Development of screening methods for functional characterization of UGTs from Stevia rebaudiana.

Glycosylation is a key modification that contributes to determine bioactivity and bioavailability of plant natural products, including that of terpenoids and steviol glycosides (SVglys). It is mediated by uridine-diphosphate glycosyltransferases (UGTs), that achieve their activity by transferring sugars on small molecules. Thus, the diversity of SVglys is due to the number, the position and the nature of glycosylations on the hydroxyl groups in C-13 and C-19 of steviol. Despite the intense sweetener property of SVglys and the numerous studies conducted, the SVglys biosynthetic pathway remains largely unknown. More than 60 SVglys and 68 putative UGTs have been identified in Stevia rebaudiana. This study aims to provide methods to characterize UGTs putatively involved in SVglys biosynthesis. After agroinfiltration-based transient gene expression in Nicotiana benthamiana, functionality of the recombinant UGT can be tested simply and directly in plants expressing it or from a crude extract. The combined use of binary vectors from pGWBs series to produce expression vectors containing the stevia's UGT, enables functionality testing with many substrates as well as other applications for further analysis, including subcellular localization.

Impact of polyphenols on receptor-ligand interactions by NMR: the case of neurotensin (NT)-neurotensin receptor fragment (NTS1) complex.

Ligand-receptor interactions can be implicated in many pathological events such as chronic neurodegenerative diseases. Thus, the discovery of molecules disrupting this type of interactions could be an interesting therapeutic approach. Polyphenols are well known for their affinity for proteins and several studies have characterized these direct interactions. But studying the direct influence of multi-therapeutic drugs on a ligand-receptor complex relevant to a neurodegenerative disorder is a challenging issue. Solution NMR, molecular modeling and iterative calculations were used to obtain information about the interaction between a phenolic compound, α-glucogallin (α-2) and a ligand/fragment receptor complex neurotensin (NT) and its receptor NTS1. The α-2 was shown to bind to NT and a peptidic fragment of its NTS1 receptor, independently. Although the formation of the corresponding ligand-receptor complex did not seem to be affected, this experimental modeling protocol will enable the evaluation of other anti-amyloidogenic compounds such as blockers of NT-NTS1 binding. These types of studies help in understanding the specificity and influence in binding and can provide information to develop new molecules with a putative pharmacological interest.Communicated by Ramaswamy H. Sarma.

Multi-host disease management: the why and the how to include wildlife.

In recent years, outbreaks caused by multi-host pathogens (MHP) have posed a serious challenge to public and animal health authorities. The frequent implication of wildlife in such disease systems and a lack of guidelines for mitigating these diseases within wild animal populations partially explain why the outbreaks are particularly challenging. To face these challenges, the French Ministry of Agriculture launched a multi-disciplinary group of experts that set out to discuss the main wildlife specific concepts in the management of MHP disease outbreaks and how to integrate wildlife in the disease management process.This position paper structures the primary specific concepts of wildlife disease management, as identified by the working group. It is designed to lay out these concepts for a wide audience of public and/or animal health officers who are not necessarily familiar with wildlife diseases. The group's discussions generated a possible roadmap for the management of MHP diseases. This roadmap is presented as a cycle for which the main successive step are: step 1-descriptive studies and monitoring; step 2-risk assessment; step 3-management goals; step 4-management actions and step 5-assessment of the management plan. In order to help choose the most adapted management actions for all involved epidemiological units, we integrated a decision-making framework (presented as a spreadsheet). This tool and the corresponding guidelines for disease management are designed to be used by public and health authorities when facing MHP disease outbreaks. These proposals are meant as an initial step towards a harmonized transboundary outbreak response framework that integrates current scientific understanding adapted to practical intervention.

UGT76G1 polymorphism in Stevia rebaudiana: New variants for steviol glycosides conjugation.

