High-resolution Mass Spectrometry Analysis Research Articles

Metabolites and novel compounds with anti-microbial or antiaging activities from Cordyceps fumosorosea

High-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) analysis revealed that there are 20 main components in spores and mycelia extract of Cordyceps fumosorosea strain RCEF 6672 including mannitol (1), uridine (2), adenine (3). N6-(2-hydroxyethyl)-adenosine (4). N6-(2-hydroxyethylacetate)-adenosine (5), fumosoroseanoside A (6) and B (7), ovalicin-4α-alcohol (8), 1-linoleoyl-sn-glycero-3-phosphocholine (9) and its isomer (10), fumosoroseain A (11) and its isomer (12), 5 non-ribosomal peptides (13 to 17) and 3 fatty acids (18 to 20). The compounds 5, 6, 7, 9 and 11 were prepared with preparative and semi-preparative HPLC and identified with 1D and 2D NMR. Compounds 4 and 5 were the first time identified from C. fumosorosea. Compounds 6, 7 and 11 are novel compounds. Compounds 6 and 7 showed antibacterial and antifungal activities, and 11 showed antiaging activity. All the secondary metabolites (4 to 8 and 11 to 17) have strong bioactivities indicating that the metabolites have pharmaceutical development potentiality.Graphical

Effect of Phenolic Extract from Red Beans (Phaseolus vulgaris L.) on T-2 Toxin-Induced Cytotoxicity in HepG2 Cells.

Red beans contain human bioactive compounds such as polyphenols. Several in vitro studies have proposed the natural compounds as an innovative strategy to modify the toxic effects produced by mycotoxins. Hence, in this work, a complete investigation of the polyphenolic fraction of red beans was performed using a Q-Orbitrap high-resolution mass spectrometry analysis. Notably, epicatechin and delphinidin were the most detected polyphenols found in red bean extracts (3.297 and 3.108 mg/Kg, respectively). Moreover, the red bean extract was evaluated against the T-2 toxin (T-2) induced cytotoxicity in hepatocarcinoma cells (HepG2) by direct treatment, simultaneous treatment, and pre-treatment assays. These data showed that T-2 affected the cell viability in a dose-dependent manner, as well as observing a cytotoxic effect and a significant increase in ROS production at 30 nM. The simultaneous treatment and the pre-treatment of HepG2 cells with red bean extract was not able to modify the cytotoxic T-2 effect. However, the simultaneous treatment of T-2 at 7.5 nM with the red bean extract showed a significant decrease in ROS production, with respect to the control. These results suggest that the red bean extract could modulate oxidative stress on HepG2 cells.

Anti-influenza patchoulol-type sesquiterpenoids from Pogostemon cablin

• Four new patchoulol-type sesquiterpenoids were isolated from Pogostemon cablin (Blanco) Benth. • This is the second report of patchoulol glucopyranoside from P. cablin . • Compound 10 represented as the first example of nor-patchoulol glucopyranoside. • Compounds 8 and 12 showed potent anti-influenza activity against tamiflu resistant virus. Four new patchoulol-type sesquiterpenoids, including 6 α ,9 β -dihydroxypatchoulol 6- O - β - d -glucopyranoside ( 1 ), 6 α -hydroxypatchoulol 6- O - β - d -glucopyranoside ( 2 ), 3 α ,9 β -dihydroxypathoulol ( 3 ), and 4 β -hydroxynorpatchoulol 4- O - β - d -glucopyranoside ( 10 ), were isolated from the roots of Pogostemon cablin (Blanco) Benth, together with eleven known sesquiterpenoids. Their structures were elucidated on the basis of extensive NMR spectral and high resolution mass spectrometry analysis. This is the second report of patchoulol glucopyranoside from P. cablin and compound 10 represented as the first example of nor-patchoulol glucopyranoside. The anti-influenza virus activities of 1–10 against A/WSN/33/2009 and A/Puerto Rico/8/1934 strains (tamiflu resistant viruses) were evaluated. Compounds 2 β ,12-dihydroxypathoulol ( 5 ) and (5R)-5-hydroxypatchoulol ( 8 ) exhibited moderate anti-influenza activity against A/WSN/33/2009 strain with EC 50 value of 52.7 μ M and 49.6 μ M (positive control oseltamivir, EC 50 = 6.75 μ M). Compounds 8 and pogostol ( 12 ) showed potent anti-influenza activity against A/Puerto Rico/8/1934 strain with EC 50 values of 3.06 μ M and 0.07 μ M, respectively, versus the postive control (amantadine, EC 50 = 67.9 μ M).

