Fabrication of fluorescence probe based on molecularly imprinted polymers on red emissive biomass-derived carbon dots coupled with smartphone readout for tyramine determination in fermented meat products.

A fluorescence probe based on molecularly imprinted polymers on red emissive biomass-derived carbon dots (r-BCDs@MIPs) was developed to detect tyramine in fermented meat products. The red emissive biomass-derived carbon dots (r-BCDs) were synthesized by the one-step solvothermal method using discarded passion fruit shells as raw materials. The fluorescence emission peak of r-BCDs was at 670 nm, and the relative quantum yield (QY) was about 2.44%. Molecularly imprinted sensing materials were prepared with r-BCDs as fluorescent centers for the detection of trace tyramine, which showed a good linear response in the concentration range of tyramine from 1 to 40 µg L-1. The linear correlation coefficient was 0.9837, and the limit of detection was 0.77 µg L-1. The method was successfully applied to the determination of tyramine in fermented meat products, and the recovery was 87.17-106.02%. The reliability of the results was verified through high-performance liquid chromatography (HPLC). Furthermore, we combined the r-BCDs@MIPs with smartphone-assisted signal readout to achieve real-time detection of tyramine in real samples. Considering its simplicity and convenience, the method could be used as a rapid and low-cost promising platform with broad application prospects for on-site detection of trace tyramine with smartphone-assisted signal readout.

Variation of Cyclodextrin (CD) Complexation with Biogenic Amine Tyramine: Pseudopolymorphs of ß-CD Inclusion vs. α-CD Exclusion, Deep Atomistic Insights.

Tyramine (TRM) is a biogenic catecholamine neurotransmitter, which can trigger migraines and hypertension. TRM accumulated in foods is reduced and detected using additive cyclodextrins (CDs) while their association characteristics remain unclear. Here, single-crystal X-ray diffraction and density functional theory (DFT) calculation have been performed, demonstrating the elusive pseudopolymorphs in ß-CD inclusion complexes with TRM base/HCl, ß-CD·0.5TRM·7.6H2O (1) and ß-CD·TRM HCl·4H2O (2) and the rare α-CD·0.5(TRM HCl)·10H2O (3) exclusion complex. Both 1 and 2 share the common inclusion mode with similar TRM structures in the round and elliptical ß-CD cavities, belong to the monoclinic space group P21, and have similar herringbone packing structures. Furthermore, 3 differs from 2, as the smaller twofold symmetry-related, round α-CD prefers an exclusion complex with the twofold disordered TRM-H+ sites. In the orthorhombic P21212 lattice, α-CDs are packed in a channel-type structure, where the column-like cavity is occupied by disordered water sites. DFT results indicate that ß-CD remains elliptical to suitably accommodate TRM, yielding an energetically favorable inclusion complex, which is significantly contributed by the ß-CD deformation, and the inclusion complex of α-CD with the TRM aminoethyl side chain is also energetically favorable compared to the exclusion mode. This study suggests the CD implications for food safety and drug/bioactive formulation and delivery.

Gut microbiota metabolite tyramine ameliorates high-fat diet-induced insulin resistance via increased Ca2+ signaling.

The gut microbiota and their metabolites are closely linked to obesity-related diseases, such as type 2 diabetes, but their causal relationship and underlying mechanisms remain largely elusive. Here, we found that dysbiosis-induced tyramine (TA) suppresses high-fat diet (HFD)-mediated insulin resistance in both Drosophila and mice. In Drosophila, HFD increases cytosolic Ca2+ signaling in enterocytes, which, in turn, suppresses intestinal lipid levels. 16 S rRNA sequencing and metabolomics revealed that HFD leads to increased prevalence of tyrosine decarboxylase (Tdc)-expressing bacteria and resulting tyramine production. Tyramine acts on the tyramine receptor, TyrR1, to promote cytosolic Ca2+ signaling and activation of the CRTC-CREB complex to transcriptionally suppress dietary lipid digestion and lipogenesis in enterocytes, while promoting mitochondrial biogenesis. Furthermore, the tyramine-induced cytosolic Ca2+ signaling is sufficient to suppress HFD-induced obesity and insulin resistance in Drosophila. In mice, tyramine intake also improves glucose tolerance and insulin sensitivity under HFD. These results indicate that dysbiosis-induced tyramine suppresses insulin resistance in both flies and mice under HFD, suggesting a potential therapeutic strategy for related metabolic disorders, such as diabetes.

