A comparison of human serum and plasma metabolites using untargeted <Superscript>1</Superscript>H NMR spectroscopy and UPLC-MS

Introduction

Differences in the metabolite profiles between serum and plasma are incompletely understood.

Objectives

To evaluate metabolic profile differences between serum and plasma and among plasma sample subtypes.

Methods

We analyzed serum, platelet rich plasma (PRP), platelet poor plasma (PPP), and platelet free plasma (PFP), collected from 8 non-fasting apparently healthy women, using untargeted standard 1D and CPMG ¹H NMR and reverse phase and hydrophilic (HILIC) UPLC-MS. Differences between metabolic profiles were evaluated using validated principal component and orthogonal partial least squares discriminant analysis.

Results

Explorative analysis showed the main source of variation among samples was due to inter-individual differences with no grouping by sample type. After correcting for inter-individual differences, lipoproteins, lipids in VLDL/LDL, lactate, glutamine, and glucose were found to discriminate serum from plasma in NMR analyses. In UPLC-MS analyses, lysophosphatidylethanolamine (lysoPE)(18:0) and lysophosphatidic acid(20:0) were higher in serum, and phosphatidylcholines (PC)(16:1/18:2, 20:3/18:0, O-20:0/22:4), lysoPC(16:0), PE(O-18:2/20:4), sphingomyelin(18:0/22:0), and linoleic acid were lower. In plasma subtype analyses, isoleucine, leucine, valine, phenylalanine, glutamate, and pyruvate were higher among PRP samples compared with PPP and PFP by NMR while lipids in VLDL/LDL, citrate, and glutamine were lower. By UPLC-MS, PE(18:0/18:2) and PC(P-16:0/20:4) were higher in PRP compared with PFP samples.

Conclusions

Correction for inter-individual variation was required to detect metabolite differences between serum and plasma. Our results suggest the potential importance of inter-individual effects and sample type on the results from serum and plasma metabolic phenotyping studies.

     

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.

     

NMRProcFlow: a graphical and interactive tool dedicated to 1D spectra processing for NMR-based metabolomics

Introduction

Concerning NMR-based metabolomics, 1D spectra processing often requires an expert eye for disentangling the intertwined peaks.

Objectives

The objective of NMRProcFlow is to assist the expert in this task in the best way without requirement of programming skills.

Methods

NMRProcFlow was developed to be a graphical and interactive 1D NMR (¹H & ¹³C) spectra processing tool.

Results

NMRProcFlow (http://nmrprocflow.org), dedicated to metabolic fingerprinting and targeted metabolomics, covers all spectra processing steps including baseline correction, chemical shift calibration and alignment.

Conclusion

Biologists and NMR spectroscopists can easily interact and develop synergies by visualizing the NMR spectra along with their corresponding experimental-factor levels, thus setting a bridge between experimental design and subsequent statistical analyses.

     

Fungal Keratitis Secondary to <Emphasis Type="Italic">Trametes betulina</Emphasis>: A Case Report and Review of Literature

Purpose

We report the first case of human infection and keratitis secondary to Trametes betulina, a rare filamentous fungus.

Methods

Clinical examination including external and slit-lamp examination and corneal scrapings with microbiologic evaluation were performed on a patient with chronic allergic conjunctivitis, entropion and a long-standing corneal ulcer resistant to treatment.

Results

The culture from the corneal scraping revealed a basidiomycetous fungus which was submitted for identification. DNA extraction with sequencing and analysis of the ITS and D1/D2 regions were performed on the isolate and demonstrated 100% similarity to Lenzites betulina/Trametes betulina. Susceptibility testing demonstrated potent in vitro activity of voriconazole (MIC < 0.03 μg/ml). The patient was treated with voriconazole, and the corneal ulcer and infiltrate resolved. The infection resulted in corneal thinning and a dense central corneal scar. Penetrating keratoplasty was performed 5 months after diagnosis and treatment and revealed stromal scarring without fungal elements.

Conclusion

This is the first reported case of keratitis caused by Trametes betulina. This organism should be considered in the differential diagnosis for rare filamentous fungal keratitis and its treatment with voriconazole also noted.

     

Plasma lipidomics identifies novel biomarkers in patients with hepatitis B virus-related acute-on-chronic liver failure

Introduction

Acute-on-chronic liver failure (ACLF) is a fatal syndrome that presents with acute deterioration of liver function in chronic hepatitis B virus (HBV) patients. However, reliable diagnostic and prognostic biomarkers are scarce.

Objectives

The aim of this study to identify lipid species associated with HBV infection as well as novel lipid biomarkers for HBV-ACLF.

Methods

High performance liquid chromatography–tandem mass spectrometry was used for targeted lipidomic analyses of 147 lipid species. Fasting-state plasma samples from 74 HBV-ACLF patients, 86 HBV-non-ACLF patients [30 HBV-immune tolerant (HBV-IT) and 56 chronic hepatitis B] and 20 healthy controls. Univariate and multivariate analyses examined changes in lipid species among patient groups.

Results

The HBV-ACLF and HBV-non-ACLF groups had distinctly different lipid profiles, while the HC and HBV-IT groups had similar lipid profiles. Further, lysophosphatidylcholine (LPC) 22:6, cholesterol ester (CE) 22:6, CE 20:4, CE 18:2 and CE 18:1 could be used as potential biomarkers for the early prediction of ACLF. Meanwhile, univariate and multivariate analyses identified CE 20:4, LPC 16:0, LPC 18:0, phosphatidylcholine (PC) 40:6 and PC 32:0 as putative diagnostic biomarkers of HBV-ACLF. Moreover, LPC 16:0 and LPC 18:0 were significantly associated with model for end stage liver disease (MELD) scores, and the two lipid species combined with MELD score had significant capability to predict the 6-month mortality.

Conclusions

Our study revealed that lipid metabolism disorders were significantly associated with the severity of liver inflammatory injury rather than HBV infection in patients with chronic HBV infection, and specific lipid species could be used as potentially biomarkers for diagnosis and prognosis in HBV-ACLF.

     

A metabolomics approach to uncover effects of different exercise modalities in type 1 diabetes

Introduction

Exercise-associated metabolism in type 1 diabetes (T1D) remains under-studied due to the complex interplay between exogenous insulin, counter-regulatory hormones and insulin-sensitivity.

Objective

To identify the metabolic differences induced by two exercise modalities in T1D using ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC–HRMS) based metabolomics.

Methods

Twelve T1D adults performed intermittent high-intensity (IHE) and continuous-moderate-intensity (CONT) exercise. Serum samples were analysed by UHPLC–HRMS.

Results

Metabolic profiling of IHE and CONT highlighted exercise-induced changes in purine and acylcarnitine metabolism.

Conclusion

IHE may increase beta-oxidation through higher ATP-turnover. UHPLC–HRMS based metabolomics as a data-driven approach without an a priori hypothesis may help uncover distinctive metabolic effects during exercise in T1D.Clinical trial registration number is www.clinicaltrials.gov: NCT02068638.

     

Automated metabolite identification from biological fluid <Superscript>1</Superscript>H NMR spectra

Introduction

Metabolite identification in biological samples using Nuclear Magnetic Resonance (NMR) spectra is a challenging task due to the complexity of the biological matrices.

Objectives

This paper introduces a new, automated computational scheme for the identification of metabolites in 1D ¹H NMR spectra based on the Human Metabolome Database.

Methods

The methodological scheme comprises of the sequential application of preprocessing, data reduction, metabolite screening and combination selection.

Results

The proposed scheme has been tested on the 1D ¹H NMR spectra of: (a) an amino acid mixture, (b) a serum sample spiked with the amino acid mixture, (c) 20 blood serum, (d) 20 human amniotic fluid samples, (e) 160 serum samples from publicly available database. The methodological scheme was compared against widely used software tools, exhibiting good performance in terms of correct assignment of the metabolites.

Conclusions

This new robust scheme accomplishes to automatically identify peak resonances in ¹H-NMR spectra with high accuracy and less human intervention with a wide range of applications in metabolic profiling.

