Comparison of α and ß-acid isomerization in hops and beer using HPLC, confocal microscopy, spectrofluorimetry and chemical analysis of metabolites and essential oils in flowers of different hop cultivars produced in Brazil.

We used confocal microscopy and spectrofluorescence to characterize the emission spectra in hop flowers, to follow the isomerization processes in different hop preparations, and beers, to compare with HPLC extracted samples. Flowers of different hop cultivars produced in three regions of Brazil, were quantitated by HPLC and GC-MS. The fluorescence spectra showed two characteristic emission bands evaluated from different preparations. The isomerization process leads to a gradual decrease in fluorescence intensity as the reaction progresses. This demonstrates the valuable use of confocal microscopy and fluorescence spectroscopy for analysis of the correlation between bitter acid indices with fluorescence intensity and lifetime microscopy. Such techniques can be used directly in the flowers allowing rapid monitoring of the brewing process. Twenty-nine substances were characterized in the essential oils and some cultivars presented quantities of bitter acids and essential oil levels close to those expected for plants after more than three years of cultivation.

Doped Carbon Quantum Dots/PVA Nanocomposite as a Platform to Sense Nitrite Ions in Meat.

A sensor device based on doped-carbon quantum dots is proposed herein for detection of nitrite in meat products by fluorescence quenching. For the sensing platform, carbon quantum dots doped with boron and functionalized with nitrogen (B,N-Cdot) were synthesized with an excellent 44.3% quantum yield via a one-step hydrothermal route using citric acid, boric acid, and branched polyethylenimine as carbon, boron, and nitrogen sources, respectively. After investigation of their chemical structure and fluorescent properties, the B,N-Cdot at aqueous suspensions showed high selectivity for NO2- in a linear range from 20 to 50 mmol L-1 under optimum conditions at pH 7.4 and a 340 nm excitation. Furthermore, the prepared B,N-Cdots successfully detected NO2- in a real meat sample with recovery of 91.4-104% within the analyzed range. In this manner, a B,N-Cdot/PVA nanocomposite film with blue emission under excitation at 360 nm was prepared, and a first assay detection of NO2- in meat products was tested using a smartphone application. The potential application of the newly developed sensing device containing a highly fluorescent probe should aid in the development of a rapid and inexpensive strategy for NO2- detection.

Transcellular propagation of fibrillar α-synuclein from enteroendocrine to neuronal cells requires cell-to-cell contact and is Rab35-dependent.

Parkinson's disease (PD) is a neurodegenerative condition featured by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of α-synuclein (αSyn) aggregates. Growing evidence suggests that PD diagnosis happens late in the disease progression and that the pathology may originate much earlier in the enteric nervous system (ENS) before advancing to the brain, via autonomic fibers. It was recently described that a specific cell type from the gut epithelium named enteroendocrine cells (EECs) possess many neuron-like properties including αSyn expression. By facing the gut lumen and being directly connected with αSyn-containing enteric neurons in a synaptic manner, EECs form a neural circuit between the gastrointestinal tract and the ENS, thereby being a possible key player in the outcome of PD in the gut. We have characterized the progression and the cellular mechanisms involved in αSyn pre-formed fibrils (PFFs) transfer from EECs to neuronal cells. We show that brain organoids efficiently internalize αSyn PFF seeds which triggers the formation of larger intracellular inclusions. In addition, in the enteroendocrine cell line STC-1 and in the neuronal cell line SH-SY5Y, αSyn PFFs induced intracellular calcium (Ca2+) oscillations on an extracellular Ca2+ source-dependent manner and triggered αSyn fibrils internalization by endocytosis. We characterized the spread of αSyn PFFs from enteroendocrine to neuronal cells and showed that this process is dependent on physical cell-to-cell contact and on Rab35 GTPase. Lastly, inhibition of Rab35 increases the clearance of αSyn fibrils by redirecting them to the lysosomal compartment. Therefore, our results reveal mechanisms that contribute to the understanding of how seeded αSyn fibrils promote the progression of αSyn pathology from EECs to neuronal cells shifting the focus of PD etiology to the ENS.

The Influence of Pregnancy on Female Prostate Morphophysiology in Gerbils (Meriones unguiculatus).

