Splenic sympathetic signaling contributes to acute neutrophil infiltration of the injured spinal cord.

BACKGROUND: Alterations in the immune system are a complication of spinal cord injury (SCI) and have been linked to an excessive sympathetic outflow to lymphoid organs. Still unknown is whether these peripheral immune changes also contribute for the deleterious inflammatory response mounted at the injured spinal cord. METHODS: We analyzed different molecular outputs of the splenic sympathetic signaling for the first 24 h after a thoracic compression SCI. We also analyzed the effect of ablating the splenic sympathetic signaling to the innate immune and inflammatory response at the spleen and spinal cord 24 h after injury. RESULTS: We found that norepinephrine (NE) levels were already raised at this time-point. Low doses of NE stimulation of splenocytes in vitro mainly affected the neutrophils' population promoting an increase in both frequency and numbers. Interestingly, the interruption of the sympathetic communication to the spleen, by ablating the splenic nerve, resulted in reduced frequencies and numbers of neutrophils both at the spleen and spinal cord 1 day post-injury. CONCLUSION: Collectively, our data demonstrates that the splenic sympathetic signaling is involved in the infiltration of neutrophils after spinal cord injury. Our findings give new mechanistic insights into the dysfunctional regulation of the inflammatory response mounted at the injured spinal cord.

Notch signaling dynamics in the adult healthy prostate and in prostatic tumor development.

BACKGROUND: The Notch signaling pathway has been implicated in prostate development, maintenance and tumorigenesis by its key role in cell-fate determination, differentiation and proliferation. Therefore, we proposed to analyze Notch family members transcription and expression, including ligands (Dll1, 3, 4 and Jagged1 and 2), receptors (Notch1-4) and effectors (Hes1, 2, 5 and Hey1, 2, L), in both normal and tumor bearing mouse prostates to better understand the dynamics of Notch signaling in prostate tumorigenesis. METHODS: Wild type mice and transgenic adenocarcinoma of the mouse prostate model (TRAMP) mice were sacrificed at 18, 24 or 30 weeks of age and the prostates collected and processed for either whole prostate or prostate cell specific populations mRNA analysis and for protein expression analysis by immunohistochemistry and immunofluorescence. RESULTS: We observed that Dll1 and Dll4 are expressed in the luminal compartment of the mouse healthy prostate, whereas Jagged2 expression is restricted to the basal and stromal compartment. Additionally, Notch2 and Notch4 are normally expressed in the prostate luminal compartment while Notch2 and Notch3 are also expressed in the stromal layer of the healthy prostate. As prostate tumor development takes place, there is up-regulation of Notch components. Particularly, the prostate tumor lesions have increased expression of Jagged1 and 2, of Notch3 and of Hey1. We have also detected the presence of activated Notch3 in prostatic tumors that co-express Jagged1 and ultimately the Hey1 effector. CONCLUSIONS: Taken together our results point out the Notch axis Jagged1-2/Notch3/Hey1 to be important for prostate tumor development and worthy of additional functional studies and validation in human clinical disease.

The presence of cutan limits the interpretation of cuticular chemistry and structure: Ficus elastica leaf as an example.

Plant cuticles have been traditionally classified on the basis of their ultrastructure, with certain chemical composition assumptions. However, the nature of the plant cuticle may be misinterpreted in the prevailing model, which was established more than 150 years ago. Using the adaxial leaf cuticle of Ficus elastica, a study was conducted with the aim of analyzing cuticular ultrastructure, chemical composition and the potential relationship between structure and chemistry. Gradual chemical extractions and diverse analytical and microscopic techniques were performed on isolated leaf cuticles of two different stages of development (i.e. young and mature leaves). Evidence for the presence of cutan in F. elastica leaf cuticles has been gained after chemical treatments and tissue analysis by infrared spectroscopy and electron microscopy. Significant calcium, boron and silicon concentrations were also measured in the cuticle of this species. Such mineral elements which are often found in plant cell walls may play a structural role and their presence in isolated cuticles further supports the interpretation of the cuticle as the most external region of the epidermal cell wall. The complex and heterogeneous nature of the cuticle, and constraints associated with current analytical procedures may limit the chance for establishing a relationship between cuticle chemical composition and structure also in relation to organ ontogeny.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BACKGROUND: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. RESULTS: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. CONCLUSIONS: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Stereochemistry of C18 monounsaturated cork suberin acids determined by spectroscopic techniques including (1) H-NMR multiplet analysis of olefinic protons.

