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The overexpression of aberrantly glycosylated tumor-associated mucin-1 (TA-MUC1) in human cancers makes it a major target for the development of anticancer vaccines derived from synthetic MUC1-(glyco) peptide antigens. However, glycopeptide-based subunit vaccines are weakly immunogenic, requiring adjuvants and/or additional immunopotentiating approaches to generate optimal immune responses. Among these strategies, unimolecular self-adjuvanting vaccine constructs that do not need coadministration of adjuvants or conjugation to carrier proteins emerge as a promising but still underexploited approach. Herein, we report the design, synthesis, immune-evaluation in mice, and NMR studies of new, self-adjuvanting and self-assembling vaccines based on our QS-21-derived minimal adjuvant platform covalently linked to TA-MUC1-(glyco)peptide antigens and a peptide helper T-cell epitope. We have developed a modular, chemoselective strategy that harnesses two distal attachment points on the saponin adjuvant to conjugate the respective components in unprotected form and high yields via orthogonal ligations. In mice, only tri-component candidates but not unconjugated or di- component combinations induced significant TA-MUC1-specific IgG antibodies able to recognize the TA-MUC1 on cancer cells. NMR studies revealed the formation of self-assembled aggregates, in which the more hydrophilic TA-MUC1 moiety gets exposed to the solvent, favoring B-cell recognition. While dilution of the di-component saponin–(Tn)MUC1 constructs resulted in partial aggregate disruption, this was not observed for the more stably-organized tri-component candidates. This higher structural stability in solution correlates with their increased immunogenicity and suggests a longer half-life of the construct in physiological media, which together with the enhanced antigen multivalent presentation enabled by the particulate self-assembly, points to this self-adjuvanting tri-component vaccine as a promising synthetic candidate for further development. Funding from the European Research Council (ERC-2016-STG-716878 to A. F.-T.; ERC-2017-AdG-788143 to J. J. B.) and the Spanish Ministry of Science and Innovation MCIN/AEI (PID2020-117911RB-I00, CTQ2017-87530-R, RYC-2015-17888 to A. F.-T.; RTI2018-096494-B-100 to J. A.; RTI2018-094751-B-C21 to J. J. B) is gratefully acknowledged. We thank Felix Elortza and Ibon Iloro from the CIC bioGUNE Proteomics Platform and Javier Calvo from the CIC biomaGUNE Mass Spectrometry Platform for their support with MALDI and HRMS analyses. A. F. T. thanks Raquel Fernandez for inspiration.

The direct functionalization of Si−H bonds by the nitrene insertion methodology is described. A copper(I) complex bearing a trispyrazolylborate ligand catalyzes the transfer of a nitrene group from PhINTs to the Si−H bond of silanes, disilanes, and siloxanes, leading to the exclusive formation of Si−NH moieties in the first example of this transformation. The process tolerates other functionalities in the substrate such as several C−H bonds and alkyne and alkene moieties directly bonded to the silicon center. Density functional theory (DFT) calculations provide a mechanistic interpretation consisting of a Si−H homolytic cleavage and subsequent rebound to the Si-centered radical., We thank the Ministerio de Ciencia e Innovación for Grants PID2020-113797RB-C21, PID2020-112825RB-I00, and CEX2019-000925-S as well as FEDER for “Una manera de hacer Europa” funding. We also thank Junta de Andalucía (P20-00348) and Universidad de Huelva (P.O.Feder UHU202016). A.M.R. and R.P.-S. thank Ministerio de Universidades for the FPU fellowships (FPU17/02738 and FPU18/ 01138, respectively), and J.P.-R. thanks Fondo de Garantía Juvenil for a research contract. CERCA Programme/Generalitat de Catalunya is also acknowledged. We thank Prof. T. R. Belderrain for helpful discussions on 29Si NMR spectroscopy. Funding for open access charge: Universidad de Huelva / CBUA

