Characterization of rhamnolipid biosurfactants produced by recombinant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain DAB with removal of crude oil

Objectives

To improve rhamnolipid production and its potential application in removal of crude oil, the recombinant Pseudomonas aeruginosa strain DAB was constructed to enhance yield of rhamnolipids.

Results

Strain DAB had a higher yield of 17.3 g rhamnolipids l^?1 in the removal process with crude oil as the sole carbon source than 10 g rhamnolipids l^?1 of wild-type strain DN1, where 1% crude oil was degraded more than 95% after 14 days cultivation. These rhamnolipids reduced the surface tension of water from 72.92 to 26.15 mN m^?1 with CMC of 90 mg l^?1. The predominant rhamnolipid congeners were Rha–C₁₀–C₁₀ and Rha–Rha–C₁₀–C₁₀ detected by MALDI-TOF MS analysis with approx. 70% relative abundance, although a total of 21 rhamnolipid congeners were accumulated.

Conclusion

Increasing the copy number of rhlAB genes efficiently enhanced the production of rhamnolipids by the recombinant P. aeruginosa DAB and thus presents a promising application for the bioremediation process.

     

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically-engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To engineer Escherichia coli for the heterologous production of di-rhamnolipids, which are important biosurfactants but mainly produced by opportunistic pathogen Pseudomonas aeruginosa.

Results

The codon-optimized rhlAB and rhlC genes originating from P. aeruginosa and Burkholderia pseudomallei were combinatorially expressed in E. coli to produce di-rhamnolipids with varied congeners compositions. Genes involved in endogenous upstream pathways (rhamnose and fatty acids synthesis) were co-overexpressed with rhlAB–rhlC, resulting in variations of rhamnolipids production and congeners compositions. Under the shake-flask condition, co-overexpression of rfbD with rhlAB–rhlC increased rhamnolipids production (0.64 ± 0.02 g l^?1) than that in strain only expressing rhlAB–rhlC (0.446 ± 0.009 g l^?1), which was mainly composed of di-rhamnolipids congeners Rha–Rha–C₁₀–C₁₀.

Conclusion

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically engineered E. coli strains were achieved via combiniations of mono-/di-rhamnolipids synthesis modules and endogenous upstream modules.

     

Resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants

Objectives

To develop and validate a microdilution method for measuring the minimum inhibitory concentration (MIC) of biosurfactants.

Results

A standardized microdilution method including resazurin dye has been developed for measuring the MIC of biosurfactants and its validity was established through the replication of tetracycline and gentamicin MIC determination with standard bacterial strains.

Conclusion

This new method allows the generation of accurate MIC measurements, whilst overcoming critical issues related to colour and solubility which may interfere with growth measurements for many types of biosurfactant extracts.

     

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.

     

Lipopeptide produced from <Emphasis Type="Italic">Bacillus</Emphasis> sp. W112 improves the hydrolysis of lignocellulose by specifically reducing non-productive binding of cellulases with and without CBMs

Background

Surfactants have attracted increasing interest for their capability to improve the enzymatic hydrolysis of lignocellulosic biomass. Compared to chemical surfactants, biosurfactants have a broader prospect for industrial applications because they are more environmentally friendly and more effective in some researches. Commercial cellulase preparations are mainly composed of endoglucanases (EGs) and cellobiohydrolases (CBHs) that possess carbohydrate-binding modules (CBMs). However, the effects of lipopeptide-type biosurfactants on enzymatic saccharification of lignocellulose and adsorption behaviors of cellulases with CBMs remain unclear.

Results

In this study, we found that Bacillus sp. W112 could produce a lipopeptide-type biosurfactant from untreated biomass, such as wheat bran and Jerusalem artichoke tuber. The lipopeptide could enhance the enzymatic hydrolysis of dilute acid pretreated Giant Juncao grass (DA-GJG) by fungal and bacterial enzymes. The enhancement increased over a range of temperatures from 30 to 50 °C. Lipopeptide was shown to be more effective in promoting DA-GJG saccharification than chemical surfactants at low dosages, with a best stimulatory degree of 20.8% at 2% loading of the substrates (w/w). Lipopeptide increased the thermostability of EG and CBH in commercial cellulase cocktails. Moreover, the dual effects of lipopeptide on the adsorption behaviors of cellulases were found. It specifically lowered the non-productive binding of cellulases to lignin and increased the binding of cellulases to cellulose. In addition, we investigated the influence of lipopeptide on the adsorption behaviors of CBHs with CBMs for the first time. Our results showed that lipopeptide reduced the adsorption of CBM-deleted CBH to DA-GJG to a greater extent than that of intact CBH while the non-productive binding of intact CBH to lignin was reduced more, indicating that lipopeptide decreased the binding of CBMs onto lignin but not their combination with cellulose.

Conclusions

In this study, we found that lipopeptide from Bacillus sp. W112 promoted the enzymatic hydrolysis of DA-GJG at relative low loadings. The stimulatory effect could be attributed to increasing the cellulase thermostability, reducing non-productive adsorption of cellulases with CBMs caused by lignin and enhancing the binding of cellulases to cellulose.

