Transition to ripening in tomato requires hormone-controlled genetic reprogramming initiated in gel tissue.

Ripening is the last stage of the developmental program in fleshy fruits. During this phase, fruits become edible and acquire their unique sensory qualities and post-harvest potential. Although our knowledge of the mechanisms that regulate fruit ripening has improved considerably over the past decades, the processes that trigger the transition to ripening remain poorly deciphered. While transcriptomic profiling of tomato (Solanum lycopersicum L.) fruit ripening to date has mainly focused on the changes occurring in pericarp tissues between the Mature Green and Breaker stages, our study addresses the changes between the Early Mature Green and Late Mature Green stages in the gel and pericarp separately. The data showed that the shift from an inability to initiate ripening to the capacity to undergo full ripening requires extensive transcriptomic reprogramming that takes place first in the locular tissues before extending to the pericarp. Genome-wide transcriptomic profiling revealed the wide diversity of transcription factor (TF) families engaged in the global reprogramming of gene expression and identified those specifically regulated at the Mature Green stage in the gel but not in the pericarp, thereby providing potential targets toward deciphering the initial factors and events that trigger the transition to ripening. The study also uncovered an extensive reformed homeostasis for most plant hormones, highlighting the multihormonal control of ripening initiation. Our data unveil the antagonistic roles of ethylene and auxin during the onset of ripening and show that auxin treatment delays fruit ripening via impairing the expression of genes required for System-2 autocatalytic ethylene production that is essential for climacteric ripening. This study unveils the detailed features of the transcriptomic reprogramming associated with the transition to ripening of tomato fruit and shows that the first changes occur in the locular gel before extending to pericarp and that a reformed auxin homeostasis is essential for the ripening to proceed.

Inhibition of TLR4 signaling protects mice from sensory and motor dysfunction in an animal model of autoimmune peripheral neuropathy.

BACKGROUND: While the etiology remains elusive, macrophages and T cells in peripheral nerves are considered as effector cells mediating autoimmune peripheral neuropathy (APN), such as Guillain-Barre syndrome. By recognizing both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) signals, TLRs play a central role in the initiation of both innate and adaptive immune responses. In this study, we aimed to understand the involvement of TLR4 in the pathogenesis of APN and explore the potential of TLR4 as a drug target for therapeutic use. METHODS: APN was induced by a partial ligation on one of the sciatic nerves in B7.2 (L31) transgenic mice which possess a predisposed inflammatory background. APN pathology and neurological function were evaluated on the other non-injured sciatic nerve. RESULTS: TLR4 and its endogenous ligand HMGB1 were highly expressed in L31 mice, in circulating immune cells and in peripheral nerves. Enhanced TLR4 signaling was blocked with TAK 242, a selective TLR4 inhibitor, before and after disease onset. Intraperitoneal administration of TAK 242 not only inhibited monocyte, macrophage and CD8+ T cell activation, but also reduced the release of pro-inflammatory cytokines. TAK 242 protected mice from severe myelin and axonal loss, resulting in a remarkable improvement in mouse motor and sensory functions. TAK 242 was effective in alleviating the disease in both preventive and reversal paradigms. CONCLUSION: The study identified the critical contribution of TLR4-mediated macrophage activation in disease course and provided strong evidence to support TLR4 as a useful drug target for treating inflammatory autoimmune neuropathy.

MS-CleanR: A Feature-Filtering Workflow for Untargeted LC-MS Based Metabolomics.

Untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS) is currently the gold-standard technique to determine the full chemical diversity in biological samples. However, this approach still has many limitations; notably, the difficulty of accurately estimating the number of unique metabolites profiled among the thousands of MS ion signals arising from chromatograms. Here, we describe a new workflow, MS-CleanR, based on the MS-DIAL/MS-FINDER suite, which tackles feature degeneracy and improves annotation rates. We show that implementation of MS-CleanR reduces the number of signals by nearly 80% while retaining 95% of unique metabolite features. Moreover, the annotation results from MS-FINDER can be ranked according to the database chosen by the user, which enhance identification accuracy. Application of MS-CleanR to the analysis of Arabidopsis thaliana grown in three different conditions fostered class separation resulting from multivariate data analysis and led to annotation of 75% of the final features. The full workflow was applied to metabolomic profiles from three strains of the leguminous plant Medicago truncatula that have different susceptibilities to the oomycete pathogen Aphanomyces euteiches. A group of glycosylated triterpenoids overrepresented in resistant lines were identified as candidate compounds conferring pathogen resistance. MS-CleanR is implemented through a Shiny interface for intuitive use by end-users (available at https://github.com/eMetaboHUB/MS-CleanR).