INTRODUCTION: Suberin is a biopolyester responsible for the protection of secondary plant tissues, and yet its molecular structure remains unknown. The C18:1 ω-hydroxyacid and the C18:1 α,ω-diacid are major monomers in the suberin structure, but the configuration of the double bond remains to be elucidated. OBJECTIVE: To unequivocally define the configuration of the C18:1 suberin acids. METHODS: Pure C18:1 ω-hydroxyacid and C18:1 α,ω-diacid, isolated from cork suberin, and two structurally very close C18:1 model compounds of known stereochemistry, methyl oleate and methyl elaidate, were analysed by NMR spectroscopy, Fourier transform infrared (FTIR) and Raman spectroscopy, and GC-MS. RESULTS: The GC-MS analysis showed that both acids were present in cork suberin as only one geometric isomer. The analysis of dimethyloxazoline (DMOX) and picolinyl derivatives proved the double bond position to be at C-9. The FTIR spectra were concordant with a cis-configuration for both suberin acids, but their unambiguous stereochemical assignment came from the NMR analysis: (i) the chemical shifts of the allylic (13) C carbons were shielded comparatively to the trans model compound, and (ii) the complex multiplets of the olefinic protons could be simulated only with (3) JHH and long-range (4) JHH coupling constants typical of a cis geometry. CONCLUSION: The two C18:1 suberin acids in cork are (Z)-18-hydroxyoctadec-9-enoic acid and (Z)-octadec-9-enedoic acid.

The secretome of macrophages has a differential impact on spinal cord injury recovery according to the polarization protocol.

Introduction: The inflammatory response after spinal cord injury (SCI) is an important contributor to secondary damage. Infiltrating macrophages can acquire a spectrum of activation states, however, the microenvironment at the SCI site favors macrophage polarization into a pro-inflammatory phenotype, which is one of the reasons why macrophage transplantation has failed. Methods: In this study, we investigated the therapeutic potential of the macrophage secretome for SCI recovery. We investigated the effect of the secretome in vitro using peripheral and CNS-derived neurons and human neural stem cells. Moreover, we perform a pre-clinical trial using a SCI compression mice model and analyzed the recovery of motor, sensory and autonomic functions. Instead of transplanting the cells, we injected the paracrine factors and extracellular vesicles that they secrete, avoiding the loss of the phenotype of the transplanted cells due to local environmental cues. Results: We demonstrated that different macrophage phenotypes have a distinct effect on neuronal growth and survival, namely, the alternative activation with IL-10 and TGF-ß1 (M(IL-10+TGF-ß1)) promotes significant axonal regeneration. We also observed that systemic injection of soluble factors and extracellular vesicles derived from M(IL-10+TGF-ß1) macrophages promotes significant functional recovery after compressive SCI and leads to higher survival of spinal cord neurons. Additionally, the M(IL-10+TGF-ß1) secretome supported the recovery of bladder function and decreased microglial activation, astrogliosis and fibrotic scar in the spinal cord. Proteomic analysis of the M(IL-10+TGF-ß1)-derived secretome identified clusters of proteins involved in axon extension, dendritic spine maintenance, cell polarity establishment, and regulation of astrocytic activation. Discussion: Overall, our results demonstrated that macrophages-derived soluble factors and extracellular vesicles might be a promising therapy for SCI with possible clinical applications.

Notes on the husbandry and long-term transportation of Bull ray (Pteromylaeus bovinus) and Dolphinfish (Coryphaena hippurus and Coryphaena equiselis).

