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Background: Previous studies proposed varying leucine requirements for adults ranging from 25 to 40 mg⋅kg -1 ⋅d -1 , but often these studies did not test intakes exceeding 40 mg⋅kg -1 ⋅d -1 . Data using the indicator amino acid oxidation (IAAO) method suggest a higher requirement of 55 mg⋅kg -1 ⋅d -1 on the basis of the total branched-chain amino acids requirement, but not leucine independently., Objectives: The IAAO method was used to determine the leucine requirement in healthy young adult males., Methods: Ten healthy adult males (26.9 ± 1.87 y, mean ± SEM) were studied at 7 leucine intakes; each studied over a 3-d period. Following 2-d of preadaptation to adequate protein intake (1.0 g⋅kg -1 ⋅d -1 ), subjects received experimental diets containing the randomly assigned test leucine intake (10, 20, 30, 40, 50, 65, and 75 mg⋅kg -1 ⋅d -1 ) for 8 h. The rate of the release of 13 CO 2 from the oxidation of L-[1- 13 C]phenylalanine (F 13 CO 2 ) was measured on the third day, and the leucine requirement was determined using mixed-effect change-point regression and the F 13 CO 2 data in R. The 95% confidence interval was calculated using parametric bootstrap. The effect of leucine intake on the concentration of plasma amino acids, insulin, and glucose were assessed using repeated measures analysis of variance and linear mixed effects., Results: The mean leucine requirement was 33.6 mg⋅kg -1 ⋅d -1 with a lower and upper 95% confidence of 26.16, 41.04 mg⋅kg -1 ⋅d -1 . Higher leucine intakes were associated with increased plasma leucine, and decreased valine, isoleucine, and serine concentrations., Conclusions: The leucine requirement of young adult males is ∼34 mg⋅kg -1 ⋅d -1 , which aligns with previously published tracer balance experiments. This trial was registered at http://clinicaltrials.gov (https://clinicaltrials.gov/study/NCT05394155?term=leucine%20young%20adult&rank=1) as NCT05394155., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

The identification of new genes involved in regulating cold tolerance in rice is urgent because low temperatures repress plant growth and reduce yields. Cold tolerance is controlled by multiple loci and involves a complex regulatory network. Here, we show that rice jacalin-related lectin (OsJRL) modulates cold tolerance in rice. The loss of OsJRL gene functions increased phenylalanine metabolism and flavonoid biosynthesis under cold stress. The OsJRL knock-out (KO) lines had higher phenylalanine ammonia-lyase (PAL) activity and greater flavonoid accumulation than the wild-type rice, Nipponbare (NIP), under cold stress. The leaves had lower levels of reactive oxygen species (ROS) and showed significantly enhanced cold tolerance compared to NIP. In contrast, the OsJRL overexpression (OE) lines had higher levels of ROS accumulation and showed lower cold tolerance than NIP. Additionally, the OsJRL KO lines accumulated more abscisic acid (ABA) and jasmonic acid (JA) under cold stress than NIP. The OsJRL OE lines showed increased sensitivity to ABA compared to NIP. We conclude that OsJRL negatively regulates the cold tolerance of rice via modulation of phenylalanine metabolism and flavonoid biosynthesis., (© 2024 John Wiley & Sons Ltd.)

Phenylalanine (Phe) accelerates fruit wound healing by activating phenylpropanoid metabolism. However, whether Phe affects sucrose and respiratory metabolism in fruit during wound healing remains unknown. In this research, we found that preharvest Phe spray promoted sucrose degradation and increased glucose and fructose levels by activating acid invertase (AI), neutral invertase (NI), sucrose synthase (SS) and sucrose phosphate synthase (SPS) on harvested muskmelons. The spray also activated hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), malate dehydrogenase (MDH), succinate dehydrogenase (SDH) and glucose-6-phosphate dehydrogenase (G6PDH). In addition, the spray improved energy and reducing power levels in the fruit. Taken together, preharvest Phe spray can provide carbon skeleton, energy and reducing power for wound healing by activating the sucrose metabolism, Embden-Meyerhof-Parnas (EMP) pathway, tricarboxylic acid (TCA) cycle and pentose phosphate (PPP) pathway in muskmelon wounds during healing, which is expected to be developed as a new strategy to accelerate fruit wound healing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Amino acids are important for cellular metabolism. Their uptake across the plasma membrane is mediated by transport proteins. Despite the fact that the organic anion transporting polypeptide 4C1 (OATP4C1, Uniprot: Q6ZQN7) mediates transport of l-arginine and l-arginine derivatives, other members of the OATP family have not been characterized as amino acid transporters. The OATP family member OATP3A1 (gene symbol SLCO3A1, Uniprot: Q9UIG8) is ubiquitously expressed in human cells and highly expressed in many cancer tissues and cell lines. However, only a few substrates are known for OATP3A1. Accordingly, knowledge about its biological relevance is restricted. Our aim was to identify new substrates of OATP3A1 to gain insights into its (patho-)physiological function. In an LC-MS-based untargeted metabolomics assay using untreated OATP3A1-overexpressing HEK293 cells and control cells, we identified several amino acids as potential substrates of OATP3A1. Subsequent uptake experiments using exogenously added substrates revealed OATP3A1-mediated transport of l-tryptophan, l-tyrosine, and l-phenylalanine with 194.8 ± 28.7% (P < 0.05), 226.2 ± 18.7% (P < 0.001), and 235.2 ± 13.5% (P < 0.001), respectively, in OATP3A1-overexpressing cells compared to control cells. Furthermore, kinetic transport parameters (K m values) were determined (Trp = 61.5 ± 14.2 μm, Tyr = 220.8 ± 54.5 μm, Phe = 234.7 ± 20.6 μm). In summary, we identified the amino acids l-tryptophan, l-tyrosine, and l-phenylalanine as new substrates of OATP3A1. These findings could be used for a better understanding of (patho-)physiological processes involving increased demand of amino acids, where OATP3A1 should be considered as an important uptake transporter of l-tryptophan, l-tyrosine, and l-phenylalanine., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Organic cation transporters (OCTs) can transport structurally highly diverse substrates. The molecular basis of this extensive polyspecificity has been further elucidated by cryo-EM. Apparently, in addition to negatively charged amino acids, aromatic residues may contribute to substrate binding and substrate selectivity. In this study, we provide a comprehensive characterization of phenylalanine 244 in OCT1 function. We analyzed the uptake of 144 OCT1 substrates for the phenylalanine 244 to alanine substitution compared to WTOCT1. This substitution had highly substrate-specific effects ranging from transport reduced to 10% of WT activity up to 8-fold increased transport rates. Four percent of substrates showed strongly increased uptake (>200% of WT) whereas 39% showed strongly reduced transport (<50% of WT). Particularly with larger, more hydrophobic, and more aromatic substrates, the Phe244Ala substitution resulted in higher transport rates and lower inhibition of the transporter. In contrast, substrates with a lower molecular weight and less aromatic rings showed generally decreased uptake rates. A comparison of our data to available transport kinetic data demonstrates that generally, high-affinity low-capacity substrates show increased uptake by the Phe244Ala substitution, whereas low-affinity high-capacity substrates are characterized by reduced transport rates. Altogether, our study provides the first comprehensive characterization of the functional role of an aromatic amino acid within the substrate translocation pathway of OCT1. The pleiotropic function further highlights that phenylalanine 244 interacts in a highly specific manner with OCT1 substrates and inhibitors., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Polyphenolic antioxidants have been suggested to control the generation of acrylamide during thermal reactions. However, their role in protecting against the toxicity of acrylamide and the mechanism of action regarding profile alteration of biomarkers and metabolome remains unclear. A total of 65 adults were randomized into tea polyphenols (TP) and control groups and served with potato chips, which corresponded to an intake level of 12.6 μg/kg·bw of acrylamide, followed by capsules containing 200 mg, 100 mg or 50 mg TP, or equivalent placebo. Moreover, nontargeted urinary metabolomics analysis in acrylamide exposed rats was conducted using ultra-high performance liquid chromatography linked with a quadrupole-orbitrap high-resolution mass spectrometry. Our results showed that supplementation with catechins promoted the excretion of N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-l-cysteine in both humans and rats. We also found that epigallocatechin gallate (EGCG) or epicatechin (EC) intervention attenuated the ratio of hemoglobin adduct of glycidamide to hemoglobin adduct of acrylamide in rat blood. Metabolomics analysis revealed that EGCG/EC intervention regulated the differential expressed metabolites, including l-glutamic acid, 2-oxoglutarate, citric acid, and cysteinylglycine. Kyoto Encyclopedia of Genes and Genomes pathway analysis further showed acrylamide-induced metabolic disorders were improved after EGCG/EC supplementation by glycolipid metabolism (alanine, aspartate and glutamate metabolism, and d-Glutamine and d-glutamate metabolism) and energy metabolism (tricarboxylic acid cycle). Notably, the supplement use of EGCG prevented the cardiometabolic risk after exposure to acrylamide by mediating the phenylalanine and hippuric acid in phenylalanine metabolism. Here we showed the beneficial effect of catechins as major polyphenolic antioxidant ingredients on the toxicity of acrylamide by the changes in biomarkers from metabolic profile analysis based on human and animal studies. These findings shed light into the catechins as natural polyphenolic antioxidants that could be a therapeutic ingredient for preventing acrylamide-induced cardiometabolic toxicity.

