LC–MS-based metabolome analysis on steroid metabolites from the starfish <Emphasis Type="Italic">Patiria</Emphasis> (=<Emphasis Type="Italic">Asterina</Emphasis>) <Emphasis Type="Italic">pectinifera</Emphasis> in conditions of active feeding and stresses

Introduction

Starfish are recognized as interesting source of natural steroid products with pharmaceutical potential. Polar steroid metabolites of starfish have unique chemical structures and exhibit various biological activities but their biological functions are controversial.

Objectives

The objective of this study was to investigate the response of polar steroid metabolome of the starfish Patiria (=Asterina) pectinifera on various environmental factors and stresses.

Methods

Here we first have applied MS-based environmental metabolomics to elucidate the metabolic changes of polar steroid metabolome of starfish. Using HPLC–ESI–Q/TOF–MS approach followed by statistical analysis including principal component analysis and partial least squares discriminant analysis for data classification and potential biomarkers selection, we investigated the changes induced by feeding, injury, variations in water temperature and salinity, and oxygen deficiency.

Results

According to multivariate and univariate statistical analysis the responses to feeding, injury and water heating were better expressed than the others and have some similarity in their action on the steroid metabolome of the starfish P. pectinifera. Most constituents of asterosaponin pool were reduced and most constituents of polyhydroxysteroid and related glycoside pool were increased at that.

Conclusion

Our results indicate that various metabolic changes in polar steroid constituents of P. pectinifera are induced by feeding and stresses. We believe that these responses are connected with biological multifunctionality of these compounds.

     

<Emphasis Type="Italic">massPix</Emphasis>: an R package for annotation and interpretation of mass spectrometry imaging data for lipidomics

Introduction

Mass spectrometry imaging (MSI) experiments result in complex multi-dimensional datasets, which require specialist data analysis tools.

Objectives

We have developed massPix—an R package for analysing and interpreting data from MSI of lipids in tissue.

Methods

massPix produces single ion images, performs multivariate statistics and provides putative lipid annotations based on accurate mass matching against generated lipid libraries.

Results

Classification of tissue regions with high spectral similarly can be carried out by principal components analysis (PCA) or k-means clustering.

Conclusion

massPix is an open-source tool for the analysis and statistical interpretation of MSI data, and is particularly useful for lipidomics applications.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

Metabolic response of porcine colon explants to in vitro infection by <Emphasis Type="Italic">Brachyspira hyodysenteriae</Emphasis>: a leap into disease pathophysiology

Introduction

Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.

Objective

The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.

Methods

Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.

Results

Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.

Conclusions

The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.

     

<Emphasis Type="Italic">Candida guilliermondii</Emphasis> as a potential biocatalyst for the production of long-chain α,ω-dicarboxylic acids

Objectives

To explore Candida guilliermondii for the production of long-chain dicarboxylic acids (DCA), we performed metabolic pathway engineering aiming to prevent DCA consumption during β-oxidation, but also to increase its production via the ω-oxidation pathway.

Results

We identified the major β- and ω-oxidation pathway genes in C. guilliermondii and performed first steps in the strain improvement. A double pox disruption mutant was created that slowed growth with oleic acid but showed accelerated DCA degradation. Increase in DCA production was achieved by homologous overexpression of a plasmid borne cytochrome P450 monooxygenase gene.

Conclusion

C. guilliermondii is a promising biocatalyst for DCA production but further insight into its fatty acid metabolism is necessary.

     

Modification effect of fenofibrate therapy,a longitudinal epigenomic-wide methylation study of triglycerides levels in the GOLDN study

Background

Identification of interactions between epigenetic factors and treatments might lead to personalized intervention of diseases. This paper aims to examine the modification effect of fenofibrate therapy on the association of methylation levels and fasting blood triglycerides (TG), and the related biological pathways among methylation sites.

Results

Mixed-effects models were employed to assess pre- and posttreatment associations and drug modification effects simultaneously. Five cytosine-phosphate-guanine (CpG) sites were found to be associated with TG levels before and after the fenofibrate therapy: cg00574958, cg17058475, and cg01082498 on CPT1A gene, chromosome 11; cg03725309 on SARS, chromosome 1; and cg06500161 on ABCG1, chromosome 21. In addition, fenofibrate therapy modified the methylation levels on the following 4 CpG sites: cg20015535 (gene EGLN1, chromosome 1); cg24870738 (gene RNF220, chromosome 1); cg06891775 (gene LOC283050, chromosome 10); and cg00607630 (gene USP7, chromosome 16). Further, gene set enrichment analysis (GSEA) identified cancer- and metabolism-related pathways that were associated with TG-related CpG sites.