"One-Pot" Readout Cyano-Programmable SERS-Encoded Platform Enables Ultrasensitive and Interference-Free Detection of Multitarget Bioamines.

Surface-enhanced Raman spectroscopy (SERS) detection platforms with high signal-to-noise ratio in the "biological-silent" region (1800-2800 cm-1) are presently being developed for sensing and imaging applications, overcoming the limitations of traditional SERS studies in the "fingerprint" region. Herein, a series of cyano-programmable Raman reporters (RRs) operating in the "biological-silent" region were designed based on 4-mercaptobenzonitrile derivatives and then embedded in core-shell Au@Ag nanostars using a "bottom-up" strategy to provide SERS enhancement and encapsulation protection. The approach enabled the "one-pot" readout interference-free detection of multiple bioamines (histamine, tyramine, and ß-phenethylamine) based on aptamer-driven magnetic-induced technology. Three cyano-encoded SERS tags resulted in separate SERS signals for histamine, tyramine, and ß-phenethylamine at 2220, 2251, and 2150 cm-1, respectively. A target-specific aptamer-complementary DNA competitive binding strategy allowed the formation of microscale core-satellite assemblies between Fe3O4-based magnetic beads and the SERS tags, enabling multiple SERS signals to be observed simultaneously under a 785 nm laser excitation laser. The LODs for detection of the three bioamines were 0.61 × 10-5, 2.67 × 10-5, and 1.78 × 10-5 mg L-1, respectively. The SERS-encoded platform utilizing programmable reporters provides a fast and sensitive approach for the simultaneous detection of multiple biomarkers, paving the way for routine SERS analyses of multiple analytes in complex matrices.

Multicopper Oxidase from Lactobacillus hilgardii: Mechanism of Degradation of Tyramine and Phenylethylamine in Fermented Food.

Elevated levels of biogenic amines (BAs) in fermented food can have negative effects on both the flavor and health. Mining enzymes that degrade BAs is an effective strategy for controlling their content. The study screened a strain of Lactobacillus hilgardii 1614 from fermented food system that can degrade BAs. The multiple copper oxidase genes LHMCO1614 were successfully mined after the whole genome protein sequences of homologous strains were clustered and followed by homology modeling. The enzyme molecules can interact with BAs to stabilize composite structures for catalytic degradation, as shown by molecular docking results. Ingeniously, the kinetic data showed that purified LHMCO1614 was less sensitive to the substrate inhibition of tyramine and phenylethylamine. The degradation rates of tyramine and phenylethylamine in huangjiu (18% vol) after adding LHMCO1614 were 41.35 and 40.21%, respectively. Furthermore, LHMCO1614 demonstrated universality in degrading tyramine and phenylethylamine present in other fermented foods as well. HS-SPME-GC-MS analysis revealed that, except for aldehydes, the addition of enzyme treatment did not significantly alter the levels of major flavor compounds in enzymatically treated fermented foods (p > 0.05). This study presents an enzymatic approach for regulating tyramine and phenylethylamine levels in fermented foods with potential applications both targeted and universal.

Preparation of Multifunctional Hydrogels with In Situ Dual Network Structure and Promotion of Wound Healing.