Background: Rice sheath blight, which is caused by Rhizoctonia solani, is the most destructive disease affecting rice production, but the resistance mechanism to this pathogen has not been fully elucidated. Results: In this study, we selected two rice cultivars based on their resistance to the pathogen and analyzed and compared the transcriptomic profiles of two cultivars, the moderately resistant variety Gangyuan8 and the highly susceptible variety Yanfeng47, at different time points after inoculation. The comparative transcriptome profiling showed that the expression of related genes gradually increased after pathogen inoculation. The number of differentially expressed genes (DEGs) in Yanfeng47 was higher than that in Gangyuan8, and this result revealed that Yanfeng47 was more susceptible to fungal attack. At the early stage (24 and 48 h), the accumulation of resistance genes and a resistance metabolism occurred earlier in Ganguan8 than in Yanfeng47, and the resistance enrichment entries were more abundant in Ganguan8 than in Yanfeng47. Conclusions: Based on the GO and KEGG enrichment analyses at five infection stages, we concluded that phenylalanine metabolism and the jasmonic acid pathway play a crucial role in the resistance of rice to sheath blight. Through a comparative transcriptome analysis, we preliminarily analyzed the molecular mechanism responsible for resistance to sheath blight in rice, and the results lay the foundation for the development of gene mining and functional research on rice resistance to sheath blight. [ABSTRACT FROM AUTHOR]

Maize (Zea mays L.) stalk rot is a devastating disease worldwide, causing severe yield losses. Although previous studies have focused on the genetic dissection of maize resistance to stalk rot, the mechanisms of resistance remain largely unknown. We used a comparative proteomics approach to identify candidate proteins associated with stalk rot resistance. Statistical analyses revealed 763 proteins differentially accumulated between Fusarium graminearum and mock-inoculated plants. Among them, the antioxidant protein ZmPrx5, which was up-accumulated in diseased plants, was selected for further study. ZmPrx5 transcripts were present in root, stalk, leaf, ear, and reproductive tissues. The expression of ZmPrx5 in three inbred lines increased significantly upon F. graminearum infection. ZmPrx5 was localized in the cytoplasm. Compared to control plants, maize plants overexpressing ZmPrx5 showed increased resistance to F. graminearum infection, and ZmPrx5 mutant plants were more susceptible than wild-type plants. Defense-associated pathways including plant-pathogen interactions, phenylalanine metabolism, and benzoxazinoid and flavonoid biosynthesis were suppressed in ZmPrx5 homozygous mutant plants compared with wild-type plants. We suggest that ZmPrx5 positively regulates resistance against stalk rot in maize, likely through defense-oriented transcriptome reprogramming. These results lay a foundation for further research on the roles of Prx5 subfamily proteins in resistance to plant fungal diseases, and provide a potential genetic resource for breeding disease-resistance maize lines. [ABSTRACT FROM AUTHOR]

Abstract Background The bacillus Calmette-Guérin (BCG) vaccine protects against tuberculosis and heterologous infections but elicits high inter-individual variation in specific and nonspecific, or trained, immune responses. While the gut microbiome is increasingly recognized as an important modulator of vaccine responses and immunity in general, its potential role in BCG-induced protection is largely unknown. Results Stool and blood were collected from 321 healthy adults before BCG vaccination, followed by blood sampling after 2 weeks and 3 months. Metagenomics based on de novo genome assembly reveals 43 immunomodulatory taxa. The nonspecific, trained immune response is detected by altered production of cytokines IL-6, IL-1β, and TNF-α upon ex vivo blood restimulation with Staphylococcus aureus and negatively correlates with abundance of Roseburia. The specific response, measured by IFN-γ production upon Mycobacterium tuberculosis stimulation, is associated positively with Ruminococcus and Eggerthella lenta. The identified immunomodulatory taxa also have the strongest effects on circulating metabolites, with Roseburia affecting phenylalanine metabolism. This is corroborated by abundances of relevant enzymes, suggesting alternate phenylalanine metabolism modules are activated in a Roseburia species-dependent manner. Conclusions Variability in cytokine production after BCG vaccination is associated with the abundance of microbial genomes, which in turn affect or produce metabolites in circulation. Roseburia is found to alter both trained immune responses and phenylalanine metabolism, revealing microbes and microbial products that may alter BCG-induced immunity. Together, our findings contribute to the understanding of specific and trained immune responses after BCG vaccination.

Niuhuang Shangqing Pill (NSP) is a renowned Chinese medicine prescription listed in the Chinese Pharmacopoeia (Edition, 2020; volume 1) and is utilized in clinical practice for treating headaches and acute pharyngitis (AP) associated with "Shanghuo". Despite its widespread use, the pharmacological mechanism and bioactive components underlying NSP in treating AP remain unclear. This study delved into evaluate the alleviation effect of NSP on AP and explore the mechanisms by analyzing multi-omics. UHPLC-Q Exactive Orbitrap HRMS was employed for the chemical ingredients of NSP. Multiple compositions, targets and pathways involved in the treatment of AP with NSP were predicted by network pharmacology. Additionally, wistar rat model of AP induced by capsaicin was established to evaluate the anti-AP activity of NSP in vivo. The potential mechanism of NSP to improve AP was investigated by real-time PCR, pharyngeal transcriptome analysis, non-targeted metabolomics, immunofluorescence and Western blot. 119 compounds were identified by UHPLC-Q Exactive Orbitrap HRMS. Both clinical data of Gene Expression Omnibus (GEO) and network pharmacology demonstrated that MAPK signaling pathway and TNF signaling pathway were the critical pathway for AP treatment. In rat model of AP induced by capsaicin, NSP demonstrated the ability to reduce the levels of IL-1β, TNF-α, IL-6, CGRP, SP, PGE 2 , COX-2 in serum. Moreover, Transcriptomics analysis comprehensively indicated that NSP regulated the MAPK signaling pathway, TNF signaling pathway, biosynthesis of phenylalanine, tyrosine and tryptophan, arachidonic acid metabolism in AP rats. Metabolomics analysis verified that NSP could rebalance arachidonic acid metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan and regulate metabolic profiles. Multi-omics Correlation analysis exhibited that the relative expression of Tnfrsf1b was significantly negatively correlated with 12(S)-HPETE. Immunofluorescence, real-time PCR and Western blot of pharyngeal tissue revealed that NSP inhibited the TNF/p38-MAPK/NF-κB signaling pathway. Additionally, in vitro study on RAW264.7 cells confirmed that NSP counteract LPS-induced inflammatory by inhibiting the TNF/p38-MAPK/NF-κB signaling pathway. Overall, NSP effectively ameliorated capsaicin-induced AP by modulating the arachidonic acid metabolism and TNF/p38-MAPK/NF-κB signaling pathway. NSP effectively ameliorated capsaicin-induced AP by modulating the arachidonic acid metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan, as well as the TNF/p38-MAPK/NF-κB signaling pathway. [Display omitted] • This study is the first time to analyze 119 active components in Niuhuang Shangqing Pill. • About 46 target metabolites and 3961 target genes were differentially expressed after treatment with NSP. • Integrated clinical data, network pharmacology and multi-omics analyses identified TNF/p38-MAPK/NF-κB signaling pathway as a crucial pathway for NSP to treat AP. • Mechanistic investigation in RAW264.7 cells interpreted that NSP significantly inhibited the TNF/p38-MAPK/NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease that is frequently found in athletes and those who have experienced repetitive head traumas. CTE is associated with a variety of neuropathologies, which cause cognitive and behavioral impairments in CTE patients. However, currently, CTE can only be diagnosed after death via brain autopsy, and it is challenging to distinguish it from other neurodegenerative diseases with similar clinical features. To better understand this multifaceted disease and identify metabolic differences in the postmortem brain tissues of CTE patients and control subjects, we performed ultra-high performance liquid chromatography–mass spectrometry (UPLC-MS)-based non-targeted metabolomics. Through multivariate and pathway analysis, we found that the brains of CTE patients had significant changes in the metabolites involved in astrocyte activation, phenylalanine, and tyrosine metabolism. The unique metabolic characteristics of CTE identified in this study were associated with cognitive dysfunction, amyloid-beta deposition, and neuroinflammation. Altogether, this study provided new insights into the pathogenesis of CTE and suggested appealing targets for both diagnosis and treatment for the disease. [ABSTRACT FROM AUTHOR]

