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Purpose: Obesity is a primary risk factor for knee osteoarthritis (OA). Prebiotics enhance beneficial gut microbes and can reduce body fat and inflammation. Our objective was to examine if a 6-month prebiotic intervention improved physical function in adults with knee osteoarthritis and obesity. We also measured knee pain, body composition, quality of life, gut microbiota, inflammatory markers, and serum metabolomics. Methods: Adults (n = 54, mostly women) with co-morbid obesity (BMI > 30 kg/m2) and unilateral/bilateral knee OA were randomly assigned to prebiotic (oligofructose-enriched inulin; 16 g/day; n = 31) or isocaloric placebo (maltodextrin; n = 21) for 6 months. Performance based-tests, knee pain, quality of life, serum metabolomics and inflammatory markers, and fecal microbiota and short-chain fatty acids were assessed. Results: Significant between group differences were detected for the change in timed-up-and-go test, 40 m fast paced walk test, and hand grip strength test from baseline that favored prebiotic over placebo. Prebiotic also reduced trunk fat mass (kg) at 6 months and trunk fat (%) at 3 months compared to placebo. There was a trend (p = 0.059) for reduced knee pain at 6 months with prebiotic versus placebo. In gut microbiota analysis, a total of 37 amplicon sequence variants differed between groups. Bifidobacterium abundance was positively correlated with distance walked in the 6-min walk test and hand grip strength. At 6 months, there was a significant separation of serum metabolites between groups with upregulation of phenylalanine and tyrosine metabolism with prebiotic. Conclusion: Prebiotics may hold promise for conservative management of knee osteoarthritis in adults with obesity and larger trials are warranted. Clinical Trial Registration: Clinicaltrials.gov/study/NCT04172688. [ABSTRACT FROM AUTHOR]

Linum album is a rich source of various phenolic compounds, especially lignans. Our previous studies revealed that phenylalanine (Phe), an aromatic amino acid, participates in lignans production. In this study, we tried to characterize how Phe as the precursor feeding affects metabolic fluxes in L. album cells. As expected, the results indicated that an increase in phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase activities (TAL) occurred at 250 μM. Their actions induced phenolic acids production, especially cinnamic acid and salicylic acid (SA). The low dosages of Phe stimulated hydrogen peroxide (H2O2) and nitric oxide (NO) generation, lipid peroxidation, and antioxidant enzymes activation, while the high dosages suppressed the mentioned physiological processes. A depletion in rhamnose/xylose, glucose, and mannose was observed at high concentrations of Phe compared with the control. These molecules altered amino acids profile through supplying carbon skeleton and cell required energy. Our observations also showed that amino acids contents were significantly influenced by Phe treatment. The reduction of endogenous Phe content in the treated cells may closely related to phenolics accumulation such as flavonoids and lignans, as the enhancement of catechin, myricetin, and kaempferol were observed in the treated cells. Likewise, phenylalanine increased lignans, except for lariciresionol (LARI), and the highest amounts of podophyllotoxin (PTOX), and 6-methoxy podophyllotoxin (6MPTOX) were detected at 1000 μM. In summary, metabolic and physiological changes can elucidate that Phe involves in the lignans production in L. album through a SA-dependent pathway. [ABSTRACT FROM AUTHOR]

The application of phosphorus-solubilizing bacteria is an effective method for increasing the available phosphorus content and inhibiting wheat uptake of heavy metals. However, further research is needed on the mechanism by which phosphorus-solubilizing bacteria inhibit cadmium (Cd) uptake in wheat roots and its impact on the expression of root-related genes. Here, the effects of strain Klebsiella aerogenes M2 on Cd absorption in wheat and the expression of root-related Cd detoxification and immobilization genes were determined. Compared with the control, strain M2 reduced (64.1–64.6%) Cd uptake by wheat roots. Cd fluorescence staining revealed that strain M2 blocked the entry of exogenous Cd into the root interior and enhanced the immobilization of Cd by cell walls. Forty-seven genes related to Cd detoxification, including genes encoding peroxidase, chalcone synthase, and naringenin 3-dioxygenase, were upregulated in the Cd+M2 treatment. Strain M2 enhanced the Cd resistance and detoxification activity of wheat roots through the regulation of flavonoid biosynthesis and antioxidant enzyme activity. Moreover, strain M2 regulated the expression of genes related to phenylalanine metabolism and the MAPK signaling pathway to enhance Cd immobilization in roots. These results provide a theoretical basis for the use of phosphorus-solubilizing bacteria to remediate Cd-contaminated fields and reduce Cd uptake in wheat. [ABSTRACT FROM AUTHOR]

Objective: To clarify the potential mechanism of Banxia Xiexin Decoction (BXD) on colorectal cancer (CRC) from the perspective of metabolomics. Methods: Forty male C57BL/6 mice were randomly divided into normal control (NC), azoxymethane/dextran sulfate sodium (AOM/DSS) model, low-dose BXD (L-BXD), high-dose BXD (H-BXD) and mesalamine (MS) groups according to a random number table, 8 mice in each group. Colorectal cancer model was induced by AOM/DSS. BXD was administered daily at doses of 3.915 (L-BXD) and 15.66 g/kg (H-BXD) by gavage for consecutive 21 days, and 100 mg/kg MS was used as positive control. Following the entire modeling cycle, colon length of mice was measured and quantity of colorectal tumors were counted. The spleen and thymus index were determined by calculating the spleen/thymus weight to body weight. Inflammatory cytokine and changes of serum metabolites were analyzed by enzyme-linked immunosorbent assay kits and ultra performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-Q/TOF-MS), respectively. Results: Notably, BXD supplementation protected against weight loss, mitigated tumor formation, and diminished histologic damage in mice treated with AOM/DSS (P<0.05 or P<0.01). Moreover, BXD suppressed expression of serum inflammatory enzymes, and improved the spleen and thymus index (P<0.05). Compared with the normal group, 102 kinds of differential metabolites were screened in the AOM/DSS group, including 48 potential biomarkers, involving 18 main metabolic pathways. Totally 18 potential biomarkers related to CRC were identified, and the anti-CRC mechanism of BXD was closely related to D-glutamine and D-glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, arginine biosynthesis, nitrogen metabolism and so on. Conclusion: BXD exerts partial protective effects on AOM/DSS-induced CRC by reducing inflammation, protecting organism immunity ability, and regulating amino acid metabolism. [ABSTRACT FROM AUTHOR]

Abstract Lignocellulosic biomass is currently underutilized, but it offers promise as a resource for the generation of commercial end-products, such as biofuels, detergents, and other oleochemicals. Rhodococcus opacus PD630 is an oleaginous, Gram-positive bacterium with an exceptional ability to utilize recalcitrant aromatic lignin breakdown products to produce lipid molecules such as triacylglycerols (TAGs), which are an important biofuel precursor. Lipid carbon storage molecules accumulate only under growth-limiting low nitrogen conditions, representing a significant challenge toward using bacterial biorefineries for fuel precursor production. In this work, we screened overexpression of 27 native transcriptional regulators for their abilities to improve lipid accumulation under nitrogen-rich conditions, resulting in three strains that accumulate increased lipids, unconstrained by nitrogen availability when grown in phenol or glucose. Transcriptomic analyses revealed that the best strain (#13) enhanced FA production via activation of the β-ketoadipate pathway. Gene deletion experiments confirm that lipid accumulation in nitrogen-replete conditions requires reprogramming of phenylalanine metabolism. By generating mutants decoupling carbon storage from low nitrogen environments, we move closer toward optimizing R. opacus for efficient bioproduction on lignocellulosic biomass.

Lignocellulosic biomass is currently underutilized, but it offers promise as a resource for the generation of commercial end-products, such as biofuels, detergents, and other oleochemicals. Rhodococcus opacus PD630 is an oleaginous, Gram-positive bacterium with an exceptional ability to utilize recalcitrant aromatic lignin breakdown products to produce lipid molecules such as triacylglycerols (TAGs), which are an important biofuel precursor. Lipid carbon storage molecules accumulate only under growth-limiting low nitrogen conditions, representing a significant challenge toward using bacterial biorefineries for fuel precursor production. In this work, we screened overexpression of 27 native transcriptional regulators for their abilities to improve lipid accumulation under nitrogen-rich conditions, resulting in three strains that accumulate increased lipids, unconstrained by nitrogen availability when grown in phenol or glucose. Transcriptomic analyses revealed that the best strain (#13) enhanced FA production via activation of the β-ketoadipate pathway. Gene deletion experiments confirm that lipid accumulation in nitrogen-replete conditions requires reprogramming of phenylalanine metabolism. By generating mutants decoupling carbon storage from low nitrogen environments, we move closer toward optimizing R. opacus for efficient bioproduction on lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Objective: The peripheral B cell compartment is heavily disturbed in systemic lupus erythematosus (SLE), but whether B cells develop aberrantly in the bone marrow (BM) is largely unknown. Methods: We performed single‐cell RNA/B cell receptor (BCR) sequencing and immune profiling of BM B cells and classified patients with SLE into two groups: early B cell (Pro‐B and Pre‐B) normal (EBnor) and EB defective/low (EBlo) groups. Results: The SLE‐EBlo group exhibited more severe disease activity and proinflammatory status, overaction of type I interferon signaling and metabolic pathways within the B cell compartment, and aberrant BCR repertoires compared with the SLE‐EBnor group. Moreover, in one patient with SLE who was initially classified in the SLE‐EBlo group, early B cell deficiency and associated abnormalities were largely rectified in a second BM sample at the remission phase. Conclusion: In summary, this study suggests that early B cell loss in BM defines a unique pathological state in a subset of patients with SLE that may play an active role in the dysregulated autoimmune responses. [ABSTRACT FROM AUTHOR]

Introduction: Atopic dermatitis (AD) is one of the most common inflammatory skin diseases. Skin microecological imbalance is an important factor in the pathogenesis of AD, but the underlying mechanism of its interaction with humans remains unclear. Methods: 16S rRNA gene sequencing was conducted to reveal the skin microbiota dynamics. Changes in skin metabolites were tracked by LC-MS metabolomics. We then explored the potential mechanism of interaction by analyzing the correlation between skin bacterial communities and metabolites in corresponding skin-associated samples. Results: Samples from 18 AD patients and 18 healthy volunteers (HVs) were subjected to 16S rRNA gene sequencing and LC-MS metabolomics. AD patients had dysbiosis of the skin bacterial community with decreased species richness and evenness. The relative abundance of the genus Staphylococcus increased significantly in AD, while the abundances of the genera Propionibacterium and Brevundimonas decreased significantly. The relative abundance of the genera Staphylococcus in healthy females was significantly higher than those in healthy males, while it showed no difference in AD patients with or without lesions. The effects of AD status, sex and the presence or absence of rashes on the number of differentially abundant metabolites per capita were successively reduced. Multiple metabolites involved in purine metabolism and phenylalanine metabolism pathways (such as xanthosine/xanthine and L-phenylalanine/trans-cinnamate) were increased in AD patients. These trends were much more obvious between female AD patients and female HVs. Spearman correlation analysis revealed that the genus Staphylococcus was positively correlated with various compounds involved in phenylalanine metabolism and purine metabolic pathways. The genera Brevundimonas and Lactobacillus were negatively correlated with various compounds involved in purine metabolism, phenylalanine metabolism and sphingolipid signaling pathways. Discussion: We suggest that purine metabolism and phenylalanine metabolism pathway disorders may play a certain role in the pathogenic mechanism of Staphylococcus aureus in AD. We also found that females are more likely to be colonized by the genus Staphylococcus than males. Differentially abundant metabolites involved in purine metabolism and phenylalanine metabolism pathways were more obvious in female. However, we should notice that the metabolites we detected do not necessarily derived from microbes, they may also origin from the host. [ABSTRACT FROM AUTHOR]

