Experimental design and sample preparation in forest tree matabolomics

ReviewAppropriate experimental design and sample preparation are key steps in metabolomics experiments, highly influencing the biological interpretation of the results. The sample preparation workflow for plant metabolomics studies includes several steps before metabolite extraction and analysis. These include the optimization of laboratory procedures, which should be optimized for di erent plants and tissues. This is particularly the case for trees, whose tissues are complex matrices to work with due to the presence of several interferents, such as oleoresins, cellulose. A good experimental design, tree tissue harvest conditions, and sample preparation are crucial to ensure consistency and reproducibility of the metadata among datasets. In this review, we discuss the main challenges when setting up a forest tree metabolomics experiment for mass spectrometry (MS)-based analysis covering all technical aspects from the biological question formulation and experimental design to sample processing and metabolite extraction and data acquisition. We also highlight the importance of forest tree metadata standardization in metabolomics studiesinfo:eu-repo/semantics/publishedVersio

Polarized Signaling via Purinoceptors in Normal and Cystic Fibrosis Airway Epithelia

Airway epithelia are confronted with distinct signals emanating from the luminal and/or serosal environments. This study tested whether airway epithelia exhibit polarized intracellular free calcium (Ca2+i) and anion secretory responses to 5′ triphosphate nucleotides (ATP/UTP), which may be released across both barriers of these epithelia. In both normal and cystic fibrosis (CF) airway epithelia, mucosal exposure to ATP/UTP increased Ca2+i and anion secretion, but both responses were greater in magnitude for CF epithelia. In CF epithelia, the mucosal nucleotide–induced response was mediated exclusively via Ca2+i interacting with a Ca2+-activated Cl− channel (CaCC). In normal airway epithelia (but not CF), nucleotides stimulated a component of anion secretion via a chelerythrine-sensitive, Ca2+-independent PKC activation of cystic fibrosis transmembrane conductance regulator. In normal and CF airway epithelia, serosally applied ATP or UTP were equally effective in mobilizing Ca2+i. However, serosally applied nucleotides failed to induce anion transport in CF epithelia, whereas a PKC-regulated anion secretory response was detected in normal airway epithelia. We conclude that (1) in normal nasal epithelium, apical/basolateral purinergic receptor activation by ATP/UTP regulates separate Ca2+-sensitive and Ca2+-insensitive (PKC-mediated) anion conductances; (2) in CF airway epithelia, the mucosal ATP/UTP-dependent anion secretory response is mediated exclusively via Ca2+i; and (3) Ca2+i regulation of the Ca2+-sensitive anion conductance (via CaCC) is compartmentalized in both CF and normal airway epithelia, with basolaterally released Ca2+i failing to activate CaCC in both epithelia

The Mitochondrial Barriers Segregate Agonist-induced Calcium-dependent Functions in Human Airway Epithelia

In airway epithelia, purinergic receptor (P2Y2-R) stimulation of intracellular calcium (Ca2+i)–regulated ion transport is restricted to the membrane domain ipsilateral to receptor activation, implying compartmentalization of Ca2+i signaling. Because mitochondria can spatially restrict cellular Ca2+i signals, immunocytochemical, electron microscopic, and fluorescent studies of mitochondria localization were performed in human airway epithelia. Although concentrated at the apical domain, mitochondria were found distributed at both the apical and the basolateral poles and in close association with the endoplasmic reticulum. The role of mitochondria in locally restricting P2Y2-R–induced Ca2+i signals was investigated by measuring changes in mitochondrial Ca2+ (Ca2+m) in human airway epithelial monolayers. P2Y2-R activation induced Ca2+m accumulation in mitochondria confined to the domain ipsilateral to P2Y2-R stimulation, which was blocked by mitochondrial uncoupling with 1 μM CCCP and 2.5 μg/ml oligomycin. The role of mitochondria in restricting the cellular cross-talk between basolateral P2Y2-R–dependent Ca2+i mobilization and apical membrane Ca2+-activated Cl− secretion was investigated in studies simultaneously measuring Ca2+i and Cl− secretion in cystic fibrosis human airway epithelial monolayers. Activation of basolateral P2Y2-Rs produced similar increases in Ca2+i in monolayers without and with pretreatment with uncouplers, whereas Ca2+i-activated Cl− secretion was only efficiently triggered in mitochondria-uncoupled conditions. We conclude that (a) mitochondria function as a Ca2+i-buffering system in airway epithelia, compartmentalizing Ca2+i-dependent functions to the membrane ipsilateral to receptor stimulation; and (b) the mitochondria provide structural barriers that protect the airway epithelia against nonspecific activation of Ca2+i-modulated functions associated with Ca2+i signals emanating from the apical or the basolateral membrane domains

Morphometric analysis of rat parameters during an animal model of colorectal cancer

Colorectal cancer is one of the leading causes of death by cancer worldwide. Animal models have been used in experimental research to find new solutions for old biomedical questions. This research aimed to make a contribution to characterize the rat model of colorectal cancer. Twelve male Wistar rats obtained from Charles River were randomly divided into two groups: control group and induced group. All ethical issues were considered, following the guidelines of the Portuguese Direção Geral de Alimentação e Veterinária (approval number 010535). Animals from induced group received a weekly intraperitoneal injection of N, N’-Dimethylhydrazine (DMH), for seven consecutive weeks. All rats were monitored for signs of distress, weight loss, and food and water consumption. Abdominal ultrasound examinations were performed before the first DMH administration and the animals’ sacrifice. Thirteen weeks later, all surviving animals were sacrificed, organs and blood were collected. Animals from control group showed a higher mean body weight. The mean relative weight of soleus was lower in induced animals when compared to the control animals (p<0.005), which may suggest the development of anorexia. Although there were no significant differences in colon’s weight, induced animals had shorter colon. The microhematocrit was not different between groups. The ultrasound examination showed a high vascularized abdominal mass in induced animals. Some induced animals had a swollen abdomen which difficult the ultrasound examination. The animals showed little changes in their biological parameters, suggesting that the disease was at an early stage. Histological analysis of animals’ organs will provide a better perception of the colorectal cancer induction

Simulation Model for Salmonella Typhimurium on a Farrow-to-Finish Herd

A stochastic model which simulates they dynamics of Salmonella Typhimurium in moderate to highly infeted farrow-to-finish farms in Portugal was developed. The model comprises six different stages: three at the reproductive phase (sows) and another three for pig growth

Preparation of well-dispersed chitosan/alginate hollow multilayered microcapsules for enhanced cellular internalization

Hollow multilayered capsules have shown massive potential for being used in the biomedical and biotechnology fields, in applications such as cellular internalization, intracellular trafficking, drug delivery, or tissue engineering. In particular, hollow microcapsules, developed by resorting to porous calcium carbonate sacrificial templates, natural-origin building blocks and the prominent Layer-by-Layer (LbL) technology, have attracted increasing attention owing to their key features. However, these microcapsules revealed a great tendency to aggregate, which represents a major hurdle when aiming for cellular internalization and intracellular therapeutics delivery. Herein, we report the preparation of well-dispersed polysaccharide-based hollow multilayered microcapsules by combining the LbL technique with an optimized purification process. Cationic chitosan (CHT) and anionic alginate (ALG) were chosen as the marine origin polysaccharides due to their biocompatibility and structural similarity to the extracellular matrices of living tissues. Moreover, the inexpensive and highly versatile LbL technology was used to fabricate core-shell microparticles and hollow multilayered microcapsules, with precise control over their composition and physicochemical properties, by repeating the alternate deposition of both materials. The microcapsules' synthesis procedure was optimized to extensively reduce their natural aggregation tendency, as shown by the morphological analysis monitored by advanced microscopy techniques. The well-dispersed microcapsules showed an enhanced uptake by fibroblasts, opening new perspectives for cellular internalization.publishe

The role of mutual in-feeding in maintaining problematic self-narratives: exploring one path to therapeutic failure

According to the author’s narrative model of change, clients may maintain a problematic self-stability across therapy, leading to therapeutic failure, by a mutual in-feeding process, which involves a cyclical movement between two opposing parts of the self. During innovative moments (IMs) in the therapy dialogue, clients’ dominant self-narrative is interrupted by exceptions to that self-narrative, but subsequently the dominant self-narrative returns. The authors identified return-to-the-problem markers (RPMs), which are empirical indicators of the mutual in-feeding process, in passages containing IMs in 10 cases of narrative therapy (five good-outcome cases and five poor-outcome cases) with females who were victims of intimate violence. The poor-outcome group had a significantly higher percentage of IMs with RPMs than the good-outcome group. The results suggest that therapeutic failures may reflect a systematic return to a dominant self-narrative after the emergence of novelties (IMs

Innovative moments in psychotherapy: dialogical processes in developing narratives

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Second-derivative spectrophotometry for the analysis of simvastatin in polymeric nanocapsules

Conventional spectrophotometry methods are very susceptible to the presence of interferences in complex mixtures such as nanoparticules, requiring prior treatment or extraction of the analyte, and not always providing an adequate response. Derivative spectrophotometry method is capable to eliminate its interference; it is an alternative method for drugs determination in complex matrices. This work investigated the utility of derivate spectrophotometry in assay of simvastatin in polymeric nanocapsules (SIVNC). Shimadzu® UV-1650 double-beam spectrophotometer with 1.0 cm quartz cells was used in this study. The second-order deriva­tive spectrum was obtained employing Δλ=20,000 nm and scaling factor=9.0. The determinations were made at 239 nm (2D239) by zero-crossing method. 2D239 method was validated employing the parameters: specificity, linearity, robustness, precision and accuracy. Results: The specificity test showed there was no interference of constituents commonly found in SIVNC formulation in 2D239. The standard curve showed a correlation coefficient of 0.994. The robustness was evaluated by small changes in the conditions of sample analysis and however, no significant changes were observed regarding drug quantitation. The precision was demonstrated by relative standard deviation (RSD) of intra-day (RSD=1.61-3.76) and inter-day studies (RSD=2.32). The recovery test resulted in an average of 100.66%, which confirmed the accuracy of the method. The procedure was simple and rapid; therefore this technique offers an alternative for determination of SIVNC without interferences