Background: Lentil is considered a high protein source. However, it is low in sulphur amino acids (SAA) and their metabolic availability (MA) is further affected by antinutritional factors in lentils. The combination of lentils with grains such as rice can enhance the protein quality of a lentil-based meal but the MA of SAA in lentils must first be known.Objectives: The objectives of the current study were to assess the MA of methionine in lentils and to test the effects of consumption of complementing lentils with rice in young adults.Methods: Five healthy young men [age <30 y, BMI <25 (in kg/m2)] were each studied at 8 or 10 intake amounts of methionine in random order; 4 daily intake amounts of l-methionine: 0.5, 1, 2, and 3 mg.kg-1.d-1 (reference diet), 3 daily intake amounts of methionine from lentils, and 3 daily intake amounts of the mixed meal of lentils + rice (test diets). The MA of methionine and the effects of complementation were assessed by comparing the indicator amino acid oxidation (IAAO) response to varying intakes of methionine in cooked Canadian lentils, and in rice + lentils combined, compared with the IAAO response to l-methionine intakes in the reference protein (crystalline AA mixture patterned after egg protein) using the slope ratio method. l-[1-13C] phenylalanine was used as the indicator. Data were analyzed using the procedure "MIXED" with subject as a random variable, and oxidation day as repeated measure.Results: The MA of methionine from lentils was 69%. Complementation of cooked lentils with rice decreased the oxidation of l-[1-13C] phenylalanine by up to 16% (P < 0.05).Conclusions: The content and MA of methionine are low in lentils. However, combination of lentils with rice in a 1:1 ratio can improve the protein quality of lentil-based diets, resulting in increased protein synthesis in young healthy adults. This trial was registered at www.clinical trials.gov as NCT03110913. [ABSTRACT FROM AUTHOR]

Purple sweet potato is considered as a healthy food because of its high anthocyanins. To understand the coloring mechanism and quality change between purple-fleshed sweet potato (cv. Xuzi201) and its cream fleshed mutant (M1001), a combined metabolomic and transcriptomic analysis was performed. The metabolome data showed that 4 anthocyanins, 19 flavones, 6 flavanones, and 4 flavonols dramatically decreased in M1001, while the contents of 3 isoflavones, 3 flavonols, 4 catechins, and 2 proanthocyanins increased. Transcriptomic analyses indicated that the expression of 49 structural genes in the flavonoid pathway and transcription factors (TFs) (e.g., bHLH2, R2R3-MYB, MYB1) inducting anthocyanin biosynthesis were downregulated, but the repressor MYB44 was upregulated. The IbMYB1-2 gene was detected as a mutation gene in M1001, which is responsible for anthocyanin accumulation in the storage roots. Thus, the deficiency of purple color in the mutant is due to the lack of anthocyanin accumulation which was regulated by IbMYB1. Moreover, the accumulation of starch and aromatic volatiles was significantly different between Xuzi201 and M1001. These results not only revealed the mechanism of color mutation but also uncovered certain health-promoting compounds in sweet potato. [ABSTRACT FROM AUTHOR]

Phenylketonuria (PKU) is an autosomal recessive disease caused by variants in the gene that encodes phenylalanine hydroxylase (PAH), limiting the metabolism of phenylalanine (Phe). When PAH activity is absent or hindered, Phe is not converted to tyrosine, leading to an accumulation of Phe in the blood, which can cause serious neurological complications. Once PKU is diagnosed, treatment should be started immediately, and the basis for this is dietary restriction of foods with high levels of Phe, associated with the use of protein substitutes and intake of foods with low protein content. This restriction accompanies patients throughout their lives, making their diets unpalatable and monotonous, which represents a major challenge for health professionals and patients, considering that these factors favor food transgression. In this context, the objective of this work was to carry out an integrative review based on evidence regarding the intake of fruits and vegetables, by phenylketonurics, taking into account the greater or lesser tolerance to Phe. Since, some researchers have dedicated themselves to evaluating the biochemical effect of unrestricted consumption of fruits and vegetables at PKU, unifying the information in this regard. It was observed that the intake of vegetable protein by patients with PKU has shown to be promising since the studies indicate that the intake of these proteins does not present adverse effects to the metabolic control of the Phe. [ABSTRACT FROM AUTHOR]

In this study, muskmelon plant and fruit were sequentially sprayed with 8 mM phenylalanine (Phe) four times during fruit development. The effect of preharvest Phe spraying on chilling injury (CI) of harvested muskmelons was assessed and the mechanism involved was investigated. We found that Phe spray activated NADPH oxidase (NOX), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX) and glutathione reductase (GR), and increased glutathione (GSH) and ascorbic acid (AsA) levels during fruit chilling. The spray increased endogenous Phe, total phenolic and flavonoid content, and DPPH and ABTS + scavenging capacity. In addition, the spray decreased O 2 .- production rate, H 2 O 2 levels, cell membrane permeability and malondialdehyde (MDA) content, and significantly reduced CI index in fruit, which was 16.5 %, 16.6 %, 13.5 %, 20.2 % and 26.5 % lower than the control after 28 d, respectively. In conclusion, Phe spraying alleviates CI in harvested muskmelons by maintaining ROS homeostasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purple sweet potato is considered as a healthy food because of its high anthocyanins. To understand the coloring mechanism and quality change between purple-fleshed sweet potato (cv. Xuzi201) and its cream fleshed mutant (M1001), a combined metabolomic and transcriptomic analysis was performed. The metabolome data showed that 4 anthocyanins, 19 flavones, 6 flavanones, and 4 flavonols dramatically decreased in M1001, while the contents of 3 isoflavones, 3 flavonols, 4 catechins, and 2 proanthocyanins increased. Transcriptomic analyses indicated that the expression of 49 structural genes in the flavonoid pathway and transcription factors (TFs) (e.g., bHLH2, R2R3-MYB, MYB1) inducting anthocyanin biosynthesis were downregulated, but the repressor MYB44 was upregulated. The IbMYB1-2 gene was detected as a mutation gene in M1001, which is responsible for anthocyanin accumulation in the storage roots. Thus, the deficiency of purple color in the mutant is due to the lack of anthocyanin accumulation which was regulated by IbMYB1. Moreover, the accumulation of starch and aromatic volatiles was significantly different between Xuzi201 and M1001. These results not only revealed the mechanism of color mutation but also uncovered certain health-promoting compounds in sweet potato.

Aims. Cryptococcosis is an invasive fungal disease that is associated with an increasing prevalence along with a very high fatality and is primarily caused by Cryptococcus. However, its mechanism to cause pathogenicity is not yet completely understood. In this study, we aim to screen the lncRNA markers in human monocytic (THP-1) cells infected by Cryptococcus neoformans (C. neoformans) through high-throughput sequencing technology and to explore its effects on biological functions. Methods. We initially conducted an lncRNA microarray analysis of the THP-1 cells infected by C. neoformans and normal THP-1 cells. Based upon these data, RT-qPCR was used to verify the expressions of the selected lncRNAs and mRNAs. We then performed functional and pathway enrichment analyses. Lastly, target prediction was performed by using the lncRNA target tool which was based on the differentially expressed lncRNAs. Results. We determined 81 upregulated and 96 downregulated lncRNAs using microarray. In addition, the profiling data showed 42 upregulated and 57 downregulated genes and discovered that neuroactive ligand-receptor interaction, tyrosine metabolism, and phenylalanine metabolism are extremely impaired in the regulation of C. neoformans infection. GO enrichment analysis of the 99 differentially expressed mRNAs exhibited that these modules showed different signaling pathways and biological mechanisms like protein binding and metal ion binding. Moreover, lncRNAs and mRNAs were analyzed for their coexpression relations. A qRT-PCR analysis confirmed that the expression of the top 10 differently expressed mRNA and lincRNA. The expressions of the lncRNAs after C. neoformans infection in THP-1 cells were detected by RNA-sequence, suggesting that microarray analysis could reveal lncRNAs having functional significance that might be linked with the progression of patients. Conclusion. The current study analyzed the differential lncRNAs and mRNAs in C. neoformans infection and predicted the corresponding pathways and their correlations that can offer new potential insights into the mechanistic basis of this condition. [ABSTRACT FROM AUTHOR]

