Advantages of Multiplexing Ability of the Orbitrap Mass Analyzer in the Multi-Mycotoxin Analysis.

In routine measurements, the length of the analysis time and nfumber of samples analysed during a time unit are crucial parameters, which are especially important for the food analysis, particularly in the case of mycotoxin determinations. High-resolution equipment, including time-of-flight or Orbitrap analyzators, can provide stable instrumental background for high-throughput analyses. In this report, a short, 1 min MS-based multi-mycotoxin method was developed with the application of a short column as a reduced chromatographic separation, taking advantages of the multiplexing and high-resolution capability of the QExactive Orbitrap MS possessing sub-1 ppm mass accuracy. The performance of the method was evaluated regarding selectivity, LOD, LOQ, linearity, matrix effect, and recovery, and compared to a UHPLC-MS/MS method. The final multiplexing method was able to quantify 11 mycotoxins in defined ranges (aflatoxins (corn, 2.8-600 µg/kg; wheat, 1.5-350 µg/kg), deoxynivalenol (corn, 640-9600 µg/kg; wheat, 128-3500 µg/kg), fumonisins (corn, 20-1500 µg/kg; wheat, 30-3500 µg/kg), HT-2 (corn, 64-5200 µg/kg; wheat, 61-3500 µg/kg), T-2 (corn, 10-800 µg/kg; wheat, 4-250 µg/kg), ochratoxin (corn, 4.7-600 µg/kg; wheat, 1-1000 µg/kg), zearalenone (corn, 64-4800 µg/kg; wheat, 4-500 µg/kg)) within one minute in corn and wheat matrices at the MRL levels stated by the European Union.

Comprehensive characterization of stress tolerant bacteria with plant growth-promoting potential isolated from glyphosate-treated environment.

The application of plant growth-promoting bacteria in agricultural systems is an efficient and environment-friendly strategy to improve crop yields and maintain soil quality. However, as different soils have diverse and specific ecological characteristics and may represent adverse abiotic conditions, in vivo application requires the careful selection of the desired beneficial microorganisms. In this study we report Ensifer adhaerens SZMC 25856 and Pseudomonas resinovorans SZMC 25875 isolates recovered from glyphosate-treated soil to possess yet undiscovered plant growth-enhancing potential. The strains were found to promote the growth of tomato seedlings significantly, to have the ability of synthesizing indole-3-acetic acid and siderophores, to tolerate pH in the range of 6.59-7.96, salinity up to 12.5 g L-1 NaCl and drought up to 125 g L-1 polyethylene glycol 6000, as well as to survive in the presence of various pesticides including glyphosate, diuron, chlorotoluron, carbendazim and thiabendazole, and heavy metals such as Al, Fe, Mn, Zn, Pb and Cu. The plant growth-promoting traits of the examined E. adhaerens and P. resinovorans isolates and their tolerance to numerous abiotic stress factors make them promising candidates for application in different agricultural environments, including soils polluted with glyphosate.

Verification of tuberculosis infection among Vác mummies (18th century CE, Hungary) based on lipid biomarker profiling with a new HPLC-HESI-MS approach.

Tuberculosis (TB) was a large burden of infections that peaked during the 19th century in Europe. Mummies from the 18th century CE, discovered in the crypt of a church at Vác, Hungary, had high TB prevalence, as revealed by amplification of key fragments of TB DNA and genome-wide TB analysis. Complementary methods are needed to confirm these diagnoses and one approach uses the identification of specific lipid biomarkers, such as TB mycocerosic acids (MCs). Previously, MC derivatives were profiled by specialised gas chromatography-mass spectrometry (GC-MS), so an alternative more direct approach has been developed. Underivatized MCs are extracted and analysed by high-performance liquid chromatography linked to a mass spectrometer, in heated electrospray ionisation mode (HPLC-HESI-MS). The method was validated using representatives of the Mycobacterium tuberculosis complex and other mycobacteria and tested on six Vác mummy cases, previously considered positive for TB infection. Analysing both rib and soft tissue samples, four out of six cases gave profiles of main C32 and major C29 and C39 mycocerosates correlating well with those of M. tuberculosis. Multidisciplinary methods are needed in the diagnosis of ancient tuberculosis; this new protocol accesses important confirmatory evidence, as demonstrated by the confirmation of TB in the Vác mummies.