The synthesis of polysubstituted spirocyclopropyl oxindoles using a series of rare-earth metal (REM) salts is reported. REMs, in particular Sc(OTf)3, allowed access to the target compounds by a multicomponent reaction with high diastereoselectivity (≤94:6:0:0). Density functional theory calculations on the model reaction are consistent with the observed selectivity and revealed that the special coordinating capabilities and the oxophilicity of the metal are key factors in inducing the formation of one main diastereoisomer. The authors thank Diana Cabrera, Sebastiaan van Liempd, and Juan M. Falcon-Perez from the CIC bioGUNE Metabolomics Platform for performing the UPLC-MS/MS analyses. The authors also acknowledge the Italian Ministry of University and Research (MUR) for Project SI.F.I.PA.CRO.DE. - Sviluppo e industrializzazione farmaci innovativi per terapia molecolare personalizzata PA.CRO.DE. (PON ARS01_00568, CUP: B29C20000360005) and for two doctoral grants. Moreover, the authors thank the University of Calabria and Calabria Region (PAC CALABRIA 2014–2020-Asse Prioritario 12, Azione B 10.5.12 CUP: H28D19000040006) for financial support. This research was also funded by MCIN/AEI/10.13039/501100011033 (Grant PID2021-125946OB-I00 to G.J.-O. and Severo Ochoa Excellence Accreditation CEX2021-001136-S to CIC bioGUNE).

Acidic xylooligosaccharides (XOS), also called aldouronics, are hetero-oligomers of xylose randomly branched with 4-O-methyl-D-glucuronic acid residues linked by α(1 → 2) bonds, which display bioactive properties. We have developed a simple and integrated method for the production of acidic XOS by enzymatic hydrolysis of a glucurono-xylan from beechwood. Among the enzymes screened, Depol 670L (a cellulolytic preparation from Trichoderma reesei) displayed the highest activity (70.3 U/mL, expressed in reducing xylose equivalents). High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis revealed the formation of a neutral fraction (corresponding to linear XOS, mainly xylose and xylobiose) and a group of more retained products (acidic XOS), which were separated using strong anion-exchange cartridges. The acidic fraction contained a major product, characterized by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and mono- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) as 2′-O-α-(4-O-methyl-α-D-glucuronosyl)-xylobiose (X2_MeGlcA). Starting from 2 g of beechwood xylan, 1.5 g of total XOS were obtained, from which 225 mg (11% yield) corresponded to the aldouronic X2_MeGlcA. The acidic XOS exhibited higher antioxidant activity (measured by the ABTS·+ discoloration assay) than xylan, whilst neutral XOS displayed no antioxidant activity. This work demonstrates that it is possible to obtain a safe and natural antioxidant by enzymatic biotransformation of hardwood hemicellulose.

Fixed drug eruption (FDE) is a cutaneous adverse drug reaction characterized by recurrence of lesions at the same sites each time a specific drug is taken. Oral mucosal involvement is rare. Nonsteroidal anti-inflammatory drugs are one of the most common offending drug groups in FDE; however, selective cyclooxygenase-2 inhibitors, such as etoricoxib, are rarely implicated. We present a case of oral mucosal and cutaneous FDE induced by etoricoxib that, to the best of our knowledge, is the first reported case of this nature. We describe the diagnostic challenges and review the pertinent literature. The value of drug provocation testing and patch testing in FDE is also discussed.

En el presente trabajo se estandarizó una metodología para la activación y producción de superficies silanizadas de naturaleza hidrofóbica. La metodología fue realizada mediante la activación de grupos OH de superficies de vidrio eliminando la materia inorgánica con etanol y la materia orgánica por reacciones de oxidación, mediante la construcción de una cámara de UV/ozono con dos longitudes de onda diferentes (253,7 nm y 180 nm). Se realizó la silanización de las superficies de vidrio activadas exponiéndolas con el reactivo (7-octen-1-il) trimetoxisilano/n-heptano a diferentes tiempos. La hidrofobicidad obtenida en las superficies silanizadas se determinó midiendo el ángulo de contacto (q). Los resultados permitieron concluir que existe influencia del tiempo de exposición en la formación de superficies hidrofóbicas. Se obtuvieron ángulos de contacto de 92,417° ± 0,631, tras 7 h de exposición seguidas de un proceso de limpieza de las superficies.

