Ecophysiological responses of Liolaemus arambarensis juveniles to experimental temperature variations.

Climate change increasingly influences the loss of biodiversity, especially in ectothermic organisms, which depend on environmental temperatures to obtain heat and regulate their life cycle. Studies that aim to understand the impact of temperature variation are important to better understand the possible impacts generated on the homeostasis of ectothermic organisms. Our objective was to characterize the responses of juvenile Liolaemus arambarensis lizards to abrupt changes in temperature, quantifying markers of body condition, intermediary and hormonal metabolism and oxidative balance. We collected 45 juvenile individuals of L. arambarensis (winter: 20 and summer: 25) in Barra do Ribeiro, Brazil. We transported the animals to the laboratory, where they were acclimatized for five days at a temperature of 20 °C, then divided and exposed to temperatures of 10 °C, 20 °C, 30 °C and 40 °C for 24 h. After exposure, the animals were euthanized and the brain, caudal muscle, thigh, and liver tissues were extracted for quantification of biomarkers of metabolism (glycogen and total proteins) and oxidative balance (acetylcholinesterase, superoxide dismutase, catalase, glutathione-S-transferase and lipoperoxidation) and plasma for corticosterone quantification. The results show that L. arambarensis is susceptible to sudden temperature variations, where higher temperatures caused greater activity of antioxidant enzymes, increased lipoperoxidation and higher plasma levels of corticosterone in animals eliminated in winter. The present study demonstrated that abrupt changes in temperature could significantly modify the homeostatic mechanisms of animals, which could lead to oxidative stress and a potential trade-off between survival and growth/reproduction. In this context, the organism mobilizes energy resources for survival, with possible damage to growth and reproduction. Demonstrate that a change in temperature can be a potential factor in extinction for a species given the profile of global climate change.

Coping with dry spells: Investigating oxidative balance and metabolic responses in the subtropical tree frog Boana pulchella (Hylidae) during dehydration and rehydration exposure.

In the face of climate change, understanding the metabolic responses of vulnerable animals to abiotic stressors like anurans is crucial. Water restriction and subsequent dehydration is a condition that can threaten populations and lead to species decline. This study examines metabolic variations in the subtropical frog Boana pulchella exposed to dehydration resulting in a 40% loss of body water followed by 24 h of rehydration. During dehydration, the scaled mass index decreases, and concentrations of metabolic substrates alter in the brain and liver. The activity of antioxidant enzymes increases in the muscle and heart, emphasizing the importance of catalase in the rehydration period. Glycogenesis increases in the muscle and liver, indicating a strategy to preserve tissue water through glycogen storage. These findings suggest that B. pulchella employs specific metabolic mechanisms to survive exposure to water restriction, highlighting tissue-specific variations in metabolic pathways and antioxidant defenses. These findings contribute to a deeper understanding of anuran adaptation to water stress and emphasize the importance of further research in other species to complement existing knowledge and provide physiological tools to conservation.

Ventilatory response to peripheral chemoreflex and muscle metaboreflex during static handgrip in healthy humans: evidence of hyperadditive integration.