As an emerging biomedical material, wound dressings play an important therapeutic function in the process of wound healing. It can provide an ideal healing environment while protecting the wound from a complex external environment. A hydrogel wound dressing composed of tilapia skin gelatin (Tsg) and fucoidan (Fuc) was designed in this article to enhance the microenvironment of wound treatment and stimulate wound healing. By mixing horseradish peroxidase (HRP), hydrogen peroxide (H2O2), tilapia skin gelatin-tyramine (Tsg-Tyr), and carboxylated fucoidan-tyramine in agarose (Aga), using the catalytic cross-linking of HRP/H2O2 and the sol-gel transformation of Aga, a novel gelatin-fucoidan (TF) double network hydrogel wound dressing was constructed. The TF hydrogels have a fast and adjustable gelation time, and the addition of Aga further enhances the stability of the hydrogels. Moreover, Tsg and Fuc are coordinated with each other in terms of biological efficacy, and the TF hydrogel demonstrated excellent antioxidant properties and biocompatibility in vitro. Also, in vivo wound healing experiments showed that the TF hydrogel could effectively accelerate wound healing, reduce wound microbial colonization, alleviate inflammation, and promote collagen deposition and angiogenesis. In conclusion, TF hydrogel wound dressings have the potential to replace traditional dressings in wound healing.

Biodegradable Tyramine Functional Gelatin/6 Arms-PLA Inks Compatible with 3D Two Photon-Polymerization Printing and Meniscus Tissue Regeneration.

The meniscus regeneration can present major challenges such as mimicking tissue microstructuration or triggering cell regeneration. In the case of lesions that require a personalized approach, photoprinting offers the possibility of designing resolutive biomaterial structures. The photo-cross-linkable ink composition determines the process ease and the final network properties. In this study, we designed a range of hybrid inks composed of gelatin(G) and 6-PLA arms(P) that were photo-cross-linked using tyramine groups. The photo-cross-linking efficiency, mechanical properties, degradation, and biological interactions of inks with different G/P mass ratios were studied. The G50P50 network properties were suitable for meniscus regeneration, with Young's modulus of 6.5 MPa, degradation in 2 months, and good cell proliferation. We then confirmed the potential of these inks to produce high-resolution microstructures by printing well-defined microstructures using two-photon polymerization. These hybrid inks offer new perspectives for biocompatible, degradable, and microstructured tissue engineering scaffold creation.

Exploring factors influencing the levels of biogenic amines in wine and microbiological strategies for controlling their occurrence in winemaking.

Fermented beverages, including wine, can accumulate high concentrations of biogenic amines (BAs), which can pose potential health risks. BAs are produced by various yeasts and lactic acid bacteria (LAB) during winemaking. LAB are the main contributors to the formation of histamine and tyramine, the most toxic and food safety relevant biogenic amines. Numerous factors, ranging from agricultural and oenological practices to sanitation conditions, can contribute to the formation of BAs in wines. Moreover, organic and biodynamic wines impose limitations on the use of common food additives employed to control the proliferation of native and spoilage microorganisms during vinification and storage. To mitigate histamine production, commercial starter cultures incapable of synthesising histamine have been effectively utilised to reduce wine histamine content. Alternative fermentative microorganisms are currently under investigation to enhance the safety, quality, and typicity of wines, including indigenous LAB, non-Saccharomyces yeasts, and BAs degrading strains. Furthermore, exploration of extracts from BAs-degrading microorganisms and their purified enzymes has been undertaken to reduce BAs levels in wines. This review highlights microbial contributors to BAs in wines, factors affecting their growth and BA production, and alternative microorganisms that can degrade or avoid BAs. The aim is to lessen reliance on additives, providing consumers with safer wine choices.

Effects of pickle brine and glycine addition on biogenic amine production in pickle fermentation.

This study investigated the effects of different pickle brines and glycine additions on biogenic amine formation in pickle fermentation. The results showed that the brines with higher biogenic amine content led to the production of more biogenic amines in the simulated pickle fermentation system. This was related to the abundance of biogenic amine-producing microorganisms in the microbial communities of the brines. Metagenome analysis of the brines and metatranscriptome analysis of the fermentation systems showed that putrescine was primarily from Lactobacillus, Oenococcus, and Pichia, while histamine and tyramine were primarily from Lactobacillus and Tetragenococcus. Addition of glycine significantly reduced the accumulation of biogenic amines in the simulated pickle fermentation system by as much as 70 %. The addition of glycine had no inhibitory effect on the amine-producing microorganisms, but it down-regulated the transcription levels of the genes for enzymes related to putrescine synthesis in Pichia, Lactobacillus, and Oenococcus, as well as the histidine decarboxylase genes in Lactobacillus and Tetragenococcus. Catalytic reaction assay using crude solutions of amino acid decarboxylase extracted from Lactobacillus brevis showed that the addition of glycine inhibited 45 %-55 % of ornithine decarboxylase and tyrosine decarboxylase activities. This study may provide a reference for the study and control of the mechanism of biogenic amine formation in pickle fermentation.