This work focuses on the search and development of drugs that may become new alternatives to the commercial drugs currently available for treatment of leishmaniasis. We have designed and synthesized 12 derivatives of bis(spiropyrazolone)cyclopropanes. We then characterized their potential application in therapeutic use. For this, the in vitro biological activities against three eukaryotic models—S. cerevisiae, five cancer cell lines, and the parasite L. mexicana—were evaluated. In addition, cytotoxicity against non-cancerous mammalian cells has been evaluated and other properties of interest have been characterized, such as genotoxicity, antioxidant properties and, in silico predictive adsorption, distribution, metabolism, and excretion (ADME). The results that we present here represent a first screening, indicating two derivatives of bis(spiropyrazolone)cyclopropanes as good candidates for the treatment of leishmaniasis. They have good specificity against parasites with respect to mammalian cells.

International audience; Mucopolysaccharidosis type IIIB is a devastating neurological disease caused by a lack of the lysosomal enzyme, aN -acetylglucosaminidase (NAGLU), leading to a toxic accumulation of heparan sulfate. Herein we explored a pharmacological chaperone approach to enhance the residual activity of NAGLU in patient fibroblasts. Capitalizing on the three-dimensional structures of two modest homoiminosugar-based NAGLU inhibitors in complex with bacterial homolog of NAGLU, CpGH89, we have synthesized a library of 17 six-membered iminosugar-C-glycosides mimicking N-acetyl-Dglucosamine and bearing various pseudo-anomeric substituents of both a-and b-configuration. Elaboration with the aglycon moiety results in low micromolar selective inhibitors of human recombinant NAGLU, but surprisingly it is the nonfunctionalized and wrongly configured b-homoiminosugar that was proved to act as the most promising pharmacological chaperone, promoting a 2.4 fold activity enhancement of mutant NAGLU at its optimal concentration.

Introduction Oral cancer is the eighth most common cancer in the world and associated with a high rate of morbidity and mortality. Appropriate and early diagnosis of the condition is associated with improved patient outcomes, and an improvement in five-year survival rates.Aims We aim to ascertain if dental and medical students have similar amounts of self-confidence in the understanding and management of oral cancer within a large UK university.Methods We invited 65 final-year dental students and 101 final-year medical students to undertake our study, with 50 dental students and 59 medical students opting to participate.Results We found final-year dental students to feel significantly more confident in their understanding and management of oral cancer when compared to their medical counterparts.Conclusions Medical students may benefit from additional teaching on oral cancer. Additionally, they should be encouraged to briefly screen the oral cavity of individuals who are at high-risk of the disease.

ObjectivesGiven the similarity in symptoms between primary Sjogren’s syndrome (SjS) and non-SjS sicca syndrome (sicca), we sought to characterise clinical and proteomic predictors of symptoms in both groups in order to better understand disease mechanisms and help guide development of immunomodulatory treatments. These have not, to date, unequivocally improved symptoms in SjS clinical trials.MethodsSerum proteomics was performed using O-link inflammation and cardiovascular II panels. SjS (n=53) fulfilled 2016 ACR/European Alliance of Associations for Rheumatology (EULAR) criteria whereas sicca (n=60) were anti-Ro negative, displayed objective or subjective dryness, and either had a negative salivary gland biopsy or, in the absence of a biopsy, it was considered that a biopsy result would not change classification status. Linear regression analysis was performed to identify the key predictors of symptoms. Cluster analysis was completed using protein expression values.ResultsEULAR-Sjögren’s-Syndrome-Patient-Reported-Index (ESSPRI), EuroQoL-5 Dimension utility values, and anxiety and depression did not differ between SjS and sicca. Correlations between body mass index (BMI) and ESSPRI were found in sicca and to a lesser extent in SjS. Twenty proteins positively associated with symptoms in sicca but none in SjS. We identified two proteomically defined subgroups in sicca and two in SjS that differed in symptom burden. Within hierarchical clustering of the SjS and sicca pool, the highest symptom burden groups were the least distinct. Levels of adrenomedullin (ADM), soluble CD40 (CD40) and spondin 2 (SPON2) together explained 51% of symptom variability in sicca. ADM was strongly correlated with ESSPRI (spearman’s r=0.62; pConclusionsObesity-related metabolic factors may regulate symptoms in sicca. Further work should explore non-inflammatory drivers of high symptom burden in SjS to improve clinical trial outcomes.

