Optimal conditions for determination of bacitracin, bacitracin zinc and bacitracin methylene disalicylate in animal feed by ultra-performance liquid tandem mass spectrometry.

An integrated method combining solid-phase extraction (SPE) with ultra-performance liquid tandem mass spectrometry (UPLC-MS/MS) has been established for quantifying bacitracin (BTC), bacitracin zinc (BZ), and bacitracin methylene disalicylate (BMD) in animal feed. A pretreatment procedure that can effectively, quickly, and simultaneously extract and purify BTC, BZ, or BMD in feed was developed for the first time through the optimization of extraction and SPE conditions. After extraction with acetonitrile + methanol + 15 % ammonia solution (1:1:1, v:v:v) and dilution with EDTA solution (1.5 mmol/L, pH 7.0), a SPE procedure was carried out with C18 cartridge. Following LC-MS/MS analysis utilized a Waters Peptide BEH C18 column with a gradient elution of 0.1 % formic acid in water/acetonitrile with. This method demonstrated a strong linear correlation (R2 > 0.9980) across a 0.01-1.0 mg/L concentration span, based on a matrix-matched standard curve. Satisfactory recoveries of BTC (bacitracin A, B1, B2, and B3), BZ, and BMD in different feeds were obtained from 80.7 % to 108.4 %, with relative standard deviations below 15.7 %. Low limits of quantification ranging within 7.2-20 µg/kg were achieved for bacitracin A, B1, B2, and B3. This method provided an effective and reliable detection method to prevent the addition of BTC and different BTC formulations in feeds.

Proteomic profile of seminal plasma from Pêga donkeys (Equus asinus) with high sperm motility and vigor: Implications for assisted reproduction.

Donkeys of the Pêga breed (Equus asinus) have been used for two centuries to produce breeding stock and create hybrids for labor and transport in southeast Brazil, and for exporting meat and milk to other countries. Furthermore, they are used in competitions, as they are docile and easy to handle. However, assisted reproduction success rates for frozen donkey semen are remarkably low, with no standardized method for cryopreserving sperm after removal of seminal plasma. This work aims to reveal the biological involvement of seminal plasma proteins from Pêga donkeys in aiding the development of assisted reproduction. This study was carried out with 14 ejaculates collected every eight days, throughout the breeding season, from three healthy fertile Pêga donkeys, with an average age of four years. After confirming the high freezability of fresh semen by evaluating quality parameters, the seminal plasma was separated by centrifugation and an aliquot from each collection was microfiltered and frozen. A label-free technique followed by LC-MS/MS analysis applied to pools of seminal plasma samples from each animal revealed 522 proteins in the proteomic profile, of which 49.8 % (260 proteins) are related to cellular energy transformation, and many proteins involved in reproduction (76), spermatogenesis (38), fertilization (29), among other biological process. By comparison with literature, Pêga donkeys share many proteins with donkeys of Dezhou breed that present great potential as fertility biomarkers. Our results showed proteins positively related to fertilization for different breeds of donkeys around the world, helping to enhance the assisted reproduction of Pêga donkeys.

In vitro bioaccessibility of cyclodi-BADGE present in canned seafood: A new approach for the estimation of dietary exposure of the Spanish population.

Human dietary exposure to chemical compounds is a priority issue for public health authorities since it constitutes a key step in risk assessment, and food packaging could be an important source of contamination. In this study, the bioaccessibility of cyclodi-BADGE was evaluated in canned seafood samples using a standardized protocol of in vitro gastrointestinal digestion and an analytical method based on liquid chromatography coupled to tandem mass spectrometry. The impact of enzymes, different gastric pHs, and food-covering liquids on the bioaccessibility of cyclodi-BADGE was studied. The results highlighted that cyclodi-BADGE was available to be absorbed at the intestinal level (90.9-112.3%), and its bioaccessibility increased substantially in fat food samples. Finally, the estimated dietary exposure to cyclodi-BADGE in the Spanish adult population reached values of 14.26 µg/kg bw/day for tuna in tomato, exceeding the tolerable daily intake (1.5 µg/kg bw/day) recommended for chemicals with high toxicological risk.

Evaluation of Hedera species as herbal medicine raw materials.

