Application of microsatellite markers to parentage studies in grapevine

The use of microsatellites in genetic analysis does not only allow differentiation but also identification and parentage analysis of grapevine cultivars. Many of the cultivars which are of great economic importance, like Cabernet Sauvignon, have been selected and propagated centuries ago and often lack reliable documentation about their origins. In our study, 51 grapevine cultivars were genotyped at 24 microsatellite loci and searched for possible parent-offspring combinations. Our data confirm the origin of Cabernet Sauvignon from a cross between Cabernet franc and Sauvignon blanc. Furthermore we proved the parentage of the cultivars Neuburger (Silvaner x Veltliner rot), Blauburger (Portugieser blau x Blaufränkisch), Zweigelt (Blaufränkisch x St. Laurent) and Müller-Thurgau (Rheinriesling x Chasselas de Courtillier) at 24 SSR loci

Mycotoxin exposure and human cancer risk : a systematic review of epidemiological studies

In recent years, there has been an increasing interest in investigating the carcinogenicity of mycotoxins in humans. This systematic review aims to provide an overview of data linking exposure to different mycotoxins with human cancer risk. Publications (2019 and earlier) of case–control or longitudinal cohort studies were identified in PubMed and EMBASE. These articles were then screened by independent reviewers and their quality was assessed according to the Newcastle–Ottawa scale. Animal, cross‐sectional, and molecular studies satisfied criteria for exclusion. In total, 14 articles were included: 13 case–control studies and 1 longitudinal cohort study. Included articles focused on associations of mycotoxin exposure with primary liver, breast, and cervical cancer. Overall, a positive association between the consumption of aflatoxin‐contaminated foods and primary liver cancer risk was verified. Two case–control studies in Africa investigated the relationship between zearalenone and its metabolites and breast cancer risk, though conflicting results were reported. Two case–control studies investigated the association between hepatocellular carcinoma and fumonisin B1 exposure, but no significant associations were observed. This systematic review incorporates several clear observations of dose‐dependent associations between aflatoxins and liver cancer risk, in keeping with IARC Monograph conclusions. Only few human epidemiological studies investigated the associations between mycotoxin exposures and cancer risk. To close this gap, more in‐depth research is needed to unravel evidence for other common mycotoxins, such as deoxynivalenol and ochratoxin A. The link between mycotoxin exposures and cancer risk has mainly been established in experimental studies, and needs to be confirmed in human epidemiological studies to support the evidence‐based public health strategies

Enzymatic surface hydrolysis of PET : effect of structural diversity on kinetic properties of cutinases from thermobifida

In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their kcat and Km values on pNP–acetate were in the ranges 2.4–211.9 s–1 and 127–200 μM while on pNP–butyrate they showed kcat and Km values between 5.3 and 195.1 s–1 and between 1483 and 2133 μM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A1340/A1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.This study was performed within the Austrian Centre of Industrial Biotechnology ACIB, the MacroFun project and COST Action 868. This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. Financial support was also given from Sachsisches Staatsministerium fur Umwelt und Landwirtschaft, Germany. PET was kindly provided by Dr. Vincent Nierstrasz from Ghent University

A Dual-Approach Strategy to Optimize the Safety and Efficacy of Anti-Zika Virus Monoclonal Antibody Therapeutics.

Antibody-dependent enhancement of infection (ADE) is clinically relevant to Dengue virus (DENV) infection and poses a major risk to the application of monoclonal antibody (mAb)-based therapeutics against related flaviviruses such as the Zika virus (ZIKV). Here, we tested a two-tier approach for selecting non-cross-reactive mAbs combined with modulating Fc glycosylation as a strategy to doubly secure the elimination of ADE while preserving Fc effector functions. To this end, we selected a ZIKV-specific mAb (ZV54) and generated three ZV54 variants using Chinese hamster ovary cells and wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants as production hosts (ZV54CHO, ZV54WT, and ZV54ΔXF). The three ZV54 variants shared an identical polypeptide backbone, but each exhibited a distinct Fc N-glycosylation profile. All three ZV54 variants showed similar neutralization potency against ZIKV but no ADE activity for DENV infection, validating the importance of selecting the virus/serotype-specific mAbs for avoiding ADE by related flaviviruses. For ZIKV infection, however, ZV54CHO and ZV54ΔXF showed significant ADE activity while ZV54WT completely forwent ADE, suggesting that Fc glycan modulation may yield mAb glycoforms that abrogate ADE even for homologous viruses. In contrast to the current strategies for Fc mutations that abrogate all effector functions along with ADE, our approach allowed the preservation of effector functions as all ZV54 glycovariants retained antibody-dependent cellular cytotoxicity (ADCC) against the ZIKV-infected cells. Furthermore, the ADE-free ZV54WT demonstrated in vivo efficacy in a ZIKV-infection mouse model. Collectively, our study provides further support for the hypothesis that antibody-viral surface antigen and Fc-mediated host cell interactions are both prerequisites for ADE, and that a dual-approach strategy, as shown herein, contributes to the development of highly safe and efficacious anti-ZIKV mAb therapeutics. Our findings may be impactful to other ADE-prone viruses, including SARS-CoV-2

Narrow genetic base in forest restoration with holm oak (Quercus ilex L.) in Sicily