Background: Sorghum is the fifth most consumed cereal grain but limiting in the indispensable amino acid lysine. Complementing sorghum with lentils can improve the quality of sorghum-based diets. However, knowledge of lysine bioavailability in sorghum is lacking.Objectives: The study objectives were to determine the bioavailability of lysine in sorghum and to assess the effect of complementation of sorghum and lentils in a mixed-meal format.Methods: We studied 5 healthy young men (≤30 years; BMI <25 kg/m2) in a repeated-measure design using the indicator amino acid oxidation (IAAO) method, with L-[1-13C] phenylalanine as the indicator. Each subject participated in 8 determinations in random order. On the reference diet, subjects received 4 amounts of L-lysine (5, 8, 12, and 15 mg. kg-1 . d-1) from a crystalline amino acid mixture patterned after egg protein. On the test diet, they received 3 levels of lysine (8.2, 12.5, and 15.7 mg. kg-1 . d-1) from sorghum, and on the complementation diet they received 1 level of lysine from a mixed meal of sorghum and lentils. The bioavailability of lysine in sorghum was estimated by comparing the IAAO response to the test diet with the IAAO response to the reference diet using the slope-ratio method. Effectiveness of complementation was assessed by comparing the IAAO response to the mixed meal to the IAAO response to the test protein.Results: The bioavailability of lysine from sorghum was 94%. Upon complementation with lentils, there was a decline in the oxidation of L-[1-13C] phenylalanine by 19% (P < 0.0495), reflecting an improvement in available lysine in the mixed meal due to increased lysine intake.Conclusions: Although the bioavailability of lysine in sorghum is high, its lysine content is limiting. Complementation with lentils in a 1:1 ratio is recommended to achieve the lysine requirement for adult men consuming a sorghum-based diet. This trial was registered at clinicaltrials.gov as NCT03411005. [ABSTRACT FROM AUTHOR]

Background There remains a limited understanding of the metabolic perturbations, beyond phenylalanine (Phe) metabolism, that contribute to phenotypic variability in phenylketonuria (PKU). Objectives This study aimed to characterize changes in the PKU plasma metabolome following a 5-d metabolic camp intervention and to compare PKU profiles with those of matched healthy controls. Methods In 28 females (aged 12–57 y), fasting plasma samples were collected on the first (day 1) and final (day 5) days of camp to measure metabolic control and to complete untargeted metabolomic profiling. Three-day dietary records were collected to assess changes in dietary adherence and composition. Univariate (Wilcoxon signed-rank and Mann–Whitney U test) and multivariate (random forest, hierarchical clustering) analyses were performed to identify clinical and metabolic features that were associated with the intervention and disease state. Results Relative to healthy controls, Phe catabolites, ketones, and carnitine- and glycine-conjugated fatty acids were elevated in females with PKU at baseline, whereas fatty acylcholine metabolites were substantially lower. After the camp intervention, plasma Phe concentrations decreased [median change: –173 µmol/L (IQR: –325, –28 µmol/L)] and 70% of PKU participants demonstrated improved dietary adherence by decreasing Phe intake and/or increasing medical food consumption. This was accompanied by a shift in abundance for 223 metabolites (q  < 0.05). Compounds associated with the metabolism of Phe, fatty acids, and choline contributed most to profile differences between camp days 1 and 5. Conclusions In females with PKU, untargeted metabolomics identified prominent perturbations in amino acid and lipid metabolites associated with bioenergetic impairment and oxidative stress. Choline-conjugated lipids could have fundamental roles in these pathways and they have not been previously evaluated in PKU. A short-term camp intervention was effective for improving or fully normalizing the abundance of the identified discriminatory metabolites. [ABSTRACT FROM AUTHOR]

Objective This study was conducted to reveal potential metabolic differences of dairy cows fed corn stover (CS) and rice straw (RS) instead of alfalfa hay (AH) as main forage source. Methods Thirty multiparous mid-late lactation Holstein dairy cows were selected and randomly assigned to three diets, AH, CS, or RS (n = 10). After 13 weeks of the feeding trial, coccygeal arterial and superficial epigastric venous plasma samples were collected before morning feeding for gas chromatography time-of-flight/mass spectrometry analyses. Results In the artery, 8 and 13 metabolites were detected as differential metabolites between AH and CS, and between AH and RS, respectively. The relative abundance of phenylpropanoate (log2fold change [FC]) = 1.30, 1.09), panthenol (log2FC = 2.36, 2.20), threitol (log2FC = 1.00, 1.07), and 3,7,12-trihydroxycoprostane (log2FC = 0.79, 0.78) were greater in both CS and RS than in AH, and tyrosine (log2FC = −0.32), phenylalanine (log2FC = −0.30), and pyruvic acid (log2FC = −0.30) were lower in RS than in AH. In the vein, 1 and 7 metabolites were detected as differential metabolites between AH and CS, and between AH and RS, respectively. By comparing AH and RS, we found that metabolic pathways of phenylalanine, tyrosine, and tryptophan biosynthesis and phenylalanine metabolism were enriched by integrative artery and vein analysis. Furthermore, AH and RS, arterial phenylpropanoate and 4-hydroxyproline were positively, and phenylalanine was negatively correlated with milk urea nitrogen. Finally, in AH and CS, arterial panthenol was negatively correlated with feed efficiency. Conclusion Arterial metabolic profiles changed more than those in the veins from animals on three forage diets, differing in amino acids. We found that phenylalanine, tyrosine, and tryptophan biosynthesis and phenylalanine metabolism were restricted when cows were fed low-quality cereal straw diets.

Abstract Background There is a dearth of drug therapies available for the treatment of acute respiratory distress syndrome (ARDS). Certain metabolites play a key role in ARDS and could serve as potential targets for developing therapies against this respiratory disorder. The present study was designed to determine such “functional metabolites” in ARDS using metabolomics and in vivo experiments in a mouse model. Methods Metabolomic profiles of blood plasma from 42 ARDS patients and 28 healthy controls were captured using Ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) assay. Univariate and multivariate statistical analysis were performed on metabolomic profiles from blood plasma of ARDS patients and healthy controls to screen for “functional metabolites”, which were determined by variable importance in projection (VIP) scores and P value. Pathway analysis of all the metabolites was performed. The mouse model of ARDS was established to investigate the role of “functional metabolites” in the lung injury and mortality caused by the respiratory disorder. Results The metabolomic profiles of patients with ARDS were significantly different from healthy controls, difference was also observed between metabolomic profiles of the non-survivors and the survivors among the ARDS patient pool. Levels of Phenylalanine, D-Phenylalanine and Phenylacetylglutamine were significantly increased in non-survivors compared to the survivors of ARDS. Phenylalanine metabolism was the most notably altered pathway between the non-survivors and survivors of ARDS patients. In vivo animal experiments demonstrated that high levels of Phenylalanine might be associated with the severer lung injury and increased mortality of ARDS. Conclusion Increased mortality of acute respiratory distress syndrome was associated with high levels of plasma Phenylalanine. Trial registration Chinese Clinical Trial Registry, ChiCTR1800015930 . Registered 29 April 2018, http://www.chictr.org.cn/edit.aspx?pid=25609&htm=4

Gut microbial metabolites have been demonstrated to play a role in diabetes mellitus and gestational diabetes mellitus (GDM). This study aimed to investigate gut microbiome, fecal metabolomics, and their association in pregnant women with and without GDM. The metabolome indicated that the top 2 differential metabolic pathways between control (Con) and GDM groups were phenylalanine metabolism and nucleotide metabolism. The increased Phenylalanylglycine, m-coumaric acid, and Phenylacetic acid were among the top differential metabolites between Con and GDM groups and involved in phenylalanine metabolism. Uracil and hypoxanthine were top differential metabolites in Con vs. GDM and involved in nucleotide metabolism. The proficiently altered gut microbiota at the class level was c&#95;unclassified&#95; Firmicutes. Association analysis between gut microbiota and fecal metabolites indicated that the increased gut symbiont Clostridium belonged to Firmicutes and was linked to the dysregulation of phenylalanine metabolism in GDM. This study may provide the mechanism underlying how Clostridium-phenylalanine metabolism association contributes to GDM pathogenesis and also be a novel therapeutic strategy to treat GDM.

Many studies have focused on the effects of small molecules, such as amino acids, on metabolism under hypoxia. Recent findings have indicated that phenylalanine levels were markedly elevated in adaptation to chronic hypoxia. This raises the possibility that phenylalanine treatment could markedly improve the hypoxic endurance. However, the importance of hypoxia-regulated phenylalanine is still unclear. This study investigates the role of phenylalanine in hypoxia adaptation using a hypoxic zebrafish model and multi-omics analysis. We found that phenylalanine-related metabolic pathways are significantly up-regulated under hypoxia, contributing to enhanced hypoxic endurance. Phenylalanine treatment reduced ROS levels, improved mitochondrial oxygen consumption rate (OCR), and extracellular acidification rate (ECAR) in hypoxic cells. Western blotting revealed increased phenylalanine uptake via L-type amino transporters (LAT1), activating the LKB1/AMPK signaling pathway. This activation up-regulated peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and the Bcl-2/Bax ratio, while down-regulating uncoupling protein 2 (UCP2), thereby improving mitochondrial function under hypoxia. This is the first comprehensive multi-omics analysis to demonstrate phenylalanine's crucial role in hypoxia adaptation, providing insights for the development of anti-hypoxic drugs., (© 2024. The Author(s).)