Glycosylation of polyphenols may increase their aqueous solubility, stability, bioavailability and pharmacological activity. Herein, we used a mutant of sucrose phosphorylase from Thermoanaerobacterium thermosaccharolyticum engineered to accept large polyphenols (variant TtSPP_R134A) to produce phloretin glucosides. The reaction was performed using 10% (v/v) acetone as cosolvent. The selective formation of a monoglucoside or a diglucoside (53% and 73% maximum conversion percentage, respectively) can be kinetically controlled. MS and 2D-NMR determined that the monoglucoside was phloretin 4’-O-α-D-glucopyranoside and the diglucoside phloretin-4’-O-[α-D-glucopyranosyl-(1→3)-O-α-D-glucopyranoside], a novel compound. The molecular features that determine the specificity of this enzyme for 4’-OH phenolic group were analysed by induced-fit docking analysis of each putative derivative, using the crystal structure of TtSPP and changing the mutated residue. The mono- and diglucoside were, respectively, 71- and 1200-fold more soluble in water than phloretin at room temperature. The α-glucosylation decreased the antioxidant capacity of phloretin, measured by DPPH and ABTS assays; however, this loss was moderate and the activity could be recovered upon deglycosylation in vivo. Since phloretin attracts a great interest in dermocosmetic applications, we analyzed the percutaneous absorption of glucosides and the aglycon employing a pig skin model. Although the three compounds were detected in all skin layers (except the fluid receptor), the diglucoside was present mainly on superficial layers., We thank Manuel Ferrer and David Almendral (ICP-CSIC) for the enzyme production support. The authors would like to express their gratitude to the members of Center of Synthetic Biology, specially Koen Beerens, Jorick Franceus, Stevie Van Overtveldt, Ophelia Gevaert, Matthieu Da Costa, Shari Dhaene, Marc De Doncker, Emma De Beul, Gilles Velghe, Dries Duchi, José Manuel Salvador López, Mónica Texido and Alba López for their constructive suggestions, discussions and all help received. We thankfully acknowledge the NMR resources and the technical support provided by the Laboratorio de RMN de Euskadi (LRE) of the Spanish ICTS Red de Laboratorios de RMN de Biomoléculas (R-LRB). This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (Grants BIO2016-76601-C3-1,3-R and PID2019-105838RB−C31). We thank the support of the scholarship of Jose Luis Gonzalez from Spanish Ministry of Education, Culture and Sport through the National Program FPU (FPU17/00044) and for the help received to do the stay at Ghent University with the subprogram for mobility aids FPU 2018. The present work could not be performed without the collaboration and Contribution of the Service of Dermocosmetic Assessment from IQAC-CSIC.

Despite the presence of a rutinosyl group at 3-OH, the aqueous solubility of the flavonoid rutin is even lower than that of its aglycon quercetin. In this work, we describe a fast, simple, and easily scalable process for polyglucosylation of rutin to enhance aqueous solubility, catalyzed by a cyclodextrin glucanotransferase (CGTase). Several reaction parameters (source of enzyme, rutin/starch weight ratio, cosolvent, pH, and temperature) were assessed to optimize the transglucosylation yield. Under the best conditions (6 mg/mL rutin, 30 mg/mL soluble starch as glucosyl donor, 20% (v/v) acetonitrile, pH 9.2, 3.3 U/mL CGTase from Geobacillus sp., 60 °C), the total glucosides reached a maximum concentration of 6 mM (60% conversion yield). The glucosylated products were chemically characterized by MALDI-TOF mass spectrometry and 2D nuclear magnetic resonance. The glucosylation takes place with an α-configuration at the 4-OH position of the β-Glc moiety. A series of maltooligosyl derivatives with 1–6 residues of glucose linked by α(1 → 4) bonds was obtained. The yield of monoglucosylated product was increased 2-fold by treatment with amyloglucosidase STA1 from S. cerevisiae., Spanish Ministry of Economy and Competitiveness (PID2019-105838RB-C31 and PID2019-105838RB-C32) and Fundación Ramón Areces and Spanish Ministry of Education, Culture and Sport through the National Program FPU (FPU17/00044)

Capitalizing on a recently reported iminosugar-based aza-crown (ISAC) accessed by a double Staudinger azaWittig coupling reaction, we have expanded the structural diversity of this new family of sweet cyclam analogs. Replacement of the two secondary amines linking the iminosugar units by two amide bonds obtained a cyclodimerization by with BOP and DIPEA led to a macrocycle that did not demonstrate efficient Zn2+ chelation unlike the parent ISAC. Introduction of two pyrene moieties on the secondary amines of the parent ISAC yielded a new fluoroionophore that selectively binds Hg2+ in methanol.

