Single-stranded DNAs (ssDNAs) play major biological functions and represent an interesting biotechnological tool. They constitute a compelling alternative to RNA, because of their greater stability as compared to the latter. As for RNA, ssDNAs function depends on the specific foldings they adopt. Therefore, information about ssDNAs 3-dimensional (3D) structures is fundamental to investigate their functions. In this context, in silico 3D structure prediction can facilitate ssDNA design. This task can be addressed indirectly, by using the tools for RNA structure prediction and then converting the output in the ssDNA format, or one of the few tools capable of directly handling ssDNA. This study assessed 3 indirect RNA 3D structure prediction methods (RNAComposer, SimRNA, and Vfold3D), based on their performance in the Critical Assessment of Structure Prediction and one direct DNA prediction tool (3dDNA) to evaluate their performances in modeling ssDNAs. At this scope, a dataset of 97 experimentally determined ssDNA structures, including challenging motifs such as G-quadruplex, was built. Various metrics, namely, Root Mean Square Deviation, Global Distance Test Total Score, and Interaction Network Fidelity, were employed to benchmark the accuracy of the predictions. The 3 indirect tools showed similar and moderate performances, while the direct tool provided better results. Nevertheless, they all performed poorly in modeling G-quadruplexes and structures containing motifs increasing the intrinsic flexibility of ssDNA. Despite the recent efforts in the prediction of the 3D folding of ssDNAs, improvements in method are still needed. This should involve taking into account the conformational variability of this kind of molecules and paying attention to their specific 3D motifs.

Lyme borreliosis (LB) is the most prevalent tick-borne illness, with an estimated 700 000 cases annually in the United States and Europe. The LB diagnosis based on a two-tiered serology remains controversial due to its indirect nature and low sensitivity during the early stage of the disease. Aptamers are single-stranded DNA or RNA oligonucleotides that exhibit high selectivity and specificity for their target due to their unique three-dimensional structure. By applying cross-over-SELEX process, an enrichment of DNA oligonucleotide sequences against a surface protein of Borrelia, named CspZ, has been performed and monitored using absorbance at 260 nm, melting curves and NGS analyses. Beyond sequence enrichment, oligonucleotides binding to CspZ were observed during the selection rounds by Dot Blot and beads assays. Thirteen unique and highly redundant oligonucleotide sequences were further characterized using multiple approaches such as Dot Blot, BioLayer Interferometry and Surface Plasmon Resonance. The selected aptamers showed KD values from tens of nanomolar to the micromolar range by BLI and SPR. Two aptamers, Apta9 and Apta10, characterized by flow cytometry and epifluorescence microscopy, were able to specifically recognize Borrelia burgdorferi sensu stricto. This strategy holds promise for the development of an improved diagnostic assay.

Spirochetes of the genus Borrelia are the causative agents of vector-borne diseases, including Lyme borreliosis (LB). Clinical LB diagnosis is challenging due to the heterogeneous and nonspecific symptoms among patients further complicated by the potential for co-infection with Borrelia species and other pathogens. Current diagnosis is based on an indirect serological approach with limitations such as early-stage low sensitivity and cross reactivity. This review explores the potential of Borrelia surface proteins as biomarkers for a more accurate diagnosis of LB, with a particular interest in proteins involved in (i) tissue adhesion, essential for host colonization, (ii) antigenic variation, and (iii) immune evasion. By detailing the role of the distinct surface proteins, the review highlights their potential as biomarkers in order to improve the detection of LB, particularly in the early stages. In addition to their diagnostic value, they also represent potential targets for the development of new therapeutic strategies. • Various Borrelia surface proteins drive host colonization and immune evasion. • Novel bacterial proteins biomarkers are needed for improved diagnosis and therapy. • State of the art on Borrelia surface proteins and their associated role.