NEW FINDINGS: What is the central question of this study? What is the effect of peripheral chemoreflex and muscle metaboreflex integration on ventilation regulation, and what is the effect of integration on breathing-related sensations and emotions? What is the main finding and its importance? Peripheral chemoreflex and muscle metaboreflex coactivation during isocapnic static handgrip exercise appeared to elicit a hyperadditive effect with regard to ventilation and an additive effect with regard to breathing-related sensations and emotions. These findings reveal the nature of the integration between two neural mechanisms that operate during small-muscle static exercise performed under hypoxia. ABSTRACT: Exercise augments the hypoxia-induced ventilatory response in an exercise intensity-dependent manner. A mutual influence of hypoxia-induced peripheral chemoreflex activation and exercise-induced muscle metaboreflex activation might mediate the augmentation phenomenon. However, the nature of these reflexes' integration (i.e., hyperadditive, additive or hypoadditive) remains unclear, and the coactivation effect on breathing-related sensations and emotions has not been explored. Accordingly, we investigated the effect of peripheral chemoreflex and muscle metaboreflex coactivation on ventilatory variables and breathing-related sensations and emotions during exercise. Fourteen healthy adults performed 2-min isocapnic static handgrip, first with the non-dominant hand and immediately after with the dominant hand. During the dominant hand exercise, we (a) did not manipulate either reflex (control); (b) activated the peripheral chemoreflex by hypoxia; (c) activated the muscle metaboreflex in the non-dominant arm by post-exercise circulatory occlusion (PECO); or (d) coactivated both reflexes by simultaneous hypoxia and PECO use. Ventilation response to coactivation of reflexes (mean ± SD, 13 ± 6 l/min) was greater than the sum of responses to separated activations of reflexes (mean ± SD, 8 ± 8 l/min, P = 0.005). Breathing-related sensory and emotional responses were similar between coactivation of reflexes and the sum of separate activations of reflexes. Thus, the peripheral chemoreflex and muscle metaboreflex integration during exercise appeared to be hyperadditive with regard to ventilation and additive with regard to breathing-related sensations and emotions in healthy adults.

Multivariate optimization results in an edible extract from Ilex paraguariensis unexplored residues with a high amount of phenolic compounds.

Ilex paraguariensis A. St. Hil. plants are used for the preparation of food and drinks which are widely consumed worldwide. During the harvest season of these plants, 2-5 ton hec-1 of agricultural residue is generated, which remains underutilized. Therefore, this study aimed to obtain an edible extract with high content of bioactive compounds and antimicrobial properties from the agricultural residue of I. paraguariensis for industrial use in food applications. The extraction conditions were optimized through a multivariate experimental design using ethanol:water. The extracted compounds were characterized by HPLC-ESY-QTOF-MS. In the optimal extraction conditions, 55 compounds were extracted, including 8 compounds that were not previously reported in I. paraguariensis. The method proved to be simple, fast, economical and environmentally friendly, with the use of green solvents. This optimization allowed for the extraction of 15.07 g of phenolic compounds per 100 g of residue. The extract showed high antioxidant activity and the capacity to inhibit Staphylococcus aureus. Results indicate that it is possible to obtain an edible extract with a high content of bioactive compounds, particularly phenolic compounds, from the I. paraguariensis residue, which has high prospects for the valorization of unexplored natural resources.

Covid-19 Automated Diagnosis and Risk Assessment through Metabolomics and Machine Learning.

COVID-19 is still placing a heavy health and financial burden worldwide. Impairment in patient screening and risk management plays a fundamental role on how governments and authorities are directing resources, planning reopening, as well as sanitary countermeasures, especially in regions where poverty is a major component in the equation. An efficient diagnostic method must be highly accurate, while having a cost-effective profile. We combined a machine learning-based algorithm with mass spectrometry to create an expeditious platform that discriminate COVID-19 in plasma samples within minutes, while also providing tools for risk assessment, to assist healthcare professionals in patient management and decision-making. A cross-sectional study enrolled 815 patients (442 COVID-19, 350 controls and 23 COVID-19 suspicious) from three Brazilian epicenters from April to July 2020. We were able to elect and identify 19 molecules related to the disease's pathophysiology and several discriminating features to patient's health-related outcomes. The method applied for COVID-19 diagnosis showed specificity >96% and sensitivity >83%, and specificity >80% and sensitivity >85% during risk assessment, both from blinded data. Our method introduced a new approach for COVID-19 screening, providing the indirect detection of infection through metabolites and contextualizing the findings with the disease's pathophysiology. The pairwise analysis of biomarkers brought robustness to the model developed using machine learning algorithms, transforming this screening approach in a tool with great potential for real-world application.

Chronic exposure to ethanol alters the expression of miR-155, miR-122 and miR-217 in alcoholic liver disease in an adult zebrafish model.