Bull rays (Pteromylaeus bovinus) and Dolphinfish (Coryphaena hippurus and Coryphaena equiselis) were collected in Olhão (south of Portugal). These animals hosted multiple parasites, namely Caligus spp., and underwent a variety of treatments to remove them. Of all treatments tested, hydrogen peroxide showed the best results, although only concentrations above 100 ppm were effective in parasite removal. These high concentrations, however, proved to be highly toxic for the fish and led to the loss of some animals, especially those which had been handled before treatment. A total of 14 Bull rays were transported to Bolougne-Sur-Mer (France) by road and some animals were lost, which was attributed to excessive time in transit (>45 hr). In another transport, three Bull rays and 10 Dolphinfishes were moved to Stralsund (Germany) by road and air. The mechanical wounds suffered by one of the Bull rays during transport led to its death and, consequently, a deterioration of water quality in the tank containing two other conspecifics. This deterioration of water quality resulted in problems for the other two Bull rays, and one perished approximately 48 hr after arrival. The authors concluded that Dolphinfish can be transported with a low bioload for at least 27 hr, and Bull rays should not undergo transports longer than 35 hr. Special attention must be taken to injured animals, since this can lead to a decrease in water quality and consequently affect other animals in the same transport tank.

Glycine max (L.) Merr. (Soybean) metabolome responses to potassium availability.

Potassium (K+) has vital physiological and metabolic functions in plants and its availability can impact tolerance to biotic and abiotic stress conditions. Limited studies have investigated the effect of K+ fertilization on soybean metabolism. Using integrated omics, ionomics and metabolomics, we investigated the field-grown Glycine max (soybean) response, after four K+ soil fertilization rates. Soybean leaf and pod tissue (valves and immature seeds) extracts were analysed by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) and inductively coupled plasma optical emission spectroscopy (ICP-OES). Multivariate analyses (PCA-X&Y e O2PLS-DA) showed that 51 compounds of 19 metabolic pathways were regulated in response to K+ availability. Under very low potassium availability, soybean plants accumulated of Ca2+, Mg2+, Fe2+, Cu2+, and B in young and old leaves. Potassium fertilization upregulated carbohydrate, galactolipid, and flavonol glycoside biosynthesis in leaves and pod valves, while K+ deficient pod tissues showed increasing amino acids, oligosaccharides, benzoic acid derivatives, and isoflavones contents. Severely K+ deficient soils elicited isoflavones, coumestans, pterocarpans, and soyasaponins in trifoliate leaves, likely associated to oxidative and photodynamic stress status. Additionally, results demonstrate that L-asparagine content is higher in potassium deficient tissues, suggesting this compound as a biomarker of K+ deficiency in soybean plants. These results demonstrate that potassium soil fertilization did not linearly contribute to changes in specialised constitutive metabolites of soybean. Altogether, this work provides a reference for improving the understanding of soybean metabolism as dependent on K+ availability.

Identification of volatile organic compounds in purple and white soybean flowers by HS-SPME/GC-MS.

This study aimed to establish a method for the extraction, enrichment, and identification of volatile organic compounds (VOCs) released by the flowers of purple (BRS 399) and white (DONMARIO 6563) soybean varieties. We tested the Static Headspace (HS) and Solid Phase Microextraction (SPME) methods using various fibre types: PDMS (Polydimethylsiloxane), PDMS/DVB (Divinylbenzene), and PDMS/DVB/CAR (Carboxen). We employed gas chromatography-mass spectrometry (GC-MS) to identify the VOCs. The SPME method with PDMS/DVB and PDMS/DVB/CAR fibres yielded the highest number of extracted compounds for both soybean cultivars. Notably, 67 compounds were detected in Glycine max. L for the first time. Using the developed method, we were able to detect 52 and 57 VOCs in the purple and white soybean varieties, respectively, including ketones, alcohols, aldehydes and benzenoids. In conclusion, the method we developed effectively identified VOCs in soybean flowers, thus enriching our understanding of the interactions between soybean flowers and their pollinators.

COVID-19 vaccination challenges: from fake news to vaccine hesitancy. / Desafios da imunização contra COVID-19 na saúde pública: das fake news à hesitação vacinal.

