Specific Avenin Cross-Reactivity with G12 Antibody in a Wide Range of Current Oat Cultivars.

Current clinical studies confirm that the consumption of oats for people suffering from celiac disease is safe. Some studies have confirmed different levels of immunoreactive gluten epitopes of oats in different cultivars, while others explain these differences due to contamination with gluten-rich species or as random cross-reactivity ELISA of homologous oat epitopes with anti-wheat gliadin antibodies. The aim of our two-year study was therefore to map cross-reactive oat epitopes in a set of 132 oat cultivars using a G12-based ELISA kit. The results were focused on the varietal and annual level of cross-reactivity (interference) of avenin epitopes with the G12 antibody on the identification of potential cultivars with significantly different interferences and assessing the degree of risk of possible false-contamination with external gluten. Although repeated evaluations confirmed high year-to-year variability (RSD ≥ 30%) in approximately 2/3 of the cultivars, the content of interfering avenin epitopes with G12 did not exceed the considered safe limit (20 mg·kg-1) for celiacs. At the same time, not only annual but, above all, significant cultivar dependences in the interference of avenins to the G12 antibody were demonstrated. Genetic dependence was further confirmed in connection with the proven avenin polymorphism as well as immunoblotting with the identification of interfering peptides with the G12 antibody in the 25 and 30 kDa regions. It was the occurrence of two bands around 30 kDa that predominantly occurred in oat cultivars with a relatively higher content of cross-reactive avenins (12-16 mg·kg-1). Due to the fact that the contents of interfering avenins ranged in several cultivars even over 16 mg·kg-1, the choice of a suitable oat cultivar may be crucial for gluten-free food producers, as it reduces the risk of a possible false-response of the commercial ELISA kits when checking the real-gluten contamination.

Genetic characterization of nodular worm infections in Asian Apes.

Parasitic nematodes of Oesophagostomum spp., commonly known, as 'nodular worms' are emerging as the most widely distributed and prevalent zoonotic nematodes. Oesophagostomum infections are well documented in African non-human primates; however, the taxonomy, distribution and transmission of Oesophagostomum in Asian non-human primates are not adequately studied. To better understand which Oesophagostomum species infect Asian non-human primates and determine their phylogeny we analysed 55 faecal samples from 50 orangutan and 5 gibbon individuals from Borneo and Sumatra. Both microscopy and molecular results revealed that semi-wild animals had higher Oesophagostomum infection prevalence than free ranging animals. Based on sequence genotyping analysis targeting the Internal transcribed spacer 2 of rDNA, we report for the first time the presence of O. aculeatum in Sumatran apes. Population genetic analysis shows that there is significant genetic differentiation between Bornean and Sumatran O. aculeatum populations. Our results clearly reveal that O. aculeatum in free-ranging animals have a higher genetic variation than those in semi-wild animals, demonstrating that O. aculeatum is circulating naturally in wildlife and zoonotic transmission is possible. Further studies should be conducted to better understand the epidemiology and dynamics of Oesophagostomum transmission between humans, non-human primates and other wild species and livestock in Southeast Asia.

Optimised method for determination hypoglycine A in maple plant material by multidimensional gas chromatography/mass spectrometry.

In 2018, more than 50 cases of horse death by equine atypical myopathy (AM) were reported in the Czech Republic. This disease is often associated with the toxin hypoglycine A (HGA), which is found in several maple plant materials. To monitor this toxin in products of these trees that grow in or around horse pastures, a rapid and inexpensive analytical method that can provide the required accuracy is needed. Until now, maple samples have been prepared for gas chromatography using time-consuming methods, with preparation processes taking longer than 1 h. In this work, a shorter method (25 min) with an accuracy of 90-94 %, reproducibility of 2-5%, precision of 3-9%, and linearity, with an R2 of 0.999, is presented. This sample preparation consists of a procedure without an SPE extraction step and consumes a lower volume of solvent during the extraction. The limit of quantitation for HGA in plant material was improved from 0.5 µg/g of plant material in previous studies to 0.2 µg/g. The method was validated according to the guideline CD 2002/657/EC and ISO 17025, and was found to have good performance characteristics. This simple and rapid method was tested for the monitoring of hypoglycine A level in maple sycamore plant material (seeds, seedlings, and leaves) during the entire growth of the trees.

Comprehensive analysis of oat avenanthramides using hybrid quadrupole-Orbitrap mass spectrometry: Possible detection of new compounds.