     

Study on the Clinical Features and Prognosis of <Emphasis Type="Italic">Penicilliosis marneffei</Emphasis> Without Human Immunodeficiency Virus Infection

Objective

To improve the diagnosis and treatment of Penicilliosis marneffei without human immunodeficiency virus infection.

Methods

Analyze and review the clinical features, diagnosis and treatment of six cases of P. marneffei without human immunodeficiency virus infection at The First Affiliated Hospital of Fujian Medical University.

Results

Two cases were diagnosed in the ENT Department, three cases in the respiratory department and one case in the dermatological department. Penicillium marneffei infection was confirmed by sputum culture, blood culture and tissue biopsy. After definite diagnosis, one refused further treatment, and others showed significant improvement.

Conclusion

Penicilliosis marneffei is insidious onset and easy to be escaped and misdiagnosed. To achieve early diagnosis and appropriate treatment, doubtful cases should be alerted for the diagnoses as P. marneffei.

     

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.

     

<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.

     

rDolphin: a GUI R package for proficient automatic profiling of 1D <Superscript>1</Superscript>H-NMR spectra of study datasets

Introduction

Adoption of automatic profiling tools for ¹H-NMR-based metabolomic studies still lags behind other approaches in the absence of the flexibility and interactivity necessary to adapt to the properties of study data sets of complex matrices.

Objectives

To provide an open source tool that fully integrates these needs and enables the reproducibility of the profiling process.

Methods

rDolphin incorporates novel techniques to optimize exploratory analysis, metabolite identification, and validation of profiling output quality.

Results

The information and quality achieved in two public datasets of complex matrices are maximized.

Conclusion

rDolphin is an open-source R package (http://github.com/danielcanueto/rDolphin) able to provide the best balance between accuracy, reproducibility and ease of use.

     

HspX protein as a candidate vaccine against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>: an overview

Background

Tuberculosis (TB) is a contagious infectious disease caused by Mycobacterium tuberculosis (Mtb). This disease with two million deaths per year has the highest mortality rate among bacterial infections. The only available vaccine against TB is BCG vaccine. BCG is an effective vaccine against TB in childhood, however, due to some limitations, has not proper efficiency in adults. Also, BCG cannot produce an adequately protective response against reactivation of latent infections.

Objective

In the present study we will review the most recent findings about contribution of HspX protein in the vaccines against tuberculosis.

Methods

Therefore, many attempts have been made to improve BCG or to find its replacement. Most of the subunit vaccines for TB in various phases of clinical trials were constructed as prophylactic vaccines using Mtb proteins expressed in the replicating stage. These vaccines might prevent active TB but not reactivation of latent tuberculosis infection (LTBI). A literature search was performed on various online databases (PubMed, Scopus, and Google Scholar) regarding the roles of HspX protein in tuberculosis vaccines.

Results

Ideal subunit post-exposure vaccines should target all forms of TB infection, including active symptomatic and dormant (latent) asymptomatic forms. Among these subunit vaccines, HspX is the most important latent phase antigen of M. tuberculosis with a strong immunological response. There are many studies that have evaluated the immunogenicity of this protein to improve TB vaccine.

Conclusion

According to the studies, HspX protein is a good candidate for development of subunit vaccines against TB infection.

     

Quantitative analysis of tetrahydrofolate metabolites from <Emphasis Type="Italic">clostridium autoethanogenum</Emphasis>

Introduction

Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.

Objective

To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.

Methods

Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC–MS/MS was used for quantification.

Results

Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.

Conclusion

Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.

     

Accumulation of Fascin<Superscript>+</Superscript> cells during experimental autoimmune neuritis

Background

Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies and is characterized by inflammation and demyelination in the peripheral nervous system. Fascin is an evolutionarily highly conserved cytoskeletal protein of 55 kDa containing two actin binding domains that cross-link filamentous actin to hexagonal bundles.

Methods

Here we have studied by immunohistochemistry the spatiotemporal accumulation of Fascin?+?cells in sciatic nerves of EAN rats.

Results

A robust accumulation of Fascin?+?cell was observed in the peripheral nervous system of EAN which was correlated with the severity of neurological signs in EAN.