Effector/memory CD8+ T cells synergize with co-stimulation competent macrophages to trigger autoimmune peripheral neuropathy.

Autoimmune peripheral neuropathy (APN) such as Guillain Barre Syndrome (GBS) is a debilitating illness and sometimes life threatening. The molecular and cellular mechanisms remain elusive but exposure to environmental factors including viral/bacterial infection and injury is highly associated with disease incidence. We demonstrated previously that both male and female B7.2 (CD86) transgenic L31 and L31/CD4KO mice develop spontaneous APN. Here we further reveal that CD8+ T cells in these mice exhibit an effector/memory phenotype, which bears a resemblance to the CD8+ T cell response following persistent cytomegalovirus (CMV) infection in humans and mice, whilst CMV has been considered as one of the most relevant pathogens in APN development. These activated, peripheral myelin Ag specific CD8+ T cells are required for the disease initiation. While an injury to a peripheral nerve results in Wallerian degeneration in control littermates, the same injury accelerates the development of APN in other non-injured nerves of L31 mice which have a predisposed inflammatory background consisting of effector/memory CD8+ T (CD8+ TEM) cells. However, CD8+ TEM cells alone are not sufficient. A certain threshold of B7.2 expression on nerve macrophages is an additional requisite. Our findings reveal that indeed, the synergism between CD8+ TEM cells and co-stimulation competent macrophages is crucial in inducing autoimmune-mediated peripheral neuropathy. The identification of decisive molecular/cellular players connecting environmental triggers and the occurrence of APN provides opportunities to prevent disease onset, reduce relapses and develop new therapeutic strategies.

Arabidopsis wat1 (walls are thin1)-mediated resistance to the bacterial vascular pathogen, Ralstonia solanacearum, is accompanied by cross-regulation of salicylic acid and tryptophan metabolism.

Inactivation of Arabidopsis WAT1 (Walls Are Thin1), a gene required for secondary cell-wall deposition, conferred broad-spectrum resistance to vascular pathogens, including the bacteria Ralstonia solanacearum and Xanthomonas campestris pv. campestris, and the fungi Verticillium dahliae and Verticillium albo-atrum. Introduction of NahG, the bacterial salicylic acid (SA)-degrading salicylate hydroxylase gene, into the wat1 mutant restored full susceptibility to both R. solanacearum and X. campestris pv. campestris. Moreover, SA content was constitutively higher in wat1 roots, further supporting a role for SA in wat1-mediated resistance to vascular pathogens. By combining transcriptomic and metabolomic data, we demonstrated a general repression of indole metabolism in wat1-1 roots as shown by constitutive down-regulation of several genes encoding proteins of the indole glucosinolate biosynthetic pathway and reduced amounts of tryptophan (Trp), indole-3-acetic acid and neoglucobrassicin, the major form of indole glucosinolate in roots. Furthermore, the susceptibility of the wat1 mutant to R. solanacearum was partially restored when crossed with either the trp5 mutant, an over-accumulator of Trp, or Pro35S:AFB1-myc, in which indole-3-acetic acid signaling is constitutively activated. Our original hypothesis placed cell-wall modifications at the heart of the wat1 resistance phenotype. However, the results presented here suggest a mechanism involving root-localized metabolic channeling away from indole metabolites to SA as a central feature of wat1 resistance to R. solanacearum.

Modification of Early Response of Vitis vinifera to Pathogens Relating to Esca Disease and Biocontrol Agent Vintec® Revealed By Untargeted Metabolomics on Woody Tissues.

Esca disease is one of the most destructive grapevine trunk diseases. Phaeoacremonium minimum and Phaeomoniella chlamydospora are two of the known fungal pathogens associated with this disease. Today, biocontrol agents against Esca are mainly based on the use of the strain of the mycoparasite fungal genus Trichoderma such as the Vintec® product. The aim of this study was to investigate early response of woody tissues to Esca pathogens and identify metabolites that could be correlated with a biocontrol activity within a complex woody matrix. An untargeted liquid chromatography-high-resolution mass spectrometry metabolomic approach coupled to a spectral similarity network was used to highlight clusters of compounds associated with the plant response to pathogens and biocontrol. Dereplication highlighted the possible role of glycerophospholipids and polyphenol compounds, the latest mainly belonging to stilbenoids. Antifungal activity of some relevant biomarkers, evaluated in vitro on Phaeomoniella chlamydospora and Botrytis cinerea, suggests that some of these compounds can play a role to limit the development of Esca pathogens in planta.

CX3CR1 But Not CCR2 Expression Is Required for the Development of Autoimmune Peripheral Neuropathy in Mice.

One hallmark of Guillain-Barre syndrome (GBS), a prototypic autoimmune peripheral neuropathy (APN) is infiltration of leukocytes (macrophages and T cells) into peripheral nerves, where chemokines and their receptors play major roles. In this study, we aimed to understand the potential contribution of chemokine receptors CCR2 and CX3CR1 in APN by using a well-established mouse model, B7.2 transgenic (L31) mice, which possesses a predisposed inflammatory background. We crossbred respectively CCR2KO and CX3CR1KO mice with L31 mice. The disease was initiated by partial ligation on one of the sciatic nerves. APN pathology and neurological function were evaluated on the other non-ligated sciatic nerve/limb. Our results revealed that L31/CX3CR1KO but not L31/CCR2KO mice were resistant to APN. CX3CR1 is needed for maintaining circulating monocyte and CD8+ T cell survival. While migration of a significant number of activated CD8+ T cells to peripheral nerves is essential in autoimmune response in nerve, recruitment of monocytes into PNS seems optional. Disease onset is independent of CCR2 mediated blood-derived macrophage recruitment, which can be replaced by compensatory proliferation of resident macrophages in peripheral nerve. CX3CR1 could also contribute to APN via its critical involvement in maintaining nerve macrophage phagocytic ability. We conclude that blockade of CX3CR1 signaling may represent an interesting anti-inflammatory strategy to improve therapeutic management for GBS patients.

Corrigendum: CX3CR1 But Not CCR2 Expression Is Required for the Development of Autoimmune Peripheral Neuropathy in Mice.

[This corrects the article DOI: 10.3389/fimmu.2021.720733.].

Cannabinoids vs. whole metabolome: Relevance of cannabinomics in analyzing Cannabis varieties.

Cannabis sativa has a long history of domestication both for its bioactive compounds and its fibers. This has produced hundreds of varieties, usually characterized in the literature by chemotypes, with Δ9-THC and CBD content as the main markers. However, chemotyping could also be done based on minor compounds (phytocannabinoids and others). In this work, a workflow, which we propose to name cannabinomics, combines mass spectrometry of the whole metabolome and statistical analysis to help differentiate C. sativa varieties and deciphering their characteristic markers. By applying this cannabinomics approach to the data obtained from 20 varieties of C. sativa (classically classified as chemotype I, II, or III), we compared the results with those obtained by a targeted quantification of 11 phytocannabinoids. Cannabinomics can be considered as a complementary tool for phenotyping and genotyping, allowing the identification of minor compounds playing a key role as markers of differentiation.

Host and nonhost resistance in Medicago-Colletotrichum interactions.

Medicago truncatula lines resistant (A17) or susceptible (F83005.5) to the alfalfa pathogen Colletotrichum trifolii were used to compare defense reactions induced upon inoculation with C. trifolii or with the nonadapted pathogens C. lindemuthianum and C. higginsianum. Nonadapted Colletotrichum spp. induced a hypersensitive response (HR)-like reaction similar to the one induced during the host-incompatible interaction. Molecular analyses indicated an induction of PR10 and chalcone synthase genes in host and nonhost interactions but delayed responses were observed in the F83005.5 line. The clste12 penetration-deficient C. lindemuthianum mutant induced an HR and defense gene expression, showing that perception of nonadapted strains occurs before penetration of epidermal cells. Cytological and transcriptomic analyses performed upon inoculation of near-isogenic M. truncatula lines, differing only at the C. trifolii resistance locus, Ct1, with the nonadapted Colletotrichum strain, showed that nonhost responses are similar in the two lines. These included a localized oxidative burst, accumulation of fluorescent compounds, and transient expression of a small number of genes. Host interactions were characterized by a group of defense and signaling-related genes induced at 3 days postinoculation, associated with an accumulation of salicylic acid. Together, these results show that M. truncatula displays a rapid and transient response to nonadapted Colletotrichum strains and that this response is not linked to the C. trifolii resistance locus.

Characterization of relapsing-remitting and chronic forms of experimental autoimmune encephalomyelitis in C57BL/6 mice.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system (CNS). Like MS, the animal model experimental autoimmune encephalomyelitis (EAE) is characterized by CNS inflammation and demyelination and can follow a relapsing-remitting (RR) or chronic (CH) disease course. The molecular and pathological differences that underlie these different forms of EAE are not fully understood. We have compared the differences in RR- and CH-EAE generated in the same mouse strain (C57BL/6) using the same antigen. At the peak of disease when mice in both groups have similar clinical scores, CH-EAE is associated with increased lesion burden, myelin loss, axonal damage, and chemokine/cytokine expression when compared with RR-EAE. We further showed that inflammation and myelin loss continue to worsen in later stages of CH-EAE, whereas these features are largely resolved at the equivalent stage in RR-EAE. Additionally, axonal loss at these later stages is more severe in CH-EAE than in RR-EAE. We also demonstrated that CH-EAE is associated with a greater predominance of CD8(+) T cells in the CNS that exhibit MOG(35-55) antigen specificity. These studies therefore showed that, as early as the peak stage of disease, RR- and CH-EAE differ remarkably in their immune cell profile, chemokine/cytokine responses, and histopathological features. These data also indicated that this model of CH-EAE exhibits pathological features of a chronic-progressive disease profile and suggested that the sustained chronic phenotype is due to a combination of axonal loss, myelin loss, and continuing inflammation.

Ulvan, a sulfated polysaccharide from green algae, activates plant immunity through the jasmonic acid signaling pathway.

The industrial use of elicitors as alternative tools for disease control needs the identification of abundant sources of them. We report on an elicitor obtained from the green algae Ulva spp. A fraction containing most exclusively the sulfated polysaccharide known as ulvan-induced expression of a GUS gene placed under the control of a lipoxygenase gene promoter. Gene expression profiling was performed upon ulvan treatments on Medicago truncatula and compared to phytohormone effects. Ulvan induced a gene expression signature similar to that observed upon methyl jasmonate treatment (MeJA). Involvement of jasmonic acid (JA) in ulvan response was confirmed by detecting induction of protease inhibitory activity and by hormonal profiling of JA, salicylic acid (SA) and abscisic acid (ABA). Ulvan activity on the hormonal pathway was further consolidated by using Arabidopsis hormonal mutants. Altogether, our results demonstrate that green algae are a potential reservoir of ulvan elicitor which acts through the JA pathway.

Phytohormone production by the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Arbuscular mycorrhizal symbiosis is a mutualistic interaction between most land plants and fungi of the glomeromycotina subphylum. The initiation, development and regulation of this symbiosis involve numerous signalling events between and within the symbiotic partners. Among other signals, phytohormones are known to play important roles at various stages of the interaction. During presymbiotic steps, plant roots exude strigolactones which stimulate fungal spore germination and hyphal branching, and promote the initiation of symbiosis. At later stages, different plant hormone classes can act as positive or negative regulators of the interaction. Although the fungus is known to reciprocally emit regulatory signals, its potential contribution to the phytohormonal pool has received little attention, and has so far only been addressed by indirect assays. In this study, using mass spectrometry, we analyzed phytohormones released into the medium by germinated spores of the arbuscular mycorrhizal fungus Rhizophagus irregularis. We detected the presence of a cytokinin (isopentenyl adenosine) and an auxin (indole-acetic acid). In addition, we identified a gibberellin (gibberellin A4) in spore extracts. We also used gas chromatography to show that R. irregularis produces ethylene from methionine and the α-keto γ-methylthio butyric acid pathway. These results highlight the possibility for AM fungi to use phytohormones to interact with their host plants, or to regulate their own development.

