Ester Cross-Link Profiling of the Cutin Polymer of Wild-Type and Cutin Synthase Tomato Mutants Highlights Different Mechanisms of Polymerization

Cuticle function is closely related to the structure of the cutin polymer. However, the structure and formation of this hydrophobic polyester of glycerol and hydroxy/epoxy fatty acids has not been fully resolved. An apoplastic GDSL-lipase known as CUTIN SYNTHASE1 (CUS1) is required for cutin deposition in tomato (Solanum lycopersicum) fruit exocarp. In vitro, CUS1 catalyzes the self-transesterification of 2-monoacylglycerol of 9(10),16-dihydroxyhexadecanoic acid, the major tomato cutin monomer. This reaction releases glycerol and leads to the formation of oligomers with the secondary hydroxyl group remaining nonesterified. To check this mechanism in planta, a benzyl etherification of nonesterified hydroxyl groups of glycerol and hydroxy fatty acids was performed within cutin. Remarkably, in addition to a significant decrease in cutin deposition, mid-chain hydroxyl esterification of the dihydroxyhexadecanoic acid was affected in tomato RNA interference and ethyl methanesulfonate-cus1 mutants. Furthermore, in these mutants, the esterification of both sn-1,3 and sn-2 positions of glycerol was impacted, and their cutin contained a higher molar glycerol-to-dihydroxyhexadecanoic acid ratio. Therefore, in planta, CUS1 can catalyze the esterification of both primary and secondary alcohol groups of cutin monomers, and another enzymatic or nonenzymatic mechanism of polymerization may coexist with CUS1-catalyzed polymerization. This mechanism is poorly efficient with secondary alcohol groups and produces polyesters with lower molecular size. Confocal Raman imaging of benzyl etherified cutins showed that the polymerization is heterogenous at the fruit surface. Finally, by comparing tomato mutants either affected or not in cutin polymerization, we concluded that the level of cutin cross-linking had no significant impact on water permeance.Cuticles are ubiquitous hydrophobic barriers at the surfaces of aerial plant organs. These complex hydrophobic assemblies consist of a biopolymer, cutin, coated and filled with waxes and can also comprise embedded cell wall polysaccharides. Waxes comprise solvent-soluble aliphatic molecules with long hydrocarbon chains, terpenes, and steroids (Kunst and Samuels, 2003; Nawrath, 2006; Pollard et al., 2008; Samuels et al., 2008; Schreiber, 2010; Lee and Suh, 2015). Cutin is an insoluble polyester of ω- and mid-chain hydroxy C₁₆ and C₁₈ fatty acids. Glycerol has also been described as a ubiquitous cutin monomer (Graça et al., 2002; Pollard et al., 2008). In some cuticles, a hydrophobic polymer that is resistant to alkaline hydrolysis (i.e. cutan) has been observed (Gupta et al., 2006; Li-Beisson et al., 2010).Cutin fulfills multiple functions in plants, such as the control of nonstomatal water loss (Sieber et al., 2000) and the permeation of gases and solutes (Kersteins, 1996; Schreiber, 2010). Cutin also plays an essential role in the regulation of cell adhesion during plant development by preventing organ fusion, as observed in Arabidopsis (Arabidopsis thaliana) mutants with cuticle defects (Sieber et al., 2000; Nawrath, 2006), or by participating in hull adhesion in grains (Taketa et al., 2008). Finally, it is generally accepted that plant cuticle and its polymeric skeleton, cutin, are primary barriers to pathogens and that cutin monomers released by fungal cutinase are signaling molecules for both the pathogen and plants (Gilbert et al., 1996; Schweizer et al., 1996; Iwamoto et al., 2002; Yeats and Rose, 2013).The biological functions of cutin are closely controlled by its structure, which is determined by its monomer composition and by the number and position of its ester bonds. Cutin monomer composition can vary according to plant species, developmental stage (Baker et al., 1982; Peschel et al., 2007; Mintz-Oron et al., 2008), organs, and environmental stress (Espelie et al., 1979; Li-Beisson et al., 2009; Panikashvili et al., 2009; Bessire et al., 2011). Actually, cutin monomer composition determines the total number of hydroxyl (OH) groups that are potentially available for the formation of ester bonds and, therefore, the cross-linking of the polyester (Bonaventure et al., 2004; Franke et al., 2005; Peschel et al., 2007). The nonesterified OH groups enhance the hydrophilic character of the cutin polymer, increasing its elasticity (Bargel and Neinhuis, 2005).Whereas cutin monomer composition has been described extensively for different plant species, organs, and development stages, the macromolecular structure of the cutin polyester has been much less thoroughly investigated. In particular, the connectivity between the monomers is a key point for understanding the three-dimensional expansion of the polyester in relation to the polymerization process. Different approaches have been proposed to delineate the polymeric architecture of cutin. Linear dimers were identified after partial alkaline hydrolysis of tomato (Solanum lycopersicum) cutin (Osman et al., 1995). NMR and mass spectrometry analyses of oligomers released after partial depolymerization revealed primary and secondary ester linkages between cutin monomers (Graça et al., 2002; Stark and Tian, 2006) as well as covalent linkages between some cutin OH fatty acids and oligosaccharides (Tian et al., 2008). However, it has been shown that partial hydrolysis does not necessarily release all of the representative building blocks of the entire polymer (Deshmukh et al., 2003). Spectrometric analyses have also been developed for the polymer. Attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy analyses of the methylene and carbonyl stretching vibrations allowed the estimation of an ester cross-linking index for cutin but could not differentiate the primary from the secondary ester linkages (Girard et al., 2012; Heredia-Guerrero et al., 2014). NMR studies have provided evidence of both ω- and mid-chain esters in tomato (Deshmukh et al., 2003).In this regard, tomato fruit has proved to be an interesting model for structural studies of the cutin polymer. Indeed, its astomatous cuticle can be easily isolated and is devoid of cutan. Moreover, tomato cutin composition is dominated by a monomer with two OH groups, 9(10),16-dihydroxyhexadecanoic acid (Baker et al., 1982; Osman et al., 1999; Deshmukh et al., 2003). Accordingly, the cutin monomers can be linked by either a linear (on the primary OH) or a branched (with a secondary OH) pattern. Previous studies have demonstrated that both linear and branched cross-links occur in tomato cutin. However, the relative proportion of the linear versus branched esters remains a matter of debate. Oxidation experiments have indicated that almost all of the primary cutin OH groups (94%) were involved in ester bonds, whereas only 44% of the secondary OH groups were esterified in the cutin polymer (Deas and Holloway, 1977; Kolattukudy, 1977). Conversely, partial depolymerization coupled with NMR studies of the released oligomers indicated that the branched secondary esters were the major form of tomato cutin (Graça and Lamosa, 2010). In addition, none of these studies could decipher the ester links of the glycerol OH groups.Additionally, the role of a GDSL lipase, involved in cutin polymerization, was recently reported using two different experimental approaches (Girard et al., 2012; Yeats et al., 2012). The corresponding GDSL lipase, named SlGDSL1 (Girard et al., 2012) or SlGDSL2 (Yeats et al., 2012), is now named CUTIN SYNTHASE1 (SlCUS1; Yeats et al., 2014). Different mutants affected in the expression of SlCUS1 have been generated and constitute attractive tools to delineate the structure of the cutin polymer (Girard et al., 2012; Petit et al., 2014).It has been further demonstrated that 2-monoacylglycerol (2-MAG), a putative precursor of the cutin polymer in Arabidopsis (Yang et al., 2010), can be used by a heterologously expressed SlCUS1 (Yeats et al., 2012) to produce in vitro linear oligomers in aqueous solution (Yeats et al., 2014). Nevertheless, the question of the mechanism of cutin polymerization in planta is still open. Indeed, SlCUS1 is specifically localized within the cutin matrix (i.e. a hydrophobic environment; Girard et al., 2012; Yeats et al., 2012), which could impact the acyltransferase activity of the enzyme as observed previously for lipases (Sharma et al., 2001).By coupling O-alkylation of the nonesterified OH groups of glycerol and fatty acids in an isolated cutin matrix and by further analyses of O-alkylated and nonalkylated monomers released after depolymerization, we elucidated the ester cross-link pattern of tomato cutin. We also showed at two stages of fruit development and in two different genetic backgrounds that the modulation of SlCUS1 protein level, either through RNA interference (cherry tomato ‘West Virginia 106’ [WVa106]) or mutagenesis (miniature tomato ‘Micro-Tom’), resulted in a strong alteration of the cutin ester cross-link pattern. These results give new insights into the polyester structure. In addition, while CUS1 esterification involves mostly primary OH groups in vitro (Yeats et al., 2014), our data here indicate that, in planta, deficiencies in CUS1 also affect the secondary OH group of 9(10),16-dihydroxyhexadecanoic acid and both the primary and secondary OH groups of glycerol.     

A Novel Approach to Characterize Data Uncertainty in Material Flow Analysis and its Application to Plastics Flows in Austria

Material flow analysis (MFA) is widely used to investigate flows and stocks of resources or pollutants in a defined system. Data availability to quantify material flows on a national or global level is often limited owing to data scarcity or lacking data. MFA input data are therefore considered inherently uncertain. In this work, an approach to characterize the uncertainty of MFA input data is presented and applied to a case study on plastics flows in major Austrian consumption sectors in the year 2010. The developed approach consists of data quality assessment as a basis for estimating the uncertainty of input data. Four different implementations of the approach with respect to the translation of indicator scores to uncertainty ranges (linear‐ vs. exponential‐type functions) and underlying probability distributions (normal vs. log‐normal) are examined. The case study results indicate that the way of deriving uncertainty estimates for material flows has a stronger effect on the uncertainty ranges of the resulting plastics flows than the assumptions about the underlying probability distributions. Because these uncertainty estimates originate from data quality evaluation as well as uncertainty characterization, it is crucial to use a well‐defined approach, building on several steps to ensure the consistent translation of the data quality underlying material flow calculations into their associated uncertainties. Although subjectivity is inherent in uncertainty assessment in MFA, the proposed approach is consistent and provides a comprehensive documentation of the choices underlying the uncertainty analysis, which is essential to interpret the results and use MFA as a decision support tool.     

External Validation of Prediction Models for Pneumonia in Primary Care Patients with Lower Respiratory Tract Infection: An Individual Patient Data Meta-Analysis

Background

Pneumonia remains difficult to diagnose in primary care. Prediction models based on signs and symptoms (S&S) serve to minimize the diagnostic uncertainty. External validation of these models is essential before implementation into routine practice. In this study all published S&S models for prediction of pneumonia in primary care were externally validated in the individual patient data (IPD) of previously performed diagnostic studies.

Methods and Findings

S&S models for diagnosing pneumonia in adults presenting to primary care with lower respiratory tract infection and IPD for validation were identified through a systematical search. Six prediction models and IPD of eight diagnostic studies (N total = 5308, prevalence pneumonia 12%) were included. Models were assessed on discrimination and calibration. Discrimination was measured using the pooled Area Under the Curve (AUC) and delta AUC, representing the performance of an individual model relative to the average dataset performance. Prediction models by van Vugt et al. and Heckerling et al. demonstrated the highest pooled AUC of 0.79 (95% CI 0.74–0.85) and 0.72 (0.68–0.76), respectively. Other models by Diehr et al., Singal et al., Melbye et al., and Hopstaken et al. demonstrated pooled AUCs of 0.65 (0.61–0.68), 0.64 (0.61–0.67), 0.56 (0.49–0.63) and 0.53 (0.5–0.56), respectively. A similar ranking was present based on the delta AUCs of the models. Calibration demonstrated close agreement of observed and predicted probabilities in the models by van Vugt et al. and Singal et al., other models lacked such correspondence. The absence of predictors in the IPD on dataset level hampered a systematical comparison of model performance and could be a limitation to the study.

Conclusions

The model by van Vugt et al. demonstrated the highest discriminative accuracy coupled with reasonable to good calibration across the IPD of different study populations. This model is therefore the main candidate for primary care use.     

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance

Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M. mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1‐Δglf strain did not produce the galactofuranose‐containing glycans as shown by immunoblots and immuno‐electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also ‘leaking’ as revealed by a β‐galactosidase‐based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose‐containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance.     

VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody‐like protein, using an anti‐HER2 VHH as a model. The total chemical synthesis of the 125 amino‐acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its “antigen.” Using this combination of orthogonal techniques, it was possible to show that the three VHHs—whether synthetic or recombinant ones—were properly and similarly folded and recognized the “antigen” HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH‐drug conjugates.     

Bonding of articular cartilage using a combination of biochemical degradation and surface cross-linking

After trauma, articular cartilage often does not heal due to incomplete bonding of the fractured surfaces. In this study we investigated the ability of chemical cross-linkers to facilitate bonding of articular cartilage, either alone or in combination with a pre-treatment with surface-degrading agents. Articular cartilage blocks were harvested from the femoropatellar groove of bovine calves. Two cartilage blocks, either after pre-treatment or without, were assembled in a custom-designed chamber in partial apposition and subjected to cross-linking treatment. Subsequently, bonding of cartilage was measured as adhesive strength, that is, the maximum force at rupture of bonded cartilage blocks divided by the overlap area. In a first approach, bonding was investigated after treatment with cross-linking reagents only, employing glutaraldehyde, 1-ethyl-3-diaminopropyl-carbodiimide (EDC)/N-hydroxysuccinimide (NHS), genipin, or transglutaminase. Experiments were conducted with or without compression of the opposing surfaces. Compression during cross-linking strongly enhanced bonding, especially when applying EDC/NHS and glutaraldehyde. Therefore, all further experiments were performed under compressive conditions. Combinations of each of the four cross-linking agents with the degrading pre-treatments, pepsin, trypsin, and guanidine, led to distinct improvements in bonding compared to the use of cross-linkers alone. The highest values of adhesive strength were achieved employing combinations of pepsin or guanidine with EDC/NHS, and guanidine with glutaraldehyde. The release of extracellular matrix components, that is, glycosaminoglycans and total collagen, from cartilage blocks after pre-treatment was measured, but could not be directly correlated to the determined adhesive strength. Cytotoxicity was determined for all substances employed, that is, surface degrading agents and cross-linkers, using the resazurin assay. Taking the favourable cell vitality after treatment with pepsin and EDC/NHS and the cytotoxic effects of guanidine and glutaraldehyde into account, the combination of pepsin and EDC/NHS appeared to be the most advantageous treatment in this study. In conclusion, bonding of articular cartilage blocks was achieved by chemical fixation of their surface components using cross-linking reagents. Application of compressive forces and prior modulation of surface structures enhanced cartilage bonding significantly. Enzymatic treatment in combination with cross-linkers may represent a promising addition to current techniques for articular cartilage repair.