Candida albicans Enhances the Progression of Oral Squamous Cell Carcinoma In Vitro and In Vivo.

Oral squamous cell carcinoma (OSCC) is associated with oral Candida albicans infection, although it is unclear whether the fungus promotes the genesis and progression of OSCC or whether cancer facilitates fungal growth. In this study, we investigated whether C. albicans can potentiate OSCC tumor development and progression. In vitro, the presence of live C. albicans, but not Candida parapsilosis, enhanced the progression of OSCC by stimulating the production of matrix metalloproteinases, oncometabolites, protumor signaling pathways, and overexpression of prognostic marker genes associated with metastatic events. C. albicans also upregulated oncogenes in nonmalignant cells. Using a newly established xenograft in vivo mouse model to investigate OSCC-C. albicans interactions, oral candidiasis enhanced the progression of OSCC through inflammation and induced the overexpression of metastatic genes and significant changes in markers of the epithelial-mesenchymal transition. Finally, using the 4-nitroquinoline 1-oxide (4NQO) murine model, we directly correlate these in vitro and short-term in vivo findings with the progression of oncogenesis over the long term. Taken together, these data indicate that C. albicans upregulates oncogenes, potentiates a premalignant phenotype, and is involved in early and late stages of malignant promotion and progression of oral cancer. IMPORTANCE Oral squamous cell carcinoma (OSCC) is a serious health issue worldwide that accounts for 2% to 4% of all cancer cases. Previous studies have revealed a higher yeast carriage and diversity in oral cancer patients than in healthy individuals. Furthermore, fungal colonization in the oral cavity bearing OSCC is higher on the neoplastic epithelial surface than on adjacent healthy surfaces, indicating a positive association between oral yeast carriage and epithelial carcinoma. In addition to this, there is strong evidence supporting the idea that Candida contributes to carcinogenesis events in the oral cavity. Here, we show that an increase in Candida albicans burden promotes an oncogenic phenotype in the oral cavity.

Bile accelerates carcinogenic processes in pancreatic ductal adenocarcinoma cells through the overexpression of MUC4.

Pancreatic cancer (PC) is one of the leading causes of mortality rate globally and is usually associated with obstructive jaundice (OJ). Up to date, there is no clear consensus on whether biliary decompression should be performed prior to surgery and how high levels of serum bile affects the outcome of PC. Therefore, our study aims were to characterise the effect of bile acids (BAs) on carcinogenic processes using pancreatic ductal adenocarcinoma (PDAC) cell lines and to investigate the underlying mechanisms. Liquid chromatography-mass spectrometry was used to determine the serum concentrations of BAs. The effects of BAs on tumour progression were investigated using different assays. Mucin expressions were studied in normal and PDAC cell lines and in human samples at gene and protein levels and results were validated with gene silencing. The levels of BAs were significantly higher in the PDAC + OJ group compared to the healthy control. Treating PDAC cells with different BAs or with human serum obtained from PDAC + OJ patients enhanced the rate of proliferation, migration, adhesion, colony forming, and the expression of MUC4. In PDAC + OJ patients, MUC4 expression was higher and the 4-year survival rate was lower compare to PDAC patients. Silencing of MUC4 decreased BAs-induced carcinogenic processes in PDAC cells. Our results show that BAs promote carcinogenic process in PDAC cells, in which the increased expression of MUC4 plays an important role. Based on these results, we assume that in PC patients, where the disease is associated with OJ, the early treatment of biliary obstruction improves life expectancy.

Corrigendum: Structural Diversity and Bioactivities of Peptaibol Compounds From the Longibrachiatum Clade of the Filamentous Fungal Genus Trichoderma.

[This corrects the article DOI: 10.3389/fmicb.2019.01434.].