     

ASICS: an automatic method for identification and quantification of metabolites in complex 1D <Superscript>1</Superscript>H NMR spectra

Introduction

Experiments in metabolomics rely on the identification and quantification of metabolites in complex biological mixtures. This remains one of the major challenges in NMR/mass spectrometry analysis of metabolic profiles. These features are mandatory to make metabolomics asserting a general approach to test a priori formulated hypotheses on the basis of exhaustive metabolome characterization rather than an exploratory tool dealing with unknown metabolic features.

Objectives

In this article we propose a method, named ASICS, based on a strong statistical theory that handles automatically the metabolites identification and quantification in proton NMR spectra.

Methods

A statistical linear model is built to explain a complex spectrum using a library containing pure metabolite spectra. This model can handle local or global chemical shift variations due to experimental conditions using a warping function. A statistical lasso-type estimator identifies and quantifies the metabolites in the complex spectrum. This estimator shows good statistical properties and handles peak overlapping issues.

Results

The performances of the method were investigated on known mixtures (such as synthetic urine) and on plasma datasets from duck and human. Results show noteworthy performances, outperforming current existing methods.

Conclusion

ASICS is a completely automated procedure to identify and quantify metabolites in ¹H NMR spectra of biological mixtures. It will enable empowering NMR-based metabolomics by quickly and accurately helping experts to obtain metabolic profiles.

     

Process optimization of benzo[ghi]perylene biodegradation by yeast consortium in presence of ZnO nanoparticles and produced biosurfactant using Box-Behnken design

Background

Benzo[ghi]perylene (BghiP), a polycyclic aromatic hydrocarbon (PAH) containing six fused benzene rings is considered as priority pollutant because of its carcinogenicity, mutagenicity and acute toxicity.

Methods

The synthesis of ZnO nanoparticles was done following the standard method. Biosurfactant production by yeast consortium YC04 in MSM was confirmed by various tests viz. drop collapse test, methylene blue agar plate method and emulsification test (E24) using the standard procedures. Efficiency of YC04 was tested to remediate BghiP in presence of ZnO nanoparticles and produced biosurfactant in the growth medium.

Results

Response surface methodology (RSM), 3-level five variables Box-Behnken design (BBD) was employed to optimize the factors viz. pH 7.0, temperature 30°C, shaking speed 130 rpm, inoculum dosage 3% and ZnO nanoparticles concentration 2 g/L after a period of 6 days of incubation for the enhanced degradation of BghiP (63.83±0.01%). It was well in close agreement with the predicated value obtained by RSM model yield (63.83±0.08%). Analysis of variance (ANOVA) showed F-value of 51.70, R2 of 0.9764, probability of<0.0001 and coefficient of variation of 1.25% confirmed the validity of the model. Degradation of BghiP was assessed using GC-MS and FTIR analysis. Kinetic study demonstrated that BghiP degradation fitted first order kinetic model.

Conclusions

To the best of our knowledge, this is the first report on process optimization toward nanobioremediation of BghiP using yeast consortium in presence of ZnO nanoparticles and produced biosurfactant in medium.

     

A hybrid parameter estimation algorithm for beta mixtures and applications to methylation state classification

Background

Mixtures of beta distributions are a flexible tool for modeling data with values on the unit interval, such as methylation levels. However, maximum likelihood parameter estimation with beta distributions suffers from problems because of singularities in the log-likelihood function if some observations take the values 0 or 1.

Methods

While ad-hoc corrections have been proposed to mitigate this problem, we propose a different approach to parameter estimation for beta mixtures where such problems do not arise in the first place. Our algorithm combines latent variables with the method of moments instead of maximum likelihood, which has computational advantages over the popular EM algorithm.

Results

As an application, we demonstrate that methylation state classification is more accurate when using adaptive thresholds from beta mixtures than non-adaptive thresholds on observed methylation levels. We also demonstrate that we can accurately infer the number of mixture components.

Conclusions

The hybrid algorithm between likelihood-based component un-mixing and moment-based parameter estimation is a robust and efficient method for beta mixture estimation. We provide an implementation of the method (“betamix”) as open source software under the MIT license.

     

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.

     

Collagen and fibronectin surface modification of nanoporous anodic alumina and macroporous silicon for endothelial cell cultures

Background

The ability to direct the cellular response by means of biomaterial surface topography is important for biomedical applications. Substrate surface topography has been shown to be an effective cue for the regulation of cellular response. Here, the response of human aortic endothelial cells to nanoporous anodic alumina and macroporous silicon with collagen and fibronectin functionalization has been studied.

Methods

Confocal microscopy and scanning electron microscopy were employed to analyse the effects of the material and the porosity on the adhesion, morphology, and proliferation of the cells. Cell spreading and filopodia formation on macro- and nanoporous material was characterized by atomic force microscopy. We have also studied the influence of the protein on the adhesion.

Results

It was obtained the best results when the material is functionalized with fibronectin, regarding cells adhesion, morphology, and proliferation.

Conclusion

These results permit to obtain chemical modified 3D structures for several biotechnology applications such as tissue engineering, organ-on-chip or regenerative medicine.

