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RAS proteins control various intracellular signaling networks. Mutations at specific locations were shown to stabilize their active GTP-bound state, which is associated with the development of multiple cancers. An attractive approach to modulate RAS signaling is through its regulatory guanine nucleotide exchange factor (GEF) son of sevenless 1 (SOS1). With the recent discovery of Nanobody14, which potently enhances SOS1-catalyzed nucleotide exchange on RAS, we explored the feasibility of developing peptide mimetics by structurally mimicking the complementarity-determining region 3 (CDR3). Guided by a biochemical GEF assay and X-ray co-crystal structures, successive rounds of optimization and gradual conformational rigidification led to CDR3 mimetics showing half of the maximal activation potential of the native nanobody. Altogether, this study provides the first proof-of-concept that peptides able to functionally modulate a protein-protein interaction can be obtained by structural mimicry of a nanobody paratope.

Made available in DSpace on 2021-06-25T10:50:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 John Innes Foundation Seventh Framework Programme European Regional Development Fund Fonds Wetenschappelijk Onderzoek Plant roots are specialized belowground organs that spatiotemporally shape their development in function of varying soil conditions. This root plasticity relies on intricate molecular networks driven by phytohormones, such as auxin and jasmonate (JA). Loss-of-function of the NOVEL INTERACTOR OF JAZ (NINJA), a core component of the JA signaling pathway, leads to enhanced triterpene biosynthesis, in particular of the thalianol gene cluster, in Arabidopsis thaliana roots. We have investigated the biological role of thalianol and its derivatives by focusing on Thalianol Synthase (THAS) and Thalianol Acyltransferase 2 (THAA2), two thalianol cluster genes that are upregulated in the roots of ninja mutant plants. THAS and THAA2 activity was investigated in yeast, and metabolite and phenotype profiling of thas and thaa2 loss-of-function plants was carried out. THAA2 was shown to be responsible for the acetylation of thalianol and its derivatives, both in yeast and in planta. In addition, THAS and THAA2 activity was shown to modulate root development. Our results indicate that the thalianol pathway is not only controlled by phytohormonal cues, but also may modulate phytohormonal action itself, thereby affecting root development and interaction with the environment. Department of Plant Biotechnology and Bioinformatics Ghent University, Technologiepark 71 VIB Center for Plant Systems Biology, Technologiepark 71 Department of Metabolic Biology John Innes Centre, Norwich Research Park, Colney Lane Laboratory of Molecular Cell Biology KU Leuven, Kasteelpark Arenberg 31 VIB Center for Microbiology, Kasteelpark Arenberg 31 Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP) Laboratory of Growth Regulators Institute of Experimental Botany of the Czech Academy of Sciences and Faculty of Science of Palacký University, Šlechtitelů 27 Department of Organic Chemistry Ghent University Department of Forest Genetics and Plant Physiology Umeå Plant Science Centre Swedish University of Agricultural Sciences Lab of Plant Growth Analysis Ghent University Global Campus Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP) Seventh Framework Programme: 613692 TriForC European Regional Development Fund: CZ.02.1.01/0.0/0.0/17_048/0007323 Fonds Wetenschappelijk Onderzoek: G004515N Fonds Wetenschappelijk Onderzoek: G008417N

Pseudomonas isolates from tropical environments have been underexplored and may form an untapped reservoir of interesting secondary metabolites. In this study, we compared Pseudomonas and cyclic lipopeptide (CLP) diversity in the rhizosphere of a cocoyam root rot disease (CRRD) suppressive soil in Boteva, Cameroon with those from four conducive soils in Cameroon and Nigeria. Compared with other soils, Boteva andosols were characterized by high silt, organic matter, nitrogen and calcium. Besides, the cocoyam rhizosphere at Boteva was characterized by strains belonging mainly to the P. koreensis and P. putida (sub)groups, with representations in the P. fluorescens, P. chlororaphis, P. jessenii and P. asplenii (sub)groups. In contrast, P. putida isolates were prominent in conducive soils. Regarding CLP diversity, Boteva was characterized by strains producing 11 different CLP types with cocoyamide A producers, belonging to the P. koreensis group, being the most abundant. However, putisolvin III-V producers were the most dominant in the rhizosphere of conducive soils in both Cameroon and Nigeria. Furthermore, we elucidated the chemical structure of putisolvin derivatives-putisolvin III-V, and described its biosynthetic gene cluster. We show that high Pseudomonas and metabolic diversity may be driven by microbial competition, which likely contributes to soil suppressiveness to CRRD.

Cyclic lipopeptides (CLiPs) have many biological functions, including the selective permeabilization of target membranes, and technical and medical applications. We studied the anionic CLiP viscosin from Pseudomonas along with a neutral analog, pseudodesmin A, and the cationic viscosin-E2K to better understand electrostatic effects on target selectivity. Calcein leakage from liposomes of anionic phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) is measured in comparison with net-neutral phosphatidylcholine by time-resolved fluorescence. By contrast to the typical selectivity of cationic peptides against anionic membranes, we find viscosin more active against PG/PE at 30 μM lipid than viscosin-E2K. At very low lipid concentration, the selectivity is reversed. An equi-activity analysis reveals the reciprocal partition coefficients, 1/K, and the CLiP-to-lipid mole ratio within the membrane as leakage after 1 h reaches 50%, Re50. As expected, 1/K to PG/PE is much lower (higher affinity) for viscosin-E2K (3 μM) than viscosin (15 μM). However, the local damage to the PG/PE membrane caused by a viscosin molecule is much stronger than that of viscosin-E2K. This can be explained by the strong membrane expansion due to PG/viscosin repulsion inducing asymmetry stress between the two leaflets and, ultimately, transient limited leakage at Re50 = 0.08. PG/viscosin-E2K attraction opposes expansion and leakage starts only as the PG charges in the outer leaflet are essentially compensated by the cationic peptide (Re50 = 0.32). In the high-lipid regime (at lipid concentrations cL ≫ 1/K), virtually all CLiP is membrane bound anyway and Re50 governs selectivity, favoring viscosin. In the low-lipid regime at cL ≪ 1/K, virtually all CLiP is in solution, 1/K becomes important and the “cation attacks anionic membrane” selectivity gets restored. Overall, activity and selectivity data can only properly be interpreted if the lipid regime is known and predictions for other lipid concentrations or cell counts require knowledge of 1/K and Re50.