Characterization of the Plant Growth-Promoting Activities of Endophytic Fungi Isolated from Sophora flavescens.

Endophytic fungi in symbiotic association with their host plant are well known to improve plant growth and reduce the adverse effects of both biotic and abiotic stresses. Therefore, fungal endophytes are beginning to receive increased attention in an effort to find growth-promoting strains that could be applied to enhance crop yield and quality. In our study, the plant growth-promoting activities of endophytic fungi isolated from various parts of Sophora flavescens (a medicinally important plant in Mongolia and China) have been revealed and investigated. Fungal isolates were identified using molecular taxonomical methods, while their plant growth-promoting abilities were evaluated in plate assays. Altogether, 15 strains were isolated, representing the genera Alternaria, Didymella, Fusarium and Xylogone. Five of the isolates possessed phosphate solubilization activities and twelve secreted siderophores, while all of them were able to produce indoleacetic acid (IAA) in the presence or absence of tryptophan. The endogenous and exogenous accumulation of IAA were also monitored in liquid cultures using the HPLC-MS/MS technique to refine the plate assay results. Furthermore, for the highest IAA producer fungi, the effects of their extracts were also examined in plant bioassays. In these tests, the primary root lengths of the model Arabidopsis thaliana were increased in several cases, while the biomasses were significantly lower than the control IAA treatment. Significant alterations have also been detected in the photosynthetic pigment (chlorophyll-a, -b and carotenoids) content due to the fungal extract treatments, but these changes did not show any specific trends.

Wild Strawberry, Blackberry, and Blueberry Leaf Extracts Alleviate Starch-Induced Hyperglycemia in Prediabetic and Diabetic Mice.

Intestinal α-glucosidase and α-amylase break down nutritional poly- and oligosaccharides to monosaccharides and their activity significantly contributes to postprandial hyperglycemia. Competitive inhibitors of these enzymes, such as acarbose, are effective antidiabetic drugs, but have unpleasant side effects. In our ethnopharmacology inspired investigations, we found that wild strawberry (Fragaria vesca), blackberry (Rubus fruticosus), and European blueberry (Vaccinium myrtillus) leaf extracts inhibit α-glucosidase and α-amylase enzyme activity in vitro and are effective in preventing postprandial hyperglycemia in vivo. Toxicology tests on H9c2 rat embryonic cardiac muscle cells demonstrated that berry leaf extracts have no cytotoxic effects. Oral administration of these leaf extracts alone or as a mixture to normal (control), obese, prediabetic, and streptozotocin-induced diabetic mice attenuated the starch-induced rise of blood glucose levels. The efficiency was similar to that of acarbose on blood glucose. These results highlight berry leaf extracts as candidates for testing in clinical trials in order to assess the clinical significance of their effects on glycemic control.

Structural Diversity and Bioactivities of Peptaibol Compounds From the Longibrachiatum Clade of the Filamentous Fungal Genus Trichoderma.