Conclusion

Our results suggest a pathological role of Fascin in EAN.

Virtual slides

The virtual slides for this article can be found here: http://www.diagnosticphatology.diagnomx.eu/vs/6734593451114811

     

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.

     

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.

     

Selection and validation of reliable reference genes in <Emphasis Type="Italic">Gossypium raimondii</Emphasis>

Objective

To identify reliable reference genes for gene expression analysis in Gossypium raimondii.

Results

Five different software tools, geNorm, NormFinder, BestKeeper, ReFinder and ?Ct method were employed to analyze the qRT-PCR data systematically of 12 housekeeping genes. SAD and TUA11 showed relatively stable expression levels in all tissues (i.e. leaves, shoots, buds, and sepals). We then limited our analysis to each plant part and identified tissue-specific reference genes. Our results showed TUA11, TUB6 and EF1a, EF1a, MZA and GAPC2, MZA, GAPC2, SAD and TUA11, and UBQ and MZA were reliable reference genes in leaves, shoots, buds, and sepals, respectively.

Conclusion

Some genes were commonly identified as candidate reference genes in more than two tissue, while others were tissue-specific. Thus, our study allows choosing an appropriate control gene based on sampling for gene expression analysis.

     

Lipid response patterns in acute phase paediatric <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> malaria

Introduction

Several studies have observed serum lipid changes during malaria infection in humans. All of them were focused at analysis of lipoproteins, not specific lipid molecules. The aim of our study was to identify novel patterns of lipid species in malaria infected patients using lipidomics profiling, to enhance diagnosis of malaria and to evaluate biochemical pathways activated during parasite infection.

Methods

Using a multivariate characterization approach, 60 samples were representatively selected, 20 from each category (mild, severe and controls) of the 690 study participants between age of 0.5–6 years. Lipids from patient’s plasma were extracted with chloroform/methanol mixture and subjected to lipid profiling with application of the LCMS-QTOF method.

Results

We observed a structured plasma lipid response among the malaria-infected patients as compared to healthy controls, demonstrated by higher levels of a majority of plasma lipids with the exception of even-chain length lysophosphatidylcholines and triglycerides with lower mass and higher saturation of the fatty acid chains. An inverse lipid profile relationship was observed when plasma lipids were correlated to parasitaemia.

Conclusions

This study demonstrates how mapping the full physiological lipid response in plasma from malaria-infected individuals can be used to understand biochemical processes during infection. It also gives insights to how the levels of these molecules relate to acute immune responses.

     

A multidimensional <Superscript>1</Superscript>H NMR lipidomics workflow to address chemical food safety issues

Introduction

Although it is still at a very early stage compared to its mass spectrometry (MS) counterpart, proton nuclear magnetic resonance (NMR) lipidomics is worth being investigated as an original and complementary solution for lipidomics. Dedicated sample preparation protocols and adapted data acquisition methods have to be developed to set up an NMR lipidomics workflow; in particular, the considerable overlap observed for lipid signals on 1D spectra may hamper its applicability.

Objectives

The study describes the development of a complete proton NMR lipidomics workflow for application to serum fingerprinting. It includes the assessment of fast 2D NMR strategies, which, besides reducing signal overlap by spreading the signals along a second dimension, offer compatibility with the high-throughput requirements of food quality characterization.

Method

The robustness of the developed sample preparation protocol is assessed in terms of repeatability and ability to provide informative fingerprints; further, different NMR acquisition schemes—including classical 1D, fast 2D based on non-uniform sampling or ultrafast schemes—are evaluated and compared. Finally, as a proof of concept, the developed workflow is applied to characterize lipid profiles disruption in serum from β-agonists diet fed pigs.

Results

Our results show the ability of the workflow to discriminate efficiently sample groups based on their lipidic profile, while using fast 2D NMR methods in an automated acquisition framework.

Conclusion

This work demonstrates the potential of fast multidimensional ¹H NMR—suited with an appropriate sample preparation—for lipidomics fingerprinting as well as its applicability to address chemical food safety issues.