4-hydroxyphenylpyruvate Research Articles

Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme

Anisomycin (compound 1), a multifunctional pyrrolidine antibiotic, primarily inhibits protein biosynthesis by binding to the ribosome. Upon binding to the ribosome, the para-phenol moiety of anisomycin inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incoming aminoacyl-tRNAs, disrupting peptide bond formation. Hence, the para-methoxyphenyl group serves as a starting point for developing novel anisomycin analogs with potent antifungal and insecticidal properties. However, the activation and condensation mechanism of phenylpyruvic acid has not yet been elucidated. In this study, genetic manipulations of aniP and its homologue siAniP confirmed their indispensable role in 1 biosynthesis. Bioinformatics analysis suggested that AniP and siAniP function as transketolase. siAniP was found to catalyzed condensation between 4-hydroxyphenylpyruvic acid (3) and glyceraldehyde (GA), initiating pyrrolidine synthesis. siAniP was specific for aromatic keto acids and tolerant of aliphatic and aromatic aldehydes, and was able to catalyze the asymmetric intermolecular condensation of two keto acids, leading to the formation of 24 α-hydroxy ketone. To the best of our knowledge, siAniP is the first TK that catalyzes the transfer of a C2 ketol and symmetrical intermolecular coupling using aromatic keto acids as donor substrates. Structural analysis, docking model construction, and site-directed mutagenesis identified that I220, H275, R322 and W391 were crucial for substrate binding. Moreover, sequence similarity network (SSN)-based genome neighborhood network (GNN) analyses of AniP suggested the widespread occurrence of the AniP-like-mediated reaction in the biosynthesis of 1 and its analogs, particularly in the assembly of benzylpyrrolidine. These findings not only expand the repertoire of TKs but also provide a potent biocatalyst that could be used for the structural innovation of 1 and its derivatives.

Chlorophyll deficiency in Agave angustifolia Haw.: unveiling the impact on secondary metabolite production.

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.

From tyrosine to hydroxytyrosol: a pathway involving biologically active compounds and their determination in wines by ultra performance liquid chromatography with mass spectrometry.

Hydroxytyrosol (HT) is a bioactive compound present in a limited number of foods such as wines, olives, and olive oils. During alcoholic fermentation, yeast converts aromatic amino acids into higher alcohols such as tyrosol, which can undergo hydroxylation into HT. The aim of this study was to validate an analytical method using ultra performance liquid chromatography coupled with mass spectrometry (UPLC/MS-MS) to quantify HT and its precursors (tyrosine, hydroxyphenylpyruvic acid, hydroxyphenylacetaldehyde, 4-hydroxyphenylacetic acid, and tyrosol) in wines. Their occurrence was evaluated in a total of 108 commercial Spanish wine samples. The validated method simultaneously determined both HT and its precursors, with adequatelimits of detection between 0.065 and 21.86 ng mL-1 and quantification limits between 0.199 and 66.27 ng mL-1 in a 5 min run. The concentration of HT in red wines was significantly higher (0.12-2.24 mg L-1) than in white wines (0.01-1.27 mg L-1). The higher the alcoholic degree, the higher was the content of HT. The bioactive 4-hydroxyphenylacetic acid was identified in Spanish wines for the first time at 3.90-127.47 mg L-1, being present in all the samples. The highest HT concentrations were found in red wines and in wines with higher ethanol content. These data are useful for a further estimation of the intake of these bioactive compounds and to enlarge knowledge on chemical composition of wines. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Cytochrome P450 CYP81A104 in Eleusine indica confers resistance to multiherbicide with different modes of action.