In this study, it was evaluated whether the whey protein nitrogen index (WPNI) of a range of skim milk powder (SMP) samples was correlated with the heat stability of the corresponding recombined filled evaporated milk (RFEM), as well as with the extent of lactosylation and changes in whey proteins (WPs). Pulsed field gradient NMR results showed that the diffusion coefficients of lactose in all samples varied only to a small extent, while the diffusion coefficients of the WP fraction ranged more widely. A lower heat stability of RFEM produced from SMP with a lower WPNI was found as compared with those with a higher WPNI: principal component analysis (PCA) and correlation analysis revealed that the decreased heat stability could be at least partly explained by the increased calcium solubilization induced by the lower pH of dispersions of SMP with a lower WPNI. Although it was claimed over the years that high-heat SMP (with a low WPNI) is beneficial to ensure a better heat stability of RFEM, it is clear from our results that a low WPNI of SMP alone was not sufficient to guarantee a better RFEM-heat stabilizing functionality.

Cyclic lipopeptides (CLiPs) are secondary metabolites secreted by a range of bacterial phyla. CLiPs from Pseudomonas in particular display diverse structural variations in terms of the number of amino acid residues, macrocycle size, amino acid identity and stereochemistry (e.g. D- vs. L-amino acids). Reports detailing the discovery of novel or already characterized CLiPs from new sources appear regularly in literature. Increasingly however, the lack of detailed characterization threatens to cause considerable confusion, especially if configurational heterogeneity is present for one or more amino acids. Using Pseudomonas CLiPs from the Bananamide, Orfamide and Xantholysin groups as test cases, we demonstrate and validate that 1H and 13C NMR chemical shifts of CLiPs are sufficiently sensitive to differentiate between possible diastereomers of a particular sequence even when they only differ in a single D/L configuration. Rapid screening, involving simple comparison of the NMR fingerprint of a newly isolated CLiP with that of a reference CLiP of known stereochemistry, can then be applied to resolve dead-ends in configurational characterization and avoid the much more cumbersome chemical characterization protocols. Even when the stereochemistry of a particular reference CLiP remains to be established, NMR fingerprinting still allows verifying whether a CLiP from a novel source is already present in the reference collection, thus contributing to dereplication. To benefit research involving CLiPs, we have made a publicly available ‘knowledge base’ at https://www.rhizoclip.be, where we present an overview of published NMR fingerprint data of characterized CLiPs, together with literature data on the originally determined structures.Significance StatementPseudomonas CLiPs, are ubiquitous specialized metabolites, impacting the producer’s lifestyle and interactions with the (a)biotic environment. Consequently, they generate interest for agricultural and clinical applications. Establishing structure-activity relationships as premise to their development is hindered because full structural characterization including stereochemistry requires labor-intensive analyses, without guarantee for success. Moreover, increasing use of superficial comparison with previously characterized CLiPs introduces or propagates erroneous attributions, clouding further scientific progress. We provide a generally applicable characterization methodology for structural comparison of newly isolated CLiPs to reference compounds with (un)known stereochemistry based on NMR fingerprints. The reference compound database available for the wide scientific community promises to facilitate structural assessment and dereplication of newly isolated CLiPs, and to support genome mining for novel CLiPs.

Proline homopolymer motifs are found in many proteins; their peculiar conformational and dynamic properties are often directly involved in those proteins' functions. However, the dynamics of proline homopolymers is hard to study by NMR due to a lack of amide protons and small chemical shift dispersion. Exploiting the spectroscopic properties of fluorinated prolines opens interesting perspectives to address these issues. Fluorinated prolines are already widely used in protein structure engineering – they introduce conformational and dynamical biases – but their use as 19F NMR reporters of proline conformation has not yet been explored. In this work, we look at model peptides where Cγ-fluorinated prolines with opposite configurations of the chiral Cγ centre have been introduced at two positions in distinct polyproline segments. By looking at the effects of swapping these (4R)-fluoroproline and (4S)-fluoroproline within the polyproline segments, we were able to separate the intrinsic conformational properties of the polyproline sequence from the conformational alterations instilled by fluorination. We assess the fluoroproline 19F relaxation properties, and we exploit the latter in elucidating binding kinetics to the SH3 (Src homology 3) domain.

Monoclonal antibodies emerged as an important therapeutic drug class with remarkable specificity and binding affinity. Nonetheless, these heterotetrameric immunoglobulin proteins come with high manufacturing and therapeutic costs which can take extraordinary proportions, besides other limitations such as their limited in cellulo access imposed by their molecular size (ca. 150 kDa). These drawbacks stimulated the development of downsized functional antibody fragments (ca. 15–50 kDa), together with smaller synthetic peptides (ca. 1–3 kDa) derived from the antibodies’ crucial complementarity-determining regions (CDR). Despite the general lack of success in the literal translation of CDR loops in peptide mimetics, rational structure-based and computational approaches have shown their potential for obtaining functional CDR-based peptide mimetics. In this review, we describe the efforts made in the development of antibody and nanobody paratope-derived peptide mimetics with particular focus on the used design strategies, in addition to highlighting the challenges associated with their development.

Alginates are fairly abundant in nature and possess many interesting properties, including their biocompatibility and ability to absorb large amounts of water. Hence, increasing interest in their derivatization has been observed and the determination of the number of newly introduced functionalities has become a key issue. For this purpose, literature generally reports on conventional H-1-NMR spectra, typically recorded at elevated temperatures and/or after hydrolysis of the alginate to circumvent line broadening effects resulting from the high viscosity. The present work reports on the modification of alginate with methacrylate functionalities and determination of the resulting degree of substitution (DS), i.e. the number of introduced methacrylate moieties relative to the initial amount of hydroxyl groups along the alginate backbone, via NMR spectroscopy. Freeze-drying and low power water presaturation were applied to improve the quality of the H-1 NMR spectra. Nevertheless, it remains a qualitative method, to be used only for mutual comparisons of samples. A new and accurate method for DS determination of methacrylated alginates, based on C-13-NMR spectroscopy, is proposed. Quantitative C-13-NMR spectra were recorded with reduced measuring times by addition of a paramagnetic relaxation agent. The proposed method will also be applicable for other water-soluble functionalized alginates and polysaccharides in general. (C) 2019 Elsevier B.V. All rights reserved.

Cyclic lipopeptides (CLiPs) are secondary metabolites produced by a variety of bacteria. These compounds show a broad range of antimicrobial activities; therefore, they are studied for their potential applications in agriculture and medicine. It is generally assumed that the primary target of the CLiPs is the cellular membrane, where they can permeabilize the lipid bilayer. Model membrane systems are commonly used to investigate the effect of lipid composition on the permeabilizing activity of CLiPs, but these systems do not represent the full complexity of true biological membranes. Here, we introduce a novel method that uses sterol-auxotrophic oomycetes to investigate how the activity of membrane-active compounds is influenced by alterations in membrane sterol composition. More specifically, we investigated how ergosterol, cholesterol, beta-sitosterol and stigmasterol affect the activity of the structurally related Pseudomonas-derived CLiPs tolaasin and sessilin against the oomycete Pythium myriotylum. Both compounds were effective against oomycetes, although tolaasin was considerably more active. Interestingly, tolaasin and sessilin effects were similarly reduced by the presence of sterols, with cholesterol showing the highest reduction of activity.

Streptomyces scabies is a phytopathogen associated with common scab disease. This is mainly attributed to its ability to produce the phytotoxin thaxtomin A, the biosynthesis of which is triggered by cellobiose. During a survey of other metabolites released in the presence of cellobiose, we discovered additional compounds in the thaxtomin-containing extract from Streptomyces scabies. Structural analysis by mass spectrometry (MS) and nuclear magnetic resonance (NMR) revealed that these compounds are amino acid sequence variants of the TOR (target of rapamycin) kinase (TORK) pathway-inhibitory lipopeptide rotihibin A, and the main compounds were named rotihibins C and D. In contrast to thaxtomin, the production of rotihibins C and D was also elicited in the presence of glucose, indicating different regulation of their biosynthesis. Through a combination of shotgun and targeted proteomics, the putative rotihibin biosynthetic gene cluster rth was identified in the publicly available genome of S. scabies 87-22. This cluster spans 33 kbp and encodes 2 different nonribosomal peptide synthetases (NRPSs) and 12 additional enzymes. Homologous rth biosynthetic gene clusters were found in other publicly available and complete actinomycete genomes. Rotihibins C and D display herbicidal activity against Lemna minor and Arabidopsis thaliana at low concentrations, shown by monitoring the effects on growth and the maximal photochemistry efficiency of photosystem II. IMPORTANCE Rotihibins A and B are plant growth inhibitors acting on the TORK pathway. We report the isolation and characterization of new sequence analogues of rotihibin from Streptomyces scabies, a major cause of common scab in potato and other tuber and root vegetables. By combining proteomics data with genomic analysis, we found a cryptic biosynthetic gene cluster coding for enzyme machinery capable of rotihibin production. This work may lead to the biotechnological production of variants of this lipopeptide to investigate the exact mechanism by which it can target the plant TORK pathway in Arabidopsis thaliana. In addition, bioinformatics revealed the existence of other variants in plant-associated Streptomyces strains, both pathogenic and nonpathogenic species, raising new questions about the actual function of this lipopeptide. The discovery of a module in the nonribosomal peptide synthetase (NRPS) that incorporates the unusual citrulline residue may improve the prediction of peptides encoded by cryptic NRPS gene clusters.

In this manuscript, we compare different analytical methodologies to validate or disprove the binding capabilities of aptamer sequences. This was prompted by the lack of a universally accepted and robust quality control protocol for the characterization of aptamer performances coupled with the observation of independent yet inconsistent data sets in the literature. As an example, we chose three aptamers with a reported affinity in the nanomolar range for ampicillin, a β-lactam antibiotic, used as biorecognition elements in several detection strategies described in the literature. Application of a well-known colorimetric assay based on aggregation of gold nanoparticles (AuNPs) yielded conflicting results with respect to the original report. Therefore, ampicillin binding was evaluated in solution using isothermal titration calorimetry (ITC), native nano-electrospray ionization mass spectrometry (native nESI-MS), and 1H-nuclear magnetic resonance spectroscopy (1H NMR). By coupling the thermodynamic data obtained with ITC with the structural information on the binding event given by native nESI-MS and 1H NMR we could verify that none of the ampicillin aptamers show any specific binding with their intended target. The effect of AuNPs on the binding event was studied by both ITC and 1H NMR, again without providing positive evidence of ampicillin binding. To validate the performance of our analytical approach, we investigated two well-characterized aptamers for cocaine/quinine (MN4), chosen for its nanomolar range affinity, and l-argininamide (1OLD) to show the versatility of our approach. The results clearly indicate the need for a multifaceted analytical approach, to unequivocally establish the actual detection potential and performance of aptamers aimed at small organic molecules.

Hypothesis It is expected that low resolution (LR) NMR diffusometry enables (more) accurate water droplet size determination for solid-fat based water-in-oil (W/O) emulsions with (sub)-micron size water droplets in comparison to liquid-oil based W/O emulsions due to hindered extra-droplet water diffusion. Experiments W/O emulsions with a volume-weighed mean diameter of about 1 µm and a solid fat content (SFC) ranging from 0% to 74% were produced. The aqueous phase contained the ionic marker tetraphenylphosphonium chloride (TPPCl). The water droplet size was estimated using LR and high resolution (HR) NMR diffusometry. Findings HR-NMR diffusometry showed that the diffusion behavior of water and TPPCl was different, indicating water diffusion beyond the droplet’s interfacial boundaries. From a certain SFC onwards, a slower echo decay was observed for the water molecules, thus decreasing the overestimation of the water droplet size in (sub)micron W/O emulsions. For those emulsions, the solid fat matrix is believed to hinder extra-droplet water diffusion, which is most likely to be related to the increased tortuosity of the diffusive path in the porous fat crystal network. Using LR-NMR, it can be verified whether the water echo attenuation is mono-exponential or bi-exponential by increasing the gradient pulse duration for the maximum gradient strength, which is more convenient for routine analysis compared to HR-NMR.

Dispersions and inks based on copper nanoparticles have raised extensive interest for printed electronics as copper holds promise for attaining high electric conductivity at low cost. Here, we use the decomposition of copper formate in oleylamine to produce a nanocolloid consisting of∼4 nm copper nanocrystals, a size that is ideal to study the binding of ligands to nanocopper. Using solution 1H NMR spectroscopy, we demonstrate that oleylamine binds to the surface of as-synthesized copper nanocrystals, thus stabilizing the dispersion by steric hindrance. We find that addition of a carboxylic acid to a purified nanocolloid induces an exchange between the originally bound oleylamine and the carboxylic acid as the surface-bound ligand.We provide evidence that the carboxylic acid dissociates upon binding to the copper nanocrystals. As such a process requires an amphoteric surface, a characteristic of a metal oxide but not of an elementary metal, we argue that ligand binding is determined by residual surface oxides and not by the pristine copper surface. Finally, we demonstrate that stable copper nanocolloids can be obtained in a variety of polar solvents by replacing oleylamine as a ligand by 2-[2-(2-methoxyethoxy)ethoxy]acetic acid(MEEAA). The inevitable oxidation of the small copper nanocrystals used here can be undone by mild thermal annealing,which especially in the case of MEEAA-stabilized nanocopper leads to significant grain growth. In this way, we turn an as-synthesized dispersion of colloidal copper nanocrystals into a nano-ink that can be formulated to produce metallic copper strips by screen or inkjet printing.

International audience; Cyclic lipo(depsi)peptides (CLiPs) from Pseudomonas constitute a class of natural products involved in a broad range of biological functions for their producers. They also display interesting antimicrobial potential including activity against Gram-positive bacteria. Literature has indicated that these compounds can induce membrane permeabilization, possibly through pore-formation, leading to the general view that the cellular membrane constitutes the primary target in their mode of action. In support of this view, we previously demonstrated that the enantiomer of pseudodesmin A, a member of the viscosin group of CLiPs, shows identical activity against a test panel of six Gram-positive bacterial strains. Here, a previously developed total organic synthesis route is used and partly adapted to generate 20 novel pseudodesmin A analogs in an effort to derive links between molecular constitution, structure and activity. From these, the importance of a macrocycle closed by an ester bond as well as a critical length of β-OH fatty acid chain capping the N-terminus is conclusively demonstrated, providing further evidence for the importance of peptide-membrane interactions in the mode of action. Moreover, an alanine scan is used to unearth the contribution of specific amino acid residues to biological activity. Subsequent interpretation in terms of a structural model describing the location and orientation of pseudodesmin A in a membrane environment, allows first insight in the peptide-membrane interactions involved. The biological screening also identified residue positions that appear less sensitive to conservative modifications, allowing the introduction of a non-perturbing tryptophan residue which will pave the way toward biophysical studies using fluorescence spectroscopy.

Pseudomonas isolates from tropical environments have been underexplored and may form an untapped reservoir of interesting secondary metabolites. In this study, we compared Pseudomonas and cyclic lipopeptide (CLP) diversity in the rhizosphere of a cocoyam root rot disease (CRRD) suppressive soil in Boteva, Cameroon with those from four conducive soils in Cameroon and Nigeria. Compared with other soils, Boteva andosols were characterized by high silt, organic matter, nitrogen and calcium. Besides, the cocoyam rhizosphere at Boteva was characterized by strains belonging mainly to the P. koreensis and P. putida (sub)groups, with representations in the P. fluorescens, P. chlororaphis, P. jessenii and P. asplenii (sub)groups. In contrast, P. putida isolates were prominent in conducive soils. Regarding CLP diversity, Boteva was characterized by strains producing 11 different CLP types with cocoyamide A producers, belonging to the P. koreensis group, being the most abundant. However, putisolvin III-V producers were the most dominant in the rhizosphere of conducive soils in both Cameroon and Nigeria. Furthermore, we elucidated the chemical structure of putisolvin derivatives-putisolvin III-V, and described its biosynthetic gene cluster. We show that high Pseudomonas and metabolic diversity may be driven by microbial competition, which likely contributes to soil suppressiveness to CRRD.

Previous investigations of the impact of an imidazole-tethered thymidine in synthetic DNA duplexes, monitored using UV and NMR spectroscopy, revealed a base context dependent increase in thermal stability of these duplexes and a striking correlation with the imidazolium pKa. Unrestrained molecular dynamics (MD) simulations demonstrated the existence of a hydrogen bond between the imidazolium and the Hoogsteen side of a nearby guanosine which, together with electrostatic interactions, form the basis of the so-called pKa-motif responsible for these duplex-stabilizing and pKa-modulating properties. Here, the robustness and utility of this pKa-motif was explored by introducing multiple imidazole-tethered thymidines at different positions on the same dsDNA duplex. For all constructs, sequence based expectations as to pKa-motif formation were supported by MD simulations and experimentally validated using NOESY. Based on the analysis of the pKa values and melting temperatures, guidelines are formulated to assist in the rational design of oligonucleotides modified with imidazolium-tethered thymidines for increased thermal stability that should be generally applicable, as demonstrated through a triply modified construct. In addition, a proof-of-principle study demonstrating enhanced stability of the l-argininamide binding aptamer modified with an imidazole-tethered thymidine in the presence and absence of ligand, demonstrates its potential for the design of more stable aptamers.

The current paper focuses on the functionalization of κ-carrageenan and gelatin as extracellular matrix polysaccharide and protein mimic respectively to produce hydrogel films for adipose tissue engineering. More specifically, κ-carrageenan as well as gelatin have been functionalized with methacrylate and methacrylamide moieties respectively to enable subsequent UV-induced crosslinking in the presence of a photo-initiator. The gel fraction, the mass swelling ratio and the mechanical properties of both the one-component hydrogels and the protein/polysaccharide blends have been evaluated. The mechanical and swelling properties of the blends could be tuned by varying the hydrogel composition as well as the crosslinking method applied. The in vitro biocompatibility assays indicated a significantly higher cell viability of adipose tissue-derived mesenchymal stem cells seeded onto the blends as compared to the one-component hydrogels. The results show that the blends of gelatin and κ-carrageenan clearly outperform the one-component hydrogels in terms of adipose tissue engineering potential.

Model experiments under laboratory conditions were carried out to assess the accumulation tendency of Zr from a silica-zirconia core–shell nanoparticles, synthesised for the assay. Acute exposition tests were conducted with Daphnia magna and Danio rerio and the accumulation tendency of the nano and the molecular form was compared. Significant elevation of Zr was found in the tissue of the test organisms treated by the NP compared to the control, however, the retention of zebrafish was lower than that of the daphnids. Increased level of bioconcentration factor and the strong correlation of the redundancy analysis data suggest accumulation tendency in D. magna, yet long-term experiments are required to prove and further assess the environmental risk of food-chain bioconcentration. The BCF results were under the REACH limit for the D. rerio groups indicating the low short-term accumulation tendency of Zr from the NP. However, Zr level was significantly higher in zebrafish individuals originating from the SiO2@ZrO2 NP exposed treatments compared to the groups supplemented by the same concentration of ZrOCl2, which did not result in the elevation of Zr in fish tissue. The trace element homeostasis of D. rerio was not affected by the acute SiO2@ZrO2 NP exposition and no lethality occurred.

Dialkyl cationic surfactants, such as dioctadecyl dimethyl ammonium bromide (DODAB) form vesicles upon dispersion in water. In order to study the influence of the surfactant's counterion, the same dialkyl surfactant was prepared with a fluoride counterion (DODAF). Visually, quite transparent dispersions were prepared when fluoride was selected as counterion, whereas more turbid dispersions were obtained using the bromide surfactant. LR-NMR revealed that the permeability of the DODAF dispersions for water was much larger, which indicated that these rather contained uni- or oligolamellar vesicles, whereas the DODAB dispersions were multilamellar. These observations seemed to indicate an increasing degree of surface charge neutralization by the heavier anions, in line with the Hofmeister series, which was indeed confirmed by electrophoretic light scattering measurements. In order to further quantify the surfactant-counterion interaction, HR-NMR diffusometry measurements were performed on dispersions of a dialkyl cationic surfactant with methyl sulphate counterion in deuterated water. The diffusion data showed that the mobility of this organic anion increased when adding different sodium halides, with a stronger effect from fluoride over chloride and bromide to iodide, which clearly indicated the gradual displacement of the methyl sulphate counterion from the vesicular surface by the stronger binding anions.

Superabsorbent polymers (SAPs) based on polysaccharides, especially alginate, could offer a valuable solution in a plethora of applications going from drug delivery to self-healing concrete. This has already been proven with both calcium alginate and methacrylated alginate combined with acrylic acid. In this manuscript, the effect of varying the degree of methacrylation and use of a combination of acrylic acid and acrylamide is investigated to explore the effects on the relevant SAP characteristics. The materials showed high gel fractions and a strong swelling capacity up to 630gwater/gSAP, especially for superabsorbent polymers with a low degree of substitution. The SAPs also showed only a limited hydrolysis in aqueous and cement filtrate solutions. ispartof: CARBOHYDRATE POLYMERS vol:168 pages:44-51 ispartof: location:England status: published

ACCERBATIN is an ethylene-mimicking small molecule that affects auxin homeostasis and ROS accumulation in etiolated seedlings. In light-grown plants, it exhibits auxin-like herbicidal properties., The volatile two-carbon hormone ethylene acts in concert with an array of signals to affect etiolated seedling development. From a chemical screen, we isolated a quinoline carboxamide designated ACCERBATIN (AEX) that exacerbates the 1-aminocyclopropane-1-carboxylic acid-induced triple response, typical for ethylene-treated seedlings in darkness. Phenotypic analyses revealed distinct AEX effects including inhibition of root hair development and shortening of the root meristem. Mutant analysis and reporter studies further suggested that AEX most probably acts in parallel to ethylene signaling. We demonstrated that AEX functions at the intersection of auxin metabolism and reactive oxygen species (ROS) homeostasis. AEX inhibited auxin efflux in BY-2 cells and promoted indole-3-acetic acid (IAA) oxidation in the shoot apical meristem and cotyledons of etiolated seedlings. Gene expression studies and superoxide/hydrogen peroxide staining further revealed that the disrupted auxin homeostasis was accompanied by oxidative stress. Interestingly, in light conditions, AEX exhibited properties reminiscent of the quinoline carboxylate-type auxin-like herbicides. We propose that AEX interferes with auxin transport from its major biosynthesis sites, either as a direct consequence of poor basipetal transport from the shoot meristematic region, or indirectly, through excessive IAA oxidation and ROS accumulation. Further investigation of AEX can provide new insights into the mechanisms connecting auxin and ROS homeostasis in plant development and provide useful tools to study auxin-type herbicides.

Gelatin is frequently used in various biomedical applications. However, gelatin is generally extracted from an animal source, which can result in issues with reproducibility as well as pathogen transmittance. Therefore, we have investigated the potential of a recombinant peptide based on collagen I (RCPhC1) for tissue engineering applications and more specifically for adipose tissue regeneration. In the current paper, RCPhC1 was functionalized with photo-crosslinkable methacrylamide moieties to enable subsequent UV-induced crosslinking in the presence of a photo-initiator. The resulting biomaterial (RCPhC1-MA) was characterized by evaluating the crosslinking behaviour, the mechanical properties, the gel fraction, the swelling properties and the biocompatibility. The obtained results were compared with the data obtained for methacrylamide-modified gelatin (Gel-MA). The results indicated that the properties of RCPhC1-MA networks are comparable to those of animal-derived Gel-MA. RCPhC1-MA is thus an attractive synthetic alternative for animal-derived Gel-MA and is envisioned to be applicable for a wide range of tissue engineering purposes.

Proline is often found as a turn inducer in peptide or protein domains. Exploitation of its restricted conformational freedom led to the development of the d-Pro-l-Pro (corresponding to (R)-Pro–(S)-Pro) segment as a “templating” unit, frequently used in the design of β-hairpin peptidomimetics, in which conformational stability is, however, inherently linked to the cis–trans isomerization of the prolyl amide bonds. In this context, the stereoelectronic properties of the CF 3 group can aid in conformational control. Herein, the impact of α-trifluoromethylated proline analogues is examined for the design of enhanced β-turn inducers. A theoretical conformational study permitted the dipeptide (R)-Pro–(R)-TfmOxa (TfmOxa: 2-trifluoromethyloxazolidine-2-carboxylic acid) to be selected as a template with an increased trans–cis rotational energy barrier. NMR spectroscopic analysis of the Ac-(R)-Pro–(R)-TfmOxa–(S)-Val-OtBu β-turn model, obtained through an original synthetic pathway, validated the prevalence of a major trans–trans conformer and indicated the presence of an internal hydrogen bond. Altogether, it was shown that the (R)-Pro–(R)-TfmOxa template fulfilled all crucial β-turn-inducer criteria.

Short alphahelical peptide sequences were stabilized through Glaser-Hay couplings of propargylated l- and/or d-serine residues at positions i and i+7. NMR analysis confirmed a full stabilization of the helical structure when a d-Ser (i), l-Ser (i+7) combination was applied. In case two l-Ser residues were involved in the cyclization, the helical conformation is disrupted outside the peptide's macrocycle.

A novel technique based on high resolution (HR)-NMR diffusometry is presented to investigate oil diffusion on the (sub)-micron scale level in fat crystal networks in situ and in a non-destructive way. The use of a high-gradient probe enables the observation of the time (Delta)-dependent diffusion behavior in fat blends (FBs) of different solid fat content (SFC), as obtained by mixing soft palm mid fraction and high-oleic sunflower oil. Hereby, the NMR-diffusion data are analyzed using CONTIN to obtain a continuous distribution of diffusion coefficients. As such, the hindrance of the oil diffusion by the fat crystal network is examined. The characterization of the lipid matrices is completed with H-1-LR-NMR relaxometry, X-ray diffraction, and microstructural visualization. For the investigated FBs, the average diffusion coefficient D(Delta) decreased with increasing SFC, indicating an increased tortuosity of the diffusive path due to an increasing density of the fat crystal networks as observed by polarized light microscopy. This is also supported by an increase in crystal surface area which is indicated by T-2-relaxation analysis. This study shows that NMR diffusometry is a promising technique for investigation of structure-function relationships in fat systems. Practical Applications: The study shows that NMR diffusometry is a promising technique for investigation of structure-function relationships in fat systems. For the first time, oil diffusion in fat crystal networks has been studied by HR-NMR diffusometry with high gradients to collect the diffusional echo signals, while CONTIN-analysis has been used for the first time to process the data.

In this work, we present a combined experimental and theoretical analysis of two-component ligand shells passivating CdSe quantum dots. Using nuclear magnetic resonance spectroscopy, we first show that exposing oleate capped quantum dots to primary carboxylic acids results in a one-for-one exchange that preserves the overall ligand surface concentration. Exposure to straight-chain acids leads to a binary ligand shell that behaves as an ideal mixture and that has a composition matching the overall acid composition of the dispersion. In the case of branched-chain acids, the exchange is restricted to about 25% of the original ligands. Based on molecular dynamics simulations, we argue that this behavior reflects the more favorable packing of oleates compared to branched carboxylates on the (100) facets of CdSe quantum dots.

Triterpenoids are widespread bioactive plant defence compounds with potential use as pharmaceuticals, pesticides and other high-value products. Enzymes belonging to the cytochrome P450 family have an essential role in creating the immense structural diversity of triterpenoids across the plant kingdom. However, for many triterpenoid oxidation reactions, the corresponding enzyme remains unknown. Here we characterize CYP716 enzymes from different medicinal plant species by heterologous expression in engineered yeasts and report ten hitherto unreported triterpenoid oxidation activities, including a cyclization reaction, leading to a triterpenoid lactone. Kingdom-wide phylogenetic analysis of over 400 CYP716s from over 200 plant species reveals details of their evolution and suggests that in eudicots the CYP716s evolved specifically towards triterpenoid biosynthesis. Our findings underscore the great potential of CYP716s as a source for generating triterpenoid structural diversity and expand the toolbox available for synthetic biology programmes for sustainable production of bioactive plant triterpenoids., Cytochrome P450 family enzymes have an essential role in the creation of triterpenoid diversity in plants. Here, the authors describe triterpenoid synthesis as mediated by CYP716 enzymes in medicinal plant species, and perform phylogenetic analysis to describe CYP716 molecular evolution in plants.

Superabsorbent polymers (SAPs) have already found their way in many applications. Some of these polymers undergo major characteristic changes by small environmental variations which makes them interesting for self-healing of cracks in concrete. In the present work, polymer networks composed of dimethylaminoethyl methacrylate and N,N′-methylenebisacrylamide have been synthesized. Moisture uptake capacities up to 45% were measured at a relative humidity of 95%. They showed a desired pH-responsive swelling behavior with no degree of degradation. A small significant mortar strength reduction is found with addition up to 1 m% compared to the cement mass. All results were compared to commercially available SAPs.

The structural importance of backbone amide protons in the bacterial cyclic lipopeptide pseudodesmin A, a viscosin group member, is studied by selective introduction of N-methylated amino acids. Site-selective N-methylation was conveniently achieved by introduction of N-Me-L-Leu at positions 1 and 7 during solid phase peptide synthesis. The impact of this modification on conformation, antibiotic activity and supramolecular behaviour allowed to establish the significance of intra- and intermolecular hydrogen bonds.

Whereas Whey Protein Isolate may be used as an effective emulsifier, it suffers from limited stability upon heating. In this research, the effect of combining Whey Protein Isolate (WPI) with Low Methoxyl Pectin (LMP) on the heat stability of WPI was investigated. This was accomplished either by simple mixing or by conjugate formation by dry heat treatment. WPI-LMP mixtures and conjugates were prepared at a WPI to LMP ratio of 1:0, 4:1, 2:1, and 1:1. Conjugates were prepared by means of dry heat treatment at a temperature of 60 °C and 74% relative humidity by incubation for up to 16 days. The pH effect and brown color development of the WPI-LMP conjugates upon incubation was monitored. SDS-Page, free amino group determination, as well as diffusion coefficient analysis, all confirmed the formation of new compounds with high molecular weight. The heat stability of the conjugates was then tested and compared to the native WPI. Upon 2 min of heating at 80 °C and pH 6.5, the solubility of WPI was reduced by approximately 50% due to thermal denaturation and subsequent aggregation. However, dry heat treatment of the WPI-LMP mixtures highly improved the heat stability of WPI: as the incubation time was increased, the residual protein solubility upon heating of the WPI became higher. Upon 16 days of incubation, the protein solubility of the heated WPI-LMP conjugates was comparable to that of the conjugates before heating. Considering the emulsifying properties, it was found that WPI-LMP conjugates produced smaller oil droplet compared to either native WPI, dry heated WPI, or mixtures of WPI-LMP which were not subjected to conjugation by dry heat treatment. Heating the emulsions at 80 °C for 10 and 20 min revealed that WPI-LMP conjugates stabilized emulsions exhibited excellent stability towards heat: whereas pronounced aggregation and gelation occurred in emulsions stabilized by WPI or mixtures with LMP, the conjugate stabilized emulsions retained their original viscosity and particle size.

Bola-amphiphilic surfactants are molecules with fascinating properties. Their unique configuration consisting of a long hydrophobic spacer connecting two hydrophilic entities renders the molecule more water soluble than the average surfactant, but still allows formation of supramolecular structures. These properties make them extremely suitable for applications in in nanotechnology, electronics, and gene and drug delivery. In general, these compounds are obtained by chemical synthesis. We report here an efficient microbial production process for the fully green synthesis of bolaform surfactants. A sophorolipid-producing Starmerella bombicola yeast strain was disabled in its sophorolipid acetyltransferase and lactone esterase, which should logically result in synthesis of non-acetylated acidic sophorolipids; molecules with the classic amphiphilic structure. Surprisingly, also bolaform glycolipids were obtained, with an additional sophorose linked to the free carboxyl end of the acidic sophorolipids as confirmed by MS and NMR analysis. The obtained titers of 27.7 g/L total product are comparable to wild type values, and the novel molecules account for at least 74% of this. Bola-amphiphile biosynthesis proved to be attributed to the promiscuous activity of both UDP-glucosyltransferases UGTA1 and UGTB1 from the core sophorolipid pathway, displaying activity toward non-acetylated intermediates. The absence of acetyl groups seems to trigger formation of bolaform compounds starting from acidic sophorolipids. Hence, wild type S. bombicola produces these compounds only at marginal amounts in general not reaching detection limits. We created a strain knocked-out in its sophorolipid acetyltransferase and lactone esterase able to produce these novel compounds in economical relevant amounts, opening doors for the application of biological-derived bolaform structures. Biotechnol. Bioeng. 2016;113: 2644-2651. © 2016 Wiley Periodicals, Inc.