Metabolomic Profiling Reveals the Anti-Herbivore Mechanisms of Rice (Oryza sativa).

The use of secondary metabolites of rice to control pests has become a research hotspot, but little is known about the mechanism of rice self-resistance. In this study, metabolomics analysis was performed on two groups of rice (T1, with insect pests; T2, without pests), indicating that fatty acids, alkaloids, and phenolic acids were significantly up-regulated in T1. The up-regulated metabolites (p-value < 0.1) were enriched in linoleic acid metabolism, terpene, piperidine, and pyridine alkaloid biosynthesis, α-linolenic acid metabolism, and tryptophan metabolism. Six significantly up-regulated differential metabolites in T1 were screened out: N-trans-feruloyl-3-methoxytyramine (1), N-trans-feruloyltyramine (2), N-trans-p-coumaroyltyramine (3), N-cis-feruloyltyramine (4), N-phenylacetyl-L-glutamine (5), and benzamide (6). The insect growth inhibitory activities of these six different metabolites were determined, and the results show that compound 1 had the highest activity, which significantly inhibited the growth of Chilo suppressalis by 59.63%. Compounds 2-4 also showed a good inhibitory effect on the growth of Chilo suppressalis, while the other compounds had no significant effect. RNA-seq analyses showed that larval exposure to compound 1 up-regulated the genes that were significantly enriched in ribosome biogenesis in eukaryotes, the cell cycle, ribosomes, and other pathways. The down-regulated genes were significantly enriched in metabolic pathways, oxidative phosphorylation, the citrate cycle (TCA cycle), and other pathways. Eighteen up-regulated genes and fifteen down-regulated genes from the above significantly enriched pathways were screened out and verified by real-time quantitative PCR. The activities of detoxification enzymes (glutathione S-transferase (GST); UDP-glucuronosyltransferase (UGT); and carboxylesterase (CarE)) under larval exposure to compound 1 were measured, which indicated that the activity of GST was significantly inhibited by compound 1, while the activities of the UGT and CarE enzymes did not significantly change. As determined by UPLC-MS, the contents of compound 1 in the T1 and T2 groups were 8.55 ng/g and 0.53 ng/g, respectively, which indicated that pest insects significantly induced the synthesis of compound 1. Compound 1 may enhance rice insect resistance by inhibiting the detoxification enzyme activity and metabolism of Chilo suppressalis, as well as promoting cell proliferation to affect its normal growth and development process. The chemical-ecological mechanism of the insect resistance of rice is preliminarily clarified in this paper.

Enterococcus-derived tyramine hijacks α2A-adrenergic receptor in intestinal stem cells to exacerbate colitis.

Inflammatory bowel disease (IBD) is characterized by dysbiosis of the gut microbiota and dysfunction of intestinal stem cells (ISCs). However, the direct interactions between IBD microbial factors and ISCs are undescribed. Here, we identify α2A-adrenergic receptor (ADRA2A) as a highly expressed GPCR in ISCs. Through PRESTO-Tango screening, we demonstrate that tyramine, primarily produced by Enterococcus via tyrosine decarboxylase (tyrDC), serves as a microbial ligand for ADRA2A. Using an engineered tyrDC-deficient Enterococcus faecalis strain and intestinal epithelial cell-specific Adra2a knockout mice, we show that Enterococcus-derived tyramine suppresses ISC proliferation, thereby impairing epithelial regeneration and exacerbating DSS-induced colitis through ADRA2A. Importantly, blocking the axis with an ADRA2A antagonist, yohimbine, disrupts tyramine-mediated suppression on ISCs and alleviates colitis. Our findings highlight a microbial ligand-GPCR pair in ISCs, revealing a causal link between microbial regulation of ISCs and colitis exacerbation and yielding a targeted therapeutic approach to restore ISC function in colitis.