RATIONALE: Avenanthramides (AVNs) are constituents unique to oats and have many outstanding health benefits. AVNs are antioxidants and possess anti-inflammatory, antifungal and antibacterial activity. The number of known AVNs increased recently because of the latest developments in high-resolution tandem mass spectrometry (HRMS/MS) techniques. METHODS: Oat seed extract from 10 oat cultivars was analysed using ultra-high-performance liquid chromatography (UHPLC) and Q Exactive hybrid quadrupole-Orbitrap mass spectrometry (HRMS/MS) with positive heated electrospray ionization. RESULTS: Thirty-five AVNs were identified and characterized in seed extracts, and the structures of 10 novel AVNs were tentatively elucidated, among which were AVNs bearing a cinamoyl or sinapoyl moiety. These AVNs are reported in oats for the first time. The method was validated using AVN standards (AVNs 2c, 2f and 2p), with limits of detection and quantitation at low picomole levels. Recovery of AVN standards varied from 83% to 106%, and relative standard deviations ranged from 2% to 9%. The total AVNs in the selected oat varieties ranged from 36.0 to 302.5 µg/g (dry weight), with AVN 2c, AVN 2f and AVN 2p representing approximately 65%-70% of that total. CONCLUSIONS: Our comprehensive method for detecting the full avenanthramide spectrum can contribute to better understanding the chemical and biological properties of individual AVNs for utilization in developing new oat cultivars and novel functional foods.

Insights into the molecular systematics of Trichuris infecting captive primates based on mitochondrial DNA analysis.

Nematodes belonging to the Trichuris genus are prevalent soil-transmitted helminths with a worldwide distribution in mammals, while humans are mainly affected in areas with insufficient sanitation such as in Africa, Asia and South America. Traditionally, whipworms infecting primates are referred to Trichuris trichiura, but recent molecular and morphological evidence suggests that more than one species may be able to infect humans and non-human primates. Here, we analyzed the genetic diversity and phylogeny of Trichuris infecting five different non-human primate species kept in captivity using sequencing of three mitochondrial genes (cox1, rrnL and cob). Phylogenetic analyses of both single and concatenated datasets suggested the presence of two main evolutionary lineages and several highly supported clades likely existing as separate taxa. The first lineage included Trichuris infecting the mantled guereza (Colobus guereza kikuyensis), the chacma baboon (Papio ursinus) and the green monkeys (Chlorocebus spp.), clustering together with Trichuris suis; the second lineage included Trichuris infecting the Japanese macaque (Macaca fuscata) and the hamadryas baboon (Papio hamadryas), clustering together with Trichuris spp. infecting humans. These results were supported by the genetic distance between samples, which suggested that at least two taxa are able to infect macaques, baboons and humans. The present study improves our understanding of the taxonomy and evolutionary relationships among Trichuris spp. infecting primates. It moreover suggests that multiple Trichuris spp. may circulate among host species and that Trichuris in non human primates (NHPs) may be zoonotic. Further studies are important to better understand the epidemiology of Trichuris in primates and for implementing appropriate control and/or conservation measures.

Fur color change and hormonal development in captive females of northern white-cheeked (Nomascus leucogenys) and buff-cheeked (Nomascus gabriellae) gibbons.

Gibbons of the genus Nomascus exhibit strong sexual dichromatism in fur color. Change of fur color in sub-adult wild Nomascus females is associated with the onset of puberty and the time of their dispersal. The variability in fur change may be influenced by social factors. In this study, we determined whether in captive females of crested gibbons begin reproductive maturity prior to dispersing and with association to their fur color. We collected 287 fecal extracts to analyze pregnandiol -3- glucuronide and 17ß estradiol profiles of 4 sub-adult females (Nomascus leucogenys and Nomascus gabriellae) and 183 samples from their mothers, using enzyme immunoassays. The sub-adult females were monitored from 4 years of age. Their hormone profiles showed the onset of ovulatory cycling between 4.6 and 5.8 years. Based on the information about the estrogen influence to the secondary sex characteristic (fur color of female) the positive link between estrogen concentration and age of the sub-adult females was found. However, the amount of the estrogen can apparently be influenced by the presence of mother. If the mother was presented, the level of estrogen was higher than if the mother was missing. Our findings suggest that the probability of changing to beige fur color by the sub-adult females increased with increased age and if they were without mother. This initial study presents the maternal influence as a possible social factor affecting the fur color change of female offspring.

Sphingolipidomics of Thermotolerant Yeasts.

Mass spectrometry-based shotgun lipidomics was applied to the analysis of sphingolipids of 11 yeast strains belonging to four genera, that is Cryptococcus, Saccharomyces, Schizosaccharomyces, and Wickerhamomyces. The analysis yielded comprehensive results on both qualitative and quantitative representation of complex sphingolipids of three classes-phosphoinositol ceramide (PtdInsCer), mannosyl inositol phosphoceramide (MInsPCer), and mannosyl diinositol phosphoceramide (M(InsP)2 Cer). In total, nearly 150 molecular species of complex sphingolipids were identified. Individual strains were cultured at five different temperatures, that is 5, 10, 20, 30, and 40 °C (Wickerhamomyces genus only up to 30 °C), and the change in the culture temperature was found to affect the representation of both the sphingolipid classes and the length of the long-chain bases (LCB). Individual classes of sphingolipids differing in polar heads differed in the temperature response. The relative content of PtdInsCer increased with increasing temperature, whereas that of M(InsP)2 Cer decreased. Molecular species having C18-LCB were associated with low cultivation temperature, and a higher proportion of C20-LCB molecular species was produced at higher temperatures regardless of the type of polar head. On the other hand, the influence of temperature on the representation of very long-chain fatty acids (VLCFA) was less noticeable, the effect of the taxonomic affiliation of the strains being more pronounced than the cultivation temperature. For example, lignoceric and 2-hydrocylo-lignoceric acids were characteristic of the genera Cryptococcus and Schizosaccharomyces, and of Saccharomyces genus cultivated at high temperatures.