Morphophysiological changes of the female prostate during pregnancy are still little known. Considering that this gland is highly influenced by steroid hormones, the aim of this study was to evaluate the impact of the pregnancy on female prostate morphophysiology in gerbils. Pregnant females were timed, and the prostates were analyzed at pregnancy days 6 (P6), 12 (P12), 18 (P18), and 24 (P24). Virgin females were used as the control group (C). We observed a profound change in the hormonal profile during gestation, which was marked by a high oscillation of the progesterone (P4) hormone. P4 serum levels increased, peaking at the middle of gestation, and decreased to the end of the pregnancy. The morphology of the gland in pregnant females also changed, being marked by an increase of acini lumen, and a decrease in stroma. Indeed, the acinar changes during pregnancy were followed by a significant reduction of the epithelial height, besides a change of the smooth muscle cells' morphology that became more relaxed. The number of progesterone receptor (PR) and androgen receptor (AR)-positives cells decreased with the increase of progesterone serum levels, showing an inverse relationship. Finally, we observed a reduction of epithelial proliferation and a significant increase of gland PAS-positive secretion at the end of pregnancy. Altogether, these results showed, for the first time, that the female prostate morphophysioloy is profoundly influenced by the gestational period, suggesting that the fluctuation of the P4 serum levels is the main factor influencing the gland during this period.

Galectin-3 Expression in Pancreatic Cell Lines Under Distinct Autophagy-Inducing Stimulus.

Hypoxia and nutrient deprivation are responsible for inducing malignant behavior in neoplastic cells. In these conditions, metabolic stress leads the cells to enhance their autophagic flux and to activate key molecules for homeostasis maintenance. Galectin-3 (Gal-3) is upregulated in pancreatic cancer and it is activated under the hypoxic atmosphere. We aimed to analyze the most effective autophagic-inducing conditions in pancreatic ductal adenocarcinoma cells and the effect exerted under these conditions in association with hypoxia on the Gal-3 expression. Gal-3 and the microtubule-associated protein light chain 3 beta (LC3) were accessed through western blot and immunofluorescence. Degradative vacuole quantification was analyzed by transmission electronic microscopy, and inhibition of Gal-3 was performed using siRNA. According to the analyses, the most effective conditions in the inducement of autophagy for PANC-1 and MIA PaCa-2 cells were nutritional deprivation and complete amino acid/glucose deprivation, respectively. PANC-1 cells presented higher Gal-3 when they were submitted to 24 h of nutritional deprivation alone and simultaneously nutritional and oxygen deprivation. Inhibition of Gal-3 causes a decrease of LC3 levels in all experimental conditions. These results confirm that Gal-3 is modulated by microenvironment factors and the possibility of Gal-3 participating in an adaptive response from PDAC cells to extreme conditions.

Developmental changes induced by exogenous testosterone during early phases of prostate organogenesis.

The aim of this study was to evaluate the impact of prenatal testosterone exposure on prostate development in male and female neonatal gerbils. Pregnant females were exposed to subcutaneous injections of testosterone cypionate (500 µg/animal) at gestational days 20 and 22. Male and female pups were then euthanized at postnatal day 1. Morphological analysis showed that females were severely affected by androgen exposure. We also observed that male and female urogenital sinus (UGS) responded differentially to testosterone treatment, demonstrating heterogeneous immunostaining for the androgen receptor (AR), estrogen receptor alpha (ERα), and proliferating cell nuclear antigen (PCNA). Smooth muscle α-actin (α-SMA) analysis showed that testosterone delays the myodifferentiation, allowing buds to reach the ectopic mesenchymes of the female UGS. Our data showed that abnormal testosterone exposure disrupted prostate organogenesis, altered the expression patterns of important markers, and demonstrated that female UGS was particularly influenced by androgen exposure during a critical window in the developmental period.

The influence of hypertensive environment on adipose tissue remodeling measured by fluorescence lifetime imaging in spontaneously hypertensive rats.