Establishing an Artificial Pathway for the Biosynthesis of Octopamine and Synephrine.

In this study, we designed an artificial pathway composed of tyramine ß-hydroxylase (TBH) and phenylethanolamine N-methyltransferase (PNMT) for the biosynthesis of both octopamine and synephrine. As most TBH and PNMT originate from eukaryotic animals and plants, the heterologous expression and identification of functional TBH and PNMT are critical for establishing the pathway in mode microorganisms like Escherichia coli. Here, three TBHs were evaluated, and only TBH from Drosophila melanogaster was successfully expressed in the soluble form in E. coli. Its expression was promoted by evaluating the effects of different expression strategies. The specific enzyme activity of TBH was optimized up to 229.50 U·g-1, and the first step in the biosynthetic pathway was successfully established and converted tyramine to synthesize 0.10 g/L of octopamine. Furthermore, the second step to produce synephrine from octopamine was developed by screening PNMT, enhancing enzyme activity, and optimizing reaction conditions, with a maximum synephrine production of 2.02 g/L. Finally, based on the optimization of the reaction conditions for each individual reaction, the one-pot cascade reaction for synthesizing synephrine from tyramine was constructed by combining the TBH and PNMT. The synthetic synephrine reached 30.05 mg/L with tyramine as substrate in the two-step enzyme cascade system. With further optimization and amplification, the titers of octopamine and synephrine were increased to 0.45 and 0.20 g/L, respectively, with tyramine as substrate. This work was the first achievement of the biosynthesis of octopamine and synephrine to date.

Puerarin-containing rhein-crosslinked tyramine-modified hyaluronic acid hydrogel for antibacterial and anti-inflammatory wound dressings.

Wound infections, posing a grave risk of severe physical consequences and even mortality, exact a substantial financial toll on society, rendering them among the most formidable challenges confronting our world today. A critical imperative is the development of hydrogel dressings endowed with immune-regulating and antibacterial properties. This study is founded upon the symbiotic physical and efficacious attributes of two small natural molecules. An injectable hydrogel is meticulously crafted by encapsulating puerarin (PUE) into tyramine-modified hyaluronic acid, subsequently introducing rhein (RHE), and catalyzing the formation of inter-phenol crosslinks with H2O2/horseradish peroxidase (HA-Tyr-R@P). Exhibiting a favorable microenvironmental impact the developed hydrogel attains an antibacterial efficacy exceeding 95 %, coupled with a wound closure rate twice that of the control group. HA-Tyr-R@P hydrogels not only inhibit bacterial growth but also mitigate inflammation, fostering wound healing, owing to their harmonized physicochemical characteristics and synergistic therapeutic effects. This work underscores the creation of a singular, versatile hydrogel platform, negating the complexities and side effects associated with pharmaceutical preparations. Furthermore, it offers new ideas for the formulation of RHE-based hydrogels for wound healing, emphasizing the pivotal role of natural small molecules in advancing biological materials.

A smartphone-combined ratiometric fluorescence molecularly imprinted probe based on biomass-derived carbon dots for determination of tyramine in fermented meat products.

A ratiometric fluorescence molecularly imprinted probe employing two distinct emission wavelengths of biomass carbon dots was developed for highly selective and visual quantitative detection of tyramine in fermented meat products. The red emission biomass carbon dots were employed as responsive elements, and the blue ones were utilized as the reference elements. The molecularly imprinted polymers were incorporated in the ratiometric sensing to distinguish and adsorb tyramine. With the linear range of 1-60 µg/L, the ratiometric fluorescence molecularly imprinted probe was successfully applied to detect tyramine in real samples with the satisfactory recoveries of 79.74-112.12% and the detect limitation of 1.3 µg/kg, indicating that this probe has great potential applications for the detection of tyramine in real samples. Moreover, smartphone-based fluorescence signal recognition analysis on hand has been developed for the quantitative analysis of tyramine, providing a portable visual optical analysis terminal for rapid on-site determination of tyramine.