AIM: The aim of this study was to evaluate the hepatic and circulating expression of miR-155, miR-122 and miR-217 in a model of chronic exposure to ethanol in adult zebrafish. METHODS: Wild-type adult zebrafish were divided into two groups (n = 281): an EG (exposed to 0.5% v/v Ethanol in aquarium water) and a CG (without ethanol). After 28 days the animals were euthanized, followed by histopathological analysis, quantification of lipids, triglycerides and inflammatory cytokines in liver tissue. miR-155, miR-122 and miR-217 gene expression was quantified in liver tissue and serum. RESULTS: We observed hepatic lesions and increased accumulation of hepatic lipids in the EG. The expression of il-1ß was higher in the EG, but there were no differences in il-10 and tnf-α between groups. In the liver, expression of miR-122 and miR-155 was higher in the EG. The circulating expression of miR-155 and miR-217 was significantly higher in the EG. CONCLUSION: Chronic exposure to ethanol in zebrafish leads to altered hepatic and circulating expression of miR-155, miR-122 and miR-217. This confirms its potential as a biomarker and therapeutic target.

Does leukotriene F4 play a major role in the infection mechanism of Candida sp.?

Candidiasis is the most common fungal infection affecting hospitalized patients, especially immunocompromised and critical patients. Limitations regarding the assertive diagnosis of both Candidemia and Candidiasis not only impairs the introduction of effective treatments but also lays a heavy financial burden over the health system. Furthermore, it is still challenging to ascertain whether diagnostic methods are accurate and whether treatment is effective for patients with Candidemia. These constraints come from the uncertainty of the pathophysiological mechanism by which the pathogen establishes the opportunistic infection. Additionally, it is the reason why some patients present positive blood culture results, and others do not, and why it is very difficult during clinical routines to prove Candidemia or invasive candidiasis. Taking into account the current situation, this contribution proposes two markers that may help to understand the mechanisms of infection by the pathogen: Leukotriene F4 and 5,6-dihydroxy-eicosatetraenoic. These two lipids putatively modulate the host's immune response, and the initial data presented in this contribution suggest that these lipids allow the opportunistic infection to be installed. The study was carried out using an omics-based platform using direct-infusion high-resolution mass spectrometry and allied with bioinformatics tools to provide accurate and reliable results for biomarker candidates screening.

Grape seed proanthocyanidins improves mitochondrial function and reduces oxidative stress through an increase in sirtuin 3 expression in EA.hy926 cells in high glucose condition.

Proanthocyanidins are phenolic compounds abundant in the diet, commonly found in grapes and derivatives, foods known for their health-promoting benefits. There is previous evidence showing the antidiabetic activity of proanthocyanidins, however, their mechanisms of action have not been fully elucidated. This study evaluated the capacity of grape seed proanthocyanidins extract (GSPE) to modulate oxidative stress, nitric oxide levels, mitochondrial dysfunction, apoptosis, and sirtuin expression in endothelial cells EA.hy926 under high glucose condition. In addition, the possible toxic effects of GSPE was evaluated in a zebrafish embryos model. The results showed that GSPE was able to enhance cell viability and avoid the disturbance in redox metabolism induced by high glucose. Moreover, GSPE was able to avoid mitochondria dysfunction and the increased in p53 and poly-(ADP-ribose) polymerase expression induced by high glucose exposition. These effects were attributed to the increase in expression of sirtuin 3, a protein able to regulate mitochondrial function. GSPE in an effective concentration did not show toxic effects in zebrafish embryos model. Taken together, these data elucidate the key molecular target of GSPE for future pharmacological interventions in diabetic patients.

Morphological abnormalities in zebrafish cryopreserved sperm.

The aim of the present study was to evaluate the effect of cryopreservation on the morphology of zebrafish sperm (Danio rerio). Sperm from 30 males were collected and divided in two treatments: fresh and cryopreserved semen. The following were measured sperm morphology, motility and membrane integrity. Cryopreservation reduced motility, the number of normal cells and the membrane integrity, as well as increased the percentage of sperm abnormalities. The most frequent types of morphological changes found in cryopreserved semen were macrocephaly, loose head, degenerated head, proximal gout, curled tail and short tail. This study opens the way for further investigations on morphological changes and for a new classification of these changes in fish semen due to cryopreservation.