Developing herbicide-resistant (HR) crop cultivars is an efficient way to control weeds and minimize crop yield losses. However, widespread and long-term herbicide application has led to the evolution of resistant weeds. Here, we established a resistant (R) E. indica population, collected from imidazolinone-resistant rice cultivar fields. The R population evolved 4.5-fold resistance to imazamox. Acetolactate synthase (ALS) gene sequencing and ALS activity assays excluded the effect of target-site resistance in this population. P450 inhibitor malathion pretreatment significantly reversed resistance to imazamox. RNA sequencing showed that a P450 gene CYP81A104 was expressed higher in R versus susceptible (S) plants. Arabidopsis overexpressing CYP81A104 showed resistance to ALS inhibitors (imazamox, tribenuron-methyl, penoxsulam and flucarbazone-sodium), PSII inhibitor (bentazone), hydroxyphenyl pyruvate dioxygenase inhibitor (mesotrione) and auxin mimics (MCPA), which was generally consistent with the results presented in the R population. This study confirmed that the CYP81A104 gene endowed resistance to multiherbicides with different modes-of-action. Our findings provide an insight into the molecular characteristics of resistance and contribute to formulating an appropriate strategy for weed management in HR crops. © 2024 Society of Chemical Industry.

Recent Advances and Outlook of Benzopyran Derivatives in the Discovery of Agricultural Chemicals.

Scaffold structures, new mechanisms of action, and targets present enormous challenges in the discovery of novel pesticides. The discovery of new scaffolds is the basis for the continuous development of modern agrochemicals. Identification of a good scaffold such as triazole, carbamate, methoxy acrylate, pyrazolamide, pyrido-pyrimidinone mesoionic, and bisamide often leads to the development of a new series of pesticides. In addition, pesticides with the same target, including the inhibitors of succinate dehydrogenase (SDH), oxysterol-binding-protein, and p-hydroxyphenyl pyruvate dioxygenase (HPPD), may have the same or similar scaffold structure. Recent years have witnessed significant progress in the discovery of new pesticides using natural products as scaffolds or bridges. In recent years, there have been increasing reports on the application of natural benzopyran compounds in the discovery of new pesticides, especially osthole and coumarin. A systematic and comprehensive review of benzopyran active compounds in the discovery of new agricultural chemicals is helpful to promote the discussion and development of benzopyran active compounds. Therefore, this work systematically reviewed the research and application of benzopyran derivatives in the discovery of agricultural chemicals, summarized the antiviral, herbicidal, antibacterial, fungicidal, insecticidal, nematicidal and acaricidal activities of benzopyran active compounds, and discussed the structural-activity relationship and mechanism of action. In addition, some active fragments were recommended to further optimize the chemical structure of benzopyran active compounds based on reference information.

Hydroxy(phenyl)pyruvic acid reductase in Actaea racemosa L.: a putative enzyme in cimicifugic and fukinolic acid biosynthesis

Main conclusionHydroxy(phenyl)pyruvic acid reductase from Actaea racemosa catalyzes dual reactions in reducing 4-hydroxyphenylpyruvic acid as well as β-hydroxypyruvic acid. It thus qualifies to be part of fukinolic and cimicifugic acid biosynthesis and also photorespiration.The accumulation of fukinolic acid and cimicifugic acids is mainly restricted to Actaea racemosa (Ranunculaceae) and other species of the genus Actaea/Cimicifuga. Cimicifugic and fukinolic acids are composed of a hydroxycinnamic acid part esterified with a benzyltartaric acid moiety. The biosynthesis of the latter is unclear. We isolated cDNA encoding a hydroxy(phenyl)pyruvic acid reductase (GenBank OR393286) from suspension-cultured material of A. racemosa (ArH(P)PR) and expressed it in E. coli for protein production. The heterologously synthesized enzyme had a mass of 36.51 kDa and catalyzed the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid or β-hydroxypyruvic acid to glyceric acid, respectively. The optimal temperature was at 38 °C and the pH optimum at pH 7.5. NADPH is the preferred cosubstrate (Km 23 ± 4 µM). Several substrates are accepted by ArH(P)PR with β-hydroxypyruvic acid (Km 0.26 ± 0.12 mM) followed by 4-hydroxyphenylpyruvic acid (Km 1.13 ± 0.12 mM) as the best ones. Thus, ArH(P)PR has properties of β-hydroxypyruvic acid reductase (involved in photorespiration) as well as hydroxyphenylpyruvic acid reductase (possibly involved in benzyltartaric acid formation).