25 p.-7 fig. Carbohydrate Chemistry: Chemical and Biological Approaches.Volume 44. Editors: Amélia Pilar Rauter, Thisbe K Lindhorst, Yves Queneau, This chapter is dedicated to the presentation of different examples of the application of solution NMR to the study of conformation, dynamics of sugar molecules (oligo and polysaccharides, glycopeptides and glycomimetics) including the investigation of glycanrelated molecular recognition events. It is not our intention to be exhaustive, rather to highlight diverse examples since 2016. They are presented depending on the chemical nature of the saccharide molecule, and on the different methodologies that can be employed., We thank MINECO (Spain, Projects) and the European Union (Project RECGLYCANMR and BACTIVAX) for financial support. AF also thanksMINECO for Juan de la Cierva contract.

Non-genetic risk factors for Sjögren's syndrome (SS) are poorly understood. Adherence to a Mediterranean diet has been associated with reduction in other autoimmune diseases. We examined the association of Mediterranean diet with SS.New patients attending a single centre warranting investigation for primary SS (pSS) were recruited into the Optimising Assessment in Sjögren's Syndrome cohort established in Birmingham, UK (2014-2018). Participants were classified into pSS and non-SS sicca, considered as cases and non-cases, respectively, and asked to complete an optional food frequency questionnaire on their diet before onset of symptoms. A semi-quantitative Mediterranean diet score (MDS) was calculated (possible range=0 to 18). Using multivariate logistic regression, corrected for energy intake, body-mass index, sex, age, symptom duration, and smoking status, we examined the association of MDS with SS.Dietary data were available for 133/243 (55%) eligible patients (n=82 pSS and n=51 sicca). In the adjusted model, a higher total MDS (mean ± SD, 9.41±2.31 points) was associated with lower odds of pSS (OR 0.81, 95% CI 0.66-0.99; p=0.038) per one unit of MDS. Among MDS components, the strongest association was seen with fish with OR 0.44 (95% CI 0.24-0.83; p=0.01) in the comparison between1 portion/week and 1 to 2.5 portions/week. Higher galactose, vitamin A-retinol-equivalents and vitamin C showed associations with lower odds of pSS in multivariate analysis, where the association of vitamin C was attenuated when adjusted for MDS.When adjusted for potential confounders, adherence to the Mediterranean diet was associated with lower likelihood of having pSS.

The transglycosylation activity of a novel commercial β-galactosidase from Bifidobacteriumbifidum (Saphera) was evaluated. The optimal conditions of operation of this enzyme, measured with o-nitrophenyl-β-D-galactopyranoside, were pH 6.0 and 40 °C. Although at low lactose concentrations the character of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS). The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other β-galactosidases, for which the highest GOS yield is achieved at 40-50% lactose conversion. Using HPAEC-PAD, semipreparative HPLC-HILIC, MS, 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme. The main identified products were Gal-β(1®3)-Gal-β(1®4)-Glc (3´-O-b-galactosyl-lactose), Gal-β(1→6)-Glc (allolactose), Gal-β(1®3)-Glc (3-galactosyl-glucose), Gal-β(1→3)-Gal (3-galactobiose) and the tetrasaccharide Gal-β(1®3)-Gal-β(1®3)-Gal-β(1®4)-Glc. In general, the B. bifidum β-galactosidase showed a tendency to form β(1®3) linkages followed by β(1®6), and more scarcely β(1®4) . &nbsp

The transglycosylation activity of a novel commercial β-galactosidase from

A new family of chiral C2 symmetric tetraazamacrocycles, coined ISAC for IminoSugar Aza-Crown, incorporating two iminosugars adopting a 4C1 conformation is disclosed. Multinuclear NMR experiments on the corresponding Cd2+ complex show that the ISAC is a strong chelator in water and its tetramine cavity adopts a conformation similar to that of the parent Cd−cyclam complex. Similar behavior is observed with Cu2+ in solution, with enhanced stability compared to the Cu−cyclam complex.