Murine cytomegalovirus infection in mice results in an acute inflammatory reaction in peripheral nerves.

Human cytomegalovirus (CMV) infection is asymptomatic in immunocompetent individuals. However, it can lead to disease in immunodeficient population. Little is known of the mechanisms underlying the pathogenicity of the virus. We investigated the impact of CMV infection on mouse nervous system. Peripheral nerves but not spinal cord was permissive to MCMV during acute infection. Activated CD8+ T cells, monocytes/macrophages and cytokine expression were increased in the blood and sciatic nerves of infected mice, which exhibited transient sensory dysfunction. This study indicates that systemic MCMV infection leads to a dissemination of MCMV into peripheral nerves, which is associated with a local inflammation but not nerve tissue damage in the acute phase.

SARS coronavirus: unusual lability of the nucleocapsid protein.

The severe acute respiratory syndrome (SARS) is a contagious disease that killed hundreds and sickened thousands of people worldwide between November 2002 and July 2003. The nucleocapsid (N) protein of the coronavirus responsible for this disease plays a critical role in viral assembly and maturation and is of particular interest because of its potential as an antiviral target or vaccine candidate. Refolding of SARS N-protein during production and purification showed the presence of two additional protein bands by SDS-PAGE. Mass spectroscopy (MALDI, SELDI, and LC/MS) confirmed that the bands are proteolytic products of N-protein and the cleavage sites are four SR motifs in the serine-arginine-rich region-sites not suggestive of any known protease. Furthermore, results of subsequent testing for contaminating protease(s) were negative: cleavage appears to be due to inherent instability and/or autolysis. The importance of N-protein proteolysis to viral life cycle and thus to possible treatment directions are discussed.

A novel cross-linked RNase A dimer with enhanced enzymatic properties.

A new cross-linked ribonuclease A (RNase A) dimer composed of monomeric units covalently linked by a single amide bond between the side-chains of Lys(66) and Glu(9) is described. The dimer was prepared in the absence of water by incubating a lyophilized preparation of RNase, sealed under vacuum, in an oven at 85 degrees C. It was determined that the in vacuo procedure does not induce any significant conformational changes to the overall structure of RNase A, yet the amide cross-link has an increased acid lability, indicating that it is exposed and conformationally strained. Examination of X-ray crystallographic structures indicates that Lys(66) and Glu(9) are not close enough for the in vacuo dimer to adopt any of the known domain-swapped conformations. Therefore, the in vacuo RNase A dimer appears to be a novel dimeric structure. The in vacuo RNase A dimer also exhibits a twofold increase in activity over monomeric RNase A on a per monomer basis. This doubling of enzymatic activity was shown using dsRNA and ssRNA as substrates. In addition to this enhanced ability to degrade RNA, the dimer is not inhibited by the cellular ribonuclease inhibitor protein (cRI).

Absence of apolipoprotein E protects mice from cerebral malaria.

Cerebral malaria claims the life of millions of people each year, particularly those of children, and is a major global public health problem. Thus, the identification of novel malaria biomarkers that could be utilized as diagnostic or therapeutic targets is becoming increasingly important. Using a proteomic approach, we previously identified unique biomarkers in the sera of malaria-infected individuals, including apolipoprotein E (ApoE). ApoE is the dominant apolipoprotein in the brain and has been implicated in several neurological disorders; therefore, we were interested in the potential role of ApoE in cerebral malaria. Here we report the first demonstration that cerebral malaria is markedly attenuated in ApoE(-/-) mice. The protection provided by the absence of ApoE was associated with decreased sequestration of parasites and T cells within the brain, and was determined to be independent from the involvement of ApoE receptors and from the altered lipid metabolism associated with the knock-out mice. Importantly, we demonstrated that treatment of mice with the ApoE antagonist heparin octasaccharide significantly decreased the incidence of cerebral malaria. Overall, our study indicates that the reduction of ApoE could be utilized in the development of therapeutic treatments aimed at mitigating the neuropathology of cerebral malaria.