Purpose: Studies have suggested that women with RA tend to avoid red meat more often than women without RA, based on their perception that it exacerbates their symptoms. Therefore, the aim of this study is to investigate and compare the postprandial metabolic response following the consumption of a red meat meal in patients with RA and a matched control group. Methods: Participants were challenged with a meal with red meat and blood samples were collected before and at 0.5, 1, 2, 3 and 5 h after the meal. Serum metabolites were quantified by Nuclear Magnetic Resonance (NMR) analysis. Orthogonal Projections to Latent Structures with Discriminant Analysis (OPLS-DA) was used to evaluate separation by metabolites due to diagnosis of RA or not and to identify changes in metabolites related to RA. Incremental area under the curve was calculated for univariate comparisons for 23 metabolites. Results: The matched groups, including 22 women with RA and 22 women without RA, did not differ significantly in age, body mass index, diet quality or reported physical activity. OPLS-DA models had a limited quality indicating that there were no differences in metabolite patterns between the groups. However, phenylalanine was significantly higher in concentration in women with RA compared to controls in both fasting and postprandial samples. Conclusion: To conclude, this well-controlled postprandial intervention study found a significantly higher concentration of phenylalanine in both fasting and postprandial samples of women with RA compared to matched women without RA. These findings warrant further investigation in larger studies. Trial registration: The PIRA (Postprandial Inflammation in Rheumatoid Arthritis) trial is Registered at Clinicaltrials.gov (NCT04247009). [ABSTRACT FROM AUTHOR]

Stalk rot is a prevalent disease of maize (Zea mays L.) that severely affects maize yield and quality worldwide. The ascomycete fungus Fusarium spp. is the most common pathogen of maize stalk rot. At present, the molecular mechanism of Fusarium proliferation during the maize stalk infection that causes maize stalk rot has rarely been reported. In this study, we investigated the response of maize to F. proliferatum infestation by analyzing the phenotypic, transcriptomic, and metabolomic data of inbred lines ZC17 (resistant) and CH72 (susceptible) with different levels of resistance to stalk rot. Physiological and phenotypic results showed that the infection CH72 was significantly more severe than ZC17 after inoculation. Transcriptome analysis showed that after inoculation, the number of differentially expressed genes (DEGs) was higher in CH72 than in ZC17. Nearly half of these DEGs showed the same expression trend in the two inbred lines. Functional annotation and enrichment analyses indicated that the major pathways enriched for DEGs and DEMs included the biosynthesis of plant secondary metabolites, phenylalanine metabolism, biosynthesis of plant hormones, and plant–pathogen interactions. The comprehensive analysis of transcriptome and metabolome data indicated that phenylalanine metabolism and the phenylalanine, tyrosine, and tryptophan biosynthesis pathways played a crucial role in maize resistance to F. proliferatum infection. In addition, a transcription factor (TF) analysis of the DEGs showed that several TF families, including MYB, bHLH, NAC, and WRKY, were significantly activated after inoculation, suggesting that these TFs play important roles in the molecular regulatory network of maize disease resistance. The findings of this study provide valuable insights into the molecular basis of the response of maize to Fusarium proliferatum infection and highlight the importance of combining multiple approaches, such as phenotyping, transcriptomics, and metabolomics, to gain a comprehensive understanding of plant–pathogen interactions. [ABSTRACT FROM AUTHOR]

The application of phosphorus-solubilizing bacteria is an effective method for increasing the available phosphorus content and inhibiting wheat uptake of heavy metals. However, further research is needed on the mechanism by which phosphorus-solubilizing bacteria inhibit cadmium (Cd) uptake in wheat roots and its impact on the expression of root-related genes. Here, the effects of strain Klebsiella aerogenes M2 on Cd absorption in wheat and the expression of root-related Cd detoxification and immobilization genes were determined. Compared with the control, strain M2 reduced (64.1–64.6%) Cd uptake by wheat roots. Cd fluorescence staining revealed that strain M2 blocked the entry of exogenous Cd into the root interior and enhanced the immobilization of Cd by cell walls. Forty-seven genes related to Cd detoxification, including genes encoding peroxidase, chalcone synthase, and naringenin 3-dioxygenase, were upregulated in the Cd+M2 treatment. Strain M2 enhanced the Cd resistance and detoxification activity of wheat roots through the regulation of flavonoid biosynthesis and antioxidant enzyme activity. Moreover, strain M2 regulated the expression of genes related to phenylalanine metabolism and the MAPK signaling pathway to enhance Cd immobilization in roots. These results provide a theoretical basis for the use of phosphorus-solubilizing bacteria to remediate Cd-contaminated fields and reduce Cd uptake in wheat.