Cultivated Enterococcus faecium B6 from children with obesity promotes nonalcoholic fatty liver disease by the bioactive metabolite tyramine.

Gut microbiota plays an essential role in nonalcoholic fatty liver disease (NAFLD). However, the contribution of individual bacterial strains and their metabolites to childhood NAFLD pathogenesis remains poorly understood. Herein, the critical bacteria in children with obesity accompanied by NAFLD were identified by microbiome analysis. Bacteria abundant in the NAFLD group were systematically assessed for their lipogenic effects. The underlying mechanisms and microbial-derived metabolites in NAFLD pathogenesis were investigated using multi-omics and LC-MS/MS analysis. The roles of the crucial metabolite in NAFLD were validated in vitro and in vivo as well as in an additional cohort. The results showed that Enterococcus spp. was enriched in children with obesity and NAFLD. The patient-derived Enterococcus faecium B6 (E. faecium B6) significantly contributed to NAFLD symptoms in mice. E. faecium B6 produced a crucial bioactive metabolite, tyramine, which probably activated PPAR-γ, leading to lipid accumulation, inflammation, and fibrosis in the liver. Moreover, these findings were successfully validated in an additional cohort. This pioneering study elucidated the important functions of cultivated E. faecium B6 and its bioactive metabolite (tyramine) in exacerbating NAFLD. These findings advance the comprehensive understanding of NAFLD pathogenesis and provide new insights for the development of microbe/metabolite-based therapeutic strategies.

Hepatocellular carcinoma biomarkers screening based on hydrogel photonic barcodes with tyramine deposition amplified ELISA.

Hepatocellular carcinoma (HCC), as one of the most lethal cancers, significantly impacts human health. Attempts in this area tends to develop novel technologies with sensitive and multiplexed detection properties for early diagnosis. Here, we present novel hydrogel photonic crystal (PhC) barcodes with tyramine deposition amplified enzyme-linked immunosorbent assay (ELISA) for highly sensitive and multiplexed HCC biomarker screening. Because of the abundant amino groups of acrylic acid (AA) component, the constructed hydrogel PhC barcodes with inverse opal structure could facilitate the loading of antibody probes for subsequent detection of tumor markers. By integrating tyramine deposition amplified ELISA on the barcode, the detection signal of tumor markers has been enhanced. Based on these features, it is demonstrated that the hydrogel PhC barcodes with tyramine deposition amplified ELISA could realize highly sensitive and multiplexed detection of HCC-related biomarkers. It was found that this method is flexible, sensitive and accurate, suitable for multivariate analysis of low abundance tumor markers and future cancer diagnosis. These features make the newly developed PhC barcodes an innovation platform, which possesses tremendous potential for practical application of low abundance targets.

Silver nitrate-assisted photo-crosslinking for enhancing the mechanical properties of an alginate/silk fibroin-based 3D scaffold.

Scaffolds play a pivotal role in tissue engineering and serve as vital biological substitutes, providing structural support for cell adhesion and subsequent tissue development. An ideal scaffold must possess mechanical properties suitable for tissue function and exhibit biodegradability. Although synthetic polymer scaffolds offer high rigidity and elasticity owing to their reactive side groups, which facilitate tailored mechanical and rheological properties, they may lack biological cues and cause persistent side effects during degradation. To address these challenges, natural polymers have garnered attention owing to their inherent bioactivity and biocompatibility. However, natural polymers such as silk fibroin (SF) and tyramine-modified alginate (AT) have limitations, including uncontrolled mechanical properties and weak structural integrity. In this study, we developed a blend of SF and AT as a printable biomaterial for extrusion-based 3D printing. Using photocrosslinkable SF/AT inks facilitated the fabrication of complex scaffolds with high printability, thereby enhancing their structural stability. The incorporation of silver nitrate facilitated the tunability of mechanical and rheological behaviors. SF/AT scaffolds with varying stiffness in the physiologically relevant range for soft tissues (51-246 kPa) exhibited excellent biocompatibility, indicating their promising potential for diverse applications in tissue engineering.