Constitutive expression of a costimulatory ligand on antigen-presenting cells in the nervous system drives demyelinating disease.

It has been proposed that the activation status of antigen-presenting cells (APCs) plays a significant role in the development of autoimmune disease. Whether expression of costimulatory ligands on tissue-resident APCs controls organ-specific autoimmune responses has not been tested. We here report that transgenic mice constitutively expressing the costimulatory ligand B7.2/CD86 on microglia in the central nervous system (CNS) and on related cells in the proximal peripheral nervous tissue spontaneously develop autoimmune demyelinating disease. Disease-affected nervous tissue in transgenic mice showed infiltration characterized by a predominance of CD8+ memory-effector T cells, as well as CD4+ T cells. Transgenic animals lacking alphabeta TCR+ T cells were completely resistant to disease development. Transgenic T cells induced disease when adoptively transferred into T cell-deficient B7.2 transgenic recipients but not into non-transgenic recipients. These data provide evidence that B7/CD28 interactions within the nervous tissue are critical determinants of disease development. Our findings have important implications for understanding the etiology of nervous system autoimmune diseases such as multiple sclerosis (MS) and Guillain-Barré syndrome (GBS).

Covalent protein immobilization on glass surfaces: application to alkaline phosphatase.

Lyophilized alkaline phosphatase (ALPase) was immobilized on aminated glass surfaces using the in vacuo cross-linking process [Simons, B.L., King, M.C., Cyr, T., Hefford, M.A., Kaplan, H., 2002. Zero-length cross-linking of lyophilized proteins. Protein Sci. 11, 1558-1564]. In this procedure, amide bonds were formed between carboxyl groups on the protein and amino groups on the glass surface. After the non-covalently attached enzyme was removed the immobilized ALPase not only retained its activity but could also be used, washed and reused at least six times without significant loss of activity. An average of 1.4+/-0.6 mg of reusable ALPase per gram of glass fibre was immobilized based on the activity of the soluble equivalent.

Evidence from Human and Animal Studies: Pathological Roles of CD8(+) T Cells in Autoimmune Peripheral Neuropathies.

Autoimmune peripheral neuropathies such as Guillain-Barre Syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) affect millions of people worldwide. Despite significant advances in understanding the pathology, the molecular and cellular mechanisms of immune-mediated neuropathies remain elusive. T lymphocytes definitely play an important role in disease pathogenesis and CD4(+) T cells have been the main area of research for decades. This is partly due to the fact that the most frequent animal model to study autoimmune peripheral neuropathy is experimental allergic neuritis (EAN). As it is induced commonly by immunization with peripheral nerve proteins, EAN is driven mainly by CD4(+) T cells. However, similarly to what has been reported for patients suffering from multiple sclerosis, a significant body of evidence indicates that CD8(+) T cells may play a pathogenic role in GBS and CIDP disease development and/or progression. Here, we summarize clinical studies pertaining to the presence and potential role of CD8(+) T cells in autoimmune peripheral neuropathies. We also discuss the findings from our most recent studies using a transgenic mouse line (L31 mice) in which the T cell co-stimulator molecule B7.2 (CD86) is constitutively expressed in antigen presenting cells of the nervous tissues. L31 mice spontaneously develop peripheral neuropathy, and CD8(+) T cells are found accumulating in peripheral nerves of symptomatic animals. Interestingly, depletion of CD4(+) T cells accelerates disease onset and increases disease prevalence. Finally, we point out some unanswered questions for future research to dissect the critical roles of CD8(+) T cells in autoimmune peripheral neuropathies.