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.

     

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.

     

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.

     

High salt and fat intake,inflammation, and risk of cancer

Background

Inflammatory conditions are involved in the pathophysiology of cancer. Recent findings have revealed that excessive salt and fat intake is involved in the development of severe inflammatory reactions.

Methods

literature search was performed on various online databases (PubMed, Scopus, and Google Scholar) regarding the roles of high salt and fat intake in the induction of inflammatory reactions and their roles in the etiopathogenesis of cancer.

Results

The results indicate that high salt and fat intake can induce severe inflammatory conditions. However, various inflammatory conditions have been strongly linked to the development of cancer. Hence, high salt and fat intake might be involved in the pathogenesis of cancer progression via putative mechanisms related to inflammatory reactions.

Conclusion

Reducing salt and fat intake may decrease the risk of cancer.

     

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.

     

<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.

     

Untargeted and stable isotope-assisted metabolomic analysis of MDA-MB-231 cells under hypoxia

Introduction

Hypoxia commonly occurs in cancers and is highly related with the occurrence, development and metastasis of cancer. Treatment of triple negative breast cancer remains challenge. Knowledge about the metabolic status of triple negative breast cancer cell lines in hypoxia is valuable for the understanding of molecular mechanisms of this tumor subtype to develop effective therapeutics.

Objectives

Comprehensively characterize the metabolic profiles of triple negative breast cancer cell line MDA-MB-231 in normoxia and hypoxia and the pathways involved in metabolic changes in hypoxia.

Methods

Differences in metabolic profiles affected pathways of MDA-MB-231 cells in normoxia and hypoxia were characterized using GC–MS based untargeted and stable isotope assisted metabolomic techniques.

Results

Thirty-three metabolites were significantly changed in hypoxia and nine pathways were involved. Hypoxia increased glycolysis, inhibited TCA cycle, pentose phosphate pathway and pyruvate carboxylation, while increased glutaminolysis in MDA-MB-231 cells.

Conclusion

The current results provide metabolic differences of MDA-MB-231 cells in normoxia and hypoxia conditions as well as the involved metabolic pathways, demonstrating the power of combined use of untargeted and stable isotope-assisted metabolomic methods in comprehensive metabolomic analysis.

     

Untargeted polar metabolomics of transformed MDA-MB-231 breast cancer cells expressing varying levels of human arylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase 1

Introduction

Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. Expression of NAT1 is elevated in several cancers including breast cancer. However, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear.

Objective

This study explored polar metabolome differences between MDA-MB-231 breast cancer cells expressing varying levels of NAT1 activity using an untargeted approach.

Methods

Three MDA-MB-231 breast adenocarcinoma cell lines that stably express wild-type, increased, and decreased levels of human NAT1 were investigated for differences in polar metabolic profile using a comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOF MS) system.

Results

Increased levels of human NAT1 in the transformed cell lines resulted in a statistically significant decreased abundance of the metabolite palmitoleic acid (q = 0.0006), when compared to normal and decreased levels of human NAT1. The fatty acid synthesis pathway utilizes acetyl coenzyme A (acetyl-CoA) in the first two reactions of the pathway and eventually leads to the synthesis of palmitoleic acid.

Conclusion

These data suggest a link between increased levels of NAT1 activity and decreased flux of acetyl-CoA through this portion of the fatty acid synthesis pathway.

     

Stimulation of mouse vibrissal follicle growth by recombinant human fibroblast growth factor 20

Objectives

To explore potential effects of recombinant human fibroblast growth factor 20 (rhFGF20) in the growth of cultured mouse vibrissal follicles.

Results

The growth of cultured mouse vibrissal follicles was significantly induced by rhFGF20 in a dose dependent pattern in the in vitro vibrissal follicle organ culture model. However, too high concentration of rhFGF20 could inhibit the growth of vibrissal follicles. We further demonstrated that rhFGF20 stimulated the proliferation of hair matrix cells and activated Wnt/β-catenin signaling pathway.

Conclusions

The rhFGF20 might be a potential therapeutic agent to treat hair loss disorders.

     

Genomics and proteomics approaches to the study of cancer-stroma interactions

Background

The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.

Methods

The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.

Results

We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR.

Conclusions

A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.

     

PTEN at the interface of immune tolerance and tumor suppression

Background

PTEN is well known to function as a tumor suppressor that antagonizes oncogenic signaling and maintains genomic stability. The PTEN gene is frequently deleted or mutated in human cancers and the wide cancer spectrum associated with PTEN deficiency has been recapitulated in a variety of mouse models of Pten deletion or mutation. Pten mutations are highly penetrant in causing various types of spontaneous tumors that often exhibit resistance to anticancer therapies including immunotherapy. Recent studies demonstrate that PTEN also regulates immune functionality.

Objective

To understand the multifaceted functions of PTEN as both a tumor suppressor and an immune regulator.

Methods

This review will summarize the emerging knowledge of PTEN function in cancer immunoediting. In addition, the mechanisms underlying functional integration of various PTEN pathways in regulating cancer evolution and tumor immunity will be highlighted.