This article aims to synthesize articles addressing fake news and COVID-19 vaccine hesitancy in the context of public health. We conducted an integrative review of articles published in any language between 2019 and 2022 in journals indexed in the following databases: Latin American and the Caribbean Literature on Health Sciences, Medical Literature Analysis and Retrieval System Online, Scopus, Web of Science, and Embase. A critical analysis was performed, guided by the research question and objective of the review. Eleven articles were selected, the overwhelming majority of which were cross-sectional studies. The main factors related to vaccine take-up highlighted by the studies were gender, age, education level, political leanings, religion, trust in health authorities, and perceptions of side-effects and vaccine efficacy. The main obstacles to attaining optimal vaccination coverage were vaccine hesitancy and disinformation. All studies addressed the relationship between low vaccination intention and the use of social media as a source of information about SARS-CoV-2. It is necessary to build public trust in vaccine safety and efficacy. Promoting a better understanding of the benefits of COVID-19 vaccination is essential to combat vaccine hesitancy and improve vaccine take-up.

O objetivo deste artigo é sintetizar artigos que abordam fake news e hesitação vacinal contra a COVID-19 no contexto de saúde pública. Revisão integrativa que incluiu estudos originais indexados nas bases de dados Literatura Latino Americana e do Caribe em Ciências da Saúde; Medical Literature Analysis and Retrieval System Online; Scopus; Web of Science e Embase, publicados em qualquer idioma, entre 2019 e 2022. A análise crítica foi realizada na forma descritiva, consoante à pergunta de pesquisa e ao objetivo da revisão. Foram selecionados 11 artigos, com predomínio de estudos transversais. Relacionaram-se ao processo de adesão à vacinação: gênero, idade, estado civil, escolaridade, posicionamento político, religião, confiança em autoridades de saúde, percepção de efeitos colaterais e eficácia das vacinas, entre outros. Hesitação e desinformação são os principais entraves para se alcançar a cobertura vacinal em muitos países. Todos os estudos abordam a relação entre baixa intenção de imunização e uso de mídias sociais como fonte de informação sobre o SARS-CoV-2. É necessário aumentar a confiança na segurança e eficácia das vacinas. A melhor compreensão dos benefícios da vacinação para COVID-19 é imprescindível para combater a hesitação e ampliar a adesão vacinal.

Epigenetics at the crossroads of secondary growth regulation.

The development of plant tissues and organs during post-embryonic growth occurs through the activity of both primary and secondary meristems. While primary meristems (root and shoot apical meristems) promote axial plant growth, secondary meristems (vascular and cork cambium or phellogen) promote radial thickening and plant axes strengthening. The vascular cambium forms the secondary xylem and phloem, whereas the cork cambium gives rise to the periderm that envelops stems and roots. Periderm takes on an increasingly important role in plant survival under climate change scenarios, but it is also a forest product with unique features, constituting the basis of a sustainable and profitable cork industry. There is established evidence that epigenetic mechanisms involving histone post-translational modifications, DNA methylation, and small RNAs play important roles in the activity of primary meristem cells, their maintenance, and differentiation of progeny cells. Here, we review the current knowledge on the epigenetic regulation of secondary meristems, particularly focusing on the phellogen activity. We also discuss the possible involvement of DNA methylation in the regulation of periderm contrasting phenotypes, given the potential impact of translating this knowledge into innovative breeding programs.

Beyond width and density: stable carbon and oxygen isotopes in cork-rings provide insights of physiological responses to water stress in Quercus suber L.