There is a lack of information correlating low adiposity with hypertension experienced by Spontaneous Hypertensive Rats (SHR) or overweight and normotension in Wistar-Kyoto (WKY). We aimed to investigate this lipodystrophy phenomenon by measuring fluorescence lifetime (FLIM), optical redox ratio (ORR), serum levels of hypothalamic-pituitary-adrenal (HPA) and/or hypothalamic-pituitary-thyroid (HPT) hormones axes between Wistar, WKY and SHR before and after establishment of hypertension. Under high blood pressure, we evaluated serum adipokines. Brown adipose tissue was characterized as lower ORR and shorter FLIM compared to white adipose tissue. HPT axis showed a crucial role in the SHR adipose tissue configuration by attenuating whitening. The increased adiposity in WKY may act as a preventive agent for hypertension, since SHR, with low adiposity, establishes the disease. The hypertensive environment can highlight key adipokines that may result in new therapeutic approaches to the treatment of adiposity dysfunctions and hypertension.

Motivating medical students to learn basic science concepts using chronic myeloid leukemia as an integration theme.

OBJECTIVE: To report on the use of chronic myeloid leukemia as a theme of basic clinical integration for first year medical students to motivate and enable in-depth understanding of the basic sciences of the future physician. METHODS: During the past thirteen years we have reviewed and updated the curriculum of the medical school of the Universidade Estadual de Campinas. The main objective of the new curriculum is to teach the students how to learn to learn. Since then, a case of chronic myeloid leukemia has been introduced to first year medical students and discussed in horizontal integration with all themes taught during a molecular and cell biology course. Cell structure and components, protein, chromosomes, gene organization, proliferation, cell cycle, apoptosis, signaling and so on are all themes approached during this course. At the end of every topic approached, the students prepare in advance the corresponding topic of clinical cases chosen randomly during the class, which are then presented by them. During the final class, a paper regarding mutations in the abl gene that cause resistance to tyrosine kinase inhibitors is discussed. After each class, three tests are solved in an interactive evaluation. RESULTS: The course has been successful since its beginning, 13 years ago. Great motivation of those who participated in the course was observed. There were less than 20% absences in the classes. At least three (and as many as nine) students every year were interested in starting research training in the field of hematology. At the end of each class, an interactive evaluation was performed and more than 70% of the answers were correct in each evaluation. Moreover, for the final evaluation, the students summarized, in a written report, the molecular and therapeutic basis of chronic myeloid leukemia, with scores ranging from 0 to 10. Considering all 13 years, a median of 78% of the class scored above 5 (min 74%-max 85%), and a median of 67% scored above 7. CONCLUSION: Chronic myeloid leukemia is an excellent example of a disease that can be used for clinical basic integration as this disorder involves well known protein, cytogenetic and cell function abnormalities, has well-defined diagnostic strategies and a target oriented therapy.

Motivating medical students to learn basic science concepts using chronic myeloid leukemia as an integration theme

Objective: To report on the use of chronic myeloid leukemia as a theme of basic clinical integration for first year medical students to motivate and enable in-depth understanding of the basic sciences of the future physician. Methods: During the past thirteen years we have reviewed and updated the curriculum of the medical school of the Universidade Estadual de Campinas. The main objective of the new curriculum is to teach the students how to learn to learn. Since then, a case of chronic myeloid leukemia has been introduced to first year medical students and discussed in horizontal integration with all themes taught during a molecular and cell biology course. Cell structure and components, protein, chromosomes, gene organization, proliferation, cell cycle, apoptosis, signaling and so on are all themes approached during this course. At the end of every topic approached, the students prepare in advance the corresponding topic of clinical cases chosen randomly during the class, which are then presented by them. During the final class, a paper regarding mutations in the abl gene that cause resistance to tyrosine kinase inhibitors is discussed. After each class, three tests are solved in an interactive evaluation. Results: The course has been successful since its beginning, 13 years ago. Great motivation of those who participated in the course was observed. There were less than 20% absences in the classes. At least three (and as many as nine) students every year were interested in starting research training in the field of hematology. At the end of each class, an interactive evaluation was performed and more than 70% of the answers were correct in each evaluation. Moreover, for the final evaluation, the students summarized, in a written report, the molecular and therapeutic basis of chronic myeloid leukemia, with scores ranging from 0 to 10. Considering all 13 years, a median of 78% of the class scored above ...

Menthol inhibits detrusor contractility independently of TRPM8 activation.

Agonists such as icilin and menthol can activate the cool temperature-sensitive ion channel TRPM8. However, biological responses to menