Extracellular Vesicles Produced by the Cardiac Microenvironment Carry Functional Enzymes to Produce Lipid Mediators In Situ.

The impact of the polyunsaturated fatty acids (PUFAs) at physiological concentrations on the composition of eicosanoids transported within the extracellular vesicles (EVs) of rat bone marrow mesenchymal stem cells and cardiomyoblasts was reported by our group in 2020. The aim of this article was to extend this observation to cells from the cardiac microenvironment involved in the processes of inflammation, namely mouse J774 macrophages and rat heart mesenchymal stem cells cMSCs. Moreover, to enhance our capacity to understand the paracrine exchange between these orchestrators of cardiac inflammation, we investigated some machinery involved in the eicosanoid's synthesis transported by the EVs produced by these cells (including the two formerly described cells: bone marrow mesenchymal stem cells BM-MSC and cardiomyoblasts H9c2). We analyzed the oxylipin and the enzymatic content of the EVs collected from cell cultures supplemented (or not) with PUFAs. We prove that large eicosanoid profiles are exported in the EVs by the cardiac microenvironment cells, but also that these EVs carry some critical and functional biosynthetic enzymes, allowing them to synthesize inflammation bioactive compounds by sensing their environment. Moreover, we demonstrate that these are functional. This observation reinforces the hypothesis that EVs are key factors in paracrine signaling, even in the absence of the parent cell. We also reveal a macrophage-specific behavior, as we observed a radical change in the lipid mediator profile when small EVs derived from J774 cells were exposed to PUFAs. To summarize, we prove that the EVs, due to the carried functional enzymes, can alone produce bioactive compounds, in the absence of the parent cell, by sensing their environment. This makes them potential circulating monitoring entities.

noeM, a New Nodulation Gene Involved in the Biosynthesis of Nod Factors with an Open-Chain Oxidized Terminal Residue and in the Symbiosis with Mimosa pudica.

The ß-rhizobium Cupriavidus taiwanensis is a nitrogen-fixing symbiont of Mimosa pudica. Nod factors produced by this species were previously found to be pentameric chitin-oligomers carrying common C18:1 or C16:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing terminal residue. Here, we report that, in addition, C. taiwanensis LMG19424 produces molecules where the reducing sugar is open and oxidized. We identified a novel nodulation gene located on the symbiotic plasmid pRalta, called noeM, which is involved in this atypical Nod factor structure. noeM encodes a transmembrane protein bearing a fatty acid hydroxylase domain. This gene is expressed during symbiosis with M. pudica and requires NodD and luteolin for optimal expression. The closest noeM homologs formed a separate phylogenetic clade containing rhizobial genes only, which are located on symbiosis plasmids downstream from a nod box. Corresponding proteins, referred to as NoeM, may have specialized in symbiosis via the connection to the nodulation pathway and the spread in rhizobia. noeM was mostly found in isolates of the Mimoseae tribe, and specifically detected in all tested strains able to nodulate M. pudica. A noeM deletion mutant of C. taiwanensis was affected for the nodulation of M. pudica, confirming the role of noeM in the symbiosis with this legume.

Endosymbiotic Sinorhizobium meliloti modulate Medicago root susceptibility to secondary infection via ethylene.

A complex network of pathways coordinates nodulation and epidermal root hair infection in the symbiotic interaction between rhizobia and legume plants. Whereas nodule formation was known to be autoregulated, it was so far unclear whether a similar control is exerted on the infection process. We assessed the capacity of Medicago plants nodulated by Sinorhizobium meliloti to modulate root susceptibility to secondary bacterial infection or to purified Nod factors in split-root and volatile assays using bacterial and plant mutant combinations. Ethylene implication in this process emerged from gas production measurements, use of a chemical inhibitor of ethylene biosynthesis and of a Medicago mutant affected in ethylene signal transduction. We identified a feedback mechanism that we named AOI (for Autoregulation Of Infection) by which endosymbiotic bacteria control secondary infection thread formation by their rhizospheric peers. AOI involves activation of a cyclic adenosine 3',5'-monophosphate (cAMP) cascade in endosymbiotic bacteria, which decreases both root infectiveness and root susceptibility to bacterial Nod factors. These latter two effects are mediated by ethylene. AOI is a novel component of the complex regulatory network controlling the interaction between Sinorhizobium meliloti and its host plants that emphasizes the implication of endosymbiotic bacteria in fine-tuning the interaction.

