Production of a therapeutic protein by fusing it with two fragments of the carboxyl-terminal peptide of human chorionic gonadotropin β-subunit in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objective

To produce a therapeutic protein (endostatin) by fusion with two fragments of the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin β-subunit in Pichia pastoris.

Results

Two CTP sequences were fused to the C-terminal of human endostatin, and the fusion protein (endo-CTP) was expressed by P. pastoris. Endo-CTP inhibited proliferation of endothelial cells with an IC₅₀ of 7 μg ml^?1, and 30 % of cells were annexin V-positive after treatment with 20 μg endo-CTP ml^?1 for 48 h. Migration of endothelial cells was inhibited by endo-CTP in a concentration-dependent manner. The half-life of endo-CTP in Sprague–Dawley rats was much longer than that of its commercial counterpart (Endostar).

Conclusion

A long-acting endostatin can be produced using CTP technology.

     

Novel one-pot ATP regeneration system based on three-enzyme cascade for industrial CTP production

Objectives

To develop a new one-pot polyphosphate kinase (PPK) system with low cost and high efficiency for ATP regeneration in industrial CTP production.

Results

We developed a new one-pot PPK system by applying a three-enzyme cascade (CMK, NDK and PPK) with an in vitro polyP-based ATP regeneration system. The PPK was selected from twenty sources, and was made solvable by fusion expressing with soluble protein and constructing polycistronic plasmids, or co-expressing with molecular chaperones GroES/EL. Activities of other enzymes were optimized by employing fusion expression, tac-pBAD system, Rosetta host and codon optimization. After 24 h, the concentration of CDP and CTP reached 3.8 ± 0.2 and 6.9 ± 0.3 mM l^?1 respectively with a yield of approximately 79%. The molar conversion rate of CTP was 51%, and its yield and conversion rate increased 100% from the traditional system.

Conclusions

A new one-pot ATP regeneration system applying polyphosphate kinase for CTP production was developed.

     

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.

     

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.

     

Metabolomic profiling of maternal hair suggests rapid development of intrahepatic cholestasis of pregnancy

Introduction

Intrahepatic cholestasis of pregnancy (ICP) is a common maternal liver disease; development can result in devastating consequences, including sudden fetal death and stillbirth. Currently, recognition of ICP only occurs following onset of clinical symptoms.

Objective

Investigate the maternal hair metabolome for predictive biomarkers of ICP.

Methods

The maternal hair metabolome (gestational age of sampling between 17 and 41 weeks) of 38 Chinese women with ICP and 46 pregnant controls was analysed using gas chromatography–mass spectrometry.

Results

Of 105 metabolites detected in hair, none were significantly associated with ICP.

Conclusion

Hair samples represent accumulative environmental exposure over time. Samples collected at the onset of ICP did not reveal any metabolic shifts, suggesting rapid development of the disease.

     

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.

     

Degradation of carbazole,dibenzothiophene and polyaromatic hydrocarbons by recombinant <Emphasis Type="Italic">Rhodococcus</Emphasis> sp.

Objectives

With the view of designing a single biocatalyst for biorefining, carbazole dioxygenase was cloned from Pseudomonas sp. and expressed in Rhodococcus sp.

Results

The recombinant, IGTS8, degraded both carbazole and dibenzothiophene at 400 mg/l in 24 h. Maximum carbazole degradation was in 1:1 (v/v) hexadecane/aqueous phase. Anthracene, phenanthrene, pyrene, fluoranthene and fluorine were also degraded without affecting the aliphatic component.

Conclusions

Recombinant Rhodococcus sp. IGTS8 can function as a single biocatalyst for removing major contaminants of fossil fuels viz. dibenzothiophene, carbazole and polyaromatic 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.

     

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.

