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.

     

Differential elemental uptake in three pseudo-metallophyte C<Subscript>4</Subscript> grasses in situ in the eastern USA

Background and aims

Elemental uptake in serpentine floras in eastern North America is largely unknown. The objective of this study was to determine major and trace element concentrations in soil and leaves of three native pseudo-metallophyte C₄ grasses in situ at five sites with three very different soil types, including three serpentine sites, in eastern USA.

Methods

Pseudo-total and extractible concentrations of 15 elements were measured and correlated from the soils and leaves of three species at the five sites.

Results

Element concentrations in soils of pseudo-metallophytes varied up to five orders of magnitude. Soils from metalliferous sites exhibited higher concentrations of their characteristic elements than non-metalliferous. In metallicolous populations, elemental concentrations depended on the element. Concentrations of major elements (Ca, Mg, K) in leaves were lower than typical toxicity thresholds, whereas concentrations of Zn were higher.

Conclusions

In grasses, species can maintain relatively low metal concentrations in their leaves even when soil concentrations are richer. However, in highly Zn-contaminated soil, we found evidence of a threshold concentration above which Zn uptake increases drastically. Finally, absence of main characteristics of serpentine soil at one site indicated the importance of soil survey and restoration to maintain serpentinophytes communities and avoid soil encroachment.

     

Comprehensive <Superscript>1</Superscript>H NMR metabolic profiling of body fluids for differentiation of meningitis in adults

Introduction

Meningitis, a morbidly infectious central nervous system pathology is accompanied by acute inflammation of the meninges, causing raised intracranial pressure linked with serious neurological sequelae.

Objective

To observe the variation in the metabolic profile, that may occur in serum and urine along with CSF in adults using ¹H NMR spectroscopy, with an attempt of appropriate and timely treatment regimen.

Methods

The ¹H NMR-based metabolomics has been performed in 115 adult subjects for differentiating bacterial meningitis (BM) and tubercular meningitis (TBM).

Results

The discriminant function analysis (DFA) of the three bio-fluids collectively identified 3-hydroxyisovalerate, lactate, glucose, formate, valine, alanine, ketonic bodies, malonate and choline containing compounds (choline and GPC) as significant metabolites among cases versus control group. The differentiation of bacterial meningitis and tuberculous meningitis (BM vs. TBM) can be done on the basis of identification of 3-hydroxyisovalerate, isobutyrate and formate in case of CSF (with a correct classification of 78 %), alanine in serum (correct classification 60 %), valine and acetone in case of urine (correct classification 89.1 %). The NMR spectral bins based orthogonal signal correction principal component analysis score plots of significant metabolites obtained from DFA also provided group classification among cases versus control group in CSF, serum and urine samples. The variable importance in projection scores also identified similar significant metabolites as obtained from DFA, collectively in CSF, serum and urine samples, responsible for differentiation of meningitis.

Conclusion

The CSF contained metabolites which are formed during infection and inflammation, and these were also found in significant quantity in serum and urine samples.

     

The effect of 1,25-dihydroxyvitamin D<Subscript>3</Subscript> on TSLP,IL-33 and IL-25 expression in respiratory epithelium

Background

Airway epithelium is an active and important component of the immunological response in the pathophysiology of obstructive lung diseases. Recent studies suggest an important role for vitamin D₃ in asthma severity and treatment response.

Objective

Our study evaluated the influence of an active form of vitamin D₃ on the expression of selected mediators of allergic inflammation in the respiratory epithelium.

Material and Methods

Primary nasal and bronchial epithelial cells were exposed to1,25D₃ for 1 hour and were then stimulated or not with IL-4, TNF-α, LPS, and poly I:C. After 24 hours TSLP, IL-33, and IL-25 protein levels were measured in culture supernatants usingELISAandmRNAlevels in cells by real time PCR.

Results

1,25D₃ increased TSLP concentration in unstimulated nasal epithelial cells, but did not influence IL-33 and IL-25 expression. In IL-4-stimulated epithelial cell cultures 1,25D₃ mostly inhibited TSLP and IL-33 expression. In LPS-treated cultures 1,25D₃ decreased IL-33 expression. Simultaneously 1,25D₃ augmented IL-25 production in the same model of stimulation.