A digest of capillary electrophoretic methods applied to lipid analyzes.

Lipids are naturally occurring organic compounds that can be classified into a number of types based on their solubility in nonpolar organic solvents, and are generally insoluble in water. The great structural variety of these various types of lipids has led them to be components of many different biological substances such as oils, waxes, cellular membranes, tissues and biological fluids. The use of capillary electrophoresis (CE) for the study of lipids during the past 30 years has been relatively rare when compared to its use for other classes of biomolecules, primarily due to their insolubility in water. However, a number of interesting studies have been conducted, and as part of this review, we will present the different approaches that were used, which mainly consist of micellar kinetic chromatography and non-aqueous CE. The main advantages of the use of these techniques compared to GC is the very simple sample preparation that is required and, compared to LC, the very robust and quick separations that can be obtained. In this review, we present the various methods that have been reported in the literature that have been used for the study of fatty acids, phospholipids, glycerides, eicosanoids and sterols, with the inclusion of various tables presenting descriptions of the CE methods used as well as the methods of detection, including UV absorbance, fluorescence, mass spectrometry, and conductivity. This review aims to demonstrate that CE can be easily used for the analysis of lipids.

Capillary electrophoresis/visible-LED induced fluorescence of tryptophan: What's new?

Tryptophane (Trp) labelled by 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) is very difficult to identify using CE and fluorescence detection (480 nm). Why in this article some mass spectrometry experiments show that Trp is really labelled by CBQCA as Leucine (Leu)? If the maximum of UV absorption (λmax ) is the same between Leu-CBQCA and Trp-CBQCA, the molar extinction coefficient is around 2 fold higher for Trp-CBQCA. The fluorescence of the Leu-CBQCA derivative is 50 times more important than for Trp-CBQCA. The addition of 7.5 mM of ß-cyclodextrin (ß-CD) was found to be a good mean to improve 2.1 fold the sensitivity of the Trp-CBQCA fluorescence. Using a buffer containing SDS and ß-CD in CE, a LOD of 0.7 µM of L-Trp can be reached and the ratio of the intensities between Leu, Isoleucine, Valine, Trp is 100, 21, 15, 1. Negative ESI/ MS and MS/MS of the labeled amino acids show that a loss of the carboxylate function takes place. In the presence of two enantiomers of Trp-CBQCA, we have shown that this decarboxylation is not due to the derivatization process in the solution but rather occurs in the source of the mass spectrometer.

Nonspecific Symbiosis Between Sophora flavescens and Different Rhizobia.

We explored the genetic basis of the promiscuous symbiosis of Sophora flavescens with diverse rhizobia. To determine the impact of Nod factors (NFs) on the symbiosis of S. flavescens, nodulation-related gene mutants of representative rhizobial strains were generated. Strains with mutations in common nodulation genes (nodC, nodM, and nodE) failed to nodulate S. flavescens, indicating that the promiscuous nodulation of this plant is strictly dependent on the basic NF structure. Mutations of the NF decoration genes nodH, nodS, nodZ, and noeI did not affect the nodulation of S. flavescens, but these mutations affected the nitrogen-fixation efficiency of nodules. Wild-type Bradyrhizobium diazoefficiens USDA110 cannot nodulate S. flavescens, but we obtained 14 Tn5 mutants of B. diazoefficiens that nodulated S. flavescens. This suggested that the mutations had disrupted a negative regulator that prevents nodulation of S. flavescens, leading to nonspecific nodulation. For Ensifer fredii CCBAU 45436 mutants, the minimal NF structure was sufficient for nodulation of soybean and S. flavescens. In summary, the mechanism of promiscuous symbiosis of S. flavescens with rhizobia might be related to its nonspecific recognition of NF structures, and the host specificity of rhizobia may also be controlled by currently unknown nodulation-related genes.

Recent advances in amino acid analysis by capillary electromigration methods: June 2015-May 2017.

In the tenth edition of this article focused on recent advances in amino acid analysis using capillary electrophoresis, we describe the most important research articles published on this topic during the period from June 2015 to May 2017. This article follows the format of the previous articles published in Electrophoresis. The new developments in amino acid analysis with CE mainly describe improvements in CE associated with mass spectrometry. Focusing on applications, we mostly describe clinical works, although metabolomics studies are also very important. Finally, works focusing on amino acids in food and agricultural applications are also described.

G-quadruplex aptamer selection using capillary electrophoresis-LED-induced fluorescence and Illumina sequencing.

One of the major difficulties that arises when selecting aptamers containing a G-quadruplex is the correct amplification of the ssDNA sequence. Can aptamers containing a G-quadruplex be selected from a degenerate library using non-equilibrium capillary electrophoresis (CE) of equilibrium mixtures (NECEEM) along with high-throughput Illumina sequencing? In this article, we present some mismatches of the G-quadruplex T29 aptamer specific to thrombin, which was PCR amplified and sequenced by Illumina sequencing. Then, we show the proportionality between the number of sequenced molecules of T29 added to the library and the number of sequences obtained in Illumina sequencing, and we find that T29 sequences from this aptamer can be detected in a random library of ssDNA after the sample is fractionated by NECEEM, amplified by PCR, and sequenced. Treatment of the data by the counting of double-stranded DNA T29 sequences containing a maximum of two mismatches reveals a good correlation with the enrichment factor (fE). This factor is the ratio of the number of aptamer sequences found in the collected complex sample divided by the total number of sequencing reads (aptamer and non-aptamer) plus the quantity of T29 molecules (spiked into a DNA library) injected into CE.

Capillary electrophoresis hyphenated with UV-native-laser induced fluorescence detection (CE/UV-native-LIF).

Native laser-induced fluorescence using UV lasers associated to CE offers now a large related literature, for now 30 years. The main works have been performed using very expensive Ar-ion lasers emitting at 257 and 275 nm. They are not affordable for routine analyses, but have numerous applications such as protein, catecholamine, and indolamine analysis. Some other lasers such as HeCd 325 nm have been used but only for few applications. Diode lasers, emitting at 266 nm, cheaper, are extensively used for the same topics, even if the obtained sensitivity is lower than the one observed using the costly UV-Ar-ion lasers. This review presents various CE or microchips applications and different UV lasers used for the excitation of native fluorescence. We showed that CE/Native UV laser induced fluorescence detection is very sensitive for detection as well as small aromatic biomolecules than proteins containing Trp and Tyr amino acids. Moreover, it is a simple way to analyze biomolecules without derivatization.

ssDNA degradation along capillary electrophoresis process using a Tris buffer.

Tris-Acetate buffer is currently used in the selection and the characterization of ssDNA by capillary electrophoresis (CE). By applying high voltage, the migration of ionic species into the capillary generates a current that induces water electrolysis. This phenomenon is followed by the modification of the pH and the production of Tris derivatives. By injecting ten times by capillary electrophoresis ssDNA (50 nM), the whole oligonucleotide was degraded. In this paper, we will show that the Tris buffer in the running vials is modified along the electrophoretic process by electrochemical reactions. We also observed that the composition of the metal ions changes in the running buffer vials. This phenomenon, never described in CE, is important for fluorescent ssDNA analysis using Tris buffer. The oligonucleotides are degraded by electrochemically synthesized species (present in the running Tris vials) until it disappears, even if the separation buffer in the capillary is clean. To address these issues, we propose to use a sodium phosphate buffer that we demonstrate to be electrochemically inactive.

Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza.

Arbuscular mycorrhiza (AM) is a root endosymbiosis between plants and glomeromycete fungi. It is the most widespread terrestrial plant symbiosis, improving plant uptake of water and mineral nutrients. Yet, despite its crucial role in land ecosystems, molecular mechanisms leading to its formation are just beginning to be unravelled. Recent evidence suggests that AM fungi produce diffusible symbiotic signals. Here we show that Glomus intraradices secretes symbiotic signals that are a mixture of sulphated and non-sulphated simple lipochitooligosaccharides (LCOs), which stimulate formation of AM in plant species of diverse families (Fabaceae, Asteraceae and Umbelliferae). In the legume Medicago truncatula these signals stimulate root growth and branching by the symbiotic DMI signalling pathway. These findings provide a better understanding of the evolution of signalling mechanisms involved in plant root endosymbioses and will greatly facilitate their molecular dissection. They also open the way to using these natural and very active molecules in agriculture.

Recent advances in amino acid analysis by capillary electromigration methods, 2013-2015.

We describe the most important research articles published on amino acid analysis using CE during the period from June 2013 to May 2015, and follows the format of the previous articles published in electrophoresis the new developments in amino acid analysis with CE are mainly describing improvements in detection means and injection methods. Enantiomeric separation developments are still important. Focusing the applications, we describe the neurochemical and clinical works, but also the metabolomic studies for which the publication number increase greatly. Finally, works focused on amino acids in food and agricultural applications are described.

Recent advances in amino acid analysis by capillary electromigration methods, 2011-2013.

This article describes the most important research published on amino acid (AA) analysis using CE during the period from June 2011 to May 2013, and follows the format of the previous articles of Smith (Electrophoresis 1999, 20, 3078-3083), Prata et al. (Electrophoresis 2001, 22, 4129-4138), and Poinsot et al. (Electrophoresis 2003, 24, 4047-4062; Electrophoresis 2006, 27, 176-194; Electrophoresis 2008, 29, 207-223; Electrophoresis 2010, 31, 105-121; Electrophoresis 2012, 33, 14-35). We present new developments in AA analysis with CE, mainly describing the use of MS or LEDs for detection following conventional or enantiomeric separation developments. In addition, in an application part, we describe neurochemical or clinical studies, metabolomics for plant extracts and biological fluids, and finally works focused on AAs in food and agricultural applications.

Engineered and Mimicked Extracellular Nanovesicles for Therapeutic Delivery.

Exosomes are spherical extracellular nanovesicles with an endosomal origin and unilamellar lipid-bilayer structure with sizes ranging from 30 to 100 nm. They contain a large range of proteins, lipids, and nucleic acid species, depending on the state and origin of the extracellular vesicle (EV)-secreting cell. EVs' function is to encapsulate part of the EV-producing cell content, to transport it through biological fluids to a targeted recipient, and to deliver their cargos specifically within the aimed recipient cells. Therefore, exosomes are considered to be potential biological drug-delivery systems that can stably deliver their cargo into targeted cells. Various cell-derived exosomes are produced for medical issues, but their use for therapeutic purposes still faces several problems. Some of these difficulties can be avoided by resorting to hemisynthetic approaches. We highlight here the uses of alternative exosome-mimes involving cell-membrane coatings on artificial nanocarriers or the hybridization between exosomes and liposomes. We also detail the drug-loading strategies deployed to make them drug-carrier systems and summarize the ongoing clinical trials involving exosomes or exosome-like structures. Finally, we summarize the open questions before considering exosome-like disposals for confident therapeutic delivery.

Relevance of fucose-rich extracellular polysaccharides produced by Rhizobium sullae strains nodulating Hedysarum coronarium l. legumes.

Specific and complex interactions between soil bacteria, known as rhizobia, and their leguminous host plants result in the development of root nodules. This process implies a complex dialogue between the partners. Rhizobia synthesize different classes of polysaccharides: exopolysaccharides (EPS), Kdo-rich capsular polysaccharides, lipopolysaccharides, and cyclic ß-(1,2)-glucans. These polymers are actors of a successful symbiosis with legumes. We focus here on studying the EPS produced by Rhizobium sullae bacteria that nodulate Hedysarum coronarium L., largely distributed in Algeria. We describe the influence of the carbon source on the production and on the composition of EPS produced by R. sullae A6 and RHF strains. High-molecular-weight EPS preserve the bacteria from desiccation. The structural characterization of the EPS produced by R. sullae strains has been performed through sugar analysis by gas chromatography-mass spectrometry. The low-molecular-weight EPS of one strain (RHF) has been totally elucidated using nuclear magnetic resonance and quantitative time-of-flight tandem mass spectrometry analyses. An unusual fucose-rich EPS has been characterized. The presence of this deoxy sugar seems to be related to nodulation capacity.

Biochemical and functional characterization of SpdA, a 2', 3'cyclic nucleotide phosphodiesterase from Sinorhizobium meliloti.

BACKGROUND: 3', 5'cAMP signaling in Sinorhizobium meliloti was recently shown to contribute to the autoregulation of legume infection. In planta, three adenylate cyclases CyaD1, CyaD2 and CyaK, synthesizing 3', 5'cAMP, together with the Crp-like transcriptional regulator Clr and smc02178, a gene of unknown function, are involved in controlling plant infection. RESULTS: Here we report on the characterization of a gene (smc02179, spdA) at the cyaD1 locus that we predicted to encode a class III cytoplasmic phosphodiesterase.First, we have shown that spdA had a similar pattern of expression as smc02178 in planta but did not require clr nor 3', 5'cAMP for expression.Second, biochemical characterization of the purified SpdA protein showed that, contrary to expectation, it had no detectable activity against 3', 5'cAMP and, instead, high activity against the positional isomers 2', 3'cAMP and 2', 3'cGMP.Third, we provide direct experimental evidence that the purified Clr protein was able to bind both 2', 3'cAMP and 3', 5'cAMP in vitro at high concentration. We further showed that Clr is a 3', 5'cAMP-dependent DNA-binding protein and identified a DNA-binding motif to which Clr binds. In contrast, 2', 3'cAMP was unable to promote Clr specific-binding to DNA and activate smc02178 target gene expression ex planta.Fourth, we have shown a negative impact of exogenous 2', 3'cAMP on 3', 5'cAMP-mediated signaling in vivo. A spdA null mutant was also partially affected in 3', 5'cAMP signaling. CONCLUSIONS: SpdA is a nodule-expressed 2', 3' specific phosphodiesterase whose biological function remains elusive. Circumstantial evidence suggests that SpdA may contribute insulating 3', 5'cAMP-based signaling from 2', 3' cyclic nucleotides of metabolic origin.

Determination of free amino acids in African gourd seed milks by capillary electrophoresis with light-emitting diode induced fluorescence and laser-induced fluorescence detection.

A CE technique coupled to LIF detection (488 nm) or LED-induced fluorescence detection (470 nm) has been evaluated to acquire a cheap way to analyze amino acids (AAs) whilst maintaining the best sensitivity. To quantitate AAs in milk of Cucurbitaceae of Sub-Saharan Africa, they were labeled with FITC. We used an optimized separation buffer composed of 30 mM boric acid buffer adjusted to pH 9.3 with NaOH (1 M) containing 12 mM SDS and 5% ethylene glycol v/v; prior to the injections, the derivatized samples are diluted 100 times. The LOQs in the sample are Arg: 1.1 µM, Ala: 3.5 µM, and Glu 8.9 µM. Cucumeropsis mannii (CM) Naudin and Citrullus lanatus (CL) are vegetable sources rich in proteins and AAs of high quality. Our analyses have led to the identification of 11 AAs in CL and CM milks. Phe, Trp, and Ala are predominant in the two types of lyophilized milks, while Asp and Val demonstrate very low contents. Six essential AAs (Phe, Thr, Val, Trp, Ile, and Leu) are present in both types of extracts, but lysine was not detected, indicating that this AA is missing in gourd milk. These results should be useful in efforts to complement or replace very expensive cow milk or the less-appreciated soya milk with milk from available local agroressources.

Experimental evolution of a plant pathogen into a legume symbiont.

Rhizobia are phylogenetically disparate alpha- and beta-proteobacteria that have achieved the environmentally essential function of fixing atmospheric nitrogen in symbiosis with legumes. Ample evidence indicates that horizontal transfer of symbiotic plasmids/islands has played a crucial role in rhizobia evolution. However, adaptive mechanisms that allow the recipient genomes to express symbiotic traits are unknown. Here, we report on the experimental evolution of a pathogenic Ralstonia solanacearum chimera carrying the symbiotic plasmid of the rhizobium Cupriavidus taiwanensis into Mimosa nodulating and infecting symbionts. Two types of adaptive mutations in the hrpG-controlled virulence pathway of R. solanacearum were identified that are crucial for the transition from pathogenicity towards mutualism. Inactivation of the hrcV structural gene of the type III secretion system allowed nodulation and early infection to take place, whereas inactivation of the master virulence regulator hrpG allowed intracellular infection of nodule cells. Our findings predict that natural selection of adaptive changes in the legume environment following horizontal transfer has been a major driving force in rhizobia evolution and diversification and show the potential of experimental evolution to decipher the mechanisms leading to symbiosis.

Genomic Insights and Functional Analysis Reveal Plant Growth Promotion Traits of Paenibacillus mucilaginosus G78.

Paenibacillus mucilaginosus has widely been reported as a plant growth-promoting rhizobacteria (PGPR). However, the important genomic insights into plant growth promotion in this species remain undescribed. In this study, the genome of P. mucilaginosus G78 was sequenced using Illumina NovaSeq PE150. It contains 8,576,872 bp with a GC content of 58.5%, and was taxonomically characterized. Additionally, a total of 7337 genes with 143 tRNAs, 41 rRNAs, and 5 ncRNAs were identified. This strain can prohibit the growth of the plant pathogen, but also has the capability to form biofilm, solubilize phosphate, and produce IAA. Twenty-six gene clusters encoding secondary metabolites were identified, and the genotypic characterization indirectly proved its resistant ability to ampicillin, bacitracin, polymyxin and chloramphenicol. The putative exopolysaccharide biosynthesis and biofilm formation gene clusters were explored. According to the genetic features, the potential monosaccharides of its exopolysaccharides for P. mucilaginosus G78 may include glucose, mannose, galactose, fucose, that can probably be acetylated and pyruvated. Conservation of the pelADEFG compared with other 40 Paenibacillus species suggests that Pel may be specific biofilm matrix component in P. mucilaginosus. Several genes relevant to plant growth-promoting traits, i.e., IAA production and phosphate solubilization are well conserved compared with other 40 other Paenibacillus strains. The current study can benefit for understanding the plant growth-promoting traits of P. mucilaginosus as well as its potential application in agriculture as PGPR.

Recent advances in amino acid analysis by capillary electrophoresis.

This paper describes the most important articles that have been published on amino acid analysis using CE during the period from June 2009 to May 2011 and follows the format of the previous articles of Smith (Electrophoresis 1999, 20, 3078-3083), Prata et al. (Electrophoresis 2001, 22, 4129-4138) and Poinsot et al. (Electrophoresis 2003, 24, 4047-4062; Electrophoresis 2006, 27, 176-194; Electrophoresis 2008, 29, 207-223; Electrophoresis 2010, 31, 105-121). We present new developments in amino acid analysis with CE, which are reported describing the use of lasers or light emitting diodes for fluorescence detection, conductimetry electrochemiluminescence detectors, mass spectrometry applications, and lab-on-a-chip applications using CE. In addition, we describe articles concerning clinical studies and neurochemical applications of these techniques.