A Qualitative Analysis of a USB Camera for AGV Control.

The increasing use of Automated Guided Vehicles (AGV) in the industry points to a search for better techniques and technologies to adapt to market requirements. Proper position control and movement give an AGV greater movement accuracy and greater lateral oscillations stability and vibration. It leads to smaller corridors and leaner plants, to more relaxed shipment devices, and to greater safety in the transport of fragile loads, for instance. AGV control techniques are not new, but new sensors' applications are possible, such as USB cameras. In this sense, it is necessary to ensure the sensor is adequate to control system requirements. This work addresses AGVs driven by passive floor demarcations. It presents a qualitative analysis of a USB camera as sensors for AGV control, not yet a common industrial application. We performed the experiments with a small AGV prototype on an eight-shaped lane, varying both camera parameters and AGV parameters, such as linear speed. The AGV uses a USB camera with different image processing settings-different morphological filters structuring elements shapes and sizes, and three different image resolutions-to analyze the factors that affect line detection and control processing. This paper's main contribution is a qualitative and quantitative analysis for the different sensor configurations. In addition, it discusses the influence sources on camera image as a position sensor. Furthermore, the experiments confirm sensor pertinence for the proposed control system.

Effects of dexamethasone on the Li-pilocarpine model of epilepsy: protection against hippocampal inflammation and astrogliosis.

BACKGROUND: Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy and is accompanied, in one third of cases, by resistance to antiepileptic drugs (AED). Most AED target neuronal activity modulated by ionic channels, and the steroid sensitivity of these channels has supported the use of corticosteroids as adjunctives to AED. Assuming the importance of astrocytes in neuronal activity, we investigated inflammatory and astroglial markers in the hippocampus, a key structure affected in TLE and in the Li-pilocarpine model of epilepsy. METHODS: Initially, hippocampal slices were obtained from sham rats and rats subjected to the Li-pilocarpine model of epilepsy, at 1, 14, and 56 days after status epilepticus (SE), which correspond to the acute, silent, and chronic phases. Dexamethasone was added to the incubation medium to evaluate the secretion of S100B, an astrocyte-derived protein widely used as a marker of brain injury. In the second set of experiments, we evaluated the in vivo effect of dexamethasone, administrated at 2 days after SE, on hippocampal inflammatory (COX-1/2, PGE2, and cytokines) and astroglial parameters: GFAP, S100B, glutamine synthetase (GS) and water (AQP-4), and K+ (Kir 4.1) channels. RESULTS: Basal S100B secretion and S100B secretion in high-K+ medium did not differ at 1, 14, and 56 days for the hippocampal slices from epileptic rats, in contrast to sham animal slices, where high-K+ medium decreased S100B secretion. Dexamethasone addition to the incubation medium per se induced a decrease in S100B secretion in sham and epileptic rats (1 and 56 days after SE induction). Following in vivo dexamethasone administration, inflammatory improvements were observed, astrogliosis was prevented (based on GFAP and S100B content), and astroglial dysfunction was partially abrogated (based on Kir 4.1 protein and GSH content). The GS decrease was not prevented by dexamethasone, and AQP-4 was not altered in this epileptic model. CONCLUSIONS: Changes in astroglial parameters emphasize the importance of these cells for understanding alterations and mechanisms of epileptic disorders in this model. In vivo dexamethasone administration prevented most of the parameters analyzed, reinforcing the importance of anti-inflammatory steroid therapy in the Li-pilocarpine model and possibly in other epileptic conditions in which neuroinflammation is present.

Environmental enrichment modulates the response to chronic stress in zebrafish.

Several studies have shown that manipulations to the housing environment modulate susceptibility to stress in laboratory animals, mainly in rodents. Environmental enrichment (EE) is one such manipulation that promotes neuroprotection and neurogenesis, besides affecting behaviors such as drug self-administration. Zebrafish are a popular and useful animal model for behavioral neuroscience studies; however, studies evaluating the impact of housing conditions in this species are scarce. In this study, we verified the effects of EE on behavioral (novel tank test) and biochemical [cortisol and reactive oxygen species (ROS)] parameters in zebrafish submitted to unpredictable chronic stress (UCS). Consistent with our previous findings, UCS increased anxiety-like behavior, cortisol and ROS levels in zebrafish. EE for 21 or 28 days attenuated the effects induced by UCS on behavior and cortisol, and prevented the effects on ROS levels. Our findings reinforce the idea that EE exerts neuromodulatory effects across species, reducing vulnerability to stress and its biochemical impact. Also, these results indicate that zebrafish is a suitable model animal to study the behavioral effects and neurobiological mechanisms related to EE.

Resolvin RvD2 reduces hypothalamic inflammation and rescues mice from diet-induced obesity.

BACKGROUND: Diet-induced hypothalamic inflammation is an important mechanism leading to dysfunction of neurons involved in controlling body mass. Studies have shown that polyunsaturated fats can reduce hypothalamic inflammation. Here, we evaluated the presence and function of RvD2, a resolvin produced from docosahexaenoic acid, in the hypothalamus of mice. METHODS: Male Swiss mice were fed either chow or a high-fat diet. RvD2 receptor and synthetic enzymes were evaluated by real-time PCR and immunofluorescence. RvD2 was determined by mass spectrometry. Dietary and pharmacological approaches were used to modulate the RvD2 system in the hypothalamus, and metabolic phenotype consequences were determined. RESULTS: All enzymes involved in the synthesis of RvD2 were detected in the hypothalamus and were modulated in response to the consumption of dietary saturated fats, leading to a reduction of hypothalamic RvD2. GPR18, the receptor for RvD2, which was detected in POMC and NPY neurons, was also modulated by dietary fats. The substitution of saturated by polyunsaturated fats in the diet resulted in increased hypothalamic RvD2, which was accompanied by reduced body mass and improved glucose tolerance. The intracerebroventricular treatment with docosahexaenoic acid resulted in increased expression of the RvD2 synthetic enzymes, increased expression of anti-inflammatory cytokines and improved metabolic phenotype. Finally, intracerebroventricular treatment with RvD2 resulted in reduced adiposity, improved glucose tolerance and increased hypothalamic expression of anti-inflammatory cytokines. CONCLUSIONS: Thus, RvD2 is produced in the hypothalamus, and its receptor and synthetic enzymes are modulated by dietary fats. The improved metabolic outcomes of RvD2 make this substance an attractive approach to treat obesity.

Phase-Dependent Astroglial Alterations in Li-Pilocarpine-Induced Status Epilepticus in Young Rats.

Epilepsy prevalence is high in infancy and in the elderly population. Lithium-pilocarpine is widely used to induce experimental animal models of epilepsy, leading to similar neurochemical and morphological alterations to those observed in temporal lobe epilepsy. As astrocytes have been implicated in epileptic disorders, we hypothesized that specific astroglial changes accompany and contribute to epileptogenesis. Herein, we evaluated time-dependent astroglial alterations in the hippocampus of young (27-day-old) rats at 1, 14 and 56 days after Li-pilocarpine-induced status epilepticus (SE), corresponding to different phases in this model of epilepsy. We determined specific markers of astroglial activation: GFAP, S100B, glutamine synthetase (GS), glutathione (GSH) content, aquaporin-4 (AQP-4) and potassium channel Kir 4.1; as well as epileptic behavioral, inflammatory and neurodegenerative changes. Phase-dependent signs of hippocampal astrogliosis were observed, as demonstrated by increments in GFAP, S100B and GS. Astrocyte dysfunction in the hippocampus was characterized, based on the decrease in GSH content, AQP-4 and Kir 4.1 channels. Degenerating neurons were identified by Fluoro-Jade C staining. We found a clear, early (at SE1) and persistent (at SE56) increase in cerebrospinal fluid (CSF) S100B levels. Additionally, serum S100B was found to decrease soon after SE induction, implicating a rapid-onset increase in the CSF/serum S100B ratio. However, serum S100B increased at SE14, possibly reflecting astroglial activation and/or long-term increase in cerebrovascular permeability. Moreover, we suggest that peripheral S100B levels may represent a useful marker for SE in young rats and for follow up during the chronic phases of this model of epilepsy. Together, results reinforce and extend the idea of astroglial involvement in epileptic disorders.

Analysis and characterisation of bovine oocyte and embryo biomarkers by matrix-assisted desorption ionisation mass spectrometry imaging.

In the field of 'single cell analysis', many classical strategies like immunofluorescence and electron microscopy are the primary techniques of choice. However, these methodologies are time consuming and do not permit direct identification of specific molecular classes, such as lipids. In the present study, a novel mass spectrometry-based analytical approach was applied to bovine oocytes and embryos. This new metabolomics-based application uses mass spectrometry imaging (MSI), efficient data processing and multivariate data analysis. Metabolic fingerprinting (MF) was applied to the analysis of unfertilised oocytes, 2-, 4- and 8-cell embryos and blastocysts. A semiquantitative strategy for sphingomyelin [SM (16:0)+Na](+) (m/z 725) and phosphatidylcholine [PC (32:0)+Na](+) (m/z 756) was developed, showing that lipid concentration was useful for selecting the best metabolic biomarkers. This study demonstrates that a combination of MF, MSI features and chemometric analysis can be applied to discriminate cell stages, characterising specific biomarkers and relating them to developmental pathways. This information furthers our understanding of fertilisation and preimplantation events during bovine embryo development.

Early developmental stages of Ascaris lumbricoides featured by high-resolution mass spectrometry.

Ascaris lumbricoides is responsible for a highly disseminated helminth parasitic disease, ascariosis, a relevant parasitosis that responds for great financial burden on the public health system of developing countries. In this work, metabolic fingerprinting using high-resolution mass spectrometry (HRMS) was employed to identify marker molecules from A. lumbricoides in different development stages. We have identified nine biomarkers, such as pheromones and steroidal prohormones in early stages, among other molecules in late development stages, making up four molecules for fertilized eggs, four marker molecules for first larvae (L1) and one marker molecule for third larvae (L3). Therefore, our findings indicate that this approach is suitable for biochemical characterization of A. lumbricoides development stages. Moreover, the straightforward analytical method employed, with almost no sample preparation from a complex matrix (feces) using high-resolution mass spectrometry, suggests that it is possible to seek for an easier and faster way to study animal molding processes.

Skin imprinting in silica plates: a potential diagnostic methodology for leprosy using high-resolution mass spectrometry.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which primarily infects macrophages and Schwann cells, affecting skin and peripheral nerves. Clinically, the most common form of identification is through the observation of anesthetic lesions on skin; however, up to 30% of infected patients may not present this clinical manifestation. Currently, the gold standard diagnostic test for leprosy is based on skin lesion biopsy, which is invasive and presents low sensibility for suspect cases. Therefore, the development of a fast, sensible and noninvasive method that identifies infected patients would be helpful for assertive diagnosis. The aim of this work was to identify lipid markers in leprosy patients directly from skin imprints, using a mass spectrometric analytical strategy. For skin imprint samples, a 1 cm(2) silica plate was gently pressed against the skin of patients or healthy volunteers. Imprinted silica lipids were extracted and submitted to direct-infusion electrospray ionization high-resolution mass spectrometry (ESI-HRMS). All samples were differentiated using a lipidomics-based data workup employing multivariate data analysis, which helped electing different lipid markers, for example, mycobacterial mycolic acids, inflammatory and apoptotic molecules were identified as leprosy patients' markers. Otherwise, phospholipids and gangliosides were pointed as healthy volunteers' skin lipid markers, according to normal skin composition. Results indicate that silica plate skin imprinting associated with ESI-HRMS is a promising fast and sensible leprosy diagnostic method. With a prompt leprosy diagnosis, an early and effective treatment could be feasible and thus the chain of leprosy transmission could be abbreviated.

Influence of environmental enrichment vs. time-of-day on behavioral repertoire of male albino Swiss mice.