[EN] The transglycosylation activity of a novel commercial β-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions for the operation of this enzyme, measured with o-nitrophenyl-β-d-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the property of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS). The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other β-galactosidases, for which the highest GOS yield is achieved at 40-50% lactose conversion. Using high-performance anion-exchange chromatography with pulsed amperometric detection, semipreparative high-performance liquid chromatography-hydrophilic interaction liquid chromatography, mass spectrometry, and 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme. The main identified products were Gal-β(1→3)-Gal-β(1→4)-Glc (3′-O-β-galactosyl-lactose), Gal-β(1→6)-Glc (allolactose), Gal-β(1→3)-Glc (3-galactosyl-glucose), Gal-β(1→3)-Gal (3-galactobiose), and the tetrasaccharide Gal-β(1→3)-Gal-β(1→3)-Gal-β(1→4)-Glc. In general, B. bifidum β-galactosidase showed a tendency to form β(1→3) linkages followed by β(1→6) and more scarcely β(1→4)., This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2016-76601-C3-1). The group at CIC bioGUNE thanks Agencia Estatal de Investigación (Spain) for the Severo Ochoa Excellence Accreditation (SEV-2016-0644).

Highlights • B. licheniformis produced an EPSp composed of polyglutamic acid and teichoic acid. • The negative charge content of the EPSp increased its electrostatic properties. • The EPSp was efficient at inhibiting viral entry against enveloped viruses. • The EPSp could have potential applications as a prophylactic therapeutic biomolecule., The exopolymer (EPSp) produced by the strain B. licheniformis IDN-EC was isolated and characterized using different techniques (MALDI-TOF, NMR, ATR-FTIR, TGA, DSC, SEM). The results showed that the low molecular weight EPSp contained a long polyglutamic acid and an extracellular teichoic acid polysaccharide. The latter was composed of poly(glycerol phosphate) and was substituted at the 2-position of the glycerol residues with a αGal and αGlcNH2. The αGal O-6 position was also found to be substituted by a phosphate group. The antiviral capability of this EPSp was also tested on both enveloped (herpesviruses HSV, PRV and vesicular stomatitis VSV) and non-enveloped (MVM) viruses. The EPSp was efficient at inhibiting viral entry for the herpesviruses and VSV but was not effective against non-enveloped viruses. The in vivo assay of the EPSp in mice showed no signs of toxicity which could allow for its application in the healthcare sector.

A set of bicyclic iminosugar C-glycosides, based on an octahydrofuro[3,2-b]pyridine motif, has been synthesized from a C-allyl iminosugar exploiting a debenzylative iodocycloetherification and an iodine nucleophilic displacement as the key steps. The halogen allowed the introduction of a range of aglycon moieties of different sizes bearing several functionalities such as alcohol, amine, amide and triazole. In these carbohydrate mimics the fused THF ring forces the piperidine to adopt a flattened

This work investigates the ability of the β‐fructofuranosidase pXd‐INV from the yeast Xanthophyllomyces dendrorhous to glycosylate the olive biophenol hydroxytyrosol (HT). Two fructosylated derivatives (Fru‐HT1 and Fru‐HT2) were synthesized. Under the best conditions (300 mg/mL sucrose, 25 mg/mL HT), the maximum yield was 45.6%. MS and 2D‐NMR analyses showed that the major product (Fru‐HT1) was fructosylated at the primary OH of HT. The structure of the complexes with the substrates and the product analyzed by crystallography led to the understanding of the molecular determinants regulating the enzymatic mechanism. Product‐soaked crystals revealed that the minor derivative (Fru‐HT2) was fructosylated at the phenolic p‐OH group. The two binding modes for HT at pXd‐INV active site are governed almost exclusively by packing to Trp105 (Fru‐HT1) and polar interactions with the loop Glu334‐Asn342 (Fru‐HT2), respectively. Specific mutagenesis at these residues was accomplished to tune the enzyme regiospecificity. One of the studied mutants (N342Q) was notably more specific for the fructosylation at the phenolic OH than the wild‐type., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1/3/4 and BIO2016-76601-C3-1-R/2-R/3-R), by an institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa (CBMSO), and by the European Union’s Horizon 2020 research and innovation program [Blue Growth: Unlocking the potential of Seas and Oceans] under grant agreement No 634486 (INMARE). We thank the support of COST-Action CM1303 on Systems Biocatalysts. M.G-P thank the Spanish Ministry of Education for FPU Grant.

