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The adequacy of the best storage temperature for fruits and vegetables is an essential measure that helps in maintaining quality and extending shelf life. In this sense, this work aimed to study the influence of temperature on the quality of the marolo (Annona crassiflora Mart) “in natura”, where the fruits were washed, sanitized with 1,216 µM hypochlorite solution, and stored at 4 different temperatures (0, 6, 12, and 20 ° C) controlling the relative humidity (80 to 90%). The parameters color L*a*b*, pH, titratable acidity, soluble solids, firmness, total and soluble pectins, enzymes (pectinamethylesterase and polygalacturonase), and vitamin C were analyzed at different storage times. The maximum storage period was 8 days for fruits kept at 0 and 6 °C. The use of higher temperatures (12 and 20 °C) resulted in a shorter storage time (6 and 4 days, respectively). We found that the color parameter L*a*b* was not influenced by time and temperature during the process, while firmness and soluble solids were affected only by temperature. On the other hand, the parameters pH, titratable acidity, total and soluble pectin, enzymes (pectinamethylesterase and polygalacturonase), and vitamin C were influenced by both temperature and storage time (p

Biodiesel is a renewable fuel with various advantages, such as the reduced emissions of toxic gases (CO2, SO2, CO, and HC) when compared to fossil diesel. It can be produced by esterification or transesterification reactions. In this chapter, the application of nanomaterials as a catalyst in these reactions is investigated through a critical review. Thus the catalytic activity and how the catalyst size can improve it are reported. A trend is also established between this catalytic activity and the main reactional parameters (reaction time, temperature, alcohol/oil or acid molar ratio, and catalyst load) in order to obtain the optimized process. Therefore this chapter is divided into a discussion of the state of the art of biodiesel synthesis and nanomaterials (bio-based catalysts) applied in these reactions, as well as the optimization process to obtain the best reaction conditions. Moreover, the chapter also highlights the main methodologies used in nanocatalyst synthesis and their characterization.

The Single Prolonged Stress protocol is considered a model for PTSD, as it induces long lasting changes in rat behaviour and endocrine regulation. Previous work demonstrated that some of these changes can be prevented by treatment with the glucocorticoid receptor antagonist RU486, administered a week after the stressor. The current study evaluated the effects of an earlier intervention with RU486, as evaluated 1 week after SPS-exposure. Most RU486 effects occurred independent of prior stress, except for the reversal of a stress-induced increase in locomotor behaviour. The accompanying changes in gene expression depended on gene, brain region, and time. DNA methylation of the robustly down-regulated Fkbp5 gene was dissociated of changes in mRNA expression. The findings reinforce the long term effects of GR antagonist treatment, but also emphasize the need to evaluate changes over time to allow the identification of robust correlates between gene expression and behavioural/endocrine outcome of stressful experiences.

B-type Natriuretic Peptide (BNP) is a biologically active circulating hormone. Plasma concentrations of BNP are routinely used in the diagnosis of heart failure, and the intravenous infusion of recombinant BNP can be used for heart failure treatment. Like many bioactive polypeptides, multiple plasma enzymes are known to cleave circulating BNP, and as part of the CVD-B/D-HPP mandate, we sought to develop a technique capable of profiling these catabolic processes in plasma. We used a neutral-coated capillary electrophoresis-electrospray ionization (CESI) separation system coupled with high-resolution mass spectrometry to profile the proteolysis of exogenous recombinant BNP1–32 in plasma. Our method utilizes electrokinetic injection of minimally processed plasma samples to simultaneously monitor the dynamic generation and breakdown of at least five BNP peptidoforms in plasma. By integrating multisegment injection, our method can produce a multipoint BNP proteolytic profile for one sample within an hour. We e...

Background Stressors activate a wide spectrum of interacting hormonal and neuronal systems resulting in behavioral and physiological responses, with consequences for the development of psychopathology. Several recent studies demonstrated that treatment with the glucocorticoid receptor (GR) antagonist RU486 during adulthood normalized effects of early life stress. We aimed to evaluate the potential of RU486 to reverse stress-induced changes in an animal model of adult stress. Method We employed the single-prolonged stress (SPS) model as a multimodal stress exposure protocol in male rats. SPS rats and unstressed controls were treated with RU486 on days 8, 9, 10 after stress exposure and the effects of treatment were evaluated after another 4 days. We determined body weight gain, corticosterone levels, behavioral reactivity in anxiety tests, and brain gene expression of c-fos, corticosteroid receptors, drivers of the stress response and genes (epi-)genitally linked to PTSD. Results RU486 affected body weight gain, corticosterone levels and open field behavior only in SPS rats. RU486 had history-independent effects in reducing fear in the elevated plus maze and fear conditioning behavior. Gene expression analysis showed a diversity of in- and interdependent effects of stress and RU486. Conclusion The effects of RU486 applied 1 week after stress and measured 4 days after treatment demonstrate that in the state of post-SPS the GR-dependence of homeostatic processes has changed. This suggests that GR-mediated processes are part of allostatic regulation after adult stress. The normalization of a number of SPS-effects after RU486 treatment reinforces the potential of targeting GR for treatment of stress-related psychopathologies.

Early life stress (ELS) is associated with a higher risk of psychopathologies in adulthood, such as depression, which may be related to persistent changes in the hypothalamic-pituitary-adrenal (HPA) axis. This study aimed to evaluate the effects of ELS on the functioning of the HPA axis in clinical and experimental situations. Clinically, patients with current depressive episodes, with and without ELS, and healthy controls, composed the sample. Subjects took a capsule containing placebo, fludrocortisone, prednisolone, dexamethasone or spironolactone followed by an assessment of plasma cortisol the morning after. Experimentally, male Wistar rats were submitted to ELS protocol based on variable, unpredictable stressors from postnatal day (P) 1 to P21. On P65 animals were behaviorally evaluated through the forced swimming test (FST). At P68, pharmacological challenges started, using mifepristone, dexamethasone, spironolactone or fludrocortisone, and corticosterone levels were determined 3 hours after injections. Cortisol response of the patients did not differ significantly from healthy subjects, regardless of their ELS history, and it was lower after fludrocortisone, prednisolone and dexamethasone compared to placebo, indicating the suppression of plasma cortisol by all these treatments. Animals exposed to ELS presented altered phenotype as indicated by an increased immobility time in the FST when compared to control, but no significant long-lasting effects of ELS were observed on the HPA axis response. Limitations on the way the volunteers were sampled may have contributed to the lack of ELS effects on the HPA axis, pointing out the need for further research to understand these complex phenomena Significance statement Early life stress (ELS) is associated with a higher risk of psychopathologies in adulthood, including depression, which seems related to persistent changes in the hypothalamic-pituitary-adrenal axis. We evaluated the effects of ELS on the functioning of hypothalamic–pituitary–adrenal axis combining clinical and experimental approaches, challenging GR and MR receptors with agonists and antagonists. Cortisol response of depressive patients did not differ from healthy subjects, regardless of their ELS history. Animals submitted to ELS presented altered behavior, as increased immobility time in the forced-swimming test, but no effects were observed in response to GR and MR challenges. It provides insights into the complexity of HPA axis-related mechanisms involved in the long-lasting consequences of ELS in both experimental animals and humans.

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.

The analysis of carbohydrate moieties of glycoprotein biopharmaceuticals is of high importance, especially because glycosylation changes can impact the biological effect of the drugs. In the case of monoclonal antibody therapeutics, the degree of core fucosylation significantly influences its effector function, thus should be closely monitored during the clone selection, development and manufacturing processes. MS analysis of labile sugar residues such as core fucosylation and terminal silylation may be misleading as such residues can break off during the ionization process or alter the ionization efficiency, both of which affect the results. In the case of monoclonal antibody therapeutics tailored for antibody-dependent cellular cytotoxicity function, core fucosylation should be kept minimal; therefore, close attention should be paid to the prevalence of this particular sugar residue. This paper reports on the core fucosylation analysis of some major immunoglobulin G N-glycans by direct infusion ESI-MS and CE–LIF. In comparison to the industry standard of CE–LIF, a lower degree of core fucosylated structures was found by ESI-MS analysis, emphasizing the need to use orthogonal quantitative separation methods in addition to MS.