Hepatobiliary circulation and dominant urinary excretion of homogentisic acid in a mouse model of alkaptonuria.

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.

Investigation of Urine Organic Acid Profile in Coronavirus Disease (COVID-19) Patients.

Coronavirus disease 2019 (COVID-19) is a viral infection caused by severe acute respiratory syn-drome coronavirus 2 (SARS-CoV-2). The high mortality and morbidity rate and its course with a wide variety of clinical symptoms indicate that the disease affects many metabolic pathways. The aim of our study was to investi-gate the metabolic effects of SARS-CoV-2 by evaluating the urinary organic acid profile in patients with SARS-CoV-2 infection. Can metabolites in urine guide the diagnosis, follow-up, treatment, and prognosis of COVID-19? Forty-two patients, including 30 SARS-CoV-2 RT-PCR positive patients and 12 SARS-CoV-2 RT-PCR negative controls, were studied. SARS-COV-2 RT-PCR of nasopharyngeal swab samples was studied. Urines were evaluated in the GCMS-QP2010 SE Gas Chromatograph Mass Spectrometer (SHIMADZU) device for organic acid profile in the metabolism laboratory. Urine organic acid profile was evaluated by studying 117 organic acids in each patient. Tiglylglycine, 2-hydroxybutyric acid, 4-hydroxyphenylpyruvic acid, 3-hydroxypropionic acid, erythro-4,5-dihydroxyhexanoic acid lactone, 2-hydroxyphenylacetic acid, N-acetyltyrosine, 3-phenyllactic acid, 5-hydroxyindolacetic acid, 3-hydroxysebacic acid, palmitic acid, 3-methylglutaconic acid, 3-methylglutaric acid, lactic acid, pyruvic acid-oxime, 3-hydroxysobutyric acid, and organic acids were found to be increased, compared to the control group. Tiglylglycine has been specifically identified as a potential biomarker of respiratory chain disorders. The deterioration in lipid metabolism and pyruvate pathway in COVID-19 patients was evaluated as remarkable.

Radical-relay C(sp3)-H azidation catalyzed by an engineered nonheme iron enzyme.

Nonheme iron enzymes are versatile biocatalysts for a broad range of unique and powerful transformations, such as hydroxylation, chlorination, and epimerization as well as cyclization/ring-opening of organic molecules. Beyond their native biological functions, these enzymes are robust for engineering due to their structural diversity and high evolvability. Based on enzyme promiscuity and directed evolution as well as inspired by synthetic organic chemistry, nonheme iron enzymes can be repurposed to catalyze reactions previously only accessible with synthetic catalysts. To this end, our group has engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for new-to-nature radical transformations. In particular, we have demonstrated that a nonheme iron enzyme, (4-hydroxyphenyl)pyruvate dioxygenase from streptomyces avermitilis (SavHppD), can be repurposed to enable abiological radical-relay process to access C(sp3)-H azidation products. This represents the first known instance of enzymatic radical relay azidation reactions. In this chapter, we describe the detailed experimental protocol to convert promiscuous nonheme iron enzymes into efficient and selective biocatalyst for radical relay azidation reactions. One round of directed evolution is described in detail, which includes the generation and handling of site-saturation mutagenesis, protein expression and whole-cell reactions screening in a 96-well plate. These protocol details might be useful to engineer various nonheme iron enzymes for other applications.

New α-keto acid-derived hydrazone ligands and their reaction with the {Re(CO)3}+

Two hydrazone ligands derived of phenylglyoxylic acid (HL1) and 4-hydroxyphenylpyruvic acid (HL2) were synthesized and their zwitterionic structures were determined by X-ray diffraction. The rhenium(I) complexes of formula [ReCl(HLn)(CO)3] were obtained by reaction of the ligands with [ReCl(CH3CN)2(CO)3]. The formation of the hydazonate complexes were also achieved. In the case of HL1 the trimetic complex [Re3(L1)3(CO)9] was isolated by using a basic medium. On the other hand, from storage of a solution of the [ReCl(HL2)(CO)3] complex, crystals of the [Re2(L2)2(CO)6] dimer could be obtained for their structural analysis by X-ray diffraction. The study of the crystalline structures is included. The coordination geometry around rhenium(I) can be described as octahedral with three carbon atoms in fac configuration. In all complexes a five-membered chelate ring is formed including two nitrogen atoms from the hydrazone chain and the pyridine group. In the mononuclear complexes the carboxylic group does not participates in the coordination but acts as a bridge to form the polynuclear compounds. The coordination mode of the ligands in all the complexes could be corroborated by comparative studies of the experimental and calculated (DFT) IR spectra.

Molecular Targets of Herbicides and Fungicides─Are There Useful Overlaps for Fungicide Discovery?

New fungicide modes of action are needed for fungicide resistance management strategies. Several commercial herbicide targets found in fungi that are not utilized by commercial fungicides are discussed as possible fungicide molecular targets. These are acetyl CoA carboxylase, acetolactate synthase, 5-enolpyruvylshikimate-3-phosphate synthase, glutamine synthase, phytoene desaturase, protoporphyrinogen oxidase, long-chain fatty acid synthase, dihydropteroate synthase, hydroxyphenyl pyruvate dioxygenase, and Ser/Thr protein phosphatase. Some of the inhibitors of these herbicide targets appear to be either good fungicides or good leads for new fungicides. For example, some acetolactate synthase and dihydropteroate inhibitors are excellent fungicides. There is evidence that some herbicides have indirect benefits to certain crops due to their effects on fungal crop pathogens. Using a pesticide with both herbicide and fungicide activities based on the same molecular target could reduce the total amount of pesticide used. The limitations of such a product are discussed.

Dysbiosis of gut microbiota and metabolites during AIDS: implications for CD4 + T cell reduction and immune activation.

Identifying the gut microbiota associated with host immunity in the AIDS stage. We performed a cross-sectional study. We recruited people with HIV (PWH) in the AIDS or non-AIDS stage and evaluated their gut microbiota and metabolites by using 16S ribosomal RNA (rRNA) sequencing and liquid chromatography-mass spectrometry (LC-MS). Machine learning models were used to analyze the correlations between key bacteria and CD4 + T cell count, CD4 + T cell activation, bacterial translocation, gut metabolites, and KEGG functional pathways. We recruited 114 PWH in the AIDS stage and 203 PWH in the non-AIDS stage. The α-diversity of gut microbiota was downregulated in the AIDS stage ( P < 0.05). Several machine learning models could be used to identify key gut microbiota associated with AIDS, including the logistic regression model with area under the curve (AUC), sensitivity, specificity, and Brier scores of 0.854, 0.813, 0.813, and 0.160, respectively. The decreased key bacteria ASV1 ( Bacteroides sp.), ASV8 ( Fusobacterium sp.), ASV30 ( Roseburia sp.), ASV37 ( Bacteroides sp.), and ASV41 ( Lactobacillus sp.) in the AIDS stage were positively correlated with the CD4 + T cell count, the EndoCAb IgM level, 4-hydroxyphenylpyruvic acid abundance, and the predicted cell growth pathway, and negatively correlated with the CD3 + CD4 + CD38 + HLA-DR + T cell count and the sCD14 level. Machine learning has the potential to recognize key gut microbiota related to AIDS. The key five bacteria in the AIDS stage and their metabolites might be related to CD4 + T cell reduction and immune activation.

Identification of Serum Biomarkers of Ischemic Stroke in a Hypertensive Population Based on Metabolomics and Lipidomics.