In order to empirically assess the effect of actual seed sampling strategy on genetic diversity of holm oak (Quercus ilex) forestations in Sicily, we have analysed the genetic composition of two seedling lots (nursery stock and plantation) and their known natural seed origin stand by means of six nuclear microsatellite loci. Significant reduction in genetic diversity and significant difference in genetic composition of the seedling lots compared to the seed origin stand were detected. The female and the total effective number of parents were quantified by means of maternity assignment of seedlings and temporal changes in allele frequencies. Extremely low effective maternity numbers were estimated (Nfe $\approx$ 2-4) and estimates accounting for both seed and pollen donors gave also low values (Ne $\approx$ 35-50). These values can be explained by an inappropriate forestry seed harvest strategy limited to a small number of spatially close trees

Safety of astaxanthin for its use as a novel food in food supplements

Following a request from the European Commission, the Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the safety of astaxanthin when used as a novel food in food supplements at maximum levels of 8 mg/day, taking into account the overall cumulative intake of astaxanthin from all food sources. In 2014, the NDA Panel assessed the safety of the novel astaxanthin-rich ingredient derived from microalgae Haematococcus pluvialis in the context of an application submitted under Regulation (EC) No 258/1997. In that opinion, the NDA Panel considered that the acceptable daily intake (ADI) for astaxanthin was 0.034 mg/kg body weight (bw) set by the EFSA FEEDAP Panel in 2014. In 2019, the FEEDAP Panel adopted an opinion which concerned the renewal of the authorisation of dimethyldisuccinate-astaxanthin and a new use of the additive for crustaceans and other fish than salmonids. In that assessment, the FEEDAP Panel derived a new ADI of 0.2 mg astaxanthin/kg bw which replaced the ADI of 0.034 mg/kg bw established in 2014. By taking into account an updated exposure assessment for astaxanthin from the background diet (fish and crustaceans) in combination with 8 mg from food supplements, the NDA Panel concludes that (i) such combined exposure to astaxanthin is safe for adults, (ii) 14 to < 18 years old adolescents reach the ADI, and (iii) the ADI is exceeded by 28% in children aged 10 to < 14 years and up to 524% in infants aged 4–6 months

Safety of vitamin D2 mushroom powder as a novel food pursuant to Regulation (EU) 2015/2283

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver a scientific opinion on vitamin D2 mushroom powder as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is an ingredient produced from Agaricus bisporus mushrooms that have been exposed to ultraviolet (UV) light to induce the conversion of provitamin D2 (ergosterol) to vitamin D2 (ergocalciferol). The NF contains concentrations of vitamin D provided by vitamin D2 in the ranges of 1,000\u20131,300 \u3bcg/g. The information provided on the manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant intends to add the NF in a variety of foods and beverages, including food for special medical purposes and food supplements. The target population is the general population except for food supplements, for which the target population is individuals above seven months of age. The Panel concludes that the NF, used as an ingredient, is safe for the general population at the proposed condition of use in foods and beverages and that the NF used as a food supplement, is safe for individuals above 1 year. The Panel, however, notes that the UL for infants aged 0\u20136 months may be exceeded in high consumers of infant formula (IF) and/or follow-on formula (FoF) that may also be high consumers of foods fortified with the NF and for infants aged 7\u201312 months consuming a daily vitamin D oral supplementation of 10 \u3bcg. However, the Panel considers this scenario unlikely as complementary feeding in high consumers of IF and/or FoF may be limited. Furthermore, the combined consumption of vitamin D via fortified foods and supplements does not specifically concern this NF application. The Panel concludes that the NF is safe under the proposed conditions of use for the proposed target populations

A Combined Nucleic Acid and Protein Analysis in Friedreich Ataxia: Implications for Diagnosis, Pathogenesis and Clinical Trial Design

BACKGROUND: Friedreich's ataxia (FRDA) is the most common hereditary ataxia among caucasians. The molecular defect in FRDA is the trinucleotide GAA expansion in the first intron of the FXN gene, which encodes frataxin. No studies have yet reported frataxin protein and mRNA levels in a large cohort of FRDA patients, carriers and controls. METHODOLOGY/PRINCIPAL FINDINGS: We enrolled 24 patients with classic FRDA phenotype (cFA), 6 late onset FRDA (LOFA), all homozygous for GAA expansion, 5 pFA cases who harbored the GAA expansion in compound heterozygosis with FXN point mutations (namely, p.I154F, c.482+3delA, p.R165P), 33 healthy expansion carriers, and 29 healthy controls. DNA was genotyped for GAA expansion, mRNA/FXN was quantified in real-time, and frataxin protein was measured using lateral-flow immunoassay in peripheral blood mononuclear cells (PBMCs). Mean residual levels of frataxin, compared to controls, were 35.8%, 65.6%, 33%, and 68.7% in cFA, LOFA, pFA and healthy carriers, respectively. Comparison of both cFA and pFA with controls resulted in 100% sensitivity and specificity, but there was overlap between LOFA, carriers and controls. Frataxin levels correlated inversely with GAA1 and GAA2 expansions, and directly with age at onset. Messenger RNA expression was reduced to 19.4% in cFA, 50.4% in LOFA, 52.7% in pFA, 53.0% in carriers, as compared to controls (p<0.0001). mRNA levels proved to be diagnostic when comparing cFA with controls resulting in 100% sensitivity and specificity. In cFA and LOFA patients mRNA levels correlated directly with protein levels and age at onset, and inversely with GAA1 and GAA2. CONCLUSION/SIGNIFICANCE: We report the first explorative study on combined frataxin and mRNA levels in PBMCs from a cohort of FRDA patients, carriers and healthy individuals. Lateral-flow immunoassay differentiated cFA and pFA patients from controls, whereas determination of mRNA in q-PCR was sensitive and specific only in cFA