This study examined the structural diversity and bioactivity of peptaibol compounds produced by species from the phylogenetically separated Longibrachiatum Clade of the filamentous fungal genus Trichoderma, which contains several biotechnologically, agriculturally and clinically important species. HPLC-ESI-MS investigations of crude extracts from 17 species of the Longibrachiatum Clade (T. aethiopicum, T. andinense, T. capillare, T. citrinoviride, T. effusum, T. flagellatum, T. ghanense, T. konilangbra, T. longibrachiatum, T. novae-zelandiae, T. pinnatum, T. parareesei, T. pseudokoningii, T. reesei, T. saturnisporum, T. sinensis, and T. orientale) revealed several new and recurrent 20-residue peptaibols related to trichobrachins, paracelsins, suzukacillins, saturnisporins, trichoaureocins, trichocellins, longibrachins, hyporientalins, trichokonins, trilongins, metanicins, trichosporins, gliodeliquescins, alamethicins and hypophellins, as well as eight 19-residue sequences from a new subfamily of peptaibols named brevicelsins. Non-ribosomal peptide synthetase genes were mined from the available genome sequences of the Longibrachiatum Clade. Their annotation and product prediction were performed in silico and revealed full agreement in 11 out of 20 positions regarding the amino acids predicted based on the signature sequences and the detected amino acids incorporated. Molecular dynamics simulations were performed for structural characterization of four selected peptaibol sequences: paracelsins B, H and their 19-residue counterparts brevicelsins I and IV. Loss of position R6 in brevicelsins resulted in smaller helical structures with higher atomic fluctuation for every residue than the structures formed by paracelsins. We observed the formation of highly bent, almost hairpin-like, helical structures throughout the trajectory, along with linear conformation. Bioactivity tests were performed on the purified peptaibol extract of T. reesei on clinically and phytopathologically important filamentous fungi, mammalian cells, and Arabidopsis thaliana seedlings. Porcine kidney cells and boar spermatozoa proved to be sensitive to the purified peptaibol extract. Peptaibol concentrations ≥0.3 mg ml-1 deterred the growth of A. thaliana. However, negative effects to plants were not detected at concentrations below 0.1 mg ml-1, which could still inhibit plant pathogenic filamentous fungi, suggesting that those peptaibols reported here may have applications for plant protection.

Host metabolite producing endophytic fungi isolated from Hypericum perforatum.

In the present study, endophytic fungi have been isolated from various parts of the medicinal herb Hypericum perforatum (St. John's Wort), which is known as a source of medically important metabolites. The isolated strains were cultured in liquid media and their ability to synthesize hypericin, the secondary metabolite of the host and its suspected precursor, emodin was tested analyzing the extracts of the fermentation broth and the mycelia. The HPLC-UV analysis of the chloroform/methanol extracts of the mycelia revealed that three isolates were able to produce emodin (SZMC 23771, 19.9 ng/mg; SZMC 23772, 20.8 ng/mg; SZMC 23769, 427.9 ng/mg) and one of them also could synthesize hypericin (SZMC 23769, 320.4 ng/mg). These results were also confirmed via UHPLC-HRMS technique both in full scan and MS/MS mode. The strains producing only emodin belong to the section Alternata of the genus Alternaria, while the isolate producing both metabolites was identified as Epicoccum nigrum. The mycelial extracts of E. nigrum and the Alternaria sp. SZMC 23772 showed higher inhibitory activities in the antimicrobial tests against the six selected bacteria compared to the hypericin and emodin standards in the applied concentration (100 µg/mL), while in case of the Alternaria sp. SZMC 23771 lower inhibition activities were observed on Staphylococcus aureus and Streptomyces albus than the pure compounds.

New 19-Residue Peptaibols from Trichoderma Clade Viride.

Trichoderma koningiopsis and T. gamsii belong to clade Viride of Trichoderma, the largest and most diverse group of this genus. They produce a wide range of bioactive secondary metabolites, including peptaibols with antibacterial, antifungal, and antiviral properties. The unusual amino acid residues of peptaibols, i.e., α-aminoisobutyric acid (Aib), isovaline (Iva), and the C-terminal 1,2-amino alcohol make them unique among peptides. In this study, the peptaibiomes of T. koningiopsis and T. gamsii were investigated by HPLC-ESI-MS. The examined strains appeared to produce 19-residue peptaibols, most of which are unknown from literature, but their amino acid sequences are similar to those of trikoningins, tricholongins, trichostrigocins, trichorzianins, and trichorzins. A new group of peptaibols detected in T. koningiopsis are described here under the name "Koningiopsin". Trikoningin KA V, the closest peptaibol compound to the peptaibols produced by these two strains, was selected for structural investigation by short MD simulation, which revealed that many residues show high preference for left handed helix formation. The bioactivity of the peptaibol mixtures produced by T. koningiopsis and T. gamsii was tested on agar plates against bacteria, yeasts, and filamentous fungi. The results revealed characteristic differences in bioactivities towards the different groups of target microorganisms, which can be explained with the differences in their cell wall structures.