This study aimed to evaluate the potential of Hedera colchica as an alternative to Hedera helix species for the treatment of mild inflammatory conditions of the upper respiratory tract and chronic inflammatory bronchial diseases. The H. colchica extract with the highest saponin content (C3S; 468.19 ± 16.01 mg HE/g dry weight) and the extract with the highest total phenol content (C1F; 108.60 ± 5.61 mg GAE/g dry weight). Chemical analysis and standardisation of the extract with the highest selective COX-2 inhibitory effect was performed using the LC-MS/MS technique. It was determined that the substances found in the highest ratio in the C1F extract were quinic acid (45.909 µg/g extract) and hesperidin (37.077 µg/g extract). As a result, secondary metabolites, in addition to saponins, found in Hedera species may also contribute to the extract's effectiveness, more potent extracts can be obtained compared to the total extract-containing preparations available in the market.

Comparison of transfer of different sulphonamides from contaminated beeswax to honey.

Introduction: No maximum residue limits in honey have been legislated in the EU for antimicrobial substances such as sulphonamides, and they are not permitted, therefore, for treating honey bees unless in a cascade system. Since sulphonamides are used illegally in apiculture to treat foulbrood, their residues can be found in honey and other apiculture products, including beeswax. The study aimed to assess the contamination of honey from beeswax containing residues of 10 sulphonamides (sulphadimethoxine (SDM), sulphadoxine (SDX), sulphamonomethoxine (SMM), sulphamethoxazole (SMX), sulphameter (SMT), sulphamethazine (SMZ), sulphamerazine (SMR), sulphadiazine (SDA), sulphathiazole (STZ) and sulphacetamide (SCA)). Material and Methods: Wax-based foundations fortified with 10 sulphonamides at 10,000 µg/kg were evaluated for sulphonamide concentrations and then placed in a beehive so that honey bees (Apis mellifera L.) could build honeycombs with them. Frames of capped honey were taken out of the hives one month later and honey was sampled from them. The honeycombs were subsequently incubated in a laboratory at 35°C for five months, and honey was sampled monthly. The honey sulphonamide concentrations were measured using liquid chromatography-tandem mass spectrometry and compared to the wax-based foundation concentrations. Results: The maximum transfers to honey of the initial amount of SDM, SDX, SMM, SMX, SMT, SMZ, SMR, SDA, STZ and SCA in the wax-based foundations were 42.6, 34.3, 31.7, 30.1, 29.5, 25.2, 18.7, 16.1, 9.5 and 8.6%, respectively. Conclusion: This study demonstrated that every tested sulphonamide could migrate from beeswax in antimicrobial-tainted honeycombs to honey, SDM having the highest migration potential and SCA the lowest.

Comprehensive phytochemical and toxicological analysis of Chenopodium ambrosioides (L.) fractions.

Chenopodium ambrosioides aerial parts have been historically employed in traditional medicine for addressing various ailments such as headaches, abdominal discomfort, joint issues, and respiratory disorders, alongside treatments for lice and warts. This study aimed to conduct a comprehensive phytochemical analysis of C. ambrosioides and assess the acute and subacute toxicity of oral treatments using fractions in preclinical trials. Spectrophotometric analysis via LC-MS/MS was used to characterize the plant's chemical composition. Acute toxicity evaluation followed Organisation for Economic Co-operation and Development code 42 guidelines, conducted on adult male and female Wistar strain mice. Subsequently, Swiss mice were divided into six groups for the subacute toxicity study, receiving oral doses of 200 mg/kg extracts and fractions for 28 days. Daily observations and biochemical analyses were performed, with LC-MS/MS revealing a diverse array of compounds including organic acids, flavonoids, phenolic acids, rutin, hesperidin, nicotiflorine, and fumaric acid. Results indicated no lethality or alterations in body weight in treated groups, though some organ weight changes were noted. Biochemical analyses demonstrated values within the normal range for all groups, suggesting that the treatments did not induce adverse effects. Acute and subacute treatments with fractions did not result in lethality or toxic alterations at therapeutic doses, implying the safety of the product at appropriate levels. This study underscores the potential of C. ambrosioides as a safe therapeutic option warranting further exploration.

Development and validation of a rapid and simple HILIC-MS/MS method for the determination of biogenic amines in tuna fish.

The production of biogenic amines (BAs), which are markers of both quality and safety in fish and fishery products, is influenced by the harvesting technique, handling, and other operations including those carried out on board the vessel. Scombroid dark-meat fish (e.g. tuna) are the fish species most frequently linked to histamine poisoning. The most commonly found BAs in fish are histamine, tyramine, putrescine, and cadaverine, which are produced when microbes decarboxylate the corresponding free amino acids. In this study, a rapid and cost-effective HILIC-MS/MS method was developed and validated for the determination of putrescine, cadaverine, histamine and tyramine in tuna samples. A simple sample preparation procedure was followed using the solvent mixture MeOH/H2O (50/50, v/v), 0.1 % acetic acid for protein precipitation and analyte extraction. Intra- and inter-day accuracy, expressed as %Recovery (%R), ranged from 88.0 % (Cad) to 102.7 % (Tyr) and from 85.0 % (Cad) to 99.8 % (Tyr), respectively. Intra- and inter-day precision, expressed as %Relative Standard Deviation (%RSD), ranged from 0.4 % (Tyr, Put) to 3.3 % (His) and from 0.7 % (Tyr) to 5.0 % (Cad), respectively. Limits of detection (LOD) and quantification (LOQ) varied from 0.0009 to 0.0940 mg/kg and from 0.0030 mg/kg to 0.3100 mg/kg, respectively, depending on the analyte. Regarding the potential toxic effects linked to biogenic amines in foods, samples examined in this study showed no risk. The proposed method is an important analytical tool for routine analysis of BAs in fish products.

A rapid LC-MS/MS method for the simultaneous quantification of ivacaftor, lumacaftor, elexacaftor, tezacaftor, hexyl-methyl ivacaftor and ivacaftor carboxylate in human plasma.

Cystic fibrosis is one of the most common genetic diseases among caucasian population. This disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding for the CFTR protein. Lumacaftor, elexacaftor, tezacaftor, and ivacaftor were currently used as the treatment to Cystic fibrosis. In this study, we describe a new method for the simultaneous quantification of four molecules: lumacaftor, elexacaftor, tezacaftor, and ivacaftor, alongside two metabolites of ivacaftor, specifically hexyl-methyl ivacaftor and ivacaftor carboxylate by liquid chromatography-tandem mass spectrometry. This method holds significant utility for therapeutic drug monitoring and the optimization of treatments related to CFTR modulators. Molecules were extracted from 100 µL of plasma by a simple method of protein precipitation using acetonitrile. Following extraction, chromatographic separation was carried out by reverse chromatography on a C18 analytical column, using a gradient elution of water (0.05 % formic acid, V/V) and acetonitrile (0.05 % formic acid, V/V). The run time was 7 minutes at a flow rate of 0.5 mL/min. After separation, molecules were detected by electrospray ionization on a Xevo TQD triple-quadrupole-mass-spectrometer (Waters®, Milford, USA). The calibration range were: 0.053-20.000 mg/L for elexacaftor, tezacaftor and lumacaftor, 0.075-14.000 mg/L for ivacaftor, and 0.024-6.500 mg/L for hexyl-methyl ivacaftor and ivacaftor carboxylate. The proposed method underwent throughout validation demonstrating satisfactory precision (inter- and intra-day coefficients of variation less than 14.3 %) and a good accuracy (inter- and intra-day bias ranging between -13.7 % and 14.7 %) for all the analytes. The presented method for the simultaneous quantification of CFTR modulators and their metabolites in human plasma has undergone rigorous validation process yielding good results including strong precision and accuracy for all analytes. This method has been effectively used in routine analytical analysis and clinical investigations within our laboratory.

Development and validation of a sensitive and simultaneous liquid chromatography tandem mass spectrometry method for the determination of eight phytocannabinoids in various CBD products.

In this study, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of eight phytocannabinoids in various cannabidiol (CBD) products from Japanese market. This method was combined with electrospray ionization in positive mode and sample preparation with QuEChERS. Three types of commercial products such as honey, chocolate, and gummies were used to perform accurate quantification with unified protocol of LC-MS/MS and QuEChERS. The limit of detection and quantification were 5-20 µg g-1 and 10-40 µg g-1, respectively. Reproducibility was ensured using matrices free of target foods, resulting in an accuracy within ±10 % and a precision with a relative standard deviation of less than 5 % for all targets. Finally, this analytical method was applied to 8 series of commercial samples from the Japanese market. This unified protocol will serve as a reference as an official method in Japan.

A practical approach to supported liquid extraction and measurement of 18 steroids in plasma and serum by targeted liquid chromatography tandem mass spectrometry.

Chromatography combined with mass spectrometry is a gold standard technique for steroid measurement, however the type of sample preparation, the dynamic range and reliability of the calibration curve, the chromatographic separation and mass spectrometry settings ultimately determine the success of the method. The steroid biosynthetic pathway is conserved in higher mammals and literature demonstrates that the concentration ranges of different steroid groups are relatively comparable across species. We sought to develop a robust and reliable multi steroid targeted analysis method for blood that would have wide application across higher mammals. The method was developed following bioanalytical method validation guidelines to standards typically applied to human clinical studies, including isotopically labelled internal standards where at all possible. Here we describe the practical approach to a 96-well supported liquid extraction (SLE) method of extraction from plasma (200 µL) using an Extrahera liquid handling robot (Biotage, Sweden), including quality control samples, followed by a comprehensive separation and targeted LC-MS/MS analysis of 18 steroids in plasma (pregnenolone, progesterone, 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, aldosterone, 11-deoxycortisol, 21-deoxycortisol, cortisol, cortisone, androstenedione, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, estrone, 17ß-estradiol and estriol). •SLE in a 96-well format of up to 74 biological plasma samples, enriched with multiple isotopically labelled internal standards, a 12-point aqueous calibration curve, and 6 serum quality controls, designed to monitor long-term performance of the method•Chromatographic separation of multiple steroids along the gradient, with ammonium fluoride mobile phase additive to improve sensitivity, followed by electrospray ionisation and constant polarity switching•Aqueous calibration standards that cover physiologically relevant ranges - high nanomolar glucocorticoids, low nanomolar androgens and picomolar ranges for estrogens and steroid intermediates.

Quantitative bile acid profiling in healthy adult dogs and pups from serum, plasma, urine, and feces using LC-MS/MS.

Synthesis and secretion of bile acids (BA) is a key physiological function of the liver. In pathological conditions like portosystemic shunt, hepatic insufficiency, hepatitis, or cirrhosis BA metabolism and secretion are disturbed. Quantification of total serum BA is an established diagnostic method to assess the general liver function and allows early detection of abnormalities, liver disease progression and guidance of treatment decisions. To date, data on comparative BA profiles in dogs are limited. However, BA profiles might be even better diagnostic parameters than total BA concentrations. On this background, the present study analyzed and compared individual BA profiles in serum, plasma, urine, and feces of 10 healthy pups and 40 adult healthy dogs using ultra-high performance liquid chromatography coupled to electrospray ionization mass spectrometry. Sample preparation was performed by solid-phase extraction for serum, plasma, and urine samples or by protein precipitation with methanol for the feces samples. For each dog, 22 different BA, including unconjugated BA and their glycine and taurine conjugates, were analyzed. In general, there was a great interindividual variation for the concentrations of single BA, mostly exemplified by the fact that cholic acid (CA) was by far the most prominent BA in blood and urine samples of some of the dogs (adults and pups), while in others, CA was under the detection limit. There were no significant age-related differences in the BA profiles, but pups showed generally lower absolute BA concentrations in serum, plasma, and urine. Taurine-conjugated BA were predominant in the serum and plasma of both pups (68%) and adults (74-75%), while unconjugated BA were predominant in the urine and feces of pups (64 and 95%, respectively) and adults (68 and 99%, respectively). The primary BA chenodeoxycholic acid and taurocholic acid and the secondary BA deoxycholic acid and lithocholic acid were the most robust analytes for potential diagnostic purpose. In conclusion, this study reports simultaneous BA profiling in dog serum, plasma, urine, and feces and provides valuable diagnostic data for subsequent clinical studies in dogs with different kinds of liver diseases.

Alterations to sphingolipid metabolism from antipsychotic administration in healthy volunteers are restored following the use of cannabidiol.