Lycopus maackianus Makino MeOH Extract Exhibits Antioxidant and Anti-Neuroinflammatory Effects in Neuronal Cells and Zebrafish Model.

Lycopus maackianus Makino belongs to the Labiatae family and is used in traditional medicine to manage postpartum edema and boils. However, few studies on its antioxidant and anti-inflammatory effects have been conducted. Here, the compounds in L. maackianus methanol (MeOH) extract were profiled using ultra-high-performance liquid chromatography–time-of-flight high-resolution mass spectrometry analysis. The antioxidant activity of L. maackianus MeOH extract was shown to increase in a concentration-dependent manner by investigating the 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity. Next, in lipopolysaccharide-treated BV2 cells, L. maackianus extract inactivated the nuclear factor-kappa B pathway, inhibiting nitric oxide, prostaglandin E2, interleukin-6, and tumor necrosis factor-α production and inducible nitric oxide synthase and cyclooxygenase-2 protein expression. Furthermore, L. maackianus extract protected against oxidative stress-induced cellular damage in glutamate-stimulated HT22 cells. L. maackianus MeOH extract induced heme oxygenase-1 expression and increased the translocation of nuclear factor E2-related factor 2 in the nucleus, thus exhibiting antioxidant and anti-inflammatory effects. Moreover, the in vivo antioxidant and anti-inflammatory effects of the extract were demonstrated in a zebrafish (Danio rerio) model treated with hydrogen peroxide and lipopolysaccharide. MeOH L. maackianus extract showed antioxidant and anti-neuroinflammatory effects by increasing the expression of heme oxygenase-1, establishing its therapeutic potential for neuroinflammatory diseases.

Diorganotin(IV) complexes based on tridentate ONO ligands as potential anticancer agents

Eight new diorganotin(IV) complexes (1a-2d), namely {[X-C6H4(O)C=N-N=C(Me)COO]R2Sn(CH3OH)}n (1a, 2a), {[X-C6H4(O)C=N-N=C(Me)COO]R2Sn(CH3OH)}2 (1b, 1c, 1d, 2b), and {[X-C6H4(O)C=N-N=C(Me)COO]R2Sn}2 (2c, 2d) (X = H-, p-Me-, p-OH-, p-NO2-; R = o-Cl-C6H4CH2- or o-Me-C6H4CH2-), have been synthesized by microwave “one-pot” reaction with arylformylhydrazine, pyruvic acid, and the corresponding R2SnCl2. All the complexes have been characterized by FT-IR (Fourier transform infrared spectroscopy), multinuclear NMR (1H, 13C, and 119Sn nuclear magnetic resonance spectroscopies), HRMS (high-resolution mass spectroscopy) and single-crystal X-ray structural analysis. The antiproliferative activity of all complexes was tested against the cancer cell lines NCI-H460, MCF-7 and HepG2. The diorganotin complex 1c has been shown to be more potent antitumor agents against HepG2 than other complexes and cisplatin. Flow cytometry analysis observation demonstrated that complex 1c mediated cell apoptosis of HepG2 cells and arrested cell cycle in the S phase. The single cell gel electrophoreses assay results show that the 1c induce DNA damage. The DNA binding activities of the 1c were studied by UV–visible absorption spectrometry, fluorescence competitive, circular dichroism measurements, and molecular docking, results shown 1c can be well embedded in the groove and cleave DNA.