Conclusion

Our study revealed the dual nature of vitamin D₃ manifested in both pro- and anti-inflammatory properties observed in airway epithelial cells.

     

Endophytic infection modifies organic acid and mineral element accumulation by rice under Na<Subscript>2</Subscript>CO<Subscript>3</Subscript> stress

Background and aims

Saline and alkali soils severely impact plant growth. Endophyte and plant associations are known to significantly modify plant metabolism. This study reports the effects of a type of endophyte on organic acid (OA) accumulation and ionic balance in rice under Na₂CO₃ stress.

Methods

Rice seedlings with (E+) and without (E-) endophytic infection were subjected to different levels of Na₂CO₃ stress (0, 5, 10, 15, and 20 mM) for two weeks. Organic acids and mineral elements in the leaves and roots were determined.

Results

Seedlings with endophytic infection accumulated mainly citrate and fumarate, with some malate and succinate in the leaves. In the roots, accumulation of malate and fumarate was enhanced significantly by endophytic infection, while less citrate and succinate was accumulated under Na₂CO₃ stress, which suggested that leaves and roots use different mechanisms to control OA metabolism. Endophytes reduced the total Na and Na:K ratios, but increased ST values, the percent changes of other measured nutrients, Chl content, and dry weight per plant under Na₂CO₃ stress.

Conclusions

Endophytic infection plays a key role in maintaining plant growth by improving nutrient uptake and adjusting OA accumulation under Na₂CO₃ stress. The application of endophytes can enhance the resistance of rice to salinity.

     

Effectiveness of fractional CO<Subscript>2</Subscript> laser in women with stress urinary incontinence

Background

Stress urinary incontinence (SUI) is a relatively common disorder that significantly affects the quality of life. Many conservative and surgical treatment methods have been recommended for SUI, but they have major limitations.

Aims

To assess the use of the CO₂ fractional laser in the treatment of SUI.

Methods

This clinical trial included 55 patients with confirmed SUI. Patients underwent fractional CO₂ laser treatment 3 times at 30-day intervals. Data on age, smoking history, sexual activity, menopause, and history of hormone replacement therapy (HRT) were collected. Response to treatment was assessed by SUI severity and the level of sexual satisfaction was assessed using the visual analog scale (VAS). Patients were evaluated at 3 different time points: before treatment, and 45 days and 6 months after the last laser treatment.

Results

The mean patient age was 44.4±11.4 years (range: 28 to 68 years). Smoking history was positive in 6 patients (9.1%); 19 (54.3%) were menopausal on HRT. The SUI severity score at baseline (before treatment) was 8.56±0.62 and decreased to 2.28 6 months after treatment (p<0.0001). The sexual satisfaction score was 3±0.94 at baseline and increased to 7.87±0.93 6 months after treatment (day 180) (p<0.0001, slope = + 2.2)

Conclusion

Our findings are in line with a previous study that showed the value of transvaginal CO₂ fractional laser treatment for alleviation of SUI symptoms and its potential as an alternative treatment. We also observed improved sexual satisfaction in SUI patients.

     

Structures of EccB<Subscript>1</Subscript> and EccD<Subscript>1</Subscript> from the core complex of the mycobacterial ESX-1 type VII secretion system

Background

The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system.

Results

This study characterizes the structures of the soluble domains of two conserved core ESX-1 components – EccB₁ and EccD₁. The periplasmic domain of EccB₁ consists of 4 repeat domains and a central domain, which together form a quasi 2-fold symmetrical structure. The repeat domains of EccB₁ are structurally similar to a known peptidoglycan binding protein suggesting a role in anchoring the ESX-1 system within the periplasmic space. The cytoplasmic domain of EccD₁has a ubiquitin-like fold and forms a dimer with a negatively charged groove.

Conclusions

These structures represent a major step towards resolving the molecular architecture of the entire ESX-1 assembly and may contribute to ESX-1 targeted tuberculosis intervention strategies.

     

Trade-off between C and N recycling and N<Subscript>2</Subscript>O emissions of soils with summer cover crops in subtropical agrosystems

Aims

Biological soil crusts (biocrusts) are soil-surface communities in drylands, dominated by cyanobacteria, mosses, and lichens. They provide key ecosystem functions by increasing soil stability and influencing soil hydrologic, nutrient, and carbon cycles. Because of this, methods to reestablish biocrusts in damaged drylands are needed. Here we test the reintroduction of field-collected vs. greenhouse-cultured biocrusts for rehabilitation.

Methods

We collected biocrusts for 1) direct reapplication, and 2) artificial cultivation under varying hydration regimes. We added field-collected and cultivated biocrusts (with and without hardening treatments) to bare field plots and monitored establishment.

Results

Both field-collected and cultivated cyanobacteria increased cover dramatically during the experimental period. Cultivated biocrusts established more rapidly than field-collected biocrusts, attaining ~82% cover in only one year, but addition of field-collected biocrusts led to higher species richness, biomass (as assessed by chlorophyll a) and level of development. Mosses and lichens did not establish well in either case, but late successional cover was affected by hardening and culture conditions.

Conclusions

This study provides further evidence that it is possible to culture biocrust components from later successional materials and reestablish cultured organisms in the field. However, more research is needed into effective reclamation techniques.

     

Relationships between nitrogen,dry matter accumulation and glucosinolates in <Emphasis Type="Italic">Eruca sativa</Emphasis> Mills. The applicability of the critical NO<Subscript>3</Subscript>-N levels approach

Background and Aims

Rocket salad (Eruca sativa Mills) is one of the major leafy vegetables produced worldwide and has been characterized as a rich source of chemoprotective glucosinolates (GSL). The relationship between N fertilization and the resulting plant biomass and N status with GSL quantity and quality in rocket leaves was examined.

Methods

A pot experiment was conducted, applying ten different N-rates and destructive sampling was carried out 15, 30 and 45 days after transplanting (DAT). The Mitscherlich equation was used to establish NO₃-N critical levels at each growth stage and as an indicator of N demand for relative maximum dry matter accumulation and glucosinolate content and composition was determined.

Results

Glucosinolate content was significantly influenced by N rate, growth stage and their interaction. Different GSL types showed dissimilar responses to N fertilization: aliphatic GSLs were significantly reduced under increased N rates whereas indole GSL showed the reverse. Under excess N fertilization (>1.04 g/plant), dry matter accumulation remained constant, NO₃-N was significantly increased and total GSL content was significantly reduced, factors that could lead to an anticipated product quality decline.

Conclusions

The application of the critical NO₃-N level approach used to identify optimal N fertilization rates for plant growth could serve as means to obtain optimized GSL content in the edible plant parts.

     

Sublethal concentration of H<Subscript>2</Subscript>O<Subscript>2</Subscript> enhances the protective effect of mesenchymal stem cells in rat model of spinal cord injury

Objective

To investigate the effect of H₂O₂ on the migration and antioxidant defense of mesenchymal stem cells (MSCs) and the neurotrophic effects of H₂O₂-treated MSCs on spinal cord injury (SCI).

Results

Sublethal concentrations of H₂O₂ decreased cell migration and expression of CXCR4 and CCR2 as well as Nrf2 expression in MSCs. In the second phase, transplantation of treated and untreated MSCs to SCI caused minor changes in locomotor dysfunction. There was a significantly difference between cell-treated and spinal cord injury groups in expression of BDNF (brain-derived neurotrophic factor). Transplantation of H₂O₂-treated cells caused an increase in BDNF expression compared to non-treated cells.

Conclusion

Transplantation of H₂O₂-treated stem cells may have protective effects against SCI through by increasing neurotrophic factors.

     

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.

     

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.

     

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.