Results

Recent preclinical and clinical studies revealed the essential role of PTEN in maintaining immune homeostasis, which significantly expands the repertoire of PTEN functions. Mechanistically, aberrant PTEN signaling alters the interplay between the immune system and tumors, leading to immunosuppression and tumor escape.

Conclusion

Rational design of personalized anti-cancer treatment requires mechanistic understanding of diverse PTEN signaling pathways in modulation of the crosstalk between tumor and immune cells.

     

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.

     

Male breast cancer: thirteen years experience of a single center

Background

This retrospective study analysed the epidemiological, clinical, and therapeutic profiles of breast cancer in males.

Methods

We report our experience at the Hospital of the University of Baskent, where 20 cases of male breast cancer were observed and treated between 1995–2008.

Results

Median age at presentation was 66,7 ± 10,9 years. Average follow-up was 63 ± 18,5 months. The main presenting symptom was a mass in 65% of cases (13 patients). Ýnvasive ductal carcinoma was the most frequent pathologic type (70% of cases).

Conclusion

Male breast cancer patients have an incidence of prostate cancer higher than would be predicted in the general population. Cause of men have a higher rate of ER positivity the responses with hormonal agents are good.

     

Crystal structure of <Emphasis Type="Italic">Escherichia coli</Emphasis> protein ybgI,a toroidal structure with a dinuclear metal site

Background

The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.

Results

The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.

Conclusion

The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.

     

Characterization of ankylosing spondylitis and rheumatoid arthritis using <Superscript>1</Superscript>H NMR-based metabolomics of human fecal extracts

Introduction

The differences in fecal metabolome between ankylosing spondylitis (AS)/rheumatoid arthritis (RA) patients and healthy individuals could be the reason for an autoimmune disorder.

Objectives

The study explored the fecal metabolome difference between AS/RA patients and healthy controls to clarify human immune disturbance.

Methods

Fecal samples from 109 individuals (healthy controls 34, AS 40, and RA 35) were analyzed by ¹H NMR spectroscopy. Data were analyzed with principal component analysis (PCA) and orthogonal projection to latent structure discriminant (OPLS-DA) analysis.

Results

Significant differences in the fecal metabolic profiles could distinguish AS/RA patients from healthy controls but could not distinguish between AS and RA patients. The significantly decreased metabolites in AS/RA patients were butyrate, propionate, methionine, and hypoxanthine. Significantly increased metabolites in AS/RA patients were taurine, methanol, fumarate, and tryptophan.

Conclusion

The metabolome variations in feces indicated AS and RA were two homologous diseases that could not be distinguished by ¹H NMR metabolomics.

     

SAGE detects microRNA precursors

Background

MicroRNAs (miRNAs) have been shown to play important roles in regulating gene expression. Since miRNAs are often evolutionarily conserved and their precursors can be folded into stem-loop hairpins, many miRNAs have been predicted. Yet experimental confirmation is difficult since miRNA expression is often specific to particular tissues and developmental stages.

Results

Analysis of 29 human and 230 mouse longSAGE libraries revealed the expression of 22 known and 10 predicted mammalian miRNAs. Most were detected in embryonic tissues. Four SAGE tags detected in human embryonic stem cells specifically match a cluster of four human miRNAs (mir-302a, b, c&d) known to be expressed in embryonic stem cells. LongSAGE data also suggest the existence of a mouse homolog of human and rat mir-493.

Conclusion

The observation that some orphan longSAGE tags uniquely match miRNA precursors provides information about the expression of some known and predicted miRNAs.

     

<Superscript>1</Superscript>H NMR metabolic profiling of gastric cancer patients with lymph node metastasis

Introduction

Gastric cancer (GC) is a malignant tumor worldwide. As primary pathway for metastasis, the lymphatic system is an important prognostic factor for GC patients. Although the metabolic changes of gastric cancer have been investigated in extensive studies, little effort focused on the metabolic profiling of lymph node metastasis (LNM)-positive or negative GC patients.

Objectives

We performed ¹H NMR spectrum of GC tissue samples with and without LNM to identify novel potential metabolic biomarkers in the process of LNM of GC.

Methods

¹H NMR-based untargeted metabolomics approach combined with multivariate statistical analyses were used to study the metabolic profiling of tissue samples from LNM-positive GC patients (n?=?40), LNM-negative GC patients (n?=?40) and normal controls (n?=?40).

Results

There was a clear separation between GC patients and normal controls, and 33 differential metabolites were identified in the study. Moreover, GC patients were also well-classified according to LNM-positive or negative. Totally eight distinguishing metabolites were selected in the metabolic profiling of GC patients with LNM-positive or negative, suggesting the metabolic dysfunction in the process of LNM. According to further validation and analysis, especially BCAAs metabolism (leucine, isoleucine, valine), GSH and betaine may be as potential factors of diagnose and prognosis of GC patients with or without LNM.

Conclusion

To our knowledge, this is the first metabolomics study focusing on LNM of GC. The identified distinguishing metabolites showed a promising application on clinical diagnose and therapy prediction, and understanding the mechanism underlying the carcinogenesis, invasion and metastasis of GC.