Hypertensive individuals are at a high risk of stroke, and thus, prevention of stroke in hypertensive patients is essential. Metabolomics and lipidomics can be used to identify diagnostic biomarkers and conduct early assessments of stroke risk in hypertensive populations. In this study, serum samples were collected from 30 hypertensive ischemic stroke (IS), 30 matched hypertensive and 30 matched healthy participants. Metabolomics and lipidomics analyses were conducted via liquid chromatography-tandem mass spectrometry, and the data were analyzed using multivariate and univariate statistical methods. A random forest algorithm and binary logistic regression were used to screen the biomarkers and establish diagnostic model. We detected 21 differential metabolites and 38 differential lipids between the hypertensive IS and healthy group. Moreover, we found 18 differential metabolites and 31 differential lipids between the hypertensive IS and hypertension group. In particular, the following seven metabolites or lipids distinguished the hypertensive IS from the healthy group: 4-hydroxyphenylpyruvic acid, cafestol, phosphatidylethanolamine (PE) (18:0p/18:2), PE (16:0e/20:4), (O-acyI)-1-hydroxy fatty acid (36:3), PE (16:0p/20:3) and PE (18:1p/18:2) (rep). The following seven biomarkers distinguished the hypertensive IS from the hypertension group: diglyceride (DG) (20:1/18:2), PE (18:0p/18:2), PE (16:0e/22:5), phosphatidylcholine (40:7), dimethylphosphatidylethanolamine (50:3), DG (18:1/18:2), and 4-hydroxyphenylpyruvic acid. The aforementioned panels had good diagnostic and predictive ability for hypertensive IS. Our study determines the metabolomic and lipidomic profiles of hypertensive IS patients and thereby identifies potential biomarkers of the presence of IS in hypertensive populations.

Expression Patterns and Functional Analysis of Three SmTAT Genes Encoding Tyrosine Aminotransferases in Salvia miltiorrhiza.

Tyrosine aminotransferase (TAT, E.C. 2.6.1.5) is a pyridoxal phosphate-dependent aminotransferase that is widely found in living organisms. It catalyzes the transfer of the amino group on tyrosine to α-ketoglutarate to produce 4-hydroxyphenylpyruvic acid (4-HPP) and is the first enzyme for tyrosine degradation. Three SmTATs have been identified in the genome of Salvia miltiorrhiza (a model medicinal plant), but their information is very limited. Here, the expression profiles of the three SmTAT genes (SmTAT1, SmTAT2, and SmTAT3) were studied. All three genes expressed in different tissues and responded to methyl jasmonate stimuli. SmTAT proteins are localized in the cytoplasm. The recombinant SmTATs were subjected to in vitro biochemical properties. All three recombinant enzymes had TAT activities and SmTAT1 had the highest catalytic activity for tyrosine, followed by SmTAT3. Also, SmTAT1 preferred the direction of tyrosine deamination to 4-HPP, while SmTAT2 preferred transamination of 4-HPP to tyrosine. In parallel, transient overexpression of SmTATs in tobacco leaves revealed that all three SmTAT proteins catalyzed tyrosine to 4-HPP in vivo, with SmTAT1 exhibiting the highest enzymatic activity. Overall, our results lay a foundation for the production of tyrosine-derived secondary metabolites via metabolic engineering or synthetic biology in the future.

Comparative analysis of three SmHPPR genes encoding hydroxyphenylpyruvate reductases in Salvia miltiorrhiza

Hydroxyphenylpyruvate reductase (HPPR) is an enzyme that is involved in the biosynthesis of hydrophilic phenolic acids in Salvia miltiorrhiza, which is a model medicinal plant. Three SmHPPR genes have been identified in the S. miltiorrhiza genome; however, only one has been functionally analyzed. Here, we cloned three SmHPPR genes (SmHPPR1, SmHPPR2, and SmHPPR3) from the cDNA of S. miltiorrhiza, and their expression profiles were studied. The expression levels of SmHPPR1 were significantly higher than those of SmHPPR2 and SmHPPR3, where SmHPPR1 revealed the highest level in stems, while SmHPPR2 and SmHPPR3 exhibited the highest level in flowers. SmHPPR1, SmHPPR2, and SmHPPR3 are localized in the cytoplasm. All three recombinant enzymes had HPPR activities and catalyzed the reduction of 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), with SmHPPR1 showing the highest activity. The transient over-expression of SmHPPR1, SmHPPR2, and SmHPPR3 in the leaves of Nicotiana benthamiana promoted the production of pHPL, which indicated that all three SmHPPRs had in vivo activities. Overall, between the three homologs, SmHPPR1 plays a dominant role in catalyzing pHPP to pHPL, which provides new insights into the biosynthesis of phenolic acids in S. miltiorrhiza.

B-201 Development of a Quantitative Gas Chromatography Tandem Mass Spectrometry Method for Metabolic Profiling of Urine Organic Acids

Abstract Background Urine organic acids (UOA) analysis is an essential investigation for inborn errors of metabolism (IEMs). The vast majority of methods use gas chromatography mass spectrometry (GC-MS) and are only qualitative. Gas chromatography tandem mass spectrometry (GC-MS/MS) offers greater sensitivity and selectivity, and enables the combination of qualitative screening with quantitative analysis. We describe the development and preliminary validation results of a quantitative GC-MS/MS method. Methods Urine creatinine concentrations were measured (CobasPro, Roche Diagnostics) and normalized to 1.25 mmol/L. After internal standard addition, each urine specimen, calibration standard and QC were incubated with a hydroxylamine solution followed by a liquid-liquid extraction. Further sample concentration permitted the isolated UOA to be derivatized using N,O-bis(trimethylsilyl)trifluoroacetamine (BSTFA) with 1% chlorotrimethylsilane (TMCS) prior to injection. GC-MS/MS analysis was performed using a GC-MS-TQ8050 triple quadrupole system (Shimadzu) with a 30 m DB-5MS (Agilent J&amp;W) capillary column (0.25 mm, ID of 0.25 µm). Split injection mode was deployed with an initial column temperature of 70°C. The mass spectrometer was operated in electron ionization (EI) and multiple reaction monitoring (MRM) modes. The linear concentration ranges of the targeted metabolites within the devised GC-MS/MS protocol were determined. Results The optimal temperature gradient for high-resolution UOA separations was derived and their respective retention times determined. MS/MS detection parameters were optimized to permit the identification of precursor and production ions for all (N = 38) derivatized UOA. Total analysis time was 33.8 min per injection. The achieved linear concentration ranges for each metabolite was respectively determined. Linear concentration ranges and correlation coefficients of representative UOA: 3-hydroxyisovaleric acid (range = 9 to 708 µmol/L, R2 = 0.9874); lactic acid (range = 15 to 751 µmol/L, R2 = 0.9936); 3-hydroxybutyric acid (range = 15 to 751 µmol/L, R2 = 0.9991); and 2-methyl-3-hydroxybutyric acid (range = 10 to 501 µmol/L, R2 = 0.9996). The correlation coefficients for all remaining UOA ranged from R2 = 0.9458 (4-hydroxyphenylpyruvic acid) to R2 = 0.9998 (3-methylglutanoic acid and ethylmalonic acid). Conclusion The devised GC-MS/MS protocol permits the unbiased identification and quantification of UOA used to investigate IEMs. The linear concentration ranges for all analytes were determined and are appropriate for monitoring these markers of inherited enzyme deficiencies. The analytical performance of this UOA GC-MS/MS method must be further validated and its diagnostic performance for selected IEMs characterized.

Caccinia macranthera Brand var. macranthera: Phytochemical analysis, phytotoxicity and antimicrobial investigations of essential oils with concomitant in silico molecular docking based on OPLS force-field