Fluorometric "AND" logic gate for detection of tyramine and tyrosinase based on in-situ formation of silicon-containing nanoparticles.

BACKGROUND: Tyramine is an important index of food freshness degree, and tyrosinase that can specifically oxidized monophenolamine to catecholamine plays a crucial part in the occurrence and development of melanin-related skin diseases. Therefore, it is crucial to develop sensitive and efficient methods for the detection of tyramine and tyrosinase. RESULTS: In this work, encouraged by tyrosinase-triggered specific oxidation of tyramine to dopamine and the unique fluorescent reaction between dopamine and amino silane, we have developed a one-step synthetic strategy of silicon containing nanoparticles (Si CNPs) for "turn-on" detection of tyramine and tyrosinase. The Si CNPs formed with thoroughly studied mechanism exhibit uniform structure and robust yellow-green fluorescence. The low detection limits for tyramine (1.87 µM) and tyrosinase (0.0029 U/mL) demonstrate admirable sensitivity outstripping most methods. The proposed assay achieves satisfactory results in the determination of tyramine and tyrosinase activity in real samples. Furthermore, we leverage this new fluorescent assay to enable the fabrication of an "AND" Boolean logic gate. SIGNIFICANCE: The entire process can be completed at easily available temperature and pressure with rapid response, convenient operation and visual observation. This fluorescent assay featured with excellent sensitivity, selectivity and stability has considerable prospects in the application of biosensors and disease diagnosis.

Quantitative tyramine analysis method for Apis mellifera larvae infected with Melissococcus plutonius, the causative agent of European foulbrood.

Tyramine, a trace monoamine produced from tyrosine by decarboxylation and found naturally in foods, plants, and animals, is a suspected virulence factor of Melissococcus plutonius that causes European foulbrood in honey bee brood. In the present study, we developed a method for quantitative analysis of tyramine in culture medium and honey bee larvae with a limit of quantitation of 3 ng/mL and a recovery rate of >97% using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry and deuterium-labeled tyramine, demonstrating for the first time that a highly virulent M. plutonius strain actually produces tyramine in infected larvae. This method will be an indispensable tool to elucidate the role of tyramine in European foulbrood pathogenesis in combination with exposure bioassays using artificially reared bee larvae.

False-positive MDA findings in HRMS-based screening of putrefied postmortem blood samples-Identification of the interference as N-acetyltyramine.

An unidentified compound in putrefied postmortem blood samples showed identical accurate mass and chromatographic behavior as 3,4-methylenedioxyamphetamine (MDA) and led to false-positive preliminary screening results. The aim of the study was to identify this unknown interference. Postmortem blood samples were analyzed after protein precipitation on a QExactive Focus high-resolution mass spectrometer (Thermo Fisher, Germany) coupled to a RP C18 column (Macherey-Nagel, Germany). Based on the analysis of mass spectrometry (MS) adducts and isotope ratios using fullscan (m/z 134-330) information, the empiric formula of the protonated molecule [M + H]+ of the unknown compound was found to be C10H14O2N (+ 0.6 ppm). Product ion spectra recorded using normalized collision energy 22% showed a base peak of C8H9O1 (+ 1.5 ppm) and a low-abundant water loss to C7H9 (+ 1.9 ppm), neutral losses of C2H2O and NH3 were found. Based on fullscan and MS-MS information and under consideration of the observed order of neutral losses, the compound was presumptively identified as N-acetyltyramine. This assumption was supported by SIRIUS software showing a SIRIUS score of 99.43% for N-acetyltyramine. Finally, the putative structure annotation was confirmed by a reference compound. The described false-positive MDA findings could be attributed to the presence of N-acetyltyramine in putrefied blood samples. Being an isomer of MDA, N-acetyltyramine could not be distinguished by high-resolution data of the protonated molecules. The presented results once again highlight that false-positive findings may occur even in hyphenated high-resolution mass spectrometry (HRMS) when using full-scan information only.