     

Discontinuous video recording of biopsies in the context of an integral third degree teaching program

Background

The application of Information and Communication Technologies (ICTs) in the field of university education can play an important role in order to fulfil the objectives of the European Space for Higher Education (ESHE). In the absence of an infrastructure that allows virtual preparations, the high definition video may become an alternative for many PC users and telepathology enthusiasts.

Methods

We have begun a video biopsy discontinuous recording pilot programme, also known as interval cinematography, in order to give our students pathological images to gather documentary evidence to follow their clinical cases. We have a working post available with a ZEISS Axioshop 2 microscope connected to a JVC digital camera with a double output for video, a PVM_14N1MDE Sony Triniton colour monitor and a LG RH199 HDD-DVD recorder.

Results

Pathologists can easily produce videos lasting between 45" and 5' according to the case. Later those videos are assessed all together and given to the student and/or the doctor who requested the case study.

Conclusion

Better follow-up studies of the cases can be achieved using Discontinuous video recording of biopsies. This approach has also clinical benefits since it contributes to the enrichment of integral patient care.

     

Does centrifugation matter? Centrifugal force and spinning time alter the plasma metabolome

Background

Centrifugation is an indispensable procedure for plasma sample preparation, but applied conditions can vary between labs.

Aim

Determine whether routinely used plasma centrifugation protocols (1500×g 10 min; 3000×g 5 min) influence non-targeted metabolomic analyses.

Methods

Nuclear magnetic resonance spectroscopy (NMR) and High Resolution Mass Spectrometry (HRMS) data were evaluated with sparse partial least squares discriminant analyses and compared with cell count measurements.

Results

Besides significant differences in platelet count, we identified substantial alterations in NMR and HRMS data related to the different centrifugation protocols.

Conclusion

Already minor differences in plasma centrifugation can significantly influence metabolomic patterns and potentially bias metabolomics studies.

     

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.

     

Pseudo current density maps of electrophysiological heart,nerve or brain function and their physical basis

Background

In recent years the visualization of biomagnetic measurement data by so-called pseudo current density maps or Hosaka-Cohen (HC) transformations became popular.

Methods

The physical basis of these intuitive maps is clarified by means of analytically solvable problems.

Results

Examples in magnetocardiography, magnetoencephalography and magnetoneurography demonstrate the usefulness of this method.

Conclusion

Hardware realizations of the HC-transformation and some similar transformations are discussed which could advantageously support cross-platform comparability of biomagnetic measurements.

     

From correlation to causation: analysis of metabolomics data using systems biology approaches

Introduction

Metabolomics is a well-established tool in systems biology, especially in the top–down approach. Metabolomics experiments often results in discovery studies that provide intriguing biological hypotheses but rarely offer mechanistic explanation of such findings. In this light, the interpretation of metabolomics data can be boosted by deploying systems biology approaches.

Objectives

This review aims to provide an overview of systems biology approaches that are relevant to metabolomics and to discuss some successful applications of these methods.

Methods

We review the most recent applications of systems biology tools in the field of metabolomics, such as network inference and analysis, metabolic modelling and pathways analysis.

Results

We offer an ample overview of systems biology tools that can be applied to address metabolomics problems. The characteristics and application results of these tools are discussed also in a comparative manner.

Conclusions

Systems biology-enhanced analysis of metabolomics data can provide insights into the molecular mechanisms originating the observed metabolic profiles and enhance the scientific impact of metabolomics studies.

     

Untargeted metabolomics suffers from incomplete raw data processing

Introduction

Untargeted metabolomics is a powerful tool for biological discoveries. To analyze the complex raw data, significant advances in computational approaches have been made, yet it is not clear how exhaustive and reliable the data analysis results are.

Objectives

Assessment of the quality of raw data processing in untargeted metabolomics.

Methods

Five published untargeted metabolomics studies, were reanalyzed.

Results

Omissions of at least 50 relevant compounds from the original results as well as examples of representative mistakes were reported for each study.

Conclusion

Incomplete raw data processing shows unexplored potential of current and legacy data.