This study was conducted to extract the essential oils (EOs) of Caccinia macranthera identify their phytochemicals, evaluate their phytotoxicity, antimicrobial activity and enzyme inhibition effects using in silico molecular docking technique.EOs of aerial parts, seeds, and roots of C. macranthera were extracted and analyzed via Gas chromatography-Mass Spectrometry. The antibacterial activity of EOs were determined on nine microorganisms via disk diffusion and microbroth dilution assays. In addition, the allelopathic properties of EOs were investigated by calculating the IC50s for inhibition of germination, seedling length and seedling weight growth of Cuscuta campestris seeds. In order to assess the possible inhibitory effect of major components of C. macranthera EOs on enzymes inhibiting germination and plant growth, molecular docking was employed against the glutamine synthetase (GS), acetohydroxyacid synthetase (AHAS), and 4-hydroxyphenyl pyruvate dioxygenase (HPPD) enzymes.The main compounds of EOs from aerial parts, seeds, and roots EOs were dihydrocarveol (29.5%), Trimethyl-2-Pentadecanone (13.6%), and Palmitic acid (16.8%), respectively. The maximum antibacterial effect was related to the aerial parts EO against Staphylococcus epidermidis. Phytotoxicity analysis exhibited a concentration-dependent increase (p ≤ 0.05) activity. The aerial parts EO demonstrated a substantial allelopathy effect, with IC50 values of 0.22 ± 0.026, 0.39 ± 0.021, and 0.20 ± 0.025 mg/mL, respectively, on inhibitory germination, seedling length and seedling weight growth of Cuscuta campestris seeds. Molecular docking analyzes showed that Oleic acid was suitable for dynamic stabilization of HPPD (−6.552 kJ/mol) and GS (−7.265 kJ/mol) and Eupatoriochromene had the inhibitory potential against AHAS, with docking score of −4.189 kJ/mol.The current research demonstrated that C. macranthera EOs from its aerial parts have an acceptable phytotoxic activity against Cuscuta campestris weed. The major components of EOs, Oleic acid and Eupatoriochromene, presented the strongest binding with HPPD, GS, and AHAS active sites causing disturbance in germination, photosynthesis and weed growth suggesting it as a natural herbicide for controlling the weeds.

Adaptive response of triploid Fujian oyster (Crassostrea angulata) to nanoplastic stress: Insights from physiological, metabolomic, and microbial community analyses

Triploid Fujian oyster (Crassostrea angulata) is crucial to aquaculture and coastal ecosystems because of its accelerated growth and heightened resilience against environmental stressors. In light of the increasing prevalence of nanoplastic pollution in the ocean, understanding its potential impact on this organism, particularly its adaptive responses, is of paramount importance. Despite this, the effects of nanoplastic pollution on the physiology of C. angulata remain largely unexplored. In this study, we explored the responses of triploid Fujian oysters to nanoplastic stress during a 14-day exposure period, employing an integrative methodology that included physiological, metabolomic, and 16S rRNA sequencing analyses. Our results demonstrate that the oysters exhibit a strong adaptive response to nanoplastic exposure, characterized by alterations in enzyme activity, metabolic pathways, and microbial community composition, indicative of an adaptive recovery state as opposed to a disordered state. Oysters subjected to elevated nanoplastic levels exhibited adaptive responses primarily by boosting the activity of the antioxidant enzyme catalase and elevating the levels of antioxidants such as adenosine, 3-(4-hydroxyphenyl)pyruvate, D-sorbitol, d-mannose, and unsaturated fatty acids, as well as the functional amino acids l-proline and l-lysine. Nanoplastic treatment also resulted in increased activity of succinate dehydrogenase, a key component of energy metabolism, and increased contents of intermediate metabolites or products of energy metabolism, such as adenosine monophosphate, adenosine, guanosine, creatine, and thiamine. Nanoplastic treatment led to an increase in the abundance of certain advantageous genera of gut bacteria, specifically Phaeobacter and Nautella. The observed adaptive response of triploid Fujian oysters to nanoplastic stress provides valuable insights into the mechanisms underpinning resilience in marine bivalves.