Towards Phytopathogen Diagnostics? Coconut Bud Rot Pathogen Phytophthora palmivora Mycelial Proteome Analysis Informs Genome Annotation.

Planetary agriculture stands to benefit immensely from phytopathogen diagnostics, which would enable early detection of pathogens with harmful effects on crops. For example, Phytophthora palmivora is one of the most destructive phytopathogens affecting many economically important tropical crops such as coconut. P. palmivora causes diseases in over 200 host plants, and notably, the bud rot disease in coconut and oil palm, which is often lethal because it is usually detected at advanced stages of infection. Limited availability of large-scale omics datasets for P. palmivora is an important barrier for progress toward phytopathogen diagnostics. We report here the mycelial proteome of P. palmivora using high-resolution mass spectrometry analysis. We identified 8073 proteins in the mycelium. Gene Ontology-based functional classification of detected proteins revealed 4884, 4981, and 3044 proteins, respectively, with roles in biological processes, molecular functions, and cellular components. Proteins such as P-loop, NTPase, and WD40 domains with key roles in signal transduction pathways were identified. KEGG pathway analysis annotated 2467 proteins to various signaling pathways, such as phosphatidylinositol, Ca2+, and mitogen-activated protein kinase, and autophagy and cell cycle. These molecular substrates might possess vital roles in filamentous growth, sporangia formation, degradation of damaged cellular content, and recycling of nutrients in P. palmivora. This large-scale proteomics data and analyses pave the way for new insights on biology, genome annotation, and vegetative growth of the important plant pathogen P. palmivora. They also can help accelerate research on future phytopathogen diagnostics and preventive interventions.

Scalable Rhodaelectro-Catalyzed Expedient Access to Seven-Membered Azepino[3,2,1- hi ]indoles via [5 + 2] C–H/N–H Annulation

Scalable Rhodaelectro-Catalyzed Expedient Access to Seven-Membered Azepino[3,2,1- <i>hi</i> ]indoles via [5 + 2] C–H/N–H Annulation

Methodology for Analysis of Peptide Consumption by Yeast during Fermentation of Enzymatic Protein Hydrolysate Supplemented Synthetic Medium Using UPLC-IMS-HRMS

Several studies have shown the ability of yeast to consume peptides as a nitrogen source in single-peptide containing media. However, a suitable and cost-effective methodology to study the utilization of peptides by yeast and other microorganisms in a complex peptide mixture has yet to be put forward. This article addresses this issue by presenting a screening methodology for tracking the consumption of peptides by yeast during alcoholic fermentation. As a peptide source, the methodology makes use of an in-house prepared peptide-mapped bovine serum albumin (BSA) proteolytic digest, which was applied to a synthetic grape must. The peptide uptake was analyzed using high-throughput ultra-high-pressure liquid chromatography coupled to data-independent acquisition-based ion mobility separation-enabled high-resolution mass spectrometry (UPLC-DIA-IMS-HRMS) analysis. The relative changes of abundance of 123 di- to hexapeptides were monitored and reported during fermentations with three commercial wine strains, demonstrating different uptake kinetics for individual peptides. Using the same peptide-mapped BSA hydrolysate, the applicability of an untargeted workflow was additionally assessed for peptide profiling in unelucidated matrixes. The comparison of the results from peptide mapping and untargeted analysis experiments highlighted the ability of untargeted analysis to consistently identify small molecular weight peptides on the length and amino acid composition. The proposed method, in combination with other analytical techniques, such as gene or protein expression analysis, can be a useful tool for different metabolic studies related to the consumption of complex nitrogen sources by yeast or other microorganisms.

Detecting Water Constituents Unique to Septic Tanks as a Wastewater Source in the Environment by Nontarget Analysis: South Florida's Deering Estate Rehydration Project Case Study.

