Efficient kinetic resolution of secondary alcohols using an organic solvent-tolerant esterase in non-aqueous medium

Objective

To identify an esterase-mediated kinetic resolution of secondary alcohols in non-aqueous medium.

Results

An esterase, EST4, from a marine mud metagenomic library, showed high activity and enantioselectivity for the kinetic resolution of secondary alcohols in non-aqueous medium. Using 1-phenylethanol as the model alcohol, the effects of organic solvents, acyl donors, molar ratio, temperatures and biocatalyst loading on the kinetic resolution catalyzed by the EST4 whole-cell biocatalyst were investigated and optimized. The optimized methodology was effective on resolving 16 various racemic secondary alcohols in neat n-hexane, providing excellent enantiomeric excess (up to 99.9 % ee). Moreover, EST4 exhibited a strong tolerance for high substrate concentration (up to 1 M), and the optical purity of the desired secondary alcohols was kept above 99 % ee.

Conclusion

The esterase EST4 is a promising biocatalyst for the enantioselective synthesis of various alcohols and esters with interesting practical applications.

     

Downregulation of RdDM during strawberry fruit ripening

Background

Recently, DNA methylation was proposed to regulate fleshy fruit ripening. Fleshy fruits can be distinguished by their ripening process as climacteric fruits, such as tomatoes, or non-climacteric fruits, such as strawberries. Tomatoes undergo a global decrease in DNA methylation during ripening, due to increased expression of a DNA demethylase gene. The dynamics and biological relevance of DNA methylation during the ripening of non-climacteric fruits are unknown.

Results

Here, we generate single-base resolution maps of the DNA methylome in immature and ripe strawberry. We observe an overall loss of DNA methylation during strawberry fruit ripening. Thus, ripening-induced DNA hypomethylation occurs not only in climacteric fruit, but also in non-climacteric fruit. Application of a DNA methylation inhibitor causes an early ripening phenotype, suggesting that DNA hypomethylation is important for strawberry fruit ripening. The mechanisms underlying DNA hypomethylation during the ripening of tomato and strawberry are distinct. Unlike in tomatoes, DNA demethylase genes are not upregulated during the ripening of strawberries. Instead, genes involved in RNA-directed DNA methylation are downregulated during strawberry ripening. Further, ripening-induced DNA hypomethylation is associated with decreased siRNA levels, consistent with reduced RdDM activity. Therefore, we propose that a downregulation of RdDM contributes to DNA hypomethylation during strawberry ripening.

Conclusions

Our findings provide new insight into the DNA methylation dynamics during the ripening of non-climacteric fruit and suggest a novel function of RdDM in regulating an important process in plant development.

     

Identification of a seed coat-specific promoter fragment from the Arabidopsis <Emphasis Type="Italic">MUCILAGE-MODIFIED4</Emphasis> gene

Key message

Pru p 3, a peach LTP, is located in pollinated flower styles and secreting downy hairs, transporting a derivative of camptothecin bound to phytosphingosine. Pru p 3 may inhibit a second pollination and may keep away herbivores until seed maturation.

Abstract

The allergen Pru p 3, a peach lipid transfer protein, has been well studied. However, its physiological function remains to be elucidated. Our results showed that Pru p 3 usually carries a lipid ligand that play an essential role in its function in plants. Using ESI-qToF, we observed that the ligand was a derivative of camptothecin binding to phytosphingosine, wich that is inserted into the hydrophobic tunnel of the protein. In addition, the described ligand displayed topoisomerase I activity inhibition and self-fluorescence, both recognized as camptothecin properties. During flower development, the highest expression of Pru p 3 was detected in the styles of pollinated flowers, in contrast to its non-expression in unpollinated pistils, where expression decreased after anthesis. During ripening, the expression of Pru p 3 were observed mainly in peel but not in pulp. In this sense, Pru p 3 protein was also localized in trichomes covering the fruit epidermis.

     

Ozone-induced inhibition of kiwifruit ripening is amplified by 1-methylcyclopropene and reversed by exogenous ethylene

Background

Understanding the mechanisms involved in climacteric fruit ripening is key to improve fruit harvest quality and postharvest performance. Kiwifruit (Actinidia deliciosa cv. ‘Hayward’) ripening involves a series of metabolic changes regulated by ethylene. Although 1-methylcyclopropene (1-MCP, inhibitor of ethylene action) or ozone (O₃) exposure suppresses ethylene-related kiwifruit ripening, how these molecules interact during ripening is unknown.

Results

Harvested ‘Hayward’ kiwifruits were treated with 1-MCP and exposed to ethylene-free cold storage (0?°C, RH 95%) with ambient atmosphere (control) or atmosphere enriched with O₃ (0.3?μL?L^??1) for up to 6?months. Their subsequent ripening performance at 20?°C (90% RH) was characterized. Treatment with either 1-MCP or O₃ inhibited endogenous ethylene biosynthesis and delayed fruit ripening at 20?°C. 1-MCP and O₃ in combination severely inhibited kiwifruit ripening, significantly extending fruit storage potential. To characterize ethylene sensitivity of kiwifruit following 1-MCP and O₃ treatments, fruit were exposed to exogenous ethylene (100?μL?L^??1, 24?h) upon transfer to 20?°C following 4 and 6?months of cold storage. Exogenous ethylene treatment restored ethylene biosynthesis in fruit previously exposed in an O₃-enriched atmosphere. Comparative proteomics analysis showed separate kiwifruit ripening responses, unraveled common 1-MCP- and O₃-dependent metabolic pathways and identified specific proteins associated with these different ripening behaviors. Protein components that were differentially expressed following exogenous ethylene exposure after 1-MCP or O₃ treatment were identified and their protein-protein interaction networks were determined. The expression of several kiwifruit ripening related genes, such as 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1), ethylene receptor (ETR1), lipoxygenase (LOX1), geranylgeranyl diphosphate synthase (GGP1), and expansin (EXP2), was strongly affected by O₃, 1-MCP, their combination, and exogenously applied ethylene.

Conclusions

Our findings suggest that the combination of 1-MCP and O₃ functions as a robust repressive modulator of kiwifruit ripening and provide new insight into the metabolic events underlying ethylene-induced and ethylene-independent ripening outcomes.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

DTL reconciliation repair

Background

Maximum parsimony phylogenetic tree reconciliation is an important technique for reconstructing the evolutionary histories of hosts and parasites, genes and species, and other interdependent pairs. Since the problem of finding temporally feasible maximum parsimony reconciliations is NP-complete, current methods use either exact algorithms with exponential worst-case running time or heuristics that do not guarantee optimal solutions.

Results

We offer an efficient new approach that begins with a potentially infeasible maximum parsimony reconciliation and iteratively “repairs” it until it becomes temporally feasible.

Conclusions

In a non-trivial number of cases, this approach finds solutions that are better than those found by the widely-used Jane heuristic.

     

Anchoring quartet-based phylogenetic distances and applications to species tree reconstruction

Background

Inferring species trees from gene trees using the coalescent-based summary methods has been the subject of much attention, yet new scalable and accurate methods are needed.

Results

We introduce DISTIQUE, a new statistically consistent summary method for inferring species trees from gene trees under the coalescent model. We generalize our results to arbitrary phylogenetic inference problems; we show that two arbitrarily chosen leaves, called anchors, can be used to estimate relative distances between all other pairs of leaves by inferring relevant quartet trees. This results in a family of distance-based tree inference methods, with running times ranging between quadratic to quartic in the number of leaves.

Conclusions

We show in simulated studies that DISTIQUE has comparable accuracy to leading coalescent-based summary methods and reduced running times.

     

A metabolomic,geographic, and seasonal analysis of the contribution of pollen-derived adenosine to allergic sensitization

Background

Studies on ragweed and birch pollen extracts suggested that the adenosine content is an important factor in allergic sensitization. However, exposure levels from other pollens and considerations of geographic and seasonal factors have not been evaluated.

Objective

This study compared the metabolite profile of pollen species important for allergic disease, specifically measured the adenosine content, and evaluated exposure to pollen-derived adenosine.

Methods

An NMR metabolomics approach was used to measure metabolite concentrations in 26 pollen extracts. Pollen count data was analyzed from five cities to model exposure.

Results

A principal component analysis of the various metabolites identified by NMR showed that pollen extracts could be differentiated primarily by sugar content: glucose, fructose, sucrose, and myo-inositol. In extracts of 10 mg of pollen/ml, the adenosine was highest for grasses (45 µM) followed by trees (23 µM) and weeds (19 µM). Pollen count data showed that tree pollen was typically 5–10 times the amount of other pollens. At the daily peaks of tree, grass, and weed season the pollen-derived adenosine exposure per day is likely to be only 1.1, 0.11, and 0.12 µg, respectively. Seasonal models of pollen exposure and respiration suggest that it would be a rare event limited to tree pollen season for concentrations of pollen-derived adenosine to approach physiological levels.

Conclusion

Sugar content and other metabolites may be useful in classifying pollens. Unless other factors create localized exposures that are very different from these models, pollen-derived adenosine is unlikely to be a major factor in allergic sensitization.

     

A lost opportunity for science: journals promote data sharing in metabolomics but do not enforce it

Introduction

Data sharing is being increasingly required by journals and has been heralded as a solution to the ‘replication crisis’.

Objectives

(i) Review data sharing policies of journals publishing the most metabolomics papers associated with open data and (ii) compare these journals’ policies to those that publish the most metabolomics papers.

Methods

A PubMed search was used to identify metabolomics papers. Metabolomics data repositories were manually searched for linked publications.

Results

Journals that support data sharing are not necessarily those with the most papers associated to open metabolomics data.

Conclusion

Further efforts are required to improve data sharing in metabolomics.

     

Extraction of natural products from bark of <Emphasis Type="Italic">Betula pendula</Emphasis> using ionic liquids

Introduction

Aqueous–methanol mixtures have successfully been applied to extract a broad range of metabolites from plant tissue. However, a certain amount of material remains insoluble.

Objectives

To enlarge the metabolic compendium, two ionic liquids were selected to extract the methanol insoluble part of trunk from Betula pendula.

Methods

The extracted compounds were analyzed by LC/MS and GC/MS.

Results

The results show that 1-butyl-3-methylimidazolium acetate (IL-Ac) predominantly resulted in fatty acids, whereas 1-ethyl-3-methylimidazolium tosylate (IL-Tos) mostly yielded phenolic structures. Interestingly, bark yielded more ionic liquid soluble metabolites compared to interior wood.

Conclusion

From this one can conclude that the application of ionic liquids may expand the metabolic snapshot.

     

Italian cohort of patients affected by inflammatory bowel disease is characterised by variation in glycerophospholipid,free fatty acids and amino acid levels

Background

Inflammatory bowel disease is a group of pathologies characterised by chronic inflammation of the intestine and an unclear aetiology. Its main manifestations are Crohn’s disease and ulcerative colitis. Currently, biopsies are the most used diagnostic tests for these diseases and metabolomics could represent a less invasive approach to identify biomarkers of disease presence and progression.

Objectives

The lipid and the polar metabolite profile of plasma samples of patients affected by inflammatory bowel disease have been compared with healthy individuals with the aim to find their metabolomic differences. Also, a selected sub-set of samples was analysed following solid phase extraction to further characterise differences between pathological samples.

Methods

A total of 200 plasma samples were analysed using drift tube ion mobility coupled with time of flight mass spectrometry and liquid chromatography for the lipid metabolite profile analysis, while liquid chromatography coupled with triple quadrupole mass spectrometry was used for the polar metabolite profile analysis.

Results

Variations in the lipid profile between inflammatory bowel disease and healthy individuals were highlighted. Phosphatidylcholines, lyso-phosphatidylcholines and fatty acids were significantly changed among pathological samples suggesting changes in phospholipase A₂ and arachidonic acid metabolic pathways. Variations in the levels of cholesteryl esters and glycerophospholipids were also found. Furthermore, a decrease in amino acids levels suggests mucosal damage in inflammatory bowel disease.

Conclusions

Given good statistical results and predictive power of the model produced in our study, metabolomics can be considered as a valid tool to investigate inflammatory bowel disease.

     

Locus-aware decomposition of gene trees with respect to polytomous species trees

Background

Horizontal gene transfer (HGT), a process of acquisition and fixation of foreign genetic material, is an important biological phenomenon. Several approaches to HGT inference have been proposed. However, most of them either rely on approximate, non-phylogenetic methods or on the tree reconciliation, which is computationally intensive and sensitive to parameter values.

Results

We investigate the locus tree inference problem as a possible alternative that combines the advantages of both approaches. We present several algorithms to solve the problem in the parsimony framework. We introduce a novel tree mapping, which allows us to obtain a heuristic solution to the problems of locus tree inference and duplication classification.

Conclusions

Our approach allows for faster comparisons of gene and species trees and improves known algorithms for duplication inference in the presence of polytomies in the species trees. We have implemented our algorithms in a software tool available at https://github.com/mciach/LocusTreeInference.

     

Inferring angiosperm phylogeny from EST data with widespread gene duplication

Background

Most studies inferring species phylogenies use sequences from single copy genes or sets of orthologs culled from gene families. For taxa such as plants, with very high levels of gene duplication in their nuclear genomes, this has limited the exploitation of nuclear sequences for phylogenetic studies, such as those available in large EST libraries. One rarely used method of inference, gene tree parsimony, can infer species trees from gene families undergoing duplication and loss, but its performance has not been evaluated at a phylogenomic scale for EST data in plants.

Results

A gene tree parsimony analysis based on EST data was undertaken for six angiosperm model species and Pinus, an outgroup. Although a large fraction of the tentative consensus sequences obtained from the TIGR database of ESTs was assembled into homologous clusters too small to be phylogenetically informative, some 557 clusters contained promising levels of information. Based on maximum likelihood estimates of the gene trees obtained from these clusters, gene tree parsimony correctly inferred the accepted species tree with strong statistical support. A slight variant of this species tree was obtained when maximum parsimony was used to infer the individual gene trees instead.

Conclusion

Despite the complexity of the EST data and the relatively small fraction eventually used in inferring a species tree, the gene tree parsimony method performed well in the face of very high apparent rates of duplication.

     

Profiling,isolation and structure elucidation of specialized acylsucrose metabolites accumulating in trichomes of <Emphasis Type="Italic">Petunia</Emphasis> species

Introduction

Acylsugar specialized metabolites function as defenses against insect herbivores, and are the most abundant specialized metabolites produced in Solanaceous trichomes. Metabolite profiling provides the foundation for determining the genetic basis of specialized metabolism and its evolution.

Objectives

To profile and identify acylsugar specialized metabolites in three Petunia species: P. axillaris, P. integrifolia and P. exserta.

Methods

Metabolites were profiled using ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOF MS). Metabolites were purified using solid phase extraction and HPLC, and structures were established using NMR spectroscopy.

Results

Twenty-eight distinct acylsucrose formulas, representing a sampling of more than 100 different detected chemical forms, were purified from three Petunia species and structures have been proposed based on one- and two-dimensional NMR data. 15 of the 28 purified acylsugars were sucrose pentaesters that possess a malonyl group on the fructose ring. These malonate esters can be readily distinguished from other acylsugars based on distinct masses of pseudomolecular ions and fragment ions generated using multiplexed collision-induced dissociation. Chemical diversity of acylsugars was observed between Petunia species, particularly with respect to the lengths of acyl chains and specific acylation positions.

Conclusions

These findings suggest substrate selectivity of various acyltransferases in Petunia species.

     

Metabolomics meets functional assays: coupling LC–MS and microfluidic cell-based receptor-ligand analyses

Introduction

Metabolomics has become a valuable tool in many research areas. However, generating metabolomics-based biochemical profiles without any related bioactivity is only of indirect value in understanding a biological process. Therefore, metabolomics research could greatly benefit from tools that directly determine the bioactivity of the detected compounds.

Objective

We aimed to combine LC–MS metabolomics with a cell based receptor assay. This combination could increase the understanding of biological processes and may provide novel opportunities for functional metabolomics.

Methods

We developed a flow through biosensor with human cells expressing both the TRPV1, a calcium ion channel which responds to capsaicin, and the fluorescent intracellular calcium ion reporter, YC3.6. We have analysed three contrasting Capsicum varieties. Two were selected with contrasting degrees of spiciness for characterization by HPLC coupled to high mass resolution MS. Subsequently, the biosensor was then used to link individual pepper compounds with TRPV1 activity.

Results

Among the compounds in the crude pepper fruit extracts, we confirmed capsaicin and also identified both nordihydrocapsaicin and dihydrocapsaicin as true agonists of the TRPV1 receptor. Furthermore, the biosensor was able to detect receptor activity in extracts of both Capsicum fruits as well as a commercial product. Sensitivity of the biosensor to this commercial product was similar to the sensory threshold of a human sensory panel.

Conclusion

Our results demonstrate that the TRPV1 biosensor is suitable for detecting bioactive metabolites. Novel opportunities may lie in the development of a continuous functional assay, where the biosensor is directly coupled to the LC–MS.

     

Tree development in structural soil – an empirical below-ground in-situ study of urban trees in Copenhagen,Denmark

Aims

The purpose of this study is to evaluate root and stem growth, nutrient status and soil properties of a tree planting in structural soil.

Methods

Root and stem growth were measured at an 10-year-old urban planting of Tilia x europaea L. ‘Pallida’ established in structural soil on a paved square in Copenhagen, Denmark.Root abundance was quantified and soil profiles were established at three different distances from 10 trees (1.0, 1.9, and 2.9 m). Soil and leaf samples were analysed for nutrient concentrations.

Results

Abundant root presence was measured across the entire profile of structural soil (160?×?60 cm) at all distances, provided that structural soil layers were constructed correctly. Erroneously constructed structural soil layers however, resulted in impaired tree and root growth. Coarse root morphology was affected by the confined growing space in the voids of the stone matrix. The soil was relatively alkaline with an average pH (CaCl₂) of 7.3. Foliar analysis revealed deficiencies of K and Mn. These deficiencies might be due to a combined effect of high pH and low soil concentrations of these nutrients.

Conclusions

The study documents that correctly constructed structural soils allow and encourage root growth in load bearing layers.

     

Crop metabolomics: from diagnostics to assisted breeding

Background

Until recently, plant metabolomics have provided a deep understanding on the metabolic regulation in individual plants as experimental units. The application of these techniques to agricultural systems subjected to more complex interactions is a step towards the implementation of translational metabolomics in crop breeding.

Aim of Review

We present here a review paper discussing advances in the knowledge reached in the last years derived from the application of metabolomic techniques that evolved from biomarker discovery to improve crop yield and quality.

Key Scientific Concepts of Review

Translational metabolomics applied to crop breeding programs.

     

Pseudomyxoma extraperitonei occurring 35 years after appendicectomy: a case report and review of literature

Background

Pseudomyxoma peritonei is a rare condition consisting of mucinous ascites, most commonly arising from mucinous tumors of the appendix and occasionally from the ovary. Very rarely mucinous implants arise in the retroperitoneum without any intra-peritoneal involvement. This has been termed as pseudomyxoma extraperitonei.

Case presentation

We report a case of a 57 year old man who developed pseudomyxoma extraperitonei, 35 years after undergoing an appendicectomy for a perforated appendix.

Conclusions

Pseudomyxoma extraperitonei has been previously reported, however we report the longest incubation period of 35 years for this condition.

     

The role of laterally transferred genes in adaptive evolution

Background

Bacterial genomes develop new mechanisms to tide them over the imposing conditions they encounter during the course of their evolution. Acquisition of new genes by lateral gene transfer may be one of the dominant ways of adaptation in bacterial genome evolution. Lateral gene transfer provides the bacterial genome with a new set of genes that help it to explore and adapt to new ecological niches.

Methods

A maximum likelihood analysis was done on the five sequenced corynebacterial genomes to model the rates of gene insertions/deletions at various depths of the phylogeny.

Results

The study shows that most of the laterally acquired genes are transient and the inferred rates of gene movement are higher on the external branches of the phylogeny and decrease as the phylogenetic depth increases. The newly acquired genes are under relaxed selection and evolve faster than their older counterparts. Analysis of some of the functionally characterised LGTs in each species has indicated that they may have a possible adaptive role.

Conclusion

The five Corynebacterial genomes sequenced to date have evolved by acquiring between 8 – 14% of their genomes by LGT and some of these genes may have a role in adaptation.