Randomized clinical trials substantiate cannabidiol (CBD) as a next-generation antipsychotic, effective in alleviating positive and negative symptoms associated with psychosis, while minimising the adverse effects seen with established treatments. Although the mechanisms remain debated, CBD is known to induce drug-responsive changes in lipid-based retrograde neurotransmitters. Lipid aberrations are also frequently observed with antipsychotics, which may contribute to their efficacy or increase the risk of undesirables, including metabolic dysfunction, obesity and dyslipidaemia. Our study investigated CBD's impact following lipid responses triggered by interaction with second-generation antipsychotics (SGA) in a randomized phase I safety study. Untargeted mass spectrometry assessed the lipidomic profiles of human sera, collected from 38 healthy volunteers. Serum samples were obtained prior to commencement of any medication (t = 0), 3 days after consecutive administration of one of the five, placebo-controlled, treatment arms designed to achieve steady-state concentrations of each SGA (amisulpride, 150 mg/day; quetiapine, 300 mg/day; olanzapine 10 mg/day; risperidone, 3 mg/day), and after six successive days of SGA treatment combined with CBD (800 mg/day). Receiver operating characteristics (ROC) refined 3712 features to a putative list of 15 lipids significantly altered (AUC > 0.7), classified into sphingolipids (53 %), glycerolipids (27 %) and glycerophospholipids (20 %). Targeted mass spectrometry confirmed reduced sphingomyelin and ceramide levels with antipsychotics, which mapped along their catabolic pathway and were restored by CBD. These sphingolipids inversely correlated with body weight after olanzapine, quetiapine, and risperidone treatment, where CBD appears to have arrested or attenuated these effects. Herein, we propose CBD may alleviate aberrant sphingolipid metabolism and that further investigation into sphingolipids as markers for monitoring side effects of SGAs and efficacy of CBD is warranted.

Characterizing lipid constituents of B. moojeni snake venom: a comparative approach for chemical and biological investigations.

Snake venoms are complex mixtures majorly composed of proteins with well-studied biological effects. However, the exploration of non-protein components, especially lipids, remains limited despite their potential for discovering bioactive molecules. This study compares three liquid-liquid lipid extraction methods for both chemical and biological analyses of Bothrops moojeni snake venom. The methods evaluated include the Bligh and Dyer method (methanol, chloroform, water), considered standard; the Acunha method, a modification of the Bligh and Dyer protocol; and the Matyash method (MTBE/methanol/water), featuring an organic phase less dense than the aqueous phase. Lipidomic analysis using liquid chromatography with high-resolution mass spectrometry (LC-HRMS) system revealed comparable values of lipid constituents' peak intensity across different extraction methods. Our results show that all methods effectively extracted a similar quantity of lipid species, yielding approximately 17-18 subclasses per method. However, the Matyash and Acunha methods exhibited notably higher proportions of biologically active lipids compared to the Bligh and Dyer method, particularly in extracting lipid species crucial for cellular structure and function, such as sphingomyelins and phosphatidylinositol-phosphate. In conclusion, when selecting a lipid extraction method, it is essential to consider the study's objectives. For a biological approach, it is crucial to evaluate not only the total quantity of extracted lipids but also their quality and biological activity. The Matyash and Acunha methods show promise in this regard, potentially offering a superior option for extracting biologically active lipids compared to the Bligh and Dyer method.

Cytotoxicity of Physalis minima Linn (Solanaceae) fruit against HCT116 and HT29 colorectal cancer cell lines.

The pantropical Physalis minima are traditionally used for the prevention and treatment of various illnesses, diseases, and cancers. While most earlier studies on the species have focused on the phytochemistry of the leaf and stem extracts, recent studies have indicated that its fruit may contain bioactive compounds of medical interest. In this study, we investigated the cytotoxicity of extracts from the fruit of P. minima against colorectal cancer cell lines and revealed its phytochemical profile via high-resolution tandem mass spectrometry analysis. Following a 24-h treatment with the fruit extract, cytoplasm shrinkage and nucleus condensation were observed in the colorectal cancer cell lines HCT116 and HT29, indicating the induction of programmed cell death. Phytochemically, 71 putative metabolites were identified. Some of these metabolites have been reported to inhibit cancers to varying degrees, further supporting the correlation of the putative metabolites with the cytotoxicity against colorectal cancer cells demonstrated in this study.

[An enrichment strategy based on hydrophobic tagging and reversed-phase chromatographic separation for the analysis of lysine-containing peptides].