Within pharmaceutical industry charge heterogeneity testing of biopharmaceuticals has to be reproducible and fast. It should pass method validation according to ICH Q2. Classical approaches for the analysis of the charge heterogeneity of biopharmaceuticals are ion exchange chromatography (IEC) and isoelectric focusing (IEF). As an alternative approach, also capillary zone electrophoresis (CZE) was expected to allow reliable charge heterogeneity profiling by separation according to the analyte's net charge and hydrodynamic radius. Aim of this study was to assess if CZE possesses all of the required features. Therefore, beside lab internal validation of this method also an international cross company study was organized. It was shown that CZE is applicable across a broad pI range between 7.4 and 9.5. The coefficient of correlation was above 0.99 which demonstrated linearity. Precision by repeatability was around 1% (maximum relative standard deviation per level) and accuracy by recovery was around 100% (mean recovery per level). Accuracy was further verified by direct comparison of IEC, IEF and CZE, which in this case showed comparable %CPA results for all three methods. However, best resolution for the investigated MAb was obtained with CZE. In dependence on sample concentration the detection limit was between 1 and 3%. Within the intercompany study for CZE the same stressed and non-stressed samples were analyzed in each of the 11 participating labs. The finally obtained dataset contained more than 1000 separations which provided an extended dataset for further statistical evaluation. Among the different labs no significant differences between the peak profiles were observed. Mean driver for dropouts in quantitative evaluation was linked to the performance of some participating labs while the impact of the method performance was negligible. In comparison to a 50cm capillary there was a slightly better separation of impurities and drug substance related compounds with a 30cm capillary which demonstrates that an increased stability indicating potential can be combined with the increased separation velocity and high throughput capability of a shorter capillary. Separation can be performed in as little as approx. 3min allowing high throughput applications. The intercompany study delivered precise results without explicit training of the participating labs in the method prior to the study (standard deviations in the range of 1%). It was demonstrated that CZE is an alternative platform technology for the charge heterogeneity testing of antibodies in the pharmaceutical industry.

A 65-year-old Brazilian man presented with an erythematous nodular lesion on the left forearm (Fig. 1). The patient had been treated with multidrug therapy for 8 months for lepromatous leprosy. During therapy, he developed recurrent episodes of reactions which were treated with high doses of prednisone and thalidomide. The histopathology of the cutaneous nodular lesion showed a granulomatous inflammatory infiltrate; some histiocytes contained vacuolations and others demonstrated oval-like or coma-like structures (Fig. 2). The specimen was cultivated in Sabouraud agar at room temperature. The colonies were transferred to Petri dishes containing Niger Seed Agar (NSA) (Fig. 3). The confirmed diagnosis was Cryptococcus neoformans var. neoformans based on microscopy and physiology, including the canavanine-glycine-bromothymol blue (CGB) medium (Lazera MS, Pires FDA, Camillo-Coura L, et al. Natural habitat of Cryptococcus neoformans var. neoformans in decaying wood forming hollows in living trees. J Med Vet Mycol 1996; 34: 127-131). The liquor culture was negative. Hemoculture and urine culture were also negative. Latex agglutination test was blood positive and liquor negative. The patient's hemogram revealed normocytic anemia and normal total and differential white blood count. The CD4 count was 189/m 3 and the CD8 count was 141/m 3 . Serology for anti-human immunodeficiency virus-1 (anti-HIV-1) antibodies was negative. The X-ray of the lungs showed an areolar image in the superior lobe of the right lung. Therapy with prednisone was suspended and fluconazole (300mg/day) was prescribed. The nodular cutaneous lesion regressed completely after 90 days. The patient was submitted to a second skin biopsy for treatment control. The culture of the specimen taken was still positive and the histopathology showed the same picture as before treatment. After 5 months of continued therapy with fluconazole, another biopsy was performed but no fungus was recovered from the specimen.