We report the design, synthesis, immunological evaluation, and conformational analysis of new saponin variants as promising vaccine adjuvants. These studies have provided expedient synthetic access to streamlined adjuvant-active saponins and yielded molecularlevel insights into saponin conformation that correlated with their in vivo adjuvant activities.

The glycosylation of plant polyphenols may modulate their solubility and bioavailability, and protect these molecules from oxygen, light degradation and during gastrointestinal transit. In this work, the synthesis of various α-glucosyl derivatives of (‒)-epigallocatechin gallate (EGCG), the predominant catechin in green tea, was performed in water at 50 °C by a transglycosylation reaction catalyzed by cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. The molecular weight of reaction products was determined by HPLC-MS. Using hydrolyzed potato starch as glucosyl donor, two main monoglucosides were obtained with conversion yields of 58% and 13%, respectively. The products were isolated and chemically characterized by combining 2D-NMR methods. The major derivative was epigallocatechin gallate 3’-O-α-D-glucopyranoside (1) and the minor epigallocatechin gallate 7-O-α-D-glucopyranoside (2)., This work was supported by a grants from the Spanish Ministry of Economy and Competitiveness (Grants BIO2013-48779-C4-1 and BIO2016-76601-C3-1-R). We thank the support of COST-Action CM1303 on Systems Biocatalysts.

Polysaccharides are the most abundant organic materials in nature, yet correlations between their three-dimensional structure and macroscopic properties have not been established. Automated glycan assembly enables the preparation of well-defined oligo- and polysaccharides resembling natural as well as unnatural structures. These synthetic glycans are ideal probes for the fundamental study of polysaccharides. According to molecular modeling simulations and NMR analysis, different classes of polysaccharides adopt fundamentally different conformations that are drastically altered by single-site substitutions. Larger synthetic polysaccharides are obtained via a "LEGO"-like approach as a first step toward the production of tailor-made carbohydrate-based materials.

Background: Stroke is the main cause of disability in adults; the most common and long-term sequela is upper-limb hemiparesis. Many studies support the idea that mental motor imagery, which is related to the visualization of movement patterns, activates the same areas of the cortex as if the movement occurred. Objectives: This study aims to examine the capacity to elaborate mental motor images, as well as its relationship to loss of movement in the upper limbs after a stroke. Method: An observational study, in a sample of 39 adults who suffered a stroke, was carried out. The upper limb movement and functionality, cognitive disorders, the ability to visualize mental images, and activities of daily living were examined. Results: The results depicted a statistically significant correlation between the ability to visualize upper limb mental motor images with movement, functionality, and strength. In addition, a correlation between visual–spatial skills and mental visualization of motor ability and upper limb movement was found. Conclusions: These results suggest that the rehabilitation approach focused on the improvement of mental motor imagery could be of interest for the upper limb rehabilitation of movement and functionality.

Las infusiones analgesicas continuas (IAC) son estrategias eficaces para controlar el dolor agudo post-quirurgico; sin embargo, dependiendo del farmaco empleado, la electrofisiologia cardiaca puede alterarse y evidenciar taquiarritmias, bradiarritmias, complejos ventriculares, fibrilacion y bloqueos atrioventriculares. El objetivo de este estudio fue caracterizar las anormalidades electrocardiograficas observadas luego de implementar el uso de infusiones analgesicas continuas de tramadol o lidocaina en perras post-ovariohisterectomizadas. Se emplearon 26 pacientes (26,6 ± 17,6 meses; 15,34 ± 7,81 kg) divididos en tres grupos: Grupo 1 tramadol (n= 9); Grupo 2 lidocaina (n=10) y Grupo 3 NaCl 0,9% como control (n= 7). Las variables estudiadas fueron frecuencia cardiaca media, maxima y minima en latidos por minuto (lpm), morfologia de los complejos anomalos y escala de Glasgow modificado. La frecuencia cardiaca media, maxima y minima correspondiente a cada grupo fue: Tramadol 90, 253 y 43 lpm respectivamente; lidocaina 93, 238 y 42 lpm respectivamente; control 100, 253 y 40 lpm respectivamente. El grupo control evidencio el mayor numero de eventos arritmicos, siendo la anomalia mas frecuente los complejos supraventriculares prematuros. Se concluyo que las IAC de tramadol o lidocaina a las dosis empleadas en el presente estudio no generan alteraciones electrocardiograficas de impacto clinico; siendo el protocolo lidocaina el que evidencio el menor numero de arritmias y el mejor comportamiento de la frecuencia cardiaca. Characterization of cardiac arrhythmias following the use of continuous infusions analgesic of tramadol or lidocaine in dogs ovariohysterectomised The use of continuous analgesic infusions (IACs) is an effective strategy for the control of postoperative pain in canines; however, depending on the used drug, cardiac electrophysiology can be altered by the presence of tachyarrhythmias, bradyarrhythmias, ventricular complexes, fibrillation or atrioventricular block. The aim of this study was to evaluate the heart rate and to characterize the electrocardiographic abnormalities during the use of continuous postoperative analgesic infusions of tramadol or lidocaine in ovariohysterectomized bitches using 24-hour Holter monitoring. For this purpose, 26 healthy canine females (26.6 ± 17.6 months, 15.34 ± 7.81 kg) were divided into three groups: Group 1 NaCl 0.9% as control (n = 7), Group 2 lidocaine 100μg / kg / minute (n = 10) and Group 3 tramadol 22μg / kg / minute (n = 9). As results the mean, minimum and maximum heart rate found in each group were Control 100, 40 and 253 bpm. Lidocaine 93, 42 and 238 bpm and Tramadol 90, 43 and 253 bpm; on the other hand, a total of 20/26 (76.9%) of the studied animals presented several electrocardiographic alterations that included in their order SVPC, AVB, VPC and RR pauses, however, there were no statistically significant differences (p

Our main interest is the characterization of compounds to support the development of alternatives to currently marketed drugs that are losing effectiveness due to the development of resistance. Schiff bases are promising biologically interesting compounds having a wide range of pharmaceutical properties, including anti-inflammatory, antipyretic, and antimicrobial activities, among others. In this work, we have synthesized 12 Schiff base derivatives of 4-aminoantipyrine. In vitro antimicrobial, antioxidant, and cytotoxicity properties are analyzed, as well as in silico predictive adsorption, distribution, metabolism, and excretion (ADME) and bioactivity scores. Results identify two potential Schiff bases: one effective against E. faecalis and the other with antioxidant activity. Both have reasonable ADME scores and provides a scaffold for developing more effective compounds in the future. Initial studies are usually limited to laboratory in vitro approaches, and following these initial studies, much research is needed before a drug can reach the clinic. Nevertheless, these laboratory approaches are mandatory and constitute a first filter to discriminate among potential drug candidates and chemical compounds that should be discarded.

The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because dNTP levels present in G1 phase are not sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use dNTP shortage to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.

[EN] Metschnikowia reukaufii is a widespread yeast able to grow in the plants’ floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l−1, which led us into the search for enzymatic activities involved in this sugar use/transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload‐sucrose reactions. These products were purified and structurally characterized by MS‐ESI and NMR techniques. The reaction mixture included new sugars showing a great variety of glycosidic bonds including α‐(1→1), α‐(1→3) and α‐(1→6) linkages. The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l−1, the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l−1 of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. Interestingly, the sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide with no previous NMR structure elucidation, as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications., This work was supported by the Projects BIO2016‐76601‐C3‐2‐R/‐1‐R from the Spanish Ministry of Economy and Competitiveness and Fundación Ramón Areces (XIX Call of Research Grants in Life and Material Sciences). Martin Garcia‐Gonzalez fellowship FPU16/02925 was supported by the Spanish Ministry of Education, Culture and Sport. We thank Fundación Ramón Areces for an institutional grant to the Center of Molecular Biology Severo Ochoa. We also thank Mrs. Asunción Martín‐Redondo for the technical support.