d-isomers of amino acids that included histidine, threonine, phenylalanine, and glutamic acid were examined using SiO2@Fc-CD-WE, it showed an enantioselective entry of amino acids into the cavity of ß-cyclodextrin instead of Fc, resulting in the release of Fc with signal enhancement. For histidine, glutamic acid, and threonine, l-isomers showed a higher peak current response compared with d-isomers. The peak current ratios between l- and d-isomers were 2.88, 1.21, and 1.40, respectively. At the same time, the opposite phenomenon occurred for phenylalanine with a peak current ratio of 3.19 between d- and l-isomers. In summary, we are assured that the recognition strategy based on the supramolecular interaction can enlarge the detection range of chiral compounds by electrochemical analysis.

A Review of Sensors and Biosensors Modified with Conducting Polymers and Molecularly Imprinted Polymers Used in Electrochemical Detection of Amino Acids: Phenylalanine, Tyrosine, and Tryptophan.

Recently, the studies on developing sensors and biosensors-with an obvious interdisciplinary character-have drawn the attention of many researchers specializing in various fundamental, but also complex domains such as chemistry, biochemistry, physics, biophysics, biology, bio-pharma-medicine, and bioengineering. Along these lines, the present paper is structured into three parts, and is aimed at synthesizing the most relevant studies on the construction and functioning of versatile devices, of electrochemical sensors and biosensors, respectively. The first part presents examples of the most representative scientific research focusing on the role and the importance of the phenylalanine, tyrosine, and tryptophan amino acids, selected depending on their chemical structure and their impact on the central nervous system. The second part is dedicated to presenting and exemplifying conductor polymers and molecularly imprinted polymers used as sensitive materials in achieving electrochemical sensors and biosensors. The last part of the review analyzes the sensors and biosensors developed so far to detect amino acids with the aid of conductor polymers and molecularly imprinted polymers from the point of view of the performances obtained, with emphasis on the detection methods, on the electrochemical reactions that take place upon detection, and on the electroanalytical performances. The present study was carried out with a view to highlighting, for the benefit of specialists in medicine and pharmacy, the possibility of achieving and purchasing efficient devices that might be used in the quality control of medicines, as well as in studying and monitoring diseases associated with these amino acids.

The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model.

INTRODUCTION: Management of phenylketonuria (PKU) is mainly achieved through dietary control with limited intake of phenylalanine (Phe) from food, supplemented with low protein (LP) food and a mixture of free synthetic (FS) amino acids (AA) (FSAA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese making by the action of the enzyme chymosin. Because CGMP in its pure form does not contain Phe, it is nutritionally suitable as a supplement in the diet for PKU when enriched with specific AAs. Lacprodan® CGMP-20 (= CGMP) used in this study contained only trace amounts of Phe due to minor presence of other proteins/peptides. OBJECTIVE: The aims were to address the following questions in a classical PKU mouse model: Study 1, off diet: Can pure CGMP or CGMP supplemented with Large Neutral Amino Acids (LNAA) as a supplement to normal diet significantly lower the content of Phe in the brain compared to a control group on normal diet, and does supplementation of selected LNAA results in significant lower brain Phe level?. Study 2, on diet: Does a combination of CGMP, essential (non-Phe) EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula which alone consist of FSAA, with a similar composition?. MATERIAL AND METHODS: 45 female mice homozygous for the Pahenu2 mutation were treated for 12 weeks in five different groups; G1(N-CGMP), fed on Normal (N) casein diet (75%) in combination with CGMP (25%); G2 (N-CGMP-LNAA), fed on Normal (N) casein diet (75%) in combination with CGMP (19,7%) and selected LNAA (5,3% Leu, Tyr and Trp); G3 (N), fed on normal casein diet (100%); G4 (CGMP-EAA-LP), fed on CGMP (70,4%) in combination with essential AA (19,6%) and LP diet; G5 (FSAA-LP), fed on FSAA (100%) and LP diet. The following parameters were measured during the treatment period: Plasma AA profiles including Phe and Tyr, growth, food and water intake and number of teeth cut. At the end of the treatment period, a body scan (fat and lean body mass) and a behavioral test (Barnes Maze) were performed. Finally, the brains were examined for content of Phe, Tyr, Trp, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA), and the bone density and bone mineral content were determined by dual-energy x-ray absorptiometry. RESULTS: Study 1: Mice off diet supplemented with CGMP (G1 (N-CGMP)) or supplemented with CGMP in combination with LNAA (G2 (N-CGMP-LNAA)) had significantly lower Phe in plasma and in the brain compared to mice fed only casein (G3 (N)). Extra LNAA (Tyr, Trp and Leu) to CGMP did not have any significant impact on Phe levels in the plasma and brain, but an increase in serotonin was measured in the brain of G2 mice compared to G1. Study 2: PKU mice fed with mixture of CGMP and EAA as supplement to LP diet (G4 (CGMP-EAA-LP)) demonstrated lower plasma-Phe levels but similar brain- Phe levels and growth as mice fed on an almost identical combination of FSAA (G5 (FSAA-LP)). CONCLUSION: CGMP can be a relevant supplement for the treatment of PKU.

The Novel Interplay between Commensal Gut Bacteria and Metabolites in Diet-Induced Hyperlipidemic Rats Treated with Simvastatin.