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic, complex illness characterized by severe and often disabling physical and mental fatigue. So far, scientists have not been able to fully pinpoint the biological cause of the illness and yet it affects millions of people worldwide. To gain a better understanding of ME/CFS, we compared the metabolic networks in the plasma of 38 ME/CFS patients to those of 24 healthy control participants. This involved an untargeted metabolomics approach in addition to the measurement of targeted substances including tryptophan and its metabolites, as well as tyrosine, phenylalanine, B vitamins, and hypoxanthine using liquid chromatography coupled to mass spectrometry. We observed significant alterations in several metabolic pathways, including the vitamin B3, arginine-proline, and aspartate-asparagine pathways, in the untargeted analysis. The targeted analysis revealed changes in the levels of 3-hydroxyanthranilic acid, 3-hydroxykynurenine, hypoxanthine, and phenylalanine in ME/CFS patients compared to the control group. These findings suggest potential alterations in immune system response and oxidative stress in ME/CFS patients.

Human papillomavirus (HPV) L1 capsid protein were produced in several host systems, but few studies have focused on enhancing the properties of the L1 protein. In this study, we aimed to produce recombinant Human papillomavirus (HPV) L1 capsid protein containing para -azido- L -phenylalanine (pAzF) in Escherichia coli . First, we expressed the maltose-binding protein (MBP)-fused HPV16 L1, and 5 residues in HPV16 L1 protein were selected by the in silico modeling for amber codon substitution. Among the variants of the five locations, we identified a candidate that exhibited significant differences in expression with and without pAzF via genetic code expansion (GCE). The expressed recombinant MBP-HPV16L1 protein was confirmed for incorporation of pAzF and the formation of VLPs was tested in vitro.

β-Branched aromatic α-amino acids are valuable building blocks in natural products and pharmaceutically active compounds. However, their chemical or enzymatic synthesis is challenging due to the presence of two stereocenters. We design phenylalanine ammonia lyases (PAL) variants for the direct asymmetric synthesis of β-branched aromatic α-amino acids. Based on extensive computational analyses, we unravel the enigma behind PAL's inability to accept β-methyl cinnamic acid (β-MeCA) as substrate and achieve the synthesis of the corresponding amino acids of β-MeCA and analogs using a double (PcPAL-L256V-I460V) and a triple mutant (PcPAL-F137V-L256V-I460V). The reactions are scaled-up using an optimized E. coli based whole-cell biotransformation system to produce ten β-branched phenylalanine analogs with high diastereoselectivity (dr > 20:1) and enantioselectivity (ee > 99.5%) in yields ranging from 41-71%. Moreover, we decipher the mechanism of PcPAL-L256V-I460V for the acceptance of β-MeCA and converting it with excellent stereoselectivity by computational simulations. Thus, this study offers an efficient method for synthesizing β-branched aromatic α-amino acids., (© 2024. The Author(s).)

Key Message: This study described the biosynthesis of 4-hydroxydihydrocinnamaldehyde sharing with monolignol pathway and supplemented the biosynthesis of colchicine in G. superba, 4-hydroxydihydrocinnamaldehyde produced in tobacco BY2 cells provided an important stepstone. The precursor, 4-hydroxydihydrocinnamaldehyde (4-HDCA), participates in the biosynthesis of the carbon skeleton of colchicine, which is derived from L-phenylalanine. However, one hypothesis proposed that 4-HDCA is synthesized by sharing the early part of the monolignol pathway in G. superba. In this study, we validated this prediction and identified the enzymatic functions involved in this pathway. GsDBR1 is a crucial enzyme to illustrate 4-HDCA diverging from monolignol pathway, we first confirmed its reductase activity on 4-coumaraldehyde, an important intermediate compound in monolignol biosynthesis. Then, the biochemical function of recombinant enzymes belonging to the other four families were verified to elucidate the entire process of 4-HDCA biosynthesis from L-phenylalanine. After reconstruction, the 4-HDCA was 78.4 ng/g with fresh weight (FW) of transgenic tobacco cells, and the yield increased to 168.22 ng/g·FW after improved treatment with methyl jasmonate (MeJA). The elucidation of 4-HDCA biosynthesis sharing the monolignol pathway supplemented the biosynthesis of colchicine in G. superba, and the production of 4-HDCA in tobacco cells provides an important step in the development of plant cell cultures as heterologous bio-factories for secondary metabolite production., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In this study, nine endophytic fungi capable of producing multiple phenolic compounds were screened and identified from 152 fungi isolated from pigeon pea in a natural habitat (Honghe, Yunnan Province, China). Talaromyces neorugulosus R-209 exhibited the highest potential for phenolic compound production. L-phenylalanine feeding was used to enhance phenolic compound production in T. neorugulosus R-209 cultures. Under the optimal feeding conditions (l-phenylalanine dose of 0.16 g/L and feeding phase of 6 days), the yields of genistein, apigenin, biochanin A, and cajaninstilbene acid increased by 15.59-fold, 7.20-fold, 25.93-fold, and 10.30-fold over control, respectively. T. neorugulosus R-209 fed with l-phenylalanine was found to be stable in the production of phenolic compounds during ten successive subcultures. Moreover, bioactivities of extracts of T. neorugulosus R-209 cultures were significantly increased by l-phenylalanine feeding. Overall, l-phenylalanine feeding strategy made T. neorugulosus R-209 more attractive as a promising alternative source for the production of health-beneficial phenolic compounds in the nutraceutical/medicinal industries., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Rice protein peptide (RPP) has been reported to alleviate the symptoms of dextran sulfate sodium (DSS)-induced colitis, but its potential protective effect and fundamental neurobiological mechanisms against DSS-induced inflammatory bowel disease (IBD), coupled with depression and cognitive impairment, remain unclear. In this study, RPP treatment in DSS-induced mice inhibited decreases in body weight and colon length and improved intestinal barrier function and behavioral performance. RPP treatment enhanced phenylalanine and tyrosine metabolism in the brains of mice, and it upregulated metabolites such as l-dopa, phenylethylamine, and 3,4-dihydroxyphenylacetate. Additionally, RPP treatment enhanced the brain-derived neurotrophic factor (BDNF) by upregulating the BDNF/TrkB/CREB signaling pathway. Spearman's correlation analysis revealed that the phenylalanine and tyrosine contents in the brain were significantly negatively correlated with the BDNF/TrkB/CREB signaling pathway and behavioral performance. In conclusion, this study suggested that RPP may serve as a unique nutritional strategy for preventing IBD and its associated cognitive impairment and depression symptoms.