Lysine (K) is widely used in the design of lysine-targeted crosslinkers, structural elucidation of protein complexes, and analysis of protein-protein interactions. In "shotgun" proteomics, which is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), proteins from complex samples are enzymatically digested, generating thousands of peptides and presenting significant challenges for the direct analysis of K-containing peptides. In view of the lack of effective methods for the enrichment of K-containing peptides, this work developed a method which based on a hydrophobic-tag-labeling reagent C10-S-S-NHS and reversed-phase chromatography (termed as HYTARP) to achieve the efficient enrichment and identification of K-containing peptides from complex samples. The C10-S-S-NHS synthesized in this work successfully labeled standard peptides containing various numbers of K and the labeling efficiency achieved up to 96% for HeLa cell protein tryptic digests. By investigating the retention behavior of these labeled peptides in C18 RP column, we found that most K-labeled peptides were eluted once when acetonitrile percentage reached 57.6% (v/v). Further optimization of the elution gradient enabled the efficient separation and enrichment of the K-labeled peptides in HeLa digests via a stepwise elution gradient. The K-labeled peptides accounted for 90% in the enriched peptides, representing an improvement of 35% compared with the number of peptides without the enrichment. The dynamic range of proteins quantified from the enriched K-containing peptides spans 5-6 orders of magnitude, and realized the detection of low-abundance proteins in the complex sample. In summary, the HYTARP strategy offers a straightforward and effective approach for reducing sample complexity and improving the identification coverage of K-containing peptides and low-abundance proteins.

Accumulation patterns of flavonoids and phenolic acids in different colored sweet potato flesh revealed based on untargeted metabolomics.

Sweet potatoes are rich in flavonoids and phenolic acids, showing incomparable nutritional and health value. In this investigation, we comprehensively analyzed the secondary metabolite profiles in the flesh of different-colored sweet potato flesh. We determined the metabolomic profiles of white sweet potato flesh (BS), orange sweet potato flesh (CS), and purple sweet potato flesh (ZS) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CS vs. BS, ZS vs. BS, and ZS vs. CS comparisons identified a total of 4447 secondary metabolites, including 1540, 1949, and 1931 differentially accumulated metabolites. Among them, there were significant differences in flavonoids and phenolic acids. There were 20 flavonoids and 13 phenolic acids that were common differential metabolites among the three comparison groups. The accumulation of paeoniflorin-like and delphinidin-like compounds may be responsible for the purple coloration of sweet potato flesh. These findings provide new rationale and insights for the development of functional foods for sweet potatoes. List of compounds: Kaempferol (PubChem CID: 5280863); Peonidin 3-(6"-p-coumarylglucoside) (PubChem CID: 44256849); Swerchirin (PubChem CID: 5281660); Trilobatin (PubChem CID: 6451798); 3-Geranyl-4-hydroxybenzoate (PubChem CID: 54730540); Eupatorin (PubChem CID: 97214); Icaritin (PubChem CID: 5318980); Isorhamnetin (PubChem CID: 5281654); Glucoliquiritin apioside (PubChem CID: 74819335); Brazilin (PubChem CID: 73384).

Mosquitocidal toxin-like islands in Bacillus thuringiensis S2160-1 revealed by complete-genome sequence and MS proteomic analysis.

Here, we present the whole genome sequence of Bt S2160-1, a potential alternative to the mosquitocidal model strain, Bti. One chromosome genome and four mega-plasmids were contained in Bt S2160-1, and 13 predicted genes encoding predicted insecticidal crystal proteins were identified clustered on one plasmid pS2160-1p2 containing two pathogenic islands (PAIs) designed as PAI-1 (Cry54Ba, Cry30Ea4, Cry69Aa-like, Cry50Ba2-like, Cry4Ca1-like, Cry30Ga2, Cry71Aa-like, Cry72Aa-like, Cry70Aa-like, Cyt1Da2-like and Vpb4C1-like) and PAI-2 (Cyt1Aa-like, and Tpp80Aa1-like). The clusters appear to represent mosquitocidal toxin islands similar to pathogenicity islands. Transcription/translation of 10 of the 13 predicted genes was confirmed by whole-proteome analysis using LTQ-Orbitrap LC-MS/MS. In summary, the present study identified the existence of a mosquitocidal toxin island in Bacillus thuringiensis, and provides important genomic information for understanding the insecticidal mechanism of B. thuringiensis.

Urine metabolomics unravel the effects of short-term dietary interventions on oxidative stress and inflammation: a randomized controlled crossover trial.

Dietary biomarkers in urine remain elusive when evaluating diet-induced oxidative stress and inflammation. In our previous study, we conducted a randomized controlled crossover trial to compare the short-term (4-weeks) effects of the balanced Korean diet (BKD) with Western diets, including the 2010 dietary guidelines for Americans (2010 DGA) and typical American diet (TAD), on various metabolic indices in obese Korean adults. Building on this work, the current research focuses on the impact of these dietary interventions on oxidative stress (d-ROMs and BAP) and inflammation (CRP, TNF-α, IL-6, IL-1ß, MCP-1) biomarkers in serum, and the concurrent urine metabolomes. Each dietary regimen was in silico and experimentally examined for their antioxidant levels using ABTS, DPPH, and FRAP assays, as well as total flavonoid (TFC) and total phenolic (TPC) contents. We assessed post-intervention variations in oxidative stress and inflammation biomarkers in serum, as well as the urine metabolite profiles for the participants (n = 48, average age: 41 years). Antioxidant contents and associated total antioxidant capacity (TAC) were significantly higher for the recommended diets (BKD and 2010 DGA) compared to TAD (p < 0.05). Butanol extracts from recommended diets (BKD and 2010 DGA) showed significantly higher antioxidant activity compared to TAD in ABTS (p < 0.01), DPPH, and FRAP (p < 0.05) assays. Consistent results were observed in total phenolic and flavonoid contents, mirroring their respective antioxidant activities. Following the intervention period, oxidative stress & inflammation markers in serum varied marginally, however, the urine metabolite profiles were clearly demarcated for the BKD and Western dietary groups (PC1 = 5.41%). For BKD group, the pre- and post-intervention urine metabolite profiles were clearly segregated (PLS2 = 2.93%). Compared to TAD, urine extracts from the recommended dietary group showed higher abundance of benzoic acid & phenolic derivatives (VIP > 0.7, p < 0.05). Metabolites associated with oxidative stress were observed higher in the urine samples from Western dietary groups compared to BKD. Urine metabolomics data delineated the post-intervention effects of three dietary interventions which corroborates the respective findings for their effects on metabolic indices.

A smooth vetch (Vicia villosa var.) strain endogenous to the broad-spectrum antagonist Bacillus siamensis JSZ06 alleviates banana wilt disease.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), poses a significant threat to banana production globally, thereby necessitating effective biocontrol methods to manage this devastating disease. This study investigates the potential of Bacillus siamensis strain JSZ06, isolated from smooth vetch, as a biocontrol agent against Foc TR4. To this end, we conducted a series of in vitro and in vivo experiments to evaluate the antifungal activity of strain JSZ06 and its crude extracts. Additionally, genomic analyses were performed to identify antibiotic synthesis genes, while metabolomic profiling was conducted to characterize bioactive compounds. The results demonstrated that strain JSZ06 exhibited strong inhibitory activity against Foc TR4, significantly reducing mycelial growth and spore germination. Moreover, scanning and transmission electron microscopy revealed substantial ultrastructural damage to Foc TR4 mycelia treated with JSZ06 extracts. Genomic analysis identified several antibiotic synthesis genes, and metabolomic profiling revealed numerous antifungal metabolites. Furthermore, in pot trials, the application of JSZ06 fermentation broth significantly enhanced banana plant growth and reduced disease severity, achieving biocontrol efficiencies of 76.71% and 79.25% for leaves and pseudostems, respectively. In conclusion, Bacillus siamensis JSZ06 is a promising biocontrol agent against Fusarium wilt in bananas, with its dual action of direct antifungal activity and plant growth promotion underscoring its potential for integrated disease management strategies.

Co-occurrence of mycotoxins in stored maize from southern and southwestern Ethiopia.

Maize grain samples collected from 129 small-scale farmers' stores in southern and southwestern Ethiopia were analysed by LC-MS/MS for a total of 218 mycotoxins and other fungal metabolites of which 15% were regulated mycotoxins. Mycotoxins produced by Penicillium, Aspergillus, and Fusarium accounted for 31%, 17%, and 12% of the metabolites, respectively. Most of the current samples were contaminated by masked and/or emerging mycotoxins with moniliformin being the most prevalent one, contaminating 93% of the samples. Each sample was co-contaminated by 3 to 114 mycotoxins/fungal metabolites. Zearalenone, fumonisin B1, and deoxynivalenol were the dominant mycotoxins, occurring in 78%, 61%, and 55% of the samples with mean concentrations of 243, 429, and 530 µg/kg, respectively. The widespread co-occurrence of several mycotoxins in the samples may pose serious health risks due to synergistic/additional effects.