     

Carbon catabolite repression in <Emphasis Type="Italic">Thermoanaerobacterium saccharolyticum</Emphasis>

Background

The thermophilic anaerobe Thermoanaerobacterium saccharolyticum is capable of directly fermenting xylan and the biomass-derived sugars glucose, cellobiose, xylose, mannose, galactose and arabinose. It has been metabolically engineered and developed as a biocatalyst for the production of ethanol.

Results

We report the initial characterization of the carbon catabolite repression system in this organism. We find that sugar metabolism in T. saccharolyticum is regulated by histidine-containing protein HPr. We describe a mutation in HPr, His15Asp, that leads to derepression of less-favored carbon source utilization.

Conclusion

Co-utilization of sugars can be achieved by mutation of HPr in T. saccharolyticum. Further manipulation of CCR in this organism will be instrumental in achieving complete and rapid conversion of all available sugars to ethanol.

     

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.

     

Normalization of TAM post-receptor signaling reveals a cell invasive signature for Axl tyrosine kinase

Background

Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis.

Methods

In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling.

Results

Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk.

Conclusion

These studies demonstrate that TAM show unique post-receptor signatures that impinge on distinct gene expression profiles and tumorigenic outcomes.

     

The optimum Se application time for reducing Cd uptake by rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) and its mechanism

Background and aims

Intraspecific aggregation of plant individuals can promote species coexistence by delaying competitive exclusions. However, such impacts may differ among species with contrasting spatial architecture and rely on the spatial distribution of resources.

Methods

We grew a phalanx clonal plant Carex neurocarpa (with aggregated ramets) and a guerilla one Bolboschoenus planiculmis (with diffused ramets) in monocultures or in 1:1 mixtures with an even or a clustered distribution pattern of the two species in homogeneous or heterogeneous soils.

Results

After 16 months, shoot biomass and ramet number were greater in mixtures than in monocultures in C. neurocarpa, but smaller in B. planiculmis. However, the growth of neither C. neurocarpa nor B. planiculmis differed between even and clustered mixtures. Soil nutrient heterogeneity did not significantly affect the growth of either species, but increased relative yield of B. planiculmis and decreased that of C. neurocarpa.

Conclusions

The relative importance of intra- vs. interspecific competition depends on the spatial architecture of plants, and soil nutrient heterogeneity slows down competitive exclusion by decreasing differences in competitive ability between plants. However, our results do not support the idea that intraspecific aggregation of individuals alters competitive interactions between species.

     

Progranulin derivative Atsttrin protects against early osteoarthritis in mouse and rat models

Background

Atsttrin, an engineered protein composed of three tumor necrosis factor receptor (TNFR)-binding fragments of progranulin (PGRN), shows therapeutic effect in multiple murine models of inflammatory arthritis . Additionally, intra-articular delivery of PGRN protects against osteoarthritis (OA) progression. The purpose of this study is to determine whether Atsttrin also has therapeutic effects in OA and the molecular mechanisms involved.

Methods

Surgically induced and noninvasive rupture OA models were established in mouse and rat, respectively. Cartilage degradation and OA were evaluated using Safranin O staining, immunohistochemistry, and ELISA. Additionally, expressions of pain-related markers, degenerative factors, and anabolic and catabolic markers known to be involved in OA were analyzed. Furthermore, the anabolic and anti-catabolic effects and underlying mechanisms of Atsttrin were determined using in-vitro assays with primary chondrocytes.

Results

Herein, we found Atsttrin effectively prevented the accelerated OA phenotype associated with PGRN deficiency. Additionally, Atsttrin exhibited a preventative effect in OA by protecting articular cartilage and reducing OA-associated pain in both nonsurgically induced rat and surgically induced murine OA models. Mechanistic studies revealed that Atsttrin stimulated TNFR2-Akt-Erk1/2-dependent chondrocyte anabolism, while inhibiting TNFα/TNFR1-mediated inflammatory catabolism.

Conclusions

These findings not only provide new insights into the role of PGRN and its derived engineered protein Atsttrin in cartilage homeostasis as well as OA in vivo, but may also lead to new therapeutic alternatives for OA as well as other relative degenerative joint diseases.

     

Development and application of a T7 RNA polymerase-dependent expression system for antibiotic production improvement in <Emphasis Type="Italic">Streptomyces</Emphasis>

Objective

To develop a reliable and easy to use expression system for antibiotic production improvement of Streptomyces.

Results

A two-compound T7 RNA polymerase-dependent gene expression system was developed to fulfill this demand. In this system, the T7 RNA polymerase coding sequence was optimized based on the codon usage of Streptomyces coelicolor. To evaluate the functionality of this system, we constructed an activator gene overexpression strain for enhancement of actinorhodin production. By overexpression of the positive regulator actII-ORF4 with this system, the maximum actinorhodin yield of engineered strain was 15-fold higher and the fermentation time was decreased by 48 h.

Conclusion

The modified two-compound T7 expression system improves both antibiotic production and accelerates the fermentation process in Streptomyces. This provides a general and useful strategy for strain improvement of important antibiotic producing Streptomyces strains.

     

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.

     

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.

     

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.