Although messenger RNA translation is tightly regulated to preserve protein synthesis and cellular homeostasis, chronic exposure to interferon-γ (IFN-γ) in several cancers can lead to tryptophan (Trp) shortage via the indoleamine-2,3-dioxygenase (IDO)- kynurenine pathway and therefore promotes the production of aberrant peptides by ribosomal frameshifting and tryptophan-to-phenylalanine (W>F) codon reassignment events (substitutants) specifically at Trp codons. However, the effect of Trp depletion on the generation of aberrant peptides by ribosomal mistranslation in gastric cancer (GC) is still obscure. Here, it is shows that the abundant infiltrating lymphocytes in EBV-positive GC continuously secreted IFN-γ, upregulated IDO1 expression, leading to Trp shortage and the induction of W>F substitutants. Intriguingly, the production of W>F substitutants in EBV-positive GC is linked to antigen presentation and the activation of the mTOR/eIF4E signaling pathway. Inhibiting either the mTOR/eIF4E pathway or EIF4E expression counteracted the production and antigen presentation of W>F substitutants. Thus, the mTOR/eIF4E pathway exposed the vulnerability of gastric cancer by accelerating the production of aberrant peptides and boosting immune activation through W>F substitutant events. This work proposes that EBV-positive GC patients with mTOR/eIF4E hyperactivation may benefit from anti-tumor immunotherapy., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Over fifty years have passed since the last large scale longitudinal study of individuals with PAH deficiency in the U.S. Since then, there have been significant changes in terms of treatment recommendations as well as treatment options. The Phenylalanine Families and Researchers Exploring Evidence (PHEFREE) Consortium was recently established to collect a more up-to-date and extensive longitudinal natural history in individuals with phenylketonuria across the lifespan. In the present paper, we describe the structure and methods of the PHEFREE longitudinal study protocol and report cross-sectional data from an initial sample of 73 individuals (5 months to 54 years of age) with PAH deficiency who have enrolled. Looking forward, the study holds the promise for advancing the field on several fronts including the validation of novel neurocognitive tools for assessment in individuals with PKU as well as evaluation of the long-term effects of changes in metabolic control (e.g., effects of Phe-lowering therapies) on outcome., Competing Interests: Declaration of competing interest SEC has received consulting fees from BioMarin, Jnana, and PTC; and BioMarin has funded past and current research. GA has served as a consultant for PTC Therapeutics. DKG has conducted clinical research trials for BioMarin and Synlogic related to the treatment of PAH deficiency. COH has received consulting fees from BioMarin, Jnana Therapeutics, PTC Therapeutics, Sanofi, Pfizer, Synlogic, Tessera Therapeutics, and Tome Biosciences and conducted clinical research trials for BioMarin, Jnana Therapeutics, Sanofi, and Synlogic related to treatment of PAH deficiency. EJ has served as a consultant for Jnana Therapeutics, Arcturus Therapeutics, Travere Therapeutics, and participated in advisory boards for Jnana Therapeutics and Sanofi. HL has received consulting fees from BioMarin, Sanofi, Jnana, PTC, and Tome Biosciences. NL has served on advisory boards for Amgen/Horizon, Amicus Therapeutics, Audentes/Astrellas, BioMarin, Chiesi/Protalix, Sanofi, Ipsen, Jaguar Gene Therapy, Jnana, Leadiant Biosciences, Moderna, Nestlé Pharma, PTC Therapeutics, Reneo, and Ultragenyx and has received clinical trial support from Amgen/Horizon, Amicus Therapeutics, Audentes/Astrellas, BioMarin, Chiesi/Protalix, Sanofi, Jnana, Moderna, PTC Therapeutics, Takeda, and Ultragenyx. SS has served on advisory boards and received clinical trial support from BioMarin, Synlogic, PTC Therapeutics, and Jnana Therapeutics. JT has served as a consultant for BioMarin and has received clinical trials support from PTC Therapeutics, BioMarin, and Sanofi. DAW has received clinical trial support from BioMarin., (Copyright © 2024. Published by Elsevier Inc.)

Metabolic cross-feeding is a pervasive interaction between bacteria to acquire novel phenotypes. However, our current understanding of the survival mechanism for cross-feeding in cocultured bacterial biofilms under heavy-metal conditions remains limited. Herein, we found that Comamonas sp. A23 produces L-phenylalanine to activate the L-phenylalanine degradation pathway in Enterobacter sp. A11, enhancing biofilm formation and cadmium [Cd(II)] immobilization in A11. The genes responsible for L-phenylalanine-degradation (paaK) and cell attachment and aggregation (csgAD) are essential for biofilm formation and Cd(II) immobilization in A11 induced by L-phenylalanine. The augmentation of A11 biofilms, in turn, protects A23 under Cd(II) and H 2 O 2 stresses. The plant-based experiments demonstrate that the induction of two rice Cd(II) transporters, OsCOPT4 and OsBCP1, by A11 and A23 enhances rice resistance against Cd(II) and H 2 O 2 stresses. Overall, our findings unveil the mutual dependence between bacteria and rice on L-phenylalanine cross-feeding for survival under abiotic stress., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Phenylketonuria (PKU, OMIM 261600) is a genetic disorder caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). If left untreated, PKU leads to systemic phenylalanine (Phe) accumulation, which can result in irreversible brain damage and intellectual disabilities. In the last 60 years, early and strict dietary restriction of phenylalanine (Phe) intake proved to prevent the severe clinical phenotype of untreated PKU. While the specific mechanisms through which phenylalanine causes brain damage are still poorly understood, preclinical models have been deeply explored to characterize the neurotoxic effect of Phe on neurodevelopmental processes. At the same time, that on the aging brain still needs to be explored. In the brain of untreated PAH Enu2(-/-) mouse, we previously reported a reduction of myelin basic protein (MBP) during postnatal development up to 60 PND. Later in the diseased mouse's life, a spontaneous and persistent restoration of MBP was detected. In this present longitudinal study, ranging from 14 to 540 post-natal days (PND) of untreated PAH Enu2(-/-) mice, we further investigated: a) the long-life consistency of two Phe-related brain metabolic alterations, such as large neutral amino acids (LNAA) and biogenic amine neurotransmitters' depletion; b) the outcome of locomotor functions during the same life span; c) the integrity of myelin as assessed ex vivo by central (hippocampus) and peripheral (extensor digitorum longus-sciatic nerve) action potential conduction velocities. In contrast with the results of other studies, brain Leu, Ile, and Val concentrations were not significantly altered in the brain PAH Enu2(-/-) mouse. On the other hand, 3-O-Methyldopa (3-OMD, a biomarker of L-DOPA), serotonin, and its associated metabolites were reduced throughout most of the considered time points, with consistent reductions observed prevalently from 14 to 60 PND. Normal saltatory conduction was restored after 60 PND and remained normal at the last examination at 360 PND, resulting nonetheless in a persistent locomotor impairment throughout a lifetime. These new findings contribute to laying the foundations for the preclinical characterization of aging in PKU, confirming neurotransmitter defects as consistent metabolic traits. LNAAs have a minor role, if any, in brain damage pathogenesis. Transient myelin synthesis failure may impact brain connectivity during postnatal development but not nervous signal conduction., Competing Interests: Declaration of competing interest Mauro Magnani and Luigia Rossi hold shares in EryDel SpA, a company with interests in the technology of RBC-based drug delivery. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

hERG potassium channels are critical for cardiac excitability. hERG channels have a Per-Arnt-Sim (PAS) domain at their N-terminus, and here, we examined the mechanism for PAS domain regulation of channel opening and closing (gating). We used TAG codon suppression to incorporate the noncanonical amino acid 4-benzoyl-L-phenylalanine (BZF), which is capable of forming covalent cross-links after photoactivation by ultraviolet (UV) light, at three locations (G47, F48, and E50) in the PAS domain. We found that hERG-G47BZF channels had faster closing (deactivation) when irradiated in the open state (at 0 mV) but showed no measurable changes when irradiated in the closed state (at -100 mV). hERG-F48BZF channels had slower activation, faster deactivation, and a marked rightward shift in the voltage dependence of activation when irradiated in the open (at 0 mV) or closed (at -100 mV) state. hERG-E50BZF channels had no measurable changes when irradiated in the open state (at 0 mV) but had slower activation, faster deactivation, and a rightward shift in the voltage dependence of activation when irradiated in the closed state (at -100mV), indicating that hERG-E50BZF had a state-dependent difference in UV photoactivation, which we interpret to mean that PAS underwent molecular motions between the open and closed states. Moreover, we propose that UV-dependent biophysical changes in hERG-G47BZF, F48BZF, and E50BZF were the direct result of photochemical cross-linking that reduced dynamic motions in the PAS domain and broadly stabilized the closed state relative to the open state of the channel., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Yeast phenylalanine tRNA (tRNA phe ) is a paradigmatic model in structural biology. In this work, we combine molecular dynamics simulations and spectroscopy modeling to establish a direct link between its structure, conformational dynamics, and infrared (IR) spectra. Employing recently developed vibrational frequency maps and coupling models, we apply a mixed quantum/classical treatment of the line shape theory to simulate the IR spectra of tRNA phe in the 1600-1800 cm -1 region across its folded and unfolded conformations and under varying concentrations of Mg 2+ ions. The predicted IR spectra of folded and unfolded tRNA phe are in good agreement with experimental measurements, validating our theoretical framework. We then elucidate how the characteristic L-shaped tertiary structure of the tRNA and its modulation in response to diverse chemical environments give rise to distinct IR absorption peaks and line shapes. These calculations effectively bridge IR spectroscopy experiments and atomistic molecular simulations, unraveling the molecular origins of the observed IR spectra of tRNA phe . This work presents a robust theoretical protocol for modeling the IR spectroscopy of nucleic acids, which will facilitate its application as a sensitive probe for detecting the fluctuating secondary and tertiary structures of these essential biological macromolecules.