Hyperlipidemia is one kind of metabolic syndrome for which the treatment commonly includes simvastatin (SV). Individuals vary widely in statin responses, and growing evidence implicates gut microbiome involvement in this variability. However, the associated molecular mechanisms between metabolic improvement and microbiota composition following SV treatment are still not fully understood. In this study, combinatory approaches using ultrahigh-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF MS/MS)-based metabolomic profiling, PCR-denaturing gradient gel electrophoresis (PCR-DGGE), quantitative PCR (qPCR), and 16S rRNA gene sequencing-based gut microbiota profiling were performed to investigate the interplay of endogenous metabolites and the gut microbiota related to SV treatment. A total of 6 key differential endogenous metabolites were identified that affect the metabolism of amino acids (phenylalanine and tyrosine), unsaturated fatty acids (linoleic acid and 9-hydroxyoctadecadienoic acid (9-HODE)), and the functions of gut microbial metabolism. Moreover, a total of 22 differentially abundant taxa were obtained following SV treatment. Three bacterial taxa were identified to be involved in SV treatment, namely, Bacteroidaceae, Prevotellaceae, and Porphyromonadaceae. These findings suggested that the phenylalanine and tyrosine-associated amino acid metabolism pathways, as well as the linoleic acid and 9-HODE-associated unsaturated fatty acid metabolism pathways, which are involved in gut flora interactions, might be potential therapeutic targets for improvement in SV hypolipidemic efficacy. The mass spectrometric data have been deposited to MassIVE (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp). Username: MSV000087842_reviewer. Password: hardworkingzsr.

A novel stability-indicating HPLC method for the determination of enantiomeric purity of eluxadoline drug: Amylose tris(3,5-dichlorophenyl carbamate) stationary phase.

A simple and sensitive stability-indicating chiral HPLC method has been developed and validated per International Conference on Harmonization guidelines for the determination of enantiomeric purity of eluxadoline (Exdl). The impact of different mobile phase compositions and chiral stationary phases on the separation of Exdl enantiomer along with process- and degradation-related impurities has been studied. Homogeneity of Exdl and stable results of Exdl enantiomer in all degraded samples reveal the fact that the proposed method was specific (stability indicating). Amylose tris(3,5-dichlorophenyl carbamate) stationary phase column Chiralpak IE-3 (150 × 4.6 mm, 3 µm) provided better resolution with polar organic solvents than cellulose derivative, crown ether, and zwitterion stationary phases and nonpolar solvents. The mobile phase consisted of acetonitrile, tetrahydrofuran, methanol, butylamine, and acetic acid in the ratio of 500:500:20:2:1.5 (v/v/v/v/v). Isocratic elution was performed at a flow rate of 1.0 mL/min, column temperature of 35°C, injection volume of 10 µL, and UV detection of 240 nm. The United States Pharmacopeia (USP) resolution of the Exdl enantiomer was found to be more than 4.0 within a 65-min run time. Exdl enantiomer detector response linearity over the concentration range of 0.859-4.524 µg/mL was found to be R2  = 0.9985. The limit of detection, limit of quantification, and average percentage recovery values were established as 0.283 µg/mL, 0.859 µg/mL, and 96.0, respectively.

Correlations of blood and brain biochemistry in phenylketonuria: Results from the Pah-enu2 PKU mouse.

BACKGROUND: In phenylketonuria (PKU), treatment monitoring is based on frequent blood phenylalanine (Phe) measurements, as this is the predictor of neurocognitive and behavioural outcome by reflecting brain Phe concentrations and brain biochemical changes. Despite clinical studies describing the relevance of blood Phe to outcome in PKU patients, blood Phe does not explain the variance in neurocognitive and behavioural outcome completely. METHODS: In a PKU mouse model we investigated 1) the relationship between plasma Phe and brain biochemistry (Brain Phe and monoaminergic neurotransmitter concentrations), and 2) whether blood non-Phe Large Neutral Amino Acids (LNAA) would be of additional value to blood Phe concentrations to explain brain biochemistry. To this purpose, we assessed blood amino acid concentrations and brain Phe as well as monoaminergic neurotransmitter levels in in 114 Pah-Enu2 mice on both B6 and BTBR backgrounds using (multiple) linear regression analyses. RESULTS: Plasma Phe concentrations were strongly correlated to brain Phe concentrations, significantly negatively correlated to brain serotonin and norepinephrine concentrations and only weakly correlated to brain dopamine concentrations. From all blood markers, Phe showed the strongest correlation to brain biochemistry in PKU mice. Including non-Phe LNAA concentrations to the multiple regression model, in addition to plasma Phe, did not help explain brain biochemistry. CONCLUSION: This study showed that blood Phe is still the best amino acid predictor of brain biochemistry in PKU. Nevertheless, neurocognitive and behavioural outcome cannot fully be explained by blood or brain Phe concentrations, necessitating a search for other additional parameters. TAKE-HOME MESSAGE: Blood Phe is still the best amino acid predictor of brain biochemistry in PKU. Nevertheless, neurocognitive and behavioural outcome cannot fully be explained by blood or brain Phe concentrations, necessitating a search for other additional parameters.

Fast label-free identification of bacteria by synchronous fluorescence of amino acids.

Fast identification of pathogenic bacteria is an essential need for patient's diagnostic in hospitals and environmental monitoring of water and air quality. Bacterial cells consist of a very high amount of biological molecules whose content changes in response to different environmental conditions. The similarity between the molecular compositions of different bacterial cells limits the possibility to find unique markers to enable differentiation among species. Although many biological molecules in the cells absorb at the UV-Vis region, only a few of them can be detected in whole cells by their intrinsic fluorescence. Among these molecules are the amino acids phenylalanine, tyrosine, and tryptophan. In this work, we develop a rapid method for bacterial identification by synchronous fluorescence. We show that we can quantify the concentration for the 3 amino acids without any significant interference from other fluorophores in the cells and that we can differentiate among 6 pathogenic bacterial species by using the concentrations of their amino acids as a bacterial fingerprint. Fluorescent amino acids exist in all living cells. Therefore, this method has the potential to be applicative for the rapid identification of cells from all kinds of organisms.