As climate change increasingly affects forest ecosystems, detailed understanding of major effects is important to anticipate their consequences under future climate scenarios. The Mediterranean region is a prominent climate change hotspot, and evergreen cork oak (Quercus suber L.) woodlands are particularly climatically sensitive due to cork (bark) harvesting. Cork oak's drought avoidance strategy is well-known and includes structural and physiological adaptations that maximise soil water uptake and transport and limit water use, potentially leading to reduced stem and cork growth. Trees' responses to cope with water-limited conditions have been extensively described based on cork-rings width and, more recently, on cork-rings density, in dendroecological studies. However, so far, tree functional attributes and physiological strategies, namely photosynthetic metabolism adjustments affecting cork formation, have never been addressed and/or integrated on these previous cork-rings-based studies. In this study, we address the relation between carbon and oxygen stable isotopes of cork rings and precipitation and temperature, in two distinct locations of southwestern Portugal-the (wetter) Tagus basin peneplain and the (drier) Grândola mountains. We aimed at assessing whether the two climatic factors affect cork-ring isotopic composition under contrasting conditions of water availability, and, therefore, if carbon and oxygen signatures in cork can reflect tree functional (physiological and structural) responses to stressful conditions, which might be aggravated by climate change. Our results indicate differences between the study areas. At the drier site, the stronger statistically significant negative cork δ 13C correlations were found with mean temperature, whereas strong positive cork δ 18O correlations were fewer and found only with precipitation. Moreover, at the wetter site, cork rings are enriched in 18O and depleted in 13C, indicating, respectively, shallow groundwater as the water source for physiological processes related with biosynthesis of non-photosynthetic secondary tissues, such as suberin, and a weak stomatal regulation under high water availability, consistent with non-existent water availability constrains. In contrast, at the drier site, trees use water from deeper ground layers, depleted in 18O, and strongly regulate stomatal conductance under water stress, thus reducing photosynthetic carbon uptake and probably relying on stored carbon reserves for cork ring formation. These results suggest that although stable isotopes signatures in cork rings are not proxies for net growth, they may be (fairly) robust indicators of trees' physiological and structural adjustments to climate and environmental changes in Mediterranean environments.

Immunomodulatory and regenerative effects of the full and fractioned adipose tissue derived stem cells secretome in spinal cord injury.

Adipose tissue derived stem cells (ASCs) are recognized to secret a myriad of molecules (secretome) know to modulate inflammatory response, promote axonal growth as well vascular remodeling and cellular survival. In previous works we have reported the benefit effects of ASCs transplanted to the injury site in a rat model of spinal cord injury (SCI). Emerging evidence have shown that the therapeutic actions of these cells are a consequence of their intense paracrine activity mediated by their secretome, which includes soluble bioactive molecules and vesicles. In this study, we intended to dissect the vesicular and protein individual function, comparing with whole secretome therapeutic effect. Therefore, we identified a beneficial effect of the whole secretome on neurite growth compared with protein or vesicular fraction alone and characterized their impact on microglia in vitro. Moreover, in a compression SCI mice model, from the motor tests performed, a statistical difference was found on beam balance test revealing differences in motor recovery between the use of the whole the secretome or their protein fraction. Finally, two different delivery methods, local or peripheral (IV), of ASC secretome were tested in vivo. Results indicate that when injected intravenously the secretome of ASCs has a beneficial effect on motor recovery of spinal cord injury animals compared with a single local injection and respective controls. Overall, our results showed that the whole secretome performed better than the fractions individually, raising ASC secretome mode of action as a synergy of proteic and vesicular fraction on SCI context. Also, when intravenously delivered, ASC secretome can promote SCI animal's motor recovery highlighting their therapeutic potential.

Long-term transportation, by road and air, of chub mackerel (Scomber japonicus) and atlantic bonito (Sarda sarda).

During the second semester of 2009, three trips were made from Olhão (Southern Portugal) to Stralsund (Northern Germany) carrying 2.122 animals, which included multiple teleosts, elasmobranchs and invertebrates. This group included scombrids, such as 1.869 Scomber japonicus and 9 Sarda sarda, which are notoriously difficult to transport. However, multiple adaptations to transport regimes adopted regularly have allowed the authors to successfully move these animals by road and air over a total of up to 25 hr. Such adaptations included maintaining oxygen saturation rates at approximately 200%, and also the constant addition of AmQuel(®) , sodium bicarbonate, and sodium carbonate. Different formulations were used during the three trips, with the best results corresponding to 20/30/30 ppm of the three aforementioned chemicals, respectively. The authors suggest, however, that a modified formula of 20/40/40 ppm will allow for an even more stable pH on future trips.

Cork cells in cork oak periderms undergo programmed cell death and proanthocyanidin deposition.