Conclusions

We identified modification effects of fenofibrate on the associations between blood TG levels and several CpG sites. Pathway enrichment analysis indicated the alternations in some metabolism and cancer-related pathways. Our findings have important implications for future research in pharmacoepigenetics and personalized medicine.

     

Volatile metabolic diversity of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in nutrient-replete conditions

Introduction

Microorganisms catabolize carbon-containing compounds in their environment during growth, releasing a subset of metabolic byproducts as volatile compounds. However, the relationship between growth media and the production of volatile compounds has been largely unexplored to-date.

Objectives

To assess the core and media-specific components of the Klebsiella pneumoniae volatile metabolome via growth in four in vitro culture media.

Methods

Headspace volatiles produced by cultures of K. pneumoniae after growth to stationary phase in four rich media (brain heart infusion broth, lysogeny broth, Mueller-Hinton broth, and tryptic soy broth) were analyzed using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS). Differences in the composition of headspace volatiles as a function of growth media were assessed using hierarchical clustering analysis (HCA) and principal component analysis (PCA).

Results

A total of 365 volatile compounds were associated with the growth of K. pneumoniae across all media, of which 36 (10%) were common to all growth media, and 148 (41%) were specific to a single medium. In addition, utilizing all K. pneumoniae-associated volatile compounds, strains clustered as a function of growth media, demonstrating the importance of media in determining the metabolic profile of this organism.

Conclusion

K. pneumoniae produces a core suite of volatile compounds across all growth media studied, although the volatile metabolic signature of this organism is fundamentally media-dependent.

     

<Emphasis Type="Italic">PiggyBac</Emphasis> transposon-mediated mutagenesis and application in yeast <Emphasis Type="Italic">Komagataella phaffii</Emphasis>

Objective

Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii.

Results

In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression.

Conclusions

Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.

     

Leaf extracts from <Emphasis Type="Italic">Nitraria retusa</Emphasis> promote cell population growth of human cancer cells by inducing apoptosis

Background

In this report the phytochemical profile of Nitraria. Retusa (N. Retusa) leaf extracts were identified and their ability to induce apoptosis in human chronic myelogenous erythroleukaemia (K562) was evaluated.

Methods

Apoptosis of the human chronic myelogenous erythroleukaemia (K562) was evidenced by investigating DNA fragmentation, PARP cleavage and caspases 3 and 8 inducing activities, in the presence of N. retusa extracts.

Results

Our study revealed that the tested extracts from N. Retusa contain many useful bioactive compounds. They induced in a time-dependent manner the apoptosis the tested cancerous our cell line. This result was confirmed by ladder DNA fragmentation profile and PARP cleavage, as well as a release in caspase-3 and caspase-8 level.

Conclusion

Our results indicate that the tested compounds have a significant antiproliferative effect which may be due to their involvement in the induction of the extrinsic apoptosic pathway.

     

Genotypic and metabolic approaches towards the segregation of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> strains producing different antibiotic resistant enzymes

Introduction

Genotype and metabolomic variation are important for bacterial survival and adaptation to environmental changes.

Objectives

In this study, we compared the relationship among Klebsiella pneumoniae strains based on their genotypic and metabolic profiles. In addition, we also evaluated the association of the relationship with beta-lactamase production.

Methods

A total of 53 K. pneumoniae strains isolated in 2013–2014 from a tertiary teaching hospital in Malaysia were subjected to antimicrobial susceptibility testing (AST) via disk diffusion method and beta-lactamase production confirmation. The bacterial strains were also typed genotypically and metabolically via REP-PCR and ¹H-NMR spectroscopy respectively. The concordance of the matrices derived based on genotypic and metabolic characterization was measured based on Spearman’s rank correlation.

Results

Spearman’s correlation rank showed that there is a weak but significant negative correlation between the genetic fingerprints and metabolic profiles of K. pneumoniae. Specifically, K. pneumoniae strains were clustered into five major clusters based on REP-PCR where most of the carbapenem resistant K. pneumoniae (CRKP) strains made up the major cluster. In contrast, metabolic patterns of the three groups (i.e. CRKP, extended spectrum beta-lactamase producing K. pneumoniae (ESBL), susceptible) of K. pneumoniae were clearly differentiated on PLS-DA score plots derived from ¹H-NMR spectroscopy.

Conclusion

Overall, this study showed that metabolomic profiling using ¹H-NMR spectroscopy is able to discriminate K. pneumoniae strains based on their beta-lactamase production status.

     

Plant growth-promoting bacterium <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> FR1 secrets a novel type of extracellular polyhydroxybutyrate polymerase involved in abiotic stress response in plants

Objectives

Identification of novel microbial factors contributing to plant protection against abiotic stress.

Results

The genome of plant growth-promoting bacterium Pseudomonas fluorescens FR1 contains a short mobile element encoding a novel type of extracellular polyhydroxybutyrate (PHB) polymerase (PhbC) associated with a type I secretion system. Genetic analysis using a phbC mutant strain and plants showed that this novel extracellular enzyme is related to the PHB production in planta and suggests that PHB could be a beneficial microbial compound synthesized during plant adaptation to cold stress.

Conclusion

Extracellular PhbC can be used as a new tool for improve crop production under abiotic stress.

     

AcMNPV-<Emphasis Type="Italic">Bm</Emphasis>K IT improves the progeny virus production via baculovirus GP64 envelope fusion protein

Objectives

To analyze the mechanisms underlying the impact of recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)-mediated BmK IT expression on the function of baculovirus GP64 envelope fusion protein and progeny virus production.

Results

Viral propagation assay indicated that overexpression GP64 could promote replication of AcMNPV. AcMNPV-mediated expression of BmK IT also promoted replication of AcMNPV. Immunofluorescence analysis showed BmK IT, which was regulated by very early promoter IE1 in AcMNPV, could make the GP64 protein move to the cytomembrane soon after transfection. BmK IT, which is regulated by P10 protein promoter (P10) and polyhedrosis promoter (PH), could promote the expression of GP64.

Conclusion

BmK IT, regulated by very early promoter IE1, P10 protein promoter (P10) and PH, accelerated the expression of GP64 protein, promoted its early cytomembrane localization and then triggered virus budding and progeny virus production.

     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Peroxide-dependent oxidation reactions catalyzed by CYP191A1 from <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

Objectives

To find the catalytic activities of CYP191A1 from Mycobacterium smegmatis, in which functions of most P450s are unknown, by using a set of reductase systems, peroxides, and various substrates including fatty acids and human drugs.

Results

CYP191A1 was functionally expressed in Escherichia coli and purified. Its catalytic activities were examined with fatty acids, chromogenic and fluorogenic substrates, and several human P450 substrates, in the presence of six different types of electron transfer systems, such as rat NADPH-P450 reductase, Candida NADPH-P450 reductase, ferredoxin/ferredoxin reductase, putidaredoxin/putidaredoxin reductase, and peroxides (H₂O₂ and t-butyl hydroperoxide). The reactions catalyzed by CYP191A1 included the hydroxylation and O-dealkylation of several substrates.

Conclusions

CYP191A1 preferentially catalyzes the peroxide-dependent oxidation of various substrates over the reductase-dependent reaction. Its peroxygenase activity may be used an effective biocatalytic tool to synthesize the metabolites of drugs.

     

Improvement of 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> by moderate expression of <Emphasis Type="Italic">puuC</Emphasis> (encoding an aldehyde dehydrogenase)

Objective

To improve 1,3-propanediol production in Klebsiella pneumoniae, the effects of puuC expression in lactate- and lactate/2,3-butanediol-deficient strains were assessed.

Results

Overexpression of puuC (encoding an aldehyde dehydrogenase) inhibited 1,3-propanediol production and increased 3-hydroxypropionic acid formation in both lactate- and lactate/2,3-butanediol-deficient strains. An improvement in 1,3-propanediol production was only achieved in a lactate-deficient strain via moderate expression of puuC; at the end of the fermentation, 1,3-propanediol productivity increased by 14 % compared with the control. Further comparative analysis of the metabolic flux distributions in different strains indicated that 3-hydroxypropionic acid formation could play a considerable role in cell metabolism in K. pneumoniae.

Conclusion

An improvement in 3-hydroxypropionic acid formation would be beneficial for cell metabolism, which can be accomplished by enhancing 1,3-propanediol productivity in a lactate-deficient strain via moderate expression of puuC.

     

Group-wise ANOVA simultaneous component analysis for designed <Emphasis Type="Italic">omics</Emphasis> experiments

Introduction

Modern omics experiments pertain not only to the measurement of many variables but also follow complex experimental designs where many factors are manipulated at the same time. This data can be conveniently analyzed using multivariate tools like ANOVA-simultaneous component analysis (ASCA) which allows interpretation of the variation induced by the different factors in a principal component analysis fashion. However, while in general only a subset of the measured variables may be related to the problem studied, all variables contribute to the final model and this may hamper interpretation.

Objectives

We introduce here a sparse implementation of ASCA termed group-wise ANOVA-simultaneous component analysis (GASCA) with the aim of obtaining models that are easier to interpret.

Methods

GASCA is based on the concept of group-wise sparsity introduced in group-wise principal components analysis where structure to impose sparsity is defined in terms of groups of correlated variables found in the correlation matrices calculated from the effect matrices.

Results

The GASCA model, containing only selected subsets of the original variables, is easier to interpret and describes relevant biological processes.

Conclusions

GASCA is applicable to any kind of omics data obtained through designed experiments such as, but not limited to, metabolomic, proteomic and gene expression data.