The present study has generated a workflow based on nontarget analysis (NTA) with Compound Discoverer Ver 3.1 to characterize a set of source-discriminating compounds identified in water samples from locations in South Florida (USA), particularly those describing a freshwater environment (Everglades based), urban impacted areas (septic tank driven), and coastal (Biscayne Bay) endmembers in and around the Charles Deering Estate property in the Village of Palmetto Bay. Waters from an interconnected managed canal system were assessed to evaluate the influence of localized emissions. Septic tank effluents influence the water in many Southeast Florida environments due to their diminished onsite treatment capacity based on the limestone-dominated geology and canal systems providing a relatively unobstructed connection pathway. Through a combination of high-resolution mass spectrometry and statistical analyses, a set of tracers and indicators was determined (azelaic acid, decanophenone, galaxolidone, methyl violet, monoolein, metoprolol, and 1-stearoylglycerol). Tentatively identified compounds were generally assigned to various categories such as dyes, personal care products, and pharmaceuticals. The NTA Compound Discoverer Ver 3.1 compound data (presented as principal component analysis and Kendrick mass defect plots) showed apparent differences between wastewater-influenced sites and non-wastewater-influenced sites along with the ranked "Top10" compounds found at each location. Waters from different locations were also compared using the presence of sucralose to further inform the NTA. The most septic-influenced site contained 3594 ± 94 ng/L of sucralose with concentrations declining steadily and reaching the lowest concentrations in Biscayne Bay of 122 ± 94 ng/L. The sucralose concentrations provided further evidence of septic influence on this system. Sucralose was determined to be a conservative tracer between the freshwater and coastal sources and complementary to other probable unique tracers of septic tank effluent identified by the NTA. Environ Toxicol Chem 2022;41:1165-1178. © 2022 SETAC.

Seven new 2-(2-phenylethyl) chromone derivatives from agarwood of Aquilaria agallocha with inhibitory effects on nitric oxide production

Seven new 2-(2-Phenethyl) chromone derivatives (1–7), including four 2-(2-Phenethyl) chromones (1–4), one 6, 7, 8 trihydroxy-2-(2-Phenethyl) chromone (5), one acetylated 5, 6, 7, 8-tetrahydroxy-2-(2-Phenethyl) chromone (6), and one chlorine-containing 5, 6, 7, 8-tetrahydro-2-(2-Phenethyl) chromone (7), along with eight known compounds (8–15), were isolated from agarwood originating from Aquilaria agallocha Roxb.. Their structures were determined mainly by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR) analysis. The absolute configurations of 3–7 were resolved by electronic circular dichroism (ECD) calculations. Nearly all compounds were evaluated for their anti-inflammatory activities in RAW264.7 cells. Compounds 1 and 7–11 displayed significant anti-inflammatory activities with IC50 values ranging from 3.71 to 32.04 μM.

Non-targeted screening of extracts from polyester-phenolic can coatings: Identification of new aldehyde molecules from resole-based resins

Phenolic and substituted phenol based resoles are commonly used in the formulation of can coatings. However, migration analyses of these coatings are very little described compared to other coating technologies. While epoxy and polyester have well known migrants with defined formation mechanisms, Non-Intentionally Added Substances (NIAS) specifically related to the phenolic resin are hardly studied in the literature. The goal of the publication is to further explore the influence of the phenolic resole, used in the formulation of can coatings, on extracted NIAS's nature. Six different model polyester-phenolic can coatings were formulated each with a specific phenol, cresol or tertbutylphenol-based resole. Can coating films were extracted for 24 h at 40 °C in acetonitrile before analysis. NIAS identification was done using gas chromatography separation coupled to high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy analyses. Cyclic polyester oligomers were found in all extracts, with oligomers found in a range of 10 μg/dm2 to 226 μg/dm2, without specific influence of the resole used in formulation. While very few or no peaks were detected from cresol- and phenol-based resoles, 48 peaks were specifically observed in coating extracts of formulas with tertbutylphenol-based resoles as well as in their respective resoles. The most intense peaks were identified as aldehyde compounds by HRMS and NMR analysis. These aldehydes were semi-quantified in similar proportions as polyester oligomers. The presence of such aldehydes has never been reported in the literature regarding NIAS in can coatings. Further study will then be needed to better understand the aldehyde formation mechanism and assess the toxicological profile of such chemicals.