Vascular plants with secondary growth develop a periderm mostly composed of dead suberized cork cells to face environmental hostile conditions. Cork oak has a highly active and long-living phellogen forming a remarkably thick periderm that is periodically debarked for industrial purposes. This wounding originates the quick formation of a new traumatic periderm, making cork oak an exceptional model to study the first periderm differentiation during normal development in young sprigs and traumatic (wound) periderm formation after debarking. Here, we studied the poorly known first periderm differentiation steps that involve cell wall suberization, polyphenolic accumulation and programmed cell death (PCD) by combining transmission electron microscopy, histochemical and molecular methods in periderms from young sprigs. These processes were further compared with traumatic periderms formed after wounding using molecular and histochemical techniques, such as the polyphenolic accumulation. In the first periderms from young sprigs, four distinct differentiation stages were defined according to the presence of PCD morphological features. First young and traumatic periderms showed an upregulation of genes related to suberin biosynthesis, proanthocyanidins biosynthesis and transport, autophagy, and PCD. Traumatic periderms revealed an overall upregulation of these genes, likely resulting from ontogeny differences and distinct phellogen origin associated with a faster metabolism, highlighting the impact of wounding on phellogen activity after debarking. First periderms from young sprigs showed gradual accumulation of proanthocyanidins in the vacuoles throughout PCD stages until total filled lumens, whereas in traumatic periderms, these compounds were found cell wall linked in already empty cells. This work enabled a comprehensive overview of the cork cells differentiation processes contributing to deepening the knowledge of the fundamental ontogenic program of this protective tissue, which is also a unique forest product, constituting the basis of a sustainable and profitable industry.

STRUCTURAL BASES OF GASTROINTESTINAL MOTILITY CHANGES IN PARKINSON'S DISEASE: STUDY IN RATS.

BACKGROUND: Gastrointestinal disorders are frequently reported in patients with Parkinson's disease whose disorders reduce the absorption of nutrients and drugs, worsening the clinical condition of patients. However, the mechanisms involved in modifying gastrointestinal pathophysiology have not yet been fully explained. AIM: To evaluate its effects on gastrointestinal motility and the involvement of the vagal and splanchnic pathways. METHODS: Male Wistar rats (250-300 g, n = 84) were used and divided into two groups. Group I (6-OHDA) received an intrastriatal injection of 6-hydroxydopamine (21 µg/animal). Group II (control) received a saline solution (NaCl, 0.9%) under the same conditions. The study of gastric emptying, intestinal transit, gastric compliance and operations (vagotomy and splanchnotomy) were performed 14 days after inducing neurodegeneration. Test meal (phenol red 5% glucose) was used to assess the rate of gastric emptying and intestinal transit. RESULTS: Parkinson's disease delayed gastric emptying and intestinal transit at all time periods studied; however, changes in gastric compliance were not observed. The delay in gastric emptying was reversed by pretreatment with vagotomy and splanchnotomy+celiac gangliectomy, thus suggesting the involvement of such pathways in the observed motor disorders. CONCLUSION: Parkinson's disease compromises gastric emptying, as well as intestinal transit, but does not alter gastric compliance. The delay in gastric emptying was reversed by truncal vagotomy, splanchnotomy and celiac ganglionectomy, suggesting the involvement of such pathways in delaying gastric emptying.

Untargeted Metabolomics Analysis by UHPLC-MS/MS of Soybean Plant in a Compatible Response to Phakopsora pachyrhizi Infection.

Phakopsora pachyrhizi is a biotrophic fungus, causer of the disease Asian Soybean Rust, a severe crop disease of soybean and one that demands greater investment from producers. Thus, research efforts to control this disease are still needed. We investigated the expression of metabolites in soybean plants presenting a resistant genotype inoculated with P. pachyrhizi through the untargeted metabolomics approach. The analysis was performed in control and inoculated plants with P. pachyrhizi using UHPLC-MS/MS. Principal component analysis (PCA) and the partial least squares discriminant analysis (PLS-DA), was applied to the data analysis. PCA and PLS-DA resulted in a clear separation and classification of groups between control and inoculated plants. The metabolites were putative classified and identified using the Global Natural Products Social Molecular Networking platform in flavonoids, isoflavonoids, lipids, fatty acyls, terpenes, and carboxylic acids. Flavonoids and isoflavonoids were up-regulation, while terpenes were down-regulated in response to the soybean-P. pachyrhizi interaction. Our data provide insights into the potential role of some metabolites as flavonoids and isoflavonoids in the plant resistance to ASR. This information could result in the development of resistant genotypes of soybean to P. pachyrhizi, and effective and specific products against the pathogen.