Stalk rot is a prevalent disease of maize (Zea mays L.) that severely affects maize yield and quality worldwide. The ascomycete fungus Fusarium spp. is the most common pathogen of maize stalk rot. At present, the molecular mechanism of Fusarium proliferation during the maize stalk infection that causes maize stalk rot has rarely been reported. In this study, we investigated the response of maize to F. proliferatum infestation by analyzing the phenotypic, transcriptomic, and metabolomic data of inbred lines ZC17 (resistant) and CH72 (susceptible) with different levels of resistance to stalk rot. Physiological and phenotypic results showed that the infection CH72 was significantly more severe than ZC17 after inoculation. Transcriptome analysis showed that after inoculation, the number of differentially expressed genes (DEGs) was higher in CH72 than in ZC17. Nearly half of these DEGs showed the same expression trend in the two inbred lines. Functional annotation and enrichment analyses indicated that the major pathways enriched for DEGs and DEMs included the biosynthesis of plant secondary metabolites, phenylalanine metabolism, biosynthesis of plant hormones, and plant–pathogen interactions. The comprehensive analysis of transcriptome and metabolome data indicated that phenylalanine metabolism and the phenylalanine, tyrosine, and tryptophan biosynthesis pathways played a crucial role in maize resistance to F. proliferatum infection. In addition, a transcription factor (TF) analysis of the DEGs showed that several TF families, including MYB, bHLH, NAC, and WRKY, were significantly activated after inoculation, suggesting that these TFs play important roles in the molecular regulatory network of maize disease resistance. The findings of this study provide valuable insights into the molecular basis of the response of maize to Fusarium proliferatum infection and highlight the importance of combining multiple approaches, such as phenotyping, transcriptomics, and metabolomics, to gain a comprehensive understanding of plant–pathogen interactions.

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.)

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.)

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.

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.)

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).)

This study aimed to explore the pharmacodynamics and mechanisms of different processing methods of Ligustrum lucidum Ait. (LLA) in addressing kidney-yin deficiency (KYD). Forty-eight Sprague-Dawley rats were divided into eight groups based on their weight. The KYD model was established by intragastric administration of levothyroxine sodium. Each group was administered the corresponding treatment for 15 consecutive days. The general condition of the rats during the treatment period was observed. In addition, the levels of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and the ratio of cAMP to cGMP in the serum of rats from different groups were measured. Serum samples were analyzed using the ultra-performance liquid chromatography (UPLC)-Orbitrap Fusion MS technique for metabolomics analysis. Compared with the model group, the general condition of the rats in the wine-steamed L. lucidum group (WL) and salt-steamed L. lucidum group (SSL) groups showed significant improvement. The serum levels of cAMP, cGMP, and the cAMP-to-cGMP ratio tended to return to normal. Metabolic analysis identified 38 relevant biomarkers and revealed 3 major metabolic pathways: phenylalanine, tyrosine, and tryptophan biosynthesis; phenylalanine metabolism; and sphingolipid metabolism. The different processing methods of LLA demonstrated therapeutic effects on KYD in rats, likely related to the restoration of disturbed metabolism by adjusting the levels of endogenous metabolites in the kidney. The SSL demonstrated significantly superior effects compared with the other four types of LLA processed products., (© 2024 John Wiley & Sons Ltd.)

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.

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

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.)

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.)

Being generally regarded as safe, Kluyveromyces lactis has been widely taken for food, feed, and pharmaceutical applications, owing to its ability to achieve high levels of protein secretion and hence being suitable for industrial production of heterologous proteins. Production platform strains can be created through genetic engineering; while prototrophic cells without chromosomally accumulated antibiotics resistance genes have been generally preferred, arising the need for dominant counterselection. We report here the establishment of a convenient counterselection system based on a Frs2 variant, Frs2v, which is a mutant of the alpha-subunit of phenylalanyl-tRNA synthase capable of preferentially incorporating a toxic analog of phenylalanine, r-chloro-phenylalanine (4-CP), into proteins to bring about cell growth inhibition. We demonstrated that expression of Frs2v from an episomal plasmid in K. lactis could make the host cells sensitive to 2 mM 4-CP, and a Frs2v-expressing plasmid could be efficiently removed from the cells immediately after a single round of cell culturing in a 4-CP-contianing YPD medium. This Frs2v-based counterselection helped us attain scarless gene replacement in K. lactis without any prior engineering of the host cells. More importantly, counterselection with this system was proven to be functionally efficient also in Saccharomyces cerevisiae and Komagataella phaffii, suggestive of a broader application scope of the system in various yeast hosts. Collectively, this work has developed a strategy to enable rapid, convenient, and high-efficiency construction of prototrophic strains of K. lactis and possibly many other yeast species, and provided an important reference for establishing similar methods in other industrially important eukaryotic microbes., (© 2024. The Author(s).)

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.

Copper amine oxidases (CAOs) catalyze the oxidative deamination of primary amines to aldehyde, ammonia, and hydrogen peroxide as products and are widely distributed in bacteria, plants, and eukaryotes. These enzymes initiate the single turnover, post-translational conversion of an active site tyrosine to the redox cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ), subsequently employing TPQ to catalyze steady-state amine oxidation. The mechanisms of TPQ biogenesis and steady-state amine oxidation have been studied extensively, with consensus mechanisms proposed for both reactions. One unresolved issue has been whether the Cu 2+ center must undergo formal reduction to Cu 1+ in the course of the reaction. Herein, we investigate the properties of the active site of a yeast ( Hansenula polymorpha ) amine oxidase (HPAO) that has undergone site-specific insertion of a para -aminophenylalanine (pAF) into the position of either the precursor tyrosine to TPQ (Y405) or the two strictly conserved neighboring tyrosines (Y305 and Y407). While our original intention was to interrogate cofactor biogenesis using a precursor unnatural amino acid (UAA) of altered redox potential and p K a , we instead observe an unanticipated reaction assigned to an intramolecular electron transfer from pAF to the active site copper ion. We establish the generality of the observed active site chemistry using exogenously added, aniline-containing substrates under conditions that prevent side chain amine oxidation. The results support previous proposals that the activation of the TPQ precursor occurs in the absence of a formal valence change at the active site copper site. The described reaction of pAFs with the active site redox Cu 2+ center of HPAO provides a prototype for either the engineering of the enzymatic oxidation of exogenous anilines or the insertion of site-specific free radical probes within proteins.

Tuta absoluta ("leafminer"), is a major pest of tomato crops worldwide. Controlling this insect is difficult due to its efficient infestation, rapid proliferation, and resilience to changing weather conditions. Furthermore, chemical pesticides have only a short-term effect due to rapid development of T. absoluta strains. Here, we show that a variety of tomato cultivars, treated with external phenylalanine solutions exhibit high resistance to T. absoluta, under both greenhouse and open field conditions, at different locations. A large-scale metabolomic study revealed that tomato leaves absorb and metabolize externally given Phe efficiently, resulting in a change in their volatile profile, and repellence of T. absoluta moths. The change in the volatile profile is due to an increase in three phenylalanine-derived benzenoid phenylpropanoid volatiles (BPVs), benzaldehyde, phenylacetaldehyde, and 2-phenylethanol. This treatment had no effect on terpenes and green leaf volatiles, known to contribute to the fight against insects. Phe-treated plants also increased the resistance of neighboring non-treated plants. RNAseq analysis of the neighboring non-treated plants revealed an exclusive upregulation of genes, with enrichment of genes related to the plant immune response system. Exposure of tomato plants to either benzaldehyde, phenylacetaldehyde, or 2-phenylethanol, resulted in induction of genes related to the plant immune system that were also induced due to neighboring Phe-treated plants. We suggest a novel role of phenylalanine-derived BPVs as mediators of plant-insect interactions, acting as inducers of the plant defense mechanisms., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Magnetoreceptive biology as a field remains relatively obscure; compared with the breadth of species believed to sense magnetic fields, it remains under-studied. Here, we present grounds for the expansion of magnetoreception studies among teleosts. We begin with the electromagnetic perceptive gene (EPG) from Kryptopterus vitreolus and expand to identify 72 teleosts with homologous proteins containing a conserved three-phenylalanine (3F) motif. Phylogenetic analysis provides insight as to how EPG may have evolved over time and indicates that certain clades may have experienced a loss of function driven by different fitness pressures. One potential factor is water type with freshwater fish significantly more likely to possess the functional motif version (FFF), and saltwater fish to have the non-functional variant (FXF). It was also revealed that when the 3F motif from the homologue of Brachyhypopomus gauderio (B.g.) is inserted into EPG-EPG(B.g.)-the response (as indicated by increased intracellular calcium) is faster. This indicates that EPG has the potential to be engineered to improve upon its response and increase its utility to be used as a controller for specific outcomes.

Altered activity of specific enzymes in phenylalanine-tyrosine (phe-tyr) metabolism results in incomplete breakdown of various metabolite substrates in this pathway. Increased biofluid concentration and tissue accumulation of the phe-tyr pathway metabolite homogentisic acid (HGA) is central to pathophysiology in the inherited disorder alkaptonuria (AKU). Accumulation of metabolites upstream of HGA, including tyrosine, occurs in patients on nitisinone, a licenced drug for AKU and hereditary tyrosinaemia type 1, which inhibits the enzyme responsible for HGA production. The aim of this study was to investigate the phe-tyr metabolite content of key biofluids and tissues in AKU mice on and off nitisinone to gain new insights into the biodistribution of metabolites in these altered metabolic states. The data show for the first time that HGA is present in bile in AKU (mean [±SD] = 1003[±410] μmol/L; nitisinone-treated AKU mean [±SD] = 45[±23] μmol/L). Biliary tyrosine, 3(4-hydroxyphenyl)pyruvic acid (HPPA) and 3(4-hydroxyphenyl)lactic acid (HPLA) are also increased on nitisinone. Urine was confirmed as the dominant elimination route of HGA in untreated AKU, but with indication of biliary excretion. These data provide new insights into pathways of phe-tyr metabolite biodistribution and metabolism, showing for the first time that hepatobiliary excretion contributes to the total pool of metabolites in this pathway. Our data suggest that biliary elimination of organic acids and other metabolites may play an underappreciated role in disorders of metabolism. We propose that our finding of approximately 3.8 times greater urinary HGA excretion in AKU mice compared with patients is one reason for the lack of extensive tissue ochronosis in the AKU mouse model., (© 2024 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Nuclear magnetic resonance (NMR) spectroscopy NMR is a well-established technique for probing protein structure, dynamics and conformational changes. Taking advantage of the high signal sensitivity and broad chemical shift range of 19 F nuclei, 19 F NMR has been applied to investigate protein function at atomic resolution. In this report, we extend the unnatural amino acid site-specific incorporation into V. natriegens, an alternate protein expression system. The unnatural amino acid L-4-trifluoromethylphenylalanine (tfmF) was site-specifically introduced into the mitogen-activated protein kinase MEKK3 in V. natriegens using genetically encoded technology, which will be an extensive method for in-cell protein structure and dynamic investigation., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Postharvest browning of A. bisporus is a significant concern, impacting both its marketability and economic value. This study aims to investigate the effectiveness of short-term anaerobic treatment combined with microperforated film packaging (AT-MAP) in reducing postharvest browning of A. bisporus , with a focus on elucidating its molecular mechanisms. Comprehensive physiological assessments were conducted, including analyses of browning index (BI), polyphenol oxidase (PPO) activity, total phenols, flavonoids, relative electrical conductivity, and malondialdehyde (MDA) content. Additionally, transcriptomic sequencing and real-time quantitative reverse transcription (RT-qPCR) were employed to explore gene expression alterations. Our findings reveal that, compared to both the air control (CK) and microperforated film packaging (MAP), AT-MAP yields enhancements in appearance quality, total phenol, and flavonoid content. Storage with AT-MAP demonstrates a reduction in the browning index, PPO activity, MDA content, and relative conductivity by 11%, 40%, 8%, and 6%, respectively, in comparison to the MAP treatment group. At the molecular level, our analysis identifies a total of 3403 differentially expressed genes (DEGs) in A. bisporus subjected to AT-MAP, comprising 1840 up-regulated genes and 1563 down-regulated genes. AT-MAP downregulates genes in the sphingolipid metabolism pathway, reducing ceramide accumulation and apoptosis. It also diminishes ROS production and enhances cell membrane integrity by modulating genes in the oxidative phosphorylation pathway, particularly those associated with NADH dehydrogenase and cytochrome C reductase. Moreover, AT-MAP curtails melanin and brown pigment formation by suppressing genes involved in the metabolism of phenylalanine, tyrosine, and tryptophan, notably those linked to 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase. This study contributes insights into the molecular mechanisms underpinning the efficacy of AT-MAP in mitigating postharvest browning in A. bisporus , providing a theoretical foundation for its application in mushroom preservation. • AT-MAP delays browning, reduces PPO activity in Agaricus bisporus. • AT-MAP boosts phenols, flavonoids; suppresses MDA rise. • AT-MAP/MAP reveals 3403 differentially expressed genes. • AT-MAP stabilizes cell membrane, modulates energy and amino acid metabolism. [ABSTRACT FROM AUTHOR]

Tomato is an important horticultural cash crop, and low-temperature stress has seriously affected the yield and quality of tomato. 5-Aminolevulinic acid (ALA) is widely used in agriculture as an efficient and harmless growth regulator. It is currently unclear whether exogenous ALA can cope with low-temperature stress by regulating tomato starch content and phenylalanine metabolism. In this study, exogenous ALA remarkably improved the low-temperature tolerance of tomato seedlings. RNA-sequencing results showed that exogenous ALA affected starch metabolism and phenylalanine metabolism in tomato seedling leaves under low-temperature stress. Subsequently, we used histochemical staining, observation of chloroplast microstructure, substance content determination, and qRT-PCR analysis to demonstrate that exogenous ALA could improve the low-temperature tolerance of tomato seedlings by regulating starch content and phenylalanine metabolism (SlPAL , SlPOD1 , and SlPOD2). Simultaneously, we found that exogenous ALA induced the expression of SlMYBs and SlWRKYs under low-temperature stress. In addition, dual luciferase, yeast one hybrid, and electrophoretic mobility shift assays indicate that SlMYB4 and SlMYB88 could regulate the expression of SlPOD2 in phenylalanine metabolism. We demonstrated that exogenous ALA could improve the low-temperature tolerance of tomato seedlings by regulating starch content and phenylalanine metabolism. • Exogenous 5-aminolevulinic acid (ALA) significantly improved low temperature tolerance in tomatoes. • ALA could regulating starch content and phenylalanine metabolism of tomato seedlings under low-temperature stress. • SlMYB4 and SlMYB88 could regulate the expression of SlPOD2 in phenylalanine metabolism. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy (DR) is one of the most severe complications of diabetes mellitus, diabetes belongs to the category of "emaciation-thirst disease" in traditional Chinese medicine (TCM). Bushen Huoxue Prescription (BHP) is composed of traditional Chinese materia medica, which has therapeutic effects on DR and early diabetic retinal edema (EDRE). However, the therapeutic mechanism is unclear. Exploring the mechanism of BHP against EDRE. Feeding Sprague Dawley (SD) rats a high-fat, high-sugar diet as well as providing intraperitoneal injections of streptozotocin (STZ) to promote inner blood-retinal barrier (iBRB) damage that can trigger EDRE, evaluating the therapeutic effect of BHP by the level of expressiveness of TJ proteins (ZO-1,Occludin) of the iBRB and the leakage of rhodamine B isothiocyanate (RITC) in the retina. The combination of network pharmacology and metabolomics was employed to study the mechanism of BHP in preventing of EDRE, then four proteins which were closely to the damage of iBRB were chosen for the validation by employing Western Blot (WB). Research of network pharmacology had shown that BHP had efficacy against EDRE by regulating targets such as AKT1, ALB, TNF, PPARG, etc, its potential pathways mainly involving signaling pathways such as HIF-1. In untargeted metabolomics analysis of serum, 15 differential metabolites were identified, with the metabolic pathways focusing on ketone body metabolism and synthesis, sphingolipid metabolism and phenylalanine metabolism. The conclusions of metabolomics and network pharmacology revealed that BHP can treat EDRE by alleviating hypoxia and oxidative stress and exerting protection of the iBRB. Finally, BHP's protection behavior of the iBRB was validated by WB experiments. Through integrating pharmacodynamics, network pharmacology and metabolomics, BHP was discovered to have a crucial function in EDRE therapy by preserving the integrity of iBRB. This comprehensive strategy also provided a reasonable way to reveal the multi-components, multi-targets, multi-pathways mechanism of TCM. [Display omitted] • Presentation of "early diabetic retinal edema" as a crucial therapeutic point. • Dyslipidemia is believed to play a significant influence in the progression of EDRE. • The mechanism of BHP anti-EDRE was studied. [ABSTRACT FROM AUTHOR]

The spontaneous plant landscape is a key focus in the development of urban environments. While many spontaneous plants can coexist with bryophytes to create appealing wilderness landscapes, the potential allelopathic effects of bryophytes on the growth of neighboring spontaneous plants remain uncertain. This study evaluated the allelopathic impact of Thuidium kanedae aqueous extracts on the germination and seedling growth of prevalent urban spontaneous plants by analyzing seed germination, seedling growth morphology, and associated indices. We also investigated the allelopathic potential of the predominant compounds in the extract on seed germination. Our findings reveal that the aqueous extract significantly impeded the seed germination of Ophiopogon japonicus, Taraxacum mongolicum, and Viola philippica, with the level of inhibition correlating positively with concentration. In contrast, Senecio scandens seed germination showed a concentration-dependent reaction, with low concentrations promoting and high concentrations hindering germination. The extract consistently reduced root length in all four species, yet it appeared to increase root vigor. The chlorophyll content in O. japonicus and V. philippica seedlings reached a maximum at a concentration of 5 g/L and decreased with higher extract concentrations. The treatment resulted in elevated catalase and soluble protein levels in the seedlings, indicating that the extract induced stress and enhanced the stress resistance index. L-phenylalanine and 2-phenylethanol, substances present in the extract, were notably inhibitory to seed germination across all species, except for O. japonicus. Notably, 2-phenylethanol exhibited a stronger allelopathic effect than L-phenylalanine. Allelopathy synthetical effect evaluation showed that high concentration of aqueous extract allelopathic inhibition effect on seed germination of four plant species, but allelopathic promotion effect on physiological and biochemical growth of Taraxacum mongolicum, Senecio scandens and Viola philippica. In summary, the study demonstrates that bryophytes exert allelopathic effects on neighboring spontaneous plants, with the degree of influence varying among species. This suggests that the germination and growth of spontaneous plant seeds may be selective in bryophyte-dominated habitats and that the density of bryophytes could shape the evolution of these landscapes., (© 2024. The Author(s).)

Background: Foliage color is considered an important ornamental character of Cymbidium tortisepalum (C. tortisepalum), which significantly improves its horticultural and economic value. However, little is understood on the formation mechanism underlying foliage-color variations., Methods: In this study, we applied a multi-omics approach based on transcriptomics and metabolomics, to investigate the biomolecule mechanisms of metabolites changes in C. tortisepalum colour mutation cultivars., Results: A total of 508 genes were identified as differentially expressed genes (DEGs) between wild and foliage colour mutation C. tortisepalum cultivars based on transcriptomic data. KEGG enrichment of DEGs showed that genes involved in phenylalanine metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis and brassinosteroid biosynthesis were most significantly enriched. A total of 420 metabolites were identified in C. tortisepalum using UPLC-MS/MS-based approach and 115 metabolites differentially produced by the mutation cultivars were identified. KEGG enrichment indicated that the most metabolites differentially produced by the mutation cultivars were involved in glycerophospholipid metabolism, tryptophan metabolism, isoflavonoid biosynthesis, flavone and flavonol biosynthesis. Integrated analysis of the metabolomic and transcriptomic data showed that there were four significant enrichment pathways between the two cultivars, including phenylalanine metabolism, phenylpropanoid biosynthesis, flavone and flavonol biosynthesis and flavonoid biosynthesis., Conclusion: The results of this study revealed the mechanism of metabolites changes in C. tortisepalum foliage colour mutation cultivars, which provides a new reference for breeders to improve the foliage color of C. tortisepalum., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jiang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Volatile organic compounds (VOCs) are metabolites pivotal in determining the aroma of various products. A well-known VOC producer of industrial importance is Saccharomyces cerevisiae, partially responsible for flavor of beers and wines. We identified VOCs in beers produced by yeast strains characterized by improved aroma obtained in UV-induced mutagenesis. We observed significant increase in concentration of compounds in strains: 1214uv16 (2-phenylethyl acetate, 2- phenylethanol), 1214uv31 (2-ethyl henxan-1-ol), 1214uv33 (ethyl decanoate, caryophyllene). We observed decrease in production of 2-phenyethyl acetate in strain 1214uv33. Analysis of intracellular metabolites based on 1 H NMR revealed that intracellular phenylalanine concentration was not changed in strains producing more phenylalanine related VOCs (1214uv16 and 1214uv33), so regulation of this pathway seems to be more sophisticated than is currently assumed. Metabolome analysis surprisingly showed the presence of 3-hydroxyisobutyrate, a product of valine degradation, which is considered to be absent in S. cerevisiae. Our results show that our knowledge of yeast metabolism including VOC production has gaps regarding synthesis pathways for individual metabolites and regulation mechanisms. Detailed analysis of 1214uv16 and 1214uv33 may enhance our knowledge of the regulatory mechanisms of VOC synthesis in yeast, and analysis of strain 1214uv31 may reveal the pathway of 2-ethyl henxan-1-ol biosynthesis., (© 2024. The Author(s).)

Stable isotope methods have been used to study protein metabolism in humans; however, there application in dogs has not been frequently explored. The present study compared the methods of precursor (13C-Leucine), end-products (15N-Glycine), and amino acid oxidation (13C-Phenylalanine) to determine the whole-body protein turnover rate in senior dogs. Six dogs (12.7 ± 2.6 years age, 13.6 ± 0.6 kg bodyweight) received a dry food diet for maintenance and were subjected to all the above-mentioned methods in succession. To establish 13C and 15N kinetics, according to different methodologies blood plasma, urine, and expired air were collected using a specifically designed mask. The volume of CO2 was determined using respirometry. The study included four methods viz. 13C-Leucine, 13C-Phenylalanine evaluated with expired air, 13C-Phenylalanine evaluated with urine, and 15N-Glycine, with six dogs (repetitions) per method. Data was subjected to variance analysis and means were compared using the Tukey test (P<0.05). In addition, the agreement between the methods was evaluated using Pearson correlation and Bland-Altman statistics. Protein synthesis (3.39 ± 0.33 g.kg-0,75. d-1), breakdown (3.26 ± 0.18 g.kg-0.75.d-1), and flux estimations were similar among the four methods of study (P>0.05). However, only 13C-Leucine and 13C-Phenylalanine (expired air) presented an elevated Pearson correlation and concordance. This suggested that caution should be applied while comparing the results with the other methodologies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pacheco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The pigmentation of the skin, modulated by different actors in melanogenesis, is mainly due to the melanins (protective pigments). In humans, these pigments' precursors are synthetized by an enzyme known as tyrosinase (TyH). The regulation of the enzyme activity by specific modulators (inhibitors or activators) can offer a means to fight hypo- and hyper-pigmentations responsible for medical, psychological and societal handicaps. Herein, we report the investigation of phenylalanine derivatives as TyH modulators. Interacting with the binuclear copper active site of the enzyme, phenylalanine derivatives combine effects induced by combination with known resorcinol inhibitors and natural substrate/intermediate (amino acid part). Computational studies including docking, molecular dynamics and free energy calculations combined with biological activity assays on isolated TyH and in human melanoma MNT-1 cells, and X-ray crystallography analyses with the TyH analogue Tyrp1, provide conclusive evidence of the interactions of phenylalanine derivatives with human tyrosinase. In particular, our findings indicate that an analogue of L-DOPA, namely (S)-3-amino-tyrosine, stands out as an amino phenol derivative with inhibitory properties against TyH., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

2-Phenylethanol (2-PE) is an aromatic compound with a rose-like fragrance that is widely used in food and other industries. Yeasts have been implicated in the biosynthesis of 2-PE; however, few studies have reported the involvement of filamentous fungi. In this study, 2-PE was detected in Annulohypoxylon stygium mycelia grown in both potato dextrose broth (PDB) and sawdust medium. Among the 27 A. stygium strains investigated in this study, the strain "Jinjiling" (strain S20) showed the highest production of 2-PE. Under optimal culture conditions, the concentration of 2-PE was 2.33 g/L. Each of the key genes in Saccharomyces cerevisiae shikimate and Ehrlich pathways was found to have homologous genes in A. stygium. Upon the addition of L-phenylalanine to the medium, there was an upregulation of all key genes in the Ehrlich pathway of A. stygium, which was consistent with that of S. cerevisiae. A. stygium as an associated fungus provides nutrition for the growth of Tremella fuciformis and most spent composts of T. fuciformis contain pure A. stygium mycelium. Our study on the high-efficiency biosynthesis of 2-PE in A. stygium offers a sustainable solution by utilizing the spent compost of T. fuciformis and provides an alternative option for the production of natural 2-PE. KEY POINTS: • Annulohypoxylon stygium can produce high concentration of 2-phenylethanol. • The pathways of 2-PE biosynthesis in Annulohypoxylon stygium were analyzed. • Spent compost of Tremella fuciformis is a potential source for 2-phenylethanol., (© 2024. The Author(s).)

Feeding and resistance exercise stimulate myofibrillar protein synthesis (MPS) rates in healthy adults. This anabolic characterization of "healthy adults" has been namely focused on males. Therefore, the purpose of this study was to examine the temporal responses of MPS and anabolic signaling to resistance exercise alone or combined with the ingestion of protein in postmenopausal females and compare postabsorptive rates with young females. Sixteen females [60 ± 7 yr; body mass index (BMI) = 26 ± 12 kg·m -2 ] completed an acute bout of unilateral resistance exercise before consuming either: a fortified whey protein supplement (WHEY) or water. Participants received primed continuous infusions of L-[ ring - 13 C 6 ]phenylalanine with bilateral muscle biopsies before and after treatment ingestion at 2 h and 4 h in nonexercised and exercised legs. Resistance exercise transiently increased MPS above baseline at 0-2 h in the water condition ( P = 0.007). Feeding after resistance exercise resulted in a late phase (2-4 h) increase in MPS in the WHEY condition ( P = 0.005). In both conditions, resistance exercise did not enhance the cumulative (0-4 h) MPS response. In the nonexercised leg, MPS did not differ at 0-2 h, 2-4 h, or 0-4 h of the measurement periods (all, P > 0.05). Likewise, there were no changes in the phosphorylation of p70S6K, AMPKα, or total and phosphorylated yes-associated protein on Ser127. Finally, postabsorptive MPS was lower in premenopausal versus postmenopausal females ( P = 0.023). Our results demonstrate that resistance exercise-induced changes in MPS are temporally regulated, but do not result in greater cumulative (0-4 h) MPS in postmenopausal women. NEW & NOTEWORTHY An adequate quality and quantity of skeletal muscle is relevant to support physical performance and metabolic health. Muscle protein synthesis (MPS) is an established remodeling marker, which can be hypertrophic or nonhypertrophic. Importantly, protein ingestion and resistance exercise are two strategies that support healthy muscle by stimulating MPS. Our study shows postmenopause modulates baseline MPS that may diminish the MPS response to the fundamental anabolic stimuli of protein ingestion and resistance exercise in older females.

The interaction of L-Phe with the membrane components, i.e., lipids and proteins, has been discussed in the current literature due to the interest to understand the effect of single amino acids in relation to the formation of amyloid aggregates. In the present work, it is shown that L-Phe interacts with 9:1 DMPC (1,2-dimyristoyl-sn-glycero-3 phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3 phosphocholine) mixtures but not in the 1:9 one. An important observation is that the interaction disappears when DPPC is replaced by diether PC (2-di-O-hexadecyl-sn-glycero-3-phosphocholine) a lipid lacking carbonyl groups (CO). This denotes that CO groups may interact specifically with L-Phe in accordance with the appearance of a new peak observed by Infrared spectroscopy (FTIR-ATR). The interaction of L-Phe affects the compressibility pattern of the 9:1 DMPC/DPPC mixture which is congruent with the changes observed by Raman spectra. The specific interaction of L-Phe with CO, propagates to phosphate and choline groups in this particular mixture as analyzed by FTIR-ATR spectroscopy and is absent when DMPC is dopped with diether PC., Competing Interests: Declaration of competing interest Authors declare no conflicts of interest, (Copyright © 2024 Elsevier B.V. All rights reserved.)