Single-stranded Nucleic Acids (ssNAs) play major biological functions and represent an interesting biotechnological tool. Their function depends strictly on the specific foldings they can adopt. Therefore, information about ssNAs’ tridimensional structures is fundamental to investigate their functions. In this context, in silico 3D structure prediction can facilitate ssNAs design. Many algorithms have been developed with this aim, mainly focused on RNA. However, the growing interest in single-stranded DNA (ssDNA), due to their greater stability as compared to RNA, has highlighted the need to adapt these methods for ssDNA. This study assessed three RNA 3D structure prediction methods (RNAComposer, SimRNA, and Vfold3D), based on their performance in the Critical Assessment of protein Structure Prediction 15 and/or 16, to evaluate their applicability to ssDNA. At this scope, a dataset of 93 experimentally determined ssDNA structures, including challenging motifs such as G-quadruplexes, was built. Various metrics, such as RMSD, GDT TS, and INF were employed to benchmark the accuracy of the predictions. The three tools showed similar and moderate performances. In addition, they show strong difficulties in modeling G-quadruplexes, and structures containing motifs strongly increasing the intrinsic flexibility of ssDNA. Despite the recent efforts in the prediction of the 3D folding of ssNAs, it is clear that a significant improvement of the methods is needed. This should involve taking into account the conformational variability of this kind of molecules and paying attention to their specific 3D motifs. Author summary Single-stranded oligonucleotides (ssNAs) are RNA or single-stranded DNA molecules involved in crucial biological processes, such as gene expression, DNA replication, and transcription. In addition, they represent a powerful biotechnlogical tool exploitable in therapeutics, diagnostics and biosensing. This is due to their capacity of recognizing different kind of molecular targets, thanks to the tridimensional foldings they can adopt. It is therefore clear that the knowledge of the ssNAs structure is fundamental to master these molecules. With this aim, much effort has been paid in developing computational tools for the prediction of ssNAs 3D structures. However, their application is mostly limited to RNA sequences, even if the interest in ssDNAs is rapidly increasing. Moreover, an extensive benchmark on their performances is missing. We focused this work on assessing the performances of three ssNA structure prediction tools, which best performed in the two latest Critical Assessment of protein Structure Prediction contests, on a large dataset of ssDNA, in order to establish the strengths and limits of the available tools. This knowledge will be helpful in finding new solutions for better understanding the ssNAs folding and their structure-function relationship. ### Competing Interest Statement The authors have declared no competing interest.

Borrelia, spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia, including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95

With almost 700 000 estimated cases each year in the United States and Europe, Lyme borreliosis (LB), also called Lyme disease, is the most common tick-borne illness in the world. Transmitted by ticks of the genus Ixodes and caused by bacteria Borrelia burgdorferi sensu lato, LB occurs with various symptoms, such as erythema migrans, which is characteristic, whereas others involve blurred clinical features such as fatigue, headaches, arthralgia, and myalgia. The diagnosis of Lyme borreliosis, based on a standard two-tiered serology, is the subject of many debates and controversies, since it relies on an indirect approach which suffers from a low sensitivity depending on the stage of the disease. Above all, early detection of the disease raises some issues. Inappropriate diagnosis of Lyme borreliosis leads to therapeutic wandering, inducing potential chronic infection with a strong antibody response that fails to clear the infection. Early and proper detection of Lyme disease is essential to propose an adequate treatment to patients and avoid the persistence of the pathogen. This review presents the available tests, with an emphasis on the improvements of the current diagnosis, the innovative methods and ideas which, ultimately, will allow more precise detection of LB.

Abstract Background Single-stranded nucleic acids (ssNAs) have important biological roles and a high biotechnological potential linked to their ability to bind to numerous molecular targets. This depends on the different spatial conformations they can assume. The first level of ssNAs spatial organisation corresponds to their base pairs pattern, i.e. their secondary structure. Many computational tools have been developed to predict the ssNAs secondary structures, making the choice of the appropriate tool difficult, and an up-to-date guide on the limits and applicability of current secondary structure prediction tools is missing. Therefore, we performed a comparative study of the performances of 9 freely available tools (mfold, RNAfold, CentroidFold, CONTRAfold, MC-Fold, LinearFold, UFold, SPOT-RNA, and MXfold2) on a dataset of 538 ssNAs with known experimental secondary structure. Results The minimum free energy-based tools, namely mfold and RNAfold, and some tools based on artificial intelligence, namely CONTRAfold and MXfold2, provided the best results, with $$\sim 50\%$$ ∼ 50 % of exact predictions, whilst MC-fold seemed to be the worst performing tool, with only $$\sim 11\%$$ ∼ 11 % of exact predictions. In addition, UFold and SPOT-RNA are the only options for pseudoknots prediction. Including in the analysis of mfold and RNAfold results 5–10 suboptimal solutions further improved the performances of these tools. Nevertheless, we could observe issues in predicting particular motifs, such as multiple-ways junctions and mini-dumbbells, or the ssNAs whose structure has been determined in complex with a protein. In addition, our benchmark shows that some effort has to be paid for ssDNA secondary structure predictions. Conclusions In general, Mfold, RNAfold, and MXfold2 seem to currently be the best choice for the ssNAs secondary structure prediction, although they still show some limits linked to specific structural motifs. Nevertheless, actual trends suggest that artificial intelligence has a high potential to overcome these remaining issues, for example the recently developed UFold and SPOT-RNA have a high success rate in predicting pseudoknots.