Main Conclusions: The albino phenotype of Agave angustifolia Haw. accumulates higher levels of phenylalanine and phenylpropanoids, while the green phenotype has a greater concentration of phenolic compounds. The metabolic consequences of chlorophyll deficiency in plants continue to be a captivating field of research, especially in relation to production of metabolic compounds. This study conducts a thorough analysis of the metabolome in green (G), variegated (V), and albino (A) phenotypes of Agave angustifolia Haw. Specifically, it examines the differences in the accumulation of compounds related to the phenylpropanoid and flavonoid biosynthesis pathways. Methanol extracts of leaf and meristem tissues from the three phenotypes grown in vitro were analyzed using liquid chromatography coupled with quadrupole time-of-flight high-resolution mass spectrometry (UPLC-MS-QTOF) for untargeted metabolomics and triple quadrupole (QqQ) mass spectrometry for targeted metabolomic analyses. By employing these methods, we discovered notable differences in the levels of important metabolites such as L-phenylalanine, 4-hydroxyphenylpyruvic acid, and various flavonoids among the different phenotypes. The results of our study indicate that the A phenotype shows a significant increase in the levels of phenylalanine and phenylpropanoids in both leaf and meristem tissues. This is in contrast to a decrease in flavonoids, suggesting a metabolic reprogramming to compensate for the lack of chlorophyll. Significantly, compounds such as kaempferol-3-O-glucoside and rutin exhibited significant quantitative reduction in the A leaves, suggesting a subtle modification in the production of flavonols and potentially a changed mechanism for antioxidant protection. This study emphasizes the complex metabolic changes in A. angustifolia´s chlorophyll-deficient phenotypes, providing insight into the complex interplay between primary and secondary metabolism in response to chlorophyll deficiency. Our research not only enhances the comprehension of plant metabolism in albino phenotypes but also opens new avenues for exploring the biochemical and genetic basis of such adaptations, with potential biotechnological applications of these distinct plant variants., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Homologation of amino acids is the insertion or deletion of a methylene group to their side chain, which is a relatively uncommon chemical transformation observed in peptide natural product (NP) structure. Homologated amino acids can potentially make the NP more stable in a biological system, but its biosynthesis is yet to be understood. This study biochemically characterized the first of three unexplored enzymes in the homologation pathway of l-phenylalanine and l-tyrosine. Previously proposed reactions catalyzed by HphA were confirmed by reversed-phase high-performance liquid chromatography and tandem mass spectrometry analysis. The substrate profile and kinetic parameters showed high selectivity for the natural substrates and their close analogs. The comparability of HphA to homologous enzymes in primary metabolic pathways, 2-isopropylmate synthase and homocitrate synthase which are involved in l-leucine and l-lysine biosynthesis, respectively, was validated by bioinformatical and site-directed mutagenesis studies. The knowledge obtained from this study has deepened the understanding of the homologation of amino acids, which can lead to future combinatorial biosynthesis and metabolic engineering studies., (© 2024 Wiley-VCH GmbH.)

Glabridin is a widely used product in the cosmetics and pharmaceutical industry, which is generally isolated and purified from Licorice ( Glycyrrhiza glabra ) extract in industrial production. It has wide clinical applications, but significant toxicity has also been reported. The purity of glabridin raw material is generally between 90% and 98%. We have identified a toxic impurity, glabrene, in the industrial product glabridin. Our investigation using an AB wild-type zebrafish toxicity test showed that glabrene has a significant lethal effect with an LC 10 of 2.8 μM. Glabrene induced obvious malformation and disrupted cartilage development in zebrafish larvae. Furthermore, the compound significantly reduced larval mobility and caused damage to brain neural tissues. Metabolic pathway analysis and neurotransmitter quantification via ELISA indicated abnormal activation of the phenylalanine metabolic pathway, resulting in elevated dopamine and acetylcholine levels in vivo. These findings provide insights into the potential risks of glabrene contamination and offer a new reference point for enhancing safety measures and quality controls in licorice-derived products.

Genetic code expansion (GCE) is a powerful strategy that expands the genetic code of an organism for incorporating noncanonical amino acids into proteins using engineered tRNAs and aminoacyl-tRNA synthetases (aaRSs). While GCE has opened up new possibilities for synthetic biology, little is known about the potential side effects of exogenous aaRS/tRNA pairs. In this study, we investigated the impact of exogenous aaRS and amber suppressor tRNA on gene expression in Escherichia coli . We discovered that in DH10β Δ cyaA , transformed with the F1RP/F2P two-hybrid system, the high consumption rate of cellular adenosine triphosphate by exogenous aaRS/tRNA at elevated temperatures induces temperature sensitivity in the expression of genes regulated by the cyclic AMP receptor protein (CRP). We harnessed this temperature sensitivity to create a novel biological AND gate in E. coli , responsive to both p -benzoylphenylalanine (BzF) and low temperature, using a BzF-dependent variant of E. coli chorismate mutase and split subunits of Bordetella pertussis adenylate cyclase. Our study provides new insights into the unexpected effects of exogenous aaRS/tRNA pairs and offers a new approach for constructing a biological logic gate.

N-lactoyl-phenylalanine (Lac-Phe) is a lactate-derived metabolite that suppresses food intake and body weight. Little is known about the mechanisms that mediate Lac-Phe transport across cell membranes. Here we identify SLC17A1 and SLC17A3, two kidney-restricted plasma membrane-localized solute carriers, as physiologic urine Lac-Phe transporters. In cell culture, SLC17A1/3 exhibit high Lac-Phe efflux activity. In humans, levels of Lac-Phe in urine exhibit a strong genetic association with the SLC17A1-4 locus. Urine Lac-Phe levels are increased following a Wingate sprint test. In mice, genetic ablation of either SLC17A1 or SLC17A3 reduces urine Lac-Phe levels. Despite these differences, both knockout strains have normal blood Lac-Phe and body weights, demonstrating SLC17A1/3-dependent de-coupling of urine and plasma Lac-Phe pools. Together, these data establish SLC17A1/3 family members as the physiologic urine Lac-Phe transporters and uncover a biochemical pathway for the renal excretion of this signaling metabolite., (© 2024. The Author(s).)

The microbial communities residing in the mosquito midgut play a key role in determining the outcome of mosquito pathogen infection. Elizabethkingia anophelis, originally isolated from the midgut of Anopheles gambiae possess a broad-spectrum antiviral phenotype, yet a gap in knowledge regarding the mechanistic basis of its interaction with viruses exists. The current study aims to identify pathways and genetic factors linked to E. anophelis antiviral activity. The understanding of E. anophelis antiviral mechanism could lead to novel transmission barrier tools to prevent arboviral outbreaks. We utilized a non-targeted multi-omics approach, analyzing extracellular lipids, proteins, metabolites of culture supernatants coinfected with ZIKV and E. anophelis. We observed a significant decrease in arginine and phenylalanine levels, metabolites that are essential for viral replication and progression of viral infection. This study provides insights into the molecular basis of E. anophelis antiviral phenotype. The findings lay a foundation for in-depth mechanistic studies., (© 2024. The Author(s).)

The plasma protein α 1 -acid glycoprotein (AGP) primarily affects the pharmacokinetics of basic drugs. There are two AGP variants in humans, A and F1*S, exhibiting distinct drug-binding selectivity. Elucidation of the drug-binding selectivity of human AGP variants is essential for drug development and personalized drug therapy. Herein, we aimed to establish the contribution of amino acids 112 and 114 of human AGP to drug-binding selectively. Both amino acids are located in the drug-binding region and differ between the variants. Phe112/Ser114 of the A variant and its equivalent residues in the F1*S variant (Leu112/Phe114) were swapped with each other. Binding experiments were then conducted using the antiarrhythmic drug disopyramide, which selectively binds to the A variant. A significant decrease in the bound fraction was observed in each singly mutated A protein (Phe112Leu or Ser114Phe). Moreover, the bound fraction of the double A mutant (Phe112Leu/Ser114Phe) was decreased to that of wild-type F1*S. Intriguingly, the double F1*S mutant (Leu112Phe/Phe114Ser), in which residues were swapped with those of the A variant, showed only partial restoration in binding. The triple F1*S mutant (Leu112Phe/Phe114Ser/Asp115Tyr), where position 115 is thought to contribute to the difference in pocket size between variants, showed a further recovery in binding to 70% of that of wild-type A. These results were supported by thermodynamic analysis and acridine orange binding, which selectively binds the A variant. Together, these data indicate that, in addition to direct interaction with Phe112 and Ser114, the binding pocket size contributed by Tyr115 is important for the drug-binding selectivity of the A variant.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels 1 are essential for pacemaking activity and neural signalling 2,3 . Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain 4 and epileptic seizures 5 . The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor 6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs.  7,8 ). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Phenylalanine is a crucial amino acid in the process of melanogenesis. However, the exact mechanism by which it is transported into melanocytes has not been disclosed. The aim of this study was to identify and examine the key transporters that are responsible for phenylalanine transportation and evaluate their significance in melanogenesis. The amino acid transporter SLC16A10 was found to be up-regulated in both melasma (GSE72140) and sun-exposed skin (GSE67098). The protein levels of SLC16A10 were proportional to the melanin content in melanocytic nevi, indicating that SLC16A10 was related to melanogenesis. After SLC16A10 overexpression, melanin increased significantly in MNT1 cells. Meanwhile, the expression of melanogenesis-related proteins such as TYR and TYRP1 increased, while their RNA levels did not change. Transcriptomics data indicated that SLC16A10 can enhance the function of ribosome. Furthermore, targeted metabolomics data and ELISA results demonstrated SLC16A10 mainly affected the transport of phenylalanine into the cells. Then, phenylalanine was added to the cell culture medium after SLC16A10 overexpression, melanin synthesis in cells furtherly increased, which verified that SLC16A10 enhances melanogenesis by promoting the uptake of phenylalanine. Finally, we found that SLC16A10 expression increased after UVB irradiation. Knockdown SLC16A10 reduced UVB-induced melanin production and phenylalanine uptake by cells. In summary, SLC16A10 enhances melanogenesis by promoting the uptake of phenylalanine, and upregulation SLC16A10 is likely responsible for the UVB-induced hyperpigmentation as well., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Adenylation enzymes catalyze the selective incorporation of aminoacyl building blocks in the biosynthesis of nonribosomal peptides and related natural products. Although β-amino acid units are one of the important aminoacyl building blocks in natural product biosynthesis, very little is known about the engineering of β-amino acid adenylation enzymes. In this study, we engineered the substrate specificity of the (S)-β-phenylalanine adenylation enzyme, HitB, involved in the biosynthesis of macrolactam polyketide hitachimycin. Based on the previously determined structure of HitB wild-type, we mutated Phe328 and Ser293, which are located near the meta and ortho position of the (S)-β-phenylalanine moiety, respectively. As a result, the HitB F328V and F328L mutants efficiently activated meta-substituted (S)-β-phenylalanine analogs, and the HitB T293G and T293S mutants efficiently activated ortho-substituted (S)-β-phenylalanine analogs. Structural analysis of the HitB F328L and T293G mutants with the corresponding nonhydrolyzable intermediate analogs revealed an enlarged substrate binding pocket for (S)-β-phenylalanine analogs, providing detailed insights into the structural basis for creating enzyme substrate promiscuity. Our findings may be useful for production of various β-amino acid-containing natural product analogs., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Competing Interests: Declaration of interests The authors have no interests to declare