Candidate High-Resolution Mass Spectrometry-Based Reference Method for the Quantification of Procalcitonin in Human Serum Using a Characterized Recombinant Protein as a Primary Calibrator.

Procalcitonin (PCT) is a widely used biomarker for rapid sepsis diagnosis and antibiotic stewardship. Variability of results in commercial assays has highlighted the need for standardization of PCT measurements. An antibody-free candidate reference measurement procedure (RMP) based on the isotope dilution mass spectrometry and protein calibration approach was developed and validated to quantify PCT in human serum. The method allows quantification of PCT from 0.25 to 13.74 μg/L (R > 0.998) with extension up to 132 μg/L after dilution of samples with PCT concentration above 13.74 μg/L. Intraday bias was between −3.3 and +5.7%, and interday bias was between −3.0 and −0.7%. Intraday precision was below 5.1%, and interday precision was below 4.0%. The candidate RMP was successfully applied to the absolute quantification of PCT in five frozen human serum pools. A recombinant PCT used as a primary calibrator was characterized by high-resolution mass spectrometry and amino acid analysis to establish traceability of the results to the SI units. This candidate RMP is fit to assign target values to secondary certified reference materials (CRMs) for further use in external quality assessment schemes to monitor the accuracy and comparability of the commercially available immunoassay results and to confirm the need for improving the harmonization of PCT assays. The candidate RMP will also be used to evaluate whether the correlation between the candidate RMP and immunoassays is sufficiently high. Overall, this candidate RMP will support reliable sepsis diagnosis and guide treatment decisions, patient monitoring, and outcomes.

Polyketide Derivatives from the Endophytic Fungus Phaeosphaeria sp. LF5 Isolated from Huperzia serrata and Their Acetylcholinesterase Inhibitory Activities.

The secondary metabolites of Phaeosphaeria sp. LF5, an endophytic fungus with acetylcholinesterase (AChE) inhibitory activity isolated from Huperzia serrata, were investigated. Their structures and absolute configurations were elucidated by means of extensive spectroscopic data, including one- and two-dimensional nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analyses, and calculations of electronic circular dichroism (ECD). A chemical study on the solid-cultured fungus LF5 resulted in 11 polyketide derivatives, which included three previously undescribed derivatives: aspilactonol I (4), 2-(1-hydroxyethyl)-6-methylisonicotinic acid (7), and 6,8-dihydroxy-3-(1′R, 2′R-dihydroxypropyl)-isocoumarin (9), and two new natural-source-derived aspilactonols (G, H) (2, 3). Moreover, the absolute configuration of de-O-methyldiaporthin (11) was identified for the first time. Compounds 4 and 11 exhibited inhibitory activity against AChE with half maximal inhibitory concentration (IC50) values of 6.26 and 21.18 µM, respectively. Aspilactonol I (4) is the first reported furanone AChE inhibitor (AChEI). The results indicated that Phaeosphaeria is a good source of polyketide derivatives. This study identified intriguing lead compounds for further research and development of new AChEIs.

Adsorption of organic acids from offshore produced water using microporous activated carbon from babassu pericarp: a low-cost alternative

Currently, in different regions of the world, there is a growth on the rate of produced water for each barrel of oil extracted. Among the compounds observed in this water are naphthenic acids (NAs). Efforts to remove NAs from produced water must comply with local legislation and avoid damage to the environment since NAs have great toxicity. This study aimed to investigate the ability of activated carbon from the pericarp of babassu coconut (Atallea speciosa Mart. ex Spreng.) to act as an absorbent of NAs in commercial and natural (offshore) produced waters. The activated carbon was physically and chemically characterized. The maximum adsorption capacity of commercial NAs at equilibrium obtained experimentally was 25 mg g−1. The activated carbon removed both NAs and other contaminants for produced water, as revealed by infrared and NMR techniques. In high-resolution mass spectrometry analysis, the abundance of the acids in the produced water was in the C9 and C10 range, being favorable for the adsorbent with a microporous structure. For the produced water kinetics experiments a maximum amount of adsorbed compounds of 17.91 mg g−1 was reached, and the time to achieve equilibrium was approximately 180 min. Thus, the results show that activated charcoal from babassu coconut pericarp can be used as an adsorbent and can potentially compete with other adsorbents to remove NAs in an aqueous solution. Also, the separation process can be adopted at a low cost on platforms, complementing the operations of hydrocyclones and flotation.