Resveratrol suppresses ethanol stress in winery and bottom brewery yeast by affecting superoxide dismutase, lipid peroxidation and fatty acid profile.

Mid-exponential cultures of two traditional biotechnological yeast species, winery Saccharomyces cerevisiae and the less ethanol tolerant bottom-fermenting brewery Saccharomyces pastorianus, were exposed to different concentrations of added ethanol (3, 5 and 8%) The degree of ethanol-induced cell stress was assessed by measuring the cellular activity of superoxide dismutase (SOD), level of lipid peroxidation products, changes in cell lipid content and fatty acid profile. The resveratrol as an antioxidant was found to decrease the ethanol-induced rise of SOD activity and suppress the ethanol-induced decrease in cell lipids. A lower resveratrol concentration (0.5 mg/l) even reduced the extent of lipid peroxidation in cells. Resveratrol also alleviated ethanol-induced changes in cell lipid composition in both species by strongly enhancing the proportion of saturated fatty acids and contributing thereby to membrane stabilization. Lower resveratrol concentrations could thus diminish the negative effects of ethanol stress on yeast cells and improve their physiological state. These effects may be utilized to enhance yeast vitality in high-ethanol-producing fermentations or to increase the number of yeast generations in brewery.

Blood parasites in northern goshawk (Accipiter gentilis) with an emphasis to Leucocytozoon toddi.

Haemosporidians and trypanosomes of the northern goshawk (Accipiter gentilis) population in the Czech Republic were studied by morphological and molecular methods. Despite the wide distribution of these medium-large birds of prey, virtually nothing is known about their blood parasites. During a 5-year period, altogether 88 nestlings and 15 adults were screened for haemosporidians and trypanosomes by microscopic examination of blood smears and by nested PCR. Both methods revealed consistently higher prevalence of blood protists in adults, Leucocytozoon (80.0 % in adults vs. 13.6 % in nestlings), Haemoproteus (60.0 vs. 2.3 %), Plasmodium (6.7 vs. 0 %), and Trypanosoma (60.0 vs. 2.3 %). Altogether, five haemosporidian lineages were detected by cytochrome b sequencing. Two broadly distributed and host nonspecific lineages, Plasmodium (TURDUS1) and Leucocytozoon (BT2), were detected only sporadically, while three newly described northern goshawk host-specific Leucocytozoon lineages (ACGE01-03) represent the absolute majority of the haemosporidians identified by molecular methods. Our findings support evidences that in falconiform birds the Leucocytozoon toddi group is formed by several host-specific clusters, with Leucocytozoon buteonis in buzzards and Leucocytozoon mathisi in hawks. Between-year comparisons revealed that the infection status of adults remained predominantly unchanged and individuals stayed uninfected or possessed the same parasite lineages; however, two gains and one loss of blood parasite taxa were also recorded.

Molecular phylogeny of anoplocephalid tapeworms (Cestoda: Anoplocephalidae) infecting humans and non-human primates.

Anoplocephalid tapeworms of the genus Bertiella Stiles and Hassall, 1902 and Anoplocephala Blanchard, 1848, found in the Asian, African and American non-human primates are presumed to sporadic ape-to-man transmissions. Variable nuclear (5.8S-ITS2; 28S rRNA) and mitochondrial genes (cox1; nad1) of isolates of anoplocephalids originating from different primates (Callicebus oenanthe, Gorilla beringei, Gorilla gorilla, Pan troglodytes and Pongo abelii) and humans from various regions (South America, Africa, South-East Asia) were sequenced. In most analyses, Bertiella formed a monophyletic group within the subfamily Anoplocephalinae, however, the 28S rRNA sequence-based analysis indicated paraphyletic relationship between Bertiella from primates and Australian marsupials and rodents, which should thus be regarded as different taxa. Moreover, isolate determined as Anoplocephala cf. gorillae from mountain gorilla clustered within the Bertiella clade from primates. This either indicates that A. gorillae deserves to be included into the genus Bertiella, or, that an unknown Bertiella species infects also mountain gorillas. The analyses allowed the genetic differentiation of the isolates, albeit with no obvious geographical or host-related patterns. The unexpected genetic diversity of the isolates studied suggests the existence of several Bertiella species in primates and human and calls for revision of the whole group, based both on molecular and morphological data.