     

Human health characterization factors of nano-TiO<Subscript>2</Subscript> for indoor and outdoor environments

Purpose

The increasing use of engineered nanomaterials (ENMs) in industrial applications and consumer products is leading to an inevitable release of these materials into the environment. This makes it necessary to assess the potential risks that these new materials pose to human health and the environment. Life cycle assessment (LCA) methodology has been recognized as a key tool for assessing the environmental performance of nanoproducts. Until now, the impacts of ENMs could not be included in LCA studies due to a lack of characterization factors (CFs). This paper provides a methodological framework for identifying human health CFs for ENMs.

Methods

The USEtox? model was used to identify CFs for assessing the potential carcinogenic and non-carcinogenic effects on human health caused by ENM emissions in both indoor (occupational settings) and outdoor environments. Nano-titanium dioxide (nano-TiO₂) was selected for defining the CFs in this study, as it is one of the most commonly used ENMs. For the carcinogenic effect assessment, a conservative approach was adopted; indeed, a critical dose estimate for pulmonary inflammation was assumed.

Results and discussion

We propose CFs for nano-TiO₂ from 5.5E?09 to 1.43E?02 cases/kg_emitted for both indoor and outdoor environments and for carcinogenic and non-carcinogenic effects.

Conclusions

These human health CFs for nano-TiO₂ are an important step toward the comprehensive application of LCA methodology in the field of nanomaterial technology.

     

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.

     

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

     

Comparative effect of inorganic N on plant growth and N<Subscript>2</Subscript> fixation of ten legume crops: towards a better understanding of the differential response among species

Aims

A better understanding of how plant growth, N nutrition and symbiotic nitrogen fixation (SNF) are influenced by soil inorganic N availability, for a wide range of legume species, is crucial to optimise legume productivity, N₂ fixation, while limiting environmental risks such as N leaching.

Methods

A comparative analysis was performed for ten legume crops, grown in a field experiment and supplied with four N fertiliser rates. Dry matter, N concentration and SNF were measured. In parallel, root elongation rates were studied in a greenhouse experiment.

Results

For most species, N fertilisation had little effect on plant growth and N accumulation. SNF was reduced by soil inorganic N available at sowing but with large differences in the magnitude of the response among species. The response varied according to plant N requirements for growth and plant ability to retrieve inorganic N. Accordingly, root lateral expansion rate measured in RhizoTubes was highly correlated with plant ability to retrieve inorganic N measured in the field experiment.

Conclusion

Combining SNF response to soil inorganic N, shoot N and plant ability to retrieve inorganic N, allowed a robust evaluation of differential response to soil inorganic N among a wide range of legume species.

     

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">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.

     

Uncovering the metabolic response of abalone (<Emphasis Type="Italic">Haliotis midae)</Emphasis> to environmental hypoxia through metabolomics

Introduction

Oxygen is essential for metabolic processes and in the absence thereof alternative metabolic pathways are required for energy production, as seen in marine invertebrates like abalone. Even though hypoxia has been responsible for significant losses to the aquaculture industry, the overall metabolic adaptations of abalone in response to environmental hypoxia are as yet, not fully elucidated.

Objective

To use a multiplatform metabolomics approach to characterize the metabolic changes associated with energy production in abalone (Haliotis midae) when exposed to environmental hypoxia.

Methods

Metabolomics analysis of abalone adductor and foot muscle, left and right gill, hemolymph, and epipodial tissue samples were conducted using a multiplatform approach, which included untargeted NMR spectroscopy, untargeted and targeted LC–MS spectrometry, and untargeted and semi-targeted GC-MS spectrometric analyses.

Results

Increased levels of anaerobic end-products specific to marine animals were found which include alanopine, strombine, tauropine and octopine. These were accompanied by elevated lactate, succinate and arginine, of which the latter is a product of phosphoarginine breakdown in abalone. Primarily amino acid metabolism was affected, with carbohydrate and lipid metabolism assisting with anaerobic energy production to a lesser extent. Different tissues showed varied metabolic responses to hypoxia, with the largest metabolic changes in the adductor muscle.

Conclusions

From this investigation, it becomes evident that abalone have well-developed (yet understudied) metabolic mechanisms for surviving hypoxic periods. Furthermore, metabolomics serves as a powerful tool for investigating the altered metabolic processes in abalone.