Quantitation Overcoming Matrix Effects of Lipophilic Toxins in Mytilus galloprovincialis by Liquid Chromatography-Full Scan High Resolution Mass Spectrometry Analysis (LC-HR-MS).

The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels (Mytilus galloprovincialis), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary.

Neuroprotective potential of terpenoid-rich extracts from orange juice by-products obtained by pressurized liquid extraction.

Pressurized liquid extraction (PLE) conditions were optimized to improve the recovery of orange (Citrus sinensis) by-products terpenoids. The neuroprotective potential of the PLE extracts were tested against a set of in-vitro assay (antioxidant (ABTS), reactive oxygen/nitrogen species (ROS/RNS)) as well as enzymatic tests (acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and lipoxygenase (LOX)). Gas chromatography coupled to high-resolution mass spectrometry (GC-q-TOF-MS) analysis revealed a higher enrichment in mono- and sesquiterpenoids of the PLE extracts with the highest neuroprotection capacity. In-silico molecular docking analysis showed the specific interaction of representative terpenoids with enzymes active sites. The results demonstrate that the selected extract at 100°C and 30 minutes possesses high antioxidant (ABTSIC50=13.5μgmL-1; ROSIC50 = 4.4μgmL-1), anti-cholinesterase (AChEIC50=137.1 vg L-1; BChEIC50=147.0μgmL-1) and anti-inflammatory properties (against IL-6 and LOXIC50=76.1μgmL-1), with low cytotoxicity and protection against L-glutamic acid in cell models.

Visualization and identification of single meteoritic organic molecules by atomic force microscopy.

Using high‐resolution atomic force microscopy (AFM) with CO‐functionalized tips, we atomically resolved individual molecules from Murchison meteorite samples. We analyzed powdered Murchison meteorite material directly, as well as processed extracts that we prepared to facilitate characterization by AFM. From the untreated Murchison sample, we resolved very few molecules, as the sample contained mostly small molecules that could not be identified by AFM. By contrast, using a procedure based on several trituration and extraction steps with organic solvents, we isolated a fraction enriched in larger organic compounds. The treatment increased the fraction of molecules that could be resolved by AFM, allowing us to identify organic constituents and molecular moieties, such as polycyclic aromatic hydrocarbons and aliphatic chains. The AFM measurements are complemented by high‐resolution mass spectrometry analysis of Murchison fractions. We provide a proof of principle that AFM can be used to image and identify individual organic molecules from meteorites and propose a method for extracting and preparing meteorite samples for their investigation by AFM. We discuss the challenges and prospects of this approach to study extraterrestrial samples based on single‐molecule identification.

Multiclass and multi-residue screening of mycotoxins, pharmacologically active substances, and pesticides in infant milk formulas through ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry analysis