RationaleWhile the GC-Orbitrap, marketed in 2015, represents a technological breakthrough in terms of sensitivity, resolution and mass stability, many studies have reported ion ratio modification in mass spectra using the standard 70 eV electron ionisation.MethodsWe studied the influence of the acquisition and sample parameters leading to these modifications on fatty acid methyl esters (FAMEs).ResultsFAMEs showed that these variations in relative intensities of ions were related to the acquisition parameters such as the mass range and the offset values of the C-TRAP, but also directly related to the column concentration of the sample, and especially that it was molecule-dependent. Advantageously, it is possible to use this feature to promote the molecular ions of FAMEs sometimes not present in a spectrum under electron ionisation at 70 eV.ConclusionsThe 70 eV electron ionisation mass spectra from the GC-Orbitrap were clearly molecule-dependent and could be due to metastable ions during storage states in the C-TRAP.

Early effects induced by cysteine were monitored using the model of Mimosa pudica pulvinar cells. Rapid dose dependent membrane depolarization (within seconds) and modification of proton secretion (within minutes) were triggered at cysteine concentrations higher than 0.1 mM. These effects did not result from a modification of the plasma membrane H+-ATPase activity nor from a protonophore effect as shown by assays on plasma membrane vesicles isolated from pulvinar tissues. In a 0.5-10 mM range, cysteine inhibited the ion-driven turgor-mediated seismonastic reaction of Mimosa pudica primary pulvini and the dark-induced movement of Cassia fasciculata leaflets. At concentrations higher than 1 mM, it induced a long-lasting leaflet necrosis dependent on the concentration and treatment duration. Electron microscopy showed that cysteine induced important damage in the nucleus, mitochondria, endoplasmic reticulum and Golgi of the M. pudica motor cell. Cysteine inhibited in a concentration-dependent manner, from 0.5 to 20 mM, both the mycelial growth and the spore germination of the fungal pathogens Phaeomoniella chlamydospora and Phaeoacremonium minimum implicated in esca disease of grapevines. Using [S-35] cysteine, we showed that the amino acid was absorbed following leaf spraying, translocated from leaves to other parts of grapevine cuttings and accumulated within trunks and roots. Therefore, cysteine showed relevant properties to be a candidate able to control fungal diseases either by acting as an early signal directing plant host reaction or/and by acting directly on fungal development.

Fatty acid methyl esters (FAMES), which are commonly used to characterize lipids, have several limitations to conclude on many structures. 3-Pyridylcarbinol esters (3-PCE) are used to characterize fatty acid structures [1], in particular, to identify ring and double bond positions on the carbon chain. Chromatographic separation of these esters is complex due to their polarity and high boiling points. In this study, we used a column with high resolutive power based on ionic liquids to increase the separation quality in gas chromatography (GC). In addition, we used a high-resolution detector (Orbitrap) to limit non-specific signals and improve the detection limits. This detector could be used with a mass filter at 5 ppm for the rapid determination of 3-PCE from its characteristic ions (m/z = 108.0441 and 92.0495). This filter allowed the identification of derivative fatty acids with good sensibility. Thus, it was possible to characterize 3-PCE by measuring the exact fragment masses to confirm structures such as C19:2n12cyclo Delta 9. (C) 2018 Elsevier B.V. All rights reserved.

Eutypa lata is the causal agent of a devastating disease affecting grapevines around the world. We have previously reported that the fungus secretes in its culture medium a variety of polypeptides, and we have developed serological method of detecting these. Rabbit antibodies raised against the polypeptide fraction, recognized secreted fungal proteins with high sensitivity (commonly 1 ng). These antibodies were specific since they cross-reacted with polypeptides secreted by various strains of Eutypa lata but not by other fungal pathogens involved in other wood decay diseases, namely esca (Phaeomoniella chlamydospora and Phaeoacremonium aleophilum) and black dead arm (Diplodia seriata and Neofusicoccum parvum). These results were obtained by ELISA assay and immunolocalization on ultrathin sections. The ability of the polypeptides to be transported in vines permitted development of a reliable dot-blot method of diagnosis rapid, easy to perform and not destructive for grapevines. Advantages and drawbacks of the method are discussed.