To date, the potential of several types of polymeric materials as healing agents for self-healing of concrete has already been investigated. Generally, for self-healing concrete with encapsulated polymeric healing agents, the curing mechanism is triggered upon contact with moisture/air or upon reaction with a second component provided by additional capsules. The present work explores the use of in-situ curable hydrogels formed as a result of the elevated pH of the cementitious matrix, via a Michael-type addition reaction, as potential healing/sealing materials for concrete applications. For this purpose, a variety of acrylate-endcapped urethane-based precursors were synthesized and combined with a thiol-based cross-linker. Various properties including the viscosity, the curing time, the swelling capacity and the cross-linking efficiency have been evaluated. The potential of the developed materials to seal concrete cracks was assessed through manual injection. The results indicate that the cross-linking reaction can readily occur in-situ due to the alkaline environment of the cementitious matrix and that the hydrogels exhibit favorable sealing properties. Keywords: Self-healing, Concrete, Hydrogel precursor, Michael-type addition reaction, In-situ curable hydrogels

The industrial application of whey proteins as a food emulsifier is still limited due to its susceptibility towards heating. In this study, dry heat conjugation of whey protein and lactose was investigated to improve the heat stability of WPC-stabilised oil in water (O/W) emulsions. A commercially available whey protein concentrate (WPC), containing 80 % protein and 12 % lactose, was dry heat incubated. Hereby, the effect of incubation temperature (60, 70, and 80 °C), relative humidity (64, 74, and 79 %), and preconditioning pH (4, 6, 8, 10) were determined. Temperature variation showed a pronounced effect on the required duration to obtain conjugates with excellent emulsion heat stabilising capacity. Four hours of incubation at 80 °C was sufficient to retain the original particle size and consistency after heating the emulsions at 80 °C for 20 min. Whereas circular dichroism spectroscopy revealed that this dry heat incubation condition (80 °C, 4 h) maintained the secondary structure of WPC, pulsed-field gradient NMR revealed that about 40 % of the lactose present should become protein-bound to generate conjugates with the desired functionality. The results indicated that a heat stable WPC with about 40 % of bound lactose could be obtained by some hours of dry heat treatment at 80 °C.

Whey protein–lactose conjugates were prepared by dry heat incubation (60 °C, 74% relative humidity) at 1:1 M ratio of protein amino groups to reducing sugar. Pulsed-field gradient-nuclear magnetic resonance (PFG-NMR) was introduced as a new and absolute method to determine the degree of conjugation without any sample pretreatment, based on the different diffusion behaviour of free and protein-bound lactose. Whereas the TNBS method revealed 73% conjugation, PFG-NMR results indicated that only 37% of lactose was bound to the protein after 8 h incubation. Circular dichroism spectra showed no change of WPI secondary structure upon dry heat incubation, whereas the electrophoretic mobility shifted towards more negative values. The emulsifying and, more importantly, heat stabilising properties of the conjugates were subsequently evaluated in oil/water emulsions. Eight hours of incubation was sufficient to produce conjugates that successfully stabilised the emulsion against heating (80 °C, 20 min), regarding the particle size and viscosity.

Silica@zirconia core@shell oxide nanoparticles of approx. 50 nm diameter were developed in view of an application as a novel carrier with adjuvant activities for cancer vaccines. To this end, the aim was to cover the surface of the nanocarrier by potent single stranded DNA (ssDNA) type immune stimulators used in cancer vaccination. Prior to characterizing the binding of ssDNA to the nanocarrier surface, the adsorption of deoxynucleoside monophosphate building blocks (dNMPs) was studied. After optimization of conditions for synthesis, solvent exchange and surface modification, the effects and possible interference of several buffers, such as HEPES, PIPES, MOPS and MOPSO, were also investigated. Formation of a new compound was revealed by UV–VIS spectroscopy during the recording of zeta potential vs. pH titration curves in the presence of HEPES and PIPES buffer in the neutral-acidic pH region. We identified this new species as gold ions etched from the electrode of the zeta cuvette, stabilized by the buffers via chelation, which is then followed by their conversion into gold nanoparticles.

Pseudomonas species are metabolically robust, with capacity to produce secondary metabolites including cyclic lipopeptides (CLPs). Herein we conducted a chemical analysis of a crude CLP extract from the cocoyam rhizosphere-derived biocontrol strain Pseudomonas sp. COW3. We performed in silico analyses on its whole genome, and conducted in vitro antagonistic assay using the strain and purified CLPs. Via LC-MS and NMR, we elucidated the structures of four novel members of the bananamide group, named bananamides D-G. Besides variability in fatty acid length, bananamides D-G differ from previously described bananamides A-C and MD-0066 by the presence of a serine and aspartic acid at position 6 and 2, respectively. In addition, bananamide G has valine instead of isoleucine at position 8. Kendrick mass defect (KMD) allowed the assignment of molecular formulae to bananamides D and E. We unraveled a non-ribosomal peptide synthetase cluster banA, banB and banC which encodes the novel bananamide derivatives. Furthermore, COW3 displayed antagonistic activity and mycophagy against Pythium myriotylum, while it mainly showed mycophagy on Pyricularia oryzae. Purified bananamides D-G inhibited the growth of P. myriotylum and P. oryzae and caused hyphal distortion. Our study shows the complementarity of chemical analyses and genome mining in the discovery and elucidation of novel CLPs. In addition, structurally diverse bananamides differ in their antimicrobial activity.