Defining the toxicological profile of 4-hydroxyphenylpyruvate dioxygenase-directed herbicides to Aedes aegypti and Amblyomma americanum

Inhibitors targeting the 4-hydroxyphenyl pyruvate dioxygenase (HPPD) enzyme are well established herbicides and HPPD is also a primary enzyme within the tyrosine metabolism pathway in hematophagous arthropods, which is an essential metaboilic pathway post-blood feeding to prevent tyrosine-mediated toxicity. The objective of this study was to characterize the toxicity of triketone, pyrazole, pyrazolone, isoxazole, and triazole herbicides that inhibit HPPD to blood-fed mosquitoes and ticks. Topical exposure of nitisinone to blood-fed Aedes aegypti yielded high toxicity with an LD50 of 3.81 ng/insect (95% CI: 3.09 to 4.67 ng; Hillslope: 0.97, r2: 0.99), yet was non-toxic to non-blood fed (NBF) mosquitoes. The rank order of toxicity was nitisinone > tembotrione > pyrazoxyfen > tebuconazole > mesotrione against blood-fed Ae. Aegypti, but nitisinone was approximately 30-fold more toxic than other chemicals tested. We also assessed the toxicity of HPPD-inhibiting herbicides to the lone star tick, Amblyomma americanum and similarly, nitisinone was toxic to Am. americanum with a lethal time to kill 50% of subjects (LT50) of 23 h at 10 μM. Knockdown of the gene encoding the HPPD enzyme was performed through RNA-interference led to significant mortality after blood feeding in both, Ae. aegypti and Am. americanum. Lastly, a fluorescence assay was developed to determine relative quantities of L-tyrosine in Ae. aegypti and Am. americanum treated with HPPD inhibitors. L-tyrosine levels correlated with toxicity with nitisinone exposure leading to increased tyrosine concentrations post-blood feeding. Taken together, these data support previous work suggesting HPPD-inhibitors represent a novel mode of toxicity to mosquitoes and ticks and may represent base scaffolds for development of novel insecticides specific for hematophagous arthropods.

Targeted metabolomics analysis for serum Helicobacter pylori -positive based on LC-MS/MS.

Helicobacter pylori (H. pylori), as a harmful bacteria associated with gastric cancer, can have adverse effects on human normal flora and metabolism. However, the effects of H. pylori on human metabolism have not been fully elucidated. 13 C breathing test was used as the basis for distinguishing negative and positive groups. Serum samples were collected from the two groups for targeted quantitative metabolomics detection, and multidimensional statistics were used, including partial least squares discriminant analysis (PLS-DA), principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and differential metabolites were screened. Unidimensional statistics combined with multidimensional statistics were used to further screen potential biomarkers, and finally pathway analysis was performed. SPSS 21.0 software package was used for statistical analysis of experimental data. Multivariate statistical analysis such as PLS-DA, PCA and OPLS-DA was performed using Simca-P 13.0 to search for differential metabolites. This study confirmed that Helicobacter pylori caused significant changes in human metabolism. In this experiment, 211 metabolites were detected in the serum of the two groups. Multivariate statistical analysis showed that PCA of metabolites was not significantly different between the two groups. PLS-DA indicated that the serum of the two groups were well clustered. There were significant differences in metabolites between OPLS-DA groups. By setting the VIP threshold as 1 and the corresponding P value < 0.05, a total of 40 metabolites were screened in this study. P < 0.05 and| log2FC | > 0 (FC, the Fold Change) together as a unidimensional statistical filter condition. The analysis found that the expression of 15 metabolites such as Propionic acid, Acetic acid, Adipic acid increased, and the metabolism of 6 products such as DCA, 4-Hydroxyphenylpyruvic acid, Pyruvic acid decreased. P < 0.05, FDR < 0.5,|log2FC|> 1 and OPLSDA_VIP > 1 together as a condition of filter screen potential biomarkers. Four potential biomarkers were screened, which were Sebacic acid, Isovaleric acid, (DCA, Indole-3-carboxylic acid). Finally, the different metabolites were brought into the SMPDB library for the corresponding pathway enrichment analysis. The significant abnormal metabolic pathways were taurine and subtaurine metabolism, tyrosine metabolism, glycolysis or gluconeogenesis, pyruvate metabolism, etc. CONCLUSIONS: This study shows that H. pylori has an impact on human metabolism. Not only a variety of metabolites have significant changes, but also metabolic pathways are abnormal, which may be the reason for the high risk of H. pylori causing gastric cancer.