Steviol glycosides (SVglys) are secondary metabolites derived from terpenoids exhibiting high-sweetening properties produced in Stevia rebaudiana leaves. Their great diversity is due to the number, the position and the nature of glycosylations on the steviol aglycone. Steviol conjugation is mediated by uridine-diphosphate glycosyltransferases (UGTs). Four UGTs have been clearly identified as involved in SVglys metabolism: UGT74G1, UGT85C2, UGT76G1 and UGT73E1. Natural non-functional mutants with nonsense codon have yet been observed for UGT76G1. To investigate the variability of UGT76G1 functionality, natural mutants with low or no content of rebaudioside A and C were identified in a germplasm collection of Stevia rebaudiana. These compounds are known to be the direct products of UGT76G1 and their biosynthesis is governed by a single gene at the locus Rae (Rebaudioside A enablement). Crosses were done with remarkable accessions including phenotypes with low (0-3%) and high proportions (70%) of rebaudioside A and C, to investigate the functionality of the Rae locus in the parents. Seven variants of UGT76G1 were found, among them 4 lead to a functional protein and 3 lead to non-functional isoforms. Five of these variants are new. We found that non-functionality of UGT76G1 towards SVglys is not due to a premature nonsense codon, which appears to be an extreme case to explain the loss of functionality of an UGT. Variations in steviol glycoside profile in stevia leaves is partly due to UGT76G1 polymorphism: amino acid substitutions in parts of the protein involved in the substrate specificity can be found by sequence comparison.

Tick-borne encephalitis in Europe, 2012 to 2016.

Since 2012, tick-borne encephalitis (TBE) is a notifiable in the European Union. The European Centre for Disease Prevention and Control annually collects data from 28 countries plus Iceland and Norway, based on the EU case definition. Between 2012 and 2016, 23 countries reported 12,500 TBE cases (Ireland and Spain reported none), of which 11,623 (93.0%) were confirmed cases and 878 (7.0%) probable cases. Two countries (Czech Republic and Lithuania) accounted for 38.6% of all reported cases, although their combined population represented only 2.7% of the population under surveillance. The annual notification rate fluctuated between 0.41 cases per 100,000 population in 2015 and 0.65 in 2013 with no significant trend over the period. Lithuania, Latvia and Estonia had the highest notification rates with 15.6, 9.5 and 8.7 cases per 100,000 population, respectively. At the subnational level, six regions had mean annual notification rates above 15 cases per 100,000 population, of which five were in the Baltic countries. Approximately 95% of cases were hospitalised and the overall case fatality ratio was 0.5%. Of the 11,663 cases reported with information on importation status, 156 (1.3%) were reported as imported. Less than 2% of cases had received two or more doses of TBE vaccine.

Unsupervised clustering of wildlife necropsy data for syndromic surveillance.

BACKGROUND: The importance of wildlife disease surveillance is increasing, because wild animals are playing a growing role as sources of emerging infectious disease events in humans. Syndromic surveillance methods have been developed as a complement to traditional health data analyses, to allow the early detection of unusual health events. Early detection of these events in wildlife could help to protect the health of domestic animals or humans. This paper aims to define syndromes that could be used for the syndromic surveillance of wildlife health data. Wildlife disease monitoring in France, from 1986 onward, has allowed numerous diagnostic data to be collected from wild animals found dead. The authors wanted to identify distinct pathological profiles from these historical data by a global analysis of the registered necropsy descriptions, and discuss how these profiles can be used to define syndromes. In view of the multiplicity and heterogeneity of the available information, the authors suggest constructing syndromic classes by a multivariate statistical analysis and classification procedure grouping cases that share similar pathological characteristics. RESULTS: A three-step procedure was applied: first, a multiple correspondence analysis was performed on necropsy data to reduce them to their principal components. Then hierarchical ascendant clustering was used to partition the data. Finally the k-means algorithm was applied to strengthen the partitioning. Nine clusters were identified: three were species- and disease-specific, three were suggestive of specific pathological conditions but not species-specific, two covered a broader pathological condition and one was miscellaneous. The clusters reflected the most distinct and most frequent disease entities on which the surveillance network focused. They could be used to define distinct syndromes characterised by specific post-mortem findings. CONCLUSIONS: The chosen statistical clustering method was found to be a useful tool to retrospectively group cases from our database into distinct and meaningful pathological entities. Syndrome definition from post-mortem findings is potentially useful for early outbreak detection because it uses the earliest available information on disease in wildlife. Furthermore, the proposed typology allows each case to be attributed to a syndrome, thus enabling the exhaustive surveillance of health events through time series analyses.