     

TarMet: a reactive GUI tool for efficient and confident quantification of MS based targeted metabolic and stable isotope tracer analysis

Introduction

Untargeted and targeted analyses are two classes of metabolic study. Both strategies have been advanced by high resolution mass spectrometers coupled with chromatography, which have the advantages of high mass sensitivity and accuracy. State-of-art methods for mass spectrometric data sets do not always quantify metabolites of interest in a targeted assay efficiently and accurately.

Objectives

TarMet can quantify targeted metabolites as well as their isotopologues through a reactive and user-friendly graphical user interface.

Methods

TarMet accepts vendor-neutral data files (NetCDF, mzXML and mzML) as inputs. Then it extracts ion chromatograms, detects peak position and bounds and confirms the metabolites via the isotope patterns. It can integrate peak areas for all isotopologues automatically.

Results

TarMet detects more isotopologues and quantify them better than state-of-art methods, and it can process isotope tracer assay well.

Conclusion

TarMet is a better tool for targeted metabolic and stable isotope tracer analyses.

     

Using multitask classification methods to investigate the kinase-specific phosphorylation sites

Background

Identification of phosphorylation sites by computational methods is becoming increasingly important because it reduces labor-intensive and costly experiments and can improve our understanding of the common properties and underlying mechanisms of protein phosphorylation.

Methods

A multitask learning framework for learning four kinase families simultaneously, instead of studying each kinase family of phosphorylation sites separately, is presented in the study. The framework includes two multitask classification methods: the Multi-Task Least Squares Support Vector Machines (MTLS-SVMs) and the Multi-Task Feature Selection (MT-Feat3).

Results

Using the multitask learning framework, we successfully identify 18 common features shared by four kinase families of phosphorylation sites. The reliability of selected features is demonstrated by the consistent performance in two multi-task learning methods.

Conclusions

The selected features can be used to build efficient multitask classifiers with good performance, suggesting they are important to protein phosphorylation across 4 kinase families.

     

Thaw pond development and initial vegetation succession in experimental plots at a Siberian lowland tundra site

Background and aims

Permafrost degradation has the potential to change the Arctic tundra landscape. We observed rapid local thawing of ice-rich permafrost resulting in thaw pond formation, which was triggered by removal of the shrub cover in a field experiment. This study aimed to examine the rate of permafrost thaw and the initial vegetation succession after the permafrost collapse.

Methods

In the experiment, we measured changes in soil thaw depth, plant species cover and soil subsidence over nine years (2007–2015).

Results

After abrupt initial thaw, soil subsidence in the removal plots continued indicating further thawing of permafrost albeit at a much slower pace: 1 cm y^?1 over 2012–2015 vs. 5 cm y^?1 over 2007–2012. Grass cover strongly increased after the initial shrub removal, but later declined with ponding of water in the subsiding removal plots. Sedges established and expanded in the wetter removal plots. Thereby, the removal plots have become increasingly similar to nearby ‘natural’ thaw ponds.

Conclusions

The nine years of field observations in a unique shrub removal experiment at a Siberian tundra site document possible trajectories of small-scale permafrost collapse and the initial stage of vegetation recovery, which is essential knowledge for assessing future tundra landscape changes.

     

A methodology for elucidating regulatory mechanisms leading to changes in lipid profiles

Introduction

It is difficult to elucidate the metabolic and regulatory factors causing lipidome perturbations.

Objectives

This work simplifies this process.

Methods

A method has been developed to query an online holistic lipid metabolic network (of 7923 metabolites) to extract the pathways that connect the input list of lipids.

Results

The output enables pathway visualisation and the querying of other databases to identify potential regulators. When used to a study a plasma lipidome dataset of polycystic ovary syndrome, 14 enzymes were identified, of which 3 are linked to ELAVL1—an mRNA stabiliser.

Conclusion

This method provides a simplified approach to identifying potential regulators causing lipid-profile perturbations.