Dans son numero de mars 1985, Biofutur publiait un dossier intitule Biogaz et Biocarburants. Si son contenu reste actuel sur certains points (« l'un des objectifs des biotechnologies modernes est de mieux valoriser la biomasse, gisement d'energie renouvelable et de carbone fermentescible »), de grandes evolutions se sont depuis produites, en particulier au niveau de la strategie.

Fossil fuel reserves are running out, global warming is becoming a reality, waste recycling is becoming ever more costly and problematic, and unrelenting population growth will require more and more energy and consumer products. There is now an alternative to the 100% oil economy; it is a renewable resource based on agroresources by using the whole plant. Production and development of these new products are based on biorefinery concept. Each constituent of the plant can be extracted and functionalized in order to produce non-food and food fractions, intermediate agro-industrial products and synthons. Three major industrial domains can be concerned: molecules, materials and energy. Molecules can be used as solvent surfactants or chemical intermediates in substitution of petrol derivatives. Fibers can be valorized in materials like composites. Sugars and oils are currently used to produce biofuels like bioethanol or biodiesel, but second-generation biofuels will use lignocellulosic biomass as raw material. Lipids can be used to produce a large diversity of products like solvent, lubricants, pastes or surfactants. Industrial biorefinery will be linked to the creation of new processes based on the twelve principles of green chemistry (clean processes, atom economy, renewable feedstocks…). Biotechnology, especially white biotechnology, will take a major part into these new processes with biotransformations (enzymology, micro-organisms…) and fermentation. The substitution of oil products by biobased products will develop a new bioeconomy and new industrial processes respecting the sustainable development concept. Industrial biorefinery can be developed on the principle that any residues of one can then be exploited as raw material by others in an industrial metabolism.

Eutypa dieback is a devastating disease of Vitis vinifera L. caused by the fungal pathogen Eutypa lata (Pets.: Fr.) Tul. et C. Tul., which colonizes the vascular tissues of the trunk. Symptoms observed in developing shoots and in the foliage indicate that a necrotic signal spreads at a distance from the infected area. Previous studies have shown that toxic polypeptides isolated from E. lata cultures are able to induce some modifications typical of the disease observed in grapevines. The main aim of this study was to investigate the biological effects of polypeptide sub-fractions (glycosylated and non-glycosylated) for determining the mode and site of action of these toxic compounds. Compounds in the non-glycosylated fraction induced leaf necrosis, increased vacuolar tannin synthesis, modified mitochondria, but induced only a partial damage of the cell wall. They hindered the H+ flux and nutrient uptake as a result of the modification of H+ gradient, as seen on plasma membrane vesicles, without affecting the H+-ATPase activity. Eutypine induced the same effects (i.e. on mitochondria and protonophoric action). Polypeptides of the glycosylated fraction increased anthocyanin synthesis in isolated leaves, promoted the decrease of the starch content and casused heavy damage to the cell wall. They also modified H+ flux and nutrient uptake but, in this case, these processes may be the consequence of a direct effect on the H+-ATPase activity. Our results show that the different toxic compounds secreted by E. lata present a complex frame of action on various plant cellular sites. These effects impair plant metabolism and damage cell structure thus favouring host invasion by the fungus.

Eutypa dieback is a devastating disease induced in vineyards by the fungal pathogen Eutypa lata. The fungus colonizes the xylem tissues of trunk and cordons but is never found in the annual canes. Nevertheless, dwarfed shoots and leaf necrosis observed in diseased plants indicate that a necrotic signal can spread at a distance from the infected area. Eutypine, a small cyclic molecule, and related compounds have been postulated as the toxins inducing these symptoms. In this work, we evidence that E. lata secreted other metabolites of polypeptidic nature which induced toxic effects on canes and leaves of vines, and on leaves of other plant materials. The polypeptide fraction (PF) isolated from culture medium of mycelium induced transitory H+ fluxes and membrane depolarization of plant cells. Complementary assays with plasma membrane vesicles (PMV) showed that H+‐ATPase is a primary site of action as indicated by inhibition of the enzyme activity and increase of H+ conductance of plasma membrane. The toxic effect was also obvious on respiration and photosynthesis. All these impairments led to a hindering in cell energetics and, as a consequence, to an inhibition of uptake of assimilates. Treatment with PF also triggered biological events, characteristics of elicitation as suggested by the early responses on cell membrane described above, the activation of NADPH oxidase and the activation of Phenylalanine ammonia lyase (PAL).

Eutypa dieback is a devastating disease of Vitis vinifera L. caused by the fungal pathogen Eutypa lata. This wood-inhabiting fungus degrades tissues in the trunk and cordons of infected vines and induces symptoms in the foliage. These symptoms have been attributed to the production of toxic metabolites by the pathogen, in particular eutypine. Recently, we have isolated polypeptide compounds secreted by the fungus in artificial culture. The aims of this study were to examine the effects induced in leaves by applying polypeptides and eutypine to detached canes and to compare this to the changes in leaf structure induced by E. lata in the vineyard. In leaves taken from vines infected with E. lata, the changes in mesophyll cells indicate that the fungus has an effect on tissue remote from the infected area. The size of mesophyll cells decreased by more than half, starch content was reduced and tannins were abundant. Plastids, mitochondria and cell walls were highly modified. In leaves taken from healthy canes treated with polypeptides of E. lata, the structure of mesophyll cells was also modified. The cell size did not change, but the tannin content increased and modifications in plastids and mitochondria were similar to those observed in leaves taken from infected vines. The major effect was the complete disorganisation of cell walls. Eutypine had less effect on organelle structure and did not modify the cell wall. In canes treated with polypeptides, vessel-associated cells (VACs) were also damaged. Abundant tannins occurred in the vacuoles of VACs and marked changes were noted in mitochondria, plastids and the protective layer, in particular in the pit at the vessel interface. In these pits, the protective layer, the primary wall and the middle lamella were all highly modified. In contrast, treatment with eutypine induced the development of a large transfer apparatus bordering the unmodified pectocellulose wall. These results illustrate that treatment with polypeptides produced by E. lata may cause changes in mesophyll cells in leaves and VACs in canes, that resemble changes observed in naturally infected vines. Comparatively, the differences with eutypine action were stressed. Both types of toxins may co-operate in vivo to produce the degeneration observed during the disease.

Eutypa dieback, a devastating disease in grapevines, is caused by the fungal pathogen Eutypa lata, a wood-inhabiting fungus. E. lata acts by degrading wood tissues in the colonisation areas, and produces foliar symptoms. These striking symptoms have been attributed to the production of toxic metabolites by the pathogen, the most widely studied being eutypine. The aims of the study were to compare the effects of E. lata on xylem structure at the site of infection and in remote tissues. In healthy Vitis vinifera, the vessel-associated cells (VACs) in the trunk have a protective layer that covers the entire lignified wall and forms a transfer apparatus in pits located at the VAC/vessel interface. This apparatus occurs similarly in VACs in the basal part of canes but is less developed in the apical part. In the presence of E. lata, which is found only in the trunk and the cordons, the VACs initiated a program of secretory activity that led to the enlargement of the transfer apparatus, which is formed by tightly associated fibrils. This secretory activity was followed by VAC death. Furthermore, the hypertrophy of the transfer apparatus spread according to an acropetal gradient in the canes. Treatment with eutypine also induced the development of the transfer apparatus in VACs of basal and apical parts of canes excised from healthy vines. However, this apparatus was formed by loosely packed fibrils in VACs that were not completely damaged. Therefore, metabolites other than eutypine are expected to be involved in the VAC degeneration observed in infected vines.

Eutypa dieback (dying arm disease, eutypiosis) is a very devastating disease in many grape-producing areas around the world. This vascular disease is induced by the ascomycete Eutypa lata Pers. Fr. Tul & C. Tul. invading the trunk by pruning wounds. The environmental factors and the nutritional requirements regulating fungus development are yet poorly known. This work shows that the isolated strain of E. lata was able to grow in a large temperature range (2-30 degrees C). However, a higher temperature (35 degrees C) presented inhibitory effects on mycelial growth. E. lata was able to use various osidic molecules (C5, C6, C12, C18, C24, and starch), showing thus a large adaptation to the carbon source supplied. As nitrogen source, it used salts and numerous natural amino acids. A significant result was obtained with cysteine presenting obvious antifungal properties. This effect can further be used with the aim of setting up a curative treatment of the disease. (c) 2005 Academie des sciences. Published by Elsevier SAS. All rights reserved.