Introduction: Nitisinone used in alkaptonuria (AKU) can result in keratopathy due to strongly increased tyrosine levels. Methods: This study aimed to investigate nutritional status and changes in plasma tyrosine and phenylalanine and urinary homogentisic acid (u-HGA) levels in 8 adult AKU patients (mean age, 56.3 ± 4.7 years) who were on tyrosine/phenylalanine-restricted diet together with 2 mg/day nitisinone. Results: The treatment period was 23.4 ± 6.9 months. Daily dietary protein intake was restricted to 0.8–1.0 g/kg/day. Daily tyrosine intake was restricted to 260–450 mg/day for females and 330–550 mg/day for males. Tyrosine/phenylalanine-free amino acid supplements accounted for an average of 56.1% of daily protein intake. The following assessments were performed: anthropometric and plasma tyrosine level measurements every 2 months; ophthalmological examination every 6 months, and nutritional laboratory analyses and measurements of plasma amino acids and u-HGA once in a year. It was targeted to keep the plasma tyrosine level <500 μmol/L. The plasma tyrosine level was <100 μmol/L before the treatment in all patients and around a mean of 582.5 ± 194.8 μmol/L during the treatment. The diet was rearranged if a plasma tyrosine level of >700 μmol/L was detected. The u-HGA level before and after the 1st year of treatment was 1,429.3 ± 1,073.4 mmol/mol creatinine and 33.6 ± 9.5 mmol/mol creatinine, respectively. None of the patients developed keratopathy or experienced weight loss and protein or micronutrient deficiency. Conclusion: AKU patients should receive tyrosine/phenylalanine-restricted diet for reducing plasma tyrosine level to the safe range. Tyrosine/phenylalanine-free amino acid supplements can be safely used to enhance dietary compliance. Keratopathy and nutrient deficiency should be frequently monitored. [ABSTRACT FROM AUTHOR]

Rapeseed (Brassica napus L.) is an important oilseed crop in the world. Its productivity is significantly influenced by numerous abiotic stresses, including cold stress (CS). Consequently, enhancement in CS tolerance is becoming an important area for agricultural investigation and crop improvement. Therefore, the current study aimed to identify the stress-responsive genes, metabolites, and metabolic pathways based on a combined transcriptome and metabolome analysis to understand the CS responses and tolerance mechanisms in the cold-tolerant (C18) and cold-sensitive (C6) rapeseed varieties. Based on the metabolome analysis, 31 differentially accumulated metabolites (DAMs) were identified between different comparisons of both varieties at the same time points. From the transcriptome analysis, 2,845, 3,358, and 2,819 differentially expressed genes (DEGs) were detected from the comparison of C6-0 vs. C18-0, C6-1 vs. C18-1, and C6-7 vs. C18-7. By combining the transcriptome and metabolome data sets, we found that numerous DAMs were strongly correlated with several differentially expressed genes (DEGs). A functional enrichment analysis of the DAMs and the correlated DEGs specified that most DEGs and DAMs were mainly enriched in diverse carbohydrates and amino acid metabolisms. Among them, starch and sucrose metabolism and phenylalanine metabolism were significantly enriched and played a vital role in the CS adaption of rapeseed. Six candidate genes were selected from the two pathways for controlling the adaption to low temperature. In a further validation, the T-DNA insertion mutants of their Arabidopsis homologous, including 4cl3, cel5, fruct4, ugp1, axs1, and bam2/9, were characterized and six lines differed significantly in levels of freezing tolerance. The outcome of the current study provided new prospects for the understanding of the molecular basis of CS responses and tolerance mechanisms in rapeseed and present a set of candidate genes for use in improving CS adaptability in the same plant.

Rapeseed (Brassica napus L.) is an important oilseed crop in the world. Its productivity is significantly influenced by numerous abiotic stresses, including cold stress (CS). Consequently, enhancement in CS tolerance is becoming an important area for agricultural investigation and crop improvement. Therefore, the current study aimed to identify the stress-responsive genes, metabolites, and metabolic pathways based on a combined transcriptome and metabolome analysis to understand the CS responses and tolerance mechanisms in the cold-tolerant (C18) and cold-sensitive (C6) rapeseed varieties. Based on the metabolome analysis, 31 differentially accumulated metabolites (DAMs) were identified between different comparisons of both varieties at the same time points. From the transcriptome analysis, 2,845, 3,358, and 2,819 differentially expressed genes (DEGs) were detected from the comparison of C6-0 vs. C18-0, C6-1 vs. C18-1, and C6-7 vs. C18-7. By combining the transcriptome and metabolome data sets, we found that numerous DAMs were strongly correlated with several differentially expressed genes (DEGs). A functional enrichment analysis of the DAMs and the correlated DEGs specified that most DEGs and DAMs were mainly enriched in diverse carbohydrates and amino acid metabolisms. Among them, starch and sucrose metabolism and phenylalanine metabolism were significantly enriched and played a vital role in the CS adaption of rapeseed. Six candidate genes were selected from the two pathways for controlling the adaption to low temperature. In a further validation, the T-DNA insertion mutants of their Arabidopsis homologous, including 4cl3, cel5, fruct4, ugp1, axs1 , and bam2/9 , were characterized and six lines differed significantly in levels of freezing tolerance. The outcome of the current study provided new prospects for the understanding of the molecular basis of CS responses and tolerance mechanisms in rapeseed and present a set of candidate genes for use in improving CS adaptability in the same plant. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is one of the most challenging diseases causing an increasing burden worldwide. Although the neuropathologic diagnosis of AD has been established for many years, the metabolic changes in neuropathologic diagnosed AD samples have not been fully investigated.Objective: To elucidate the potential metabolism dysregulation in the postmortem human brain samples assessed by AD related pathological examination.Methods: We performed untargeted and targeted metabolomics in 44 postmortem human brain tissues. The metabolic differences in the hippocampus between AD group and control (NC) group were compared.Results: The results show that a pervasive metabolic dysregulation including phenylalanine metabolism, valine, leucine, and isoleucine biosynthesis, biotin metabolism, and purine metabolism are associated with AD pathology. Targeted metabolomics reveal that phenylalanine, phenylpyruvic acid, and N-acetyl-L-phenylalanine are upregulated in AD samples. In addition, the enzyme IL-4I1 catalyzing transformation from phenylalanine to phenylpyruvic acid is also upregulated in AD samples.Conclusion: There is a pervasive metabolic dysregulation in hippocampus with AD-related pathological changes. Our study suggests that the dysregulation of phenylalanine metabolism in hippocampus may be an important pathogenesis for AD pathology formation. [ABSTRACT FROM AUTHOR]