Infant milk formulas are designed to substitute human milk when breastfeeding is unavailable. In addition to human milk and milk-derived products, these formulas can be a vehicle of contaminants. In this work, a multiclass method based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach was developed for the simultaneous determination of contaminants (n = 45), including mycotoxins and veterinary drug residues, occurring in infant milk formulas. By using an ultra-high-performance liquid chromatography quadrupole-Orbitrap coupled with high-resolution mass spectrometry analysis (UHPLC-Q-Orbitrap HRMS; Thermo Fisher Scientific), further retrospective analysis of 337 contaminants, including pesticides, was achieved. The method was validated in accordance with European regulations and applied for the analysis of 54 infant milk samples. Risk assessment was also performed. Dexamethasone was detected in 16.6% of samples (range: 0.905-1.131 ng/mL), and procaine benzyl penicillin in 1 sample at a concentration of 0.295 ng/mL. Zearalenone was found in 55.5% of samples (range: 0.133-0.638 ng/mL) and α-zearalenol in 16.6% of samples (range: 1.534-10.408 ng/mL). Up to 49 pesticides, 11 veterinary drug residues, and 5 mycotoxins were tentatively identified via retrospective analysis based on the mass spectral library. These findings highlight the necessity of careful evaluation of contaminants in infant formulas, considering that they are intended for a vulnerable part of the population.

Anti-Pseudomonasaeruginosa activity of a C16-terpene dilactone isolated from the endophytic fungus Neofusicoccumluteum of Kigeliaafricana (Lam.)

Fungal endophytes have the capacity to biosynthesize secondary metabolites that are produced by their host plants. In this study, a dilactone terpenoid of C16 architecture was isolated from the fungal endophytes of Kigeliaafricana, in our attempt to identify anti-Pseudomonasaeruginosa metabolites. Thirty-eight fungal isolates were cultured for biomolecule production over a period of thirty days. Extracts from three (ZF 34, ZF 52 and ZF 91) of the fungi showed good anti-P.aeruginosa activity, with ZF 52 presenting the best MIC of 19.53 µg/mL and was accordingly subjected to chromatographic separation. Based on nuclear magnetic resonance (NMR) spectroscopy, high resolution mass spectrometry and single crystal X-ray diffraction (XRD) analyses, the isolated compound was identified as a C16-terpene dilactone, with a structure consistent with that of the known diterpene, CJ-14445. The isolated dilactone showed anti-P.aeruginosa activity with MIC of 0.61 µg/mL, signifying the antibacterial potential of the biomolecule. The bioactive fungal isolate (ZF 52) was identified as Neofusicoccumluteum based on genomic DNA sequencing. This is the first report of the endophyte N.luteum from K.africana and the first reported occurrence of CJ-14445 in the fungus.

Comparative secretome analysis of Staphylococcus aureus strains with different within-herd intramammary infection prevalence

ABSTRACT Staphylococcus aureus is a major pathogen causing intramammary infection and mastitis in dairy cows. S. aureus genotypes (GT) can differ significantly in their ability to diffuse and persist in the herd; while the association of virulence gene carriage with epidemiological behavior remains unclear, a role for secreted proteins has been postulated. We characterized the secretome of six S. aureus strains belonging to two genotypes with opposite within-herd prevalence, GTB (high) and GTS (low), corresponding to sequence types (ST) 8 and 398, by high-resolution tandem mass spectrometry and differential analysis with Proteome Discoverer. Data are available via ProteomeXchange with identifier PXD029571. Out of 720 identified proteins, 98 were unique or more abundant in GTB/ST8 and 68 in GTS/ST398. GTB/ST8 released more immunoglobulin-binding proteins, complement and antimicrobial peptide inhibitors, enterotoxins, and metabolic enzymes, while GTS/ST398 released more leukocidins, hemolysins, lipases, and peptidases. Furthermore, GTB/ST8 released the von Willebrand factor protein, staphylokinase, and clumping factor B, while GTS released the staphylococcal coagulase and clumping factor A. Hence, GTB/ST8 secretomes indicated a higher propensity for immune evasion and chronicity and GTS/ST398 secretomes for cellular damage and inflammation, consistent with their epidemiological characteristics. Accordingly, GTS/ST398 secretions were significantly more cytotoxic against bovine PBMCs in vitro. Our findings confirm the crucial role of extracellular virulence factors in S. aureus pathogenesis and highlight the need to investigate their differential release adding to gene carriage for a better understanding of the relationship of S. aureus genotypes with epidemiological behavior and, possibly, disease severity.