Unraveling Asian Soybean Rust metabolomics using mass spectrometry and Molecular Networking approach.

Asian Soybean Rust (ASR), caused by the biotrophic fungus Phakopsora pachyrhizi, is a devastating disease with an estimated crop yield loss of up to 90%. Yet, there is a nerf of information on the metabolic response of soybean plants to the pathogen Untargeted metabolomics and Global Natural Products Social Molecular Networking platform approach was used to explore soybean metabolome modulation to P. pachyrhizi infection. Soybean plants susceptible to ASR was inoculated with P. pachyrhizi spore suspension and non-inoculated plants were used as controls. Leaves from both groups were collected 14 days post-inoculation and extracted using different extractor solvent mixtures. The extracts were analyzed on an ultra-high performance liquid chromatography system coupled to high-definition electrospray ionization-mass spectrometry. There was a significant production of defense secondary metabolites (phenylpropanoids, terpenoids and flavonoids) when P. pachyrhizi infected soybean plants, such as putatively identified liquiritigenin, coumestrol, formononetin, pisatin, medicarpin, biochanin A, glyoceollidin I, glyoceollidin II, glyoceollin I, glyoceolidin II, glyoceolidin III, glyoceolidin IV, glyoceolidin VI. Primary metabolites (amino acids, peptides and lipids) also were putatively identified. This is the first report using untargeted metabolomics and GNPS-Molecular Networking approach to explore ASR in soybean plants. Our data provide insights into the potential role of some metabolites in the plant resistance to ASR, which could result in the development of resistant genotypes of soybean to P. pachyrhizi, and effective and specific products against the pathogen.

Proteomic evaluation of wound-healing processes in potato (Solanum tuberosum L.) tuber tissue.

Proteins from potato (Solanum tuberosum L.) tuber slices, related to the wound-healing process, were separated by 2-DE and identified by an MS analysis in MS and MS/MS mode. Slicing triggered differentiation processes that lead to changes in metabolism, activation of defence and cell-wall reinforcement. Proteins related to storage, cell growth and division, cell structure, signal transduction, energy production, disease/defence mechanisms and secondary metabolism were detected. Image analysis of the 2-DE gels revealed a time-dependent change in the complexity of the polypeptide patterns. By microscopic observation the polyalyphatic domain of suberin was clearly visible by D4, indicating that a closing layer (primary suberisation) was formed by then. A PCA of the six sampling dates revealed two time phases, D0-D2 and D4-D8, with a border position between D2 and D4. Moreover, a PCA of differentially expressed proteins indicated the existence of a succession of proteomic events leading to wound-periderm reconstruction. Some late-expressed proteins (D6-D8), including a suberisation-associated anionic peroxidase, have also been identified in the native periderm. Despite this, protein patterns of D8 slices and native periderm were still different, suggesting that the processes of wound-periderm formation are extended in time and not fully equivalent. The information presented in this study gives clues for further work on wound healing-periderm formation processes.

Applying pyrolysis-gas chromatography/mass spectrometry to the identification of oriental lacquers: study of two lacquered shields.

Oriental lacquers have been used as coating materials for thousands of years for wooden, ceramics, leather and metal objects. Lacquers are natural polymers obtained from three species growing in different regions of Asia: Rhus vernicifera (China, Japan and Korea); Rhus succedanea (Vietnam and Taiwan); and Melanorrhoea usitate (Myanmar and Thailand). The identification of lacquer films is important for conservation and restoration purposes, as well as for art history studies because it may help in determining the origin of the lacquered objects. In this work, pyrolysis-gas chromatography/mass spectrometry using a filament-type pyrolyser was successfully applied to the characterization of oriental lacquers. A method to identify the three kinds of lacquer was developed and applied to the study of two lacquered shields imported from Asia in the sixteenth century. The materials that constitute the shields were also examined by Fourier-transform infrared microspectroscopy and details of the lacquering technique are reported.