Environmental enrichment (EE) is a non-pharmacological manipulation that promotes diverse forms of benefits in the central nervous system of captive animals. It is thought that EE influences animal behavior in a specie-(strain)-specific manner. Since rodents in general present different behaviors during distinct periods of the day, in this study we aimed to investigate the influence of time-of-day on behavioral repertoire of Swiss mice that reared in EE. Forty male Swiss mice (21days old) were housed in standard (SC) or enriched conditions (EC) for 60days. Behavioral assessments were conducted during the light phase (in presence of light) or dark phase (in absence of light) in the following tasks: open field, object recognition and elevated plus maze. First, we observed that the locomotor and exploratory activities are distinct between SC and EC groups only during the light phase. Second, we observed that "self-protective behaviors" were increased in EC group only when mice were tested during the light phase. However, "less defensive behaviors" were not affected by both housing conditions and time-of-day. Third, we showed that the performance of EE animals in object recognition task was improved in both light and dark conditions. Our findings highlight that EE-induced alterations in exploratory and emotional behaviors are just evident during light conditions. However, EE-induced cognitive benefits are remarkable even during dark conditions, when exploratory and emotional behaviors were similar between groups.

Mass spectrometry imaging: an expeditious and powerful technique for fast in situ lignin assessment in Eucalyptus.

Plant biomass has been suggested as an alternative to produce bioethanol. The recalcitrance of plant biomass to convert cellulose into simpler carbohydrates used in the fermentation process is partially due to lignin, but the standard methods used to analyze lignin composition frequently use toxic solvents and are laborious and time-consuming. MS imaging was used to study lignin in Eucalyptus, since this genus is the main source of cellulose in the world. Hand-cut sections of stems of two Eucalyptus species were covered with silica and directly analyzed by matrix-assisted laser sesorption ionization (MALDI)-imaging mass spectrometry (MS). Information available in the literature about soluble lignin subunits and structures were used to trace their distribution in the sections and using a software image a relative quantification could be made. Matrixes routinely used in MALDI-imaging analysis are not satisfactory to analyze plant material and were efficiently substituted by thin layer chromatography (TLC) grade silica. A total of 22 compounds were detected and relatively quantified. It was also possible to establish a proportion between syringyl and guaiacyl monolignols, characteristic for each species. Because of the simple way that samples are prepared, the MALDI-imaging approach presented here can replace, in routine analysis, complex and laborious MS methods in the study of lignin composition.

Cellular senescence induced by prolonged subculture adversely affects glutamate uptake in C6 lineage.

Several researchers have recently used C6 cells to evaluate functional properties of high-affinity glutamate transporters. However, it has been demonstrated that this lineage suffers several morphological and biochemical alterations according to the number of passages in culture. Currently, there are no reports showing whether functional properties of high-affinity glutamate transporters comply with these sub culturing-dependent modifications. The present study aimed to compare the functional properties of high-affinity glutamate transporters expressed in early (EPC6) and late (LPC6) passage C6 cells through a detailed pharmacological and biochemical characterization. Between 60-180 min of L-[(3)H]glu incubation, LPC6 presented an intracellular [(3)H] 55% lower than EPC6. Both cultures showed a time-dependent increase of intracellular [(3)H] reaching maximal levels at 120 min. Cultures incubated with D-[(3)H]asp showed a time-dependent increase of [(3)H] until 180 min. Moreover, LPC6 have a D-[(3)H]asp-derived intracellular [(3)H] 30-45% lower than EPC6 until 120 min. Only EAAT3 was immunodetected in cultures and its total content was equal between them. PMA-stimulated EAAT3 trafficking to membrane increased 50% of L-[(3)H]glu-derived intracellular [(3)H] in EPC6 and had no effect in LPC6. LPC6 displayed characteristics that resemble senescence, such as high ß-Gal staining, cell enlargement and increase of large and regular nuclei. Our results demonstrated that LPC6 exhibited glutamate uptake impairment, which may have occurred due to its inability to mobilize EAAT3 to cell membrane. This profile might be related to senescent process observed in this culture. Our results suggest that LPC6 cells are an inappropriate glial cellular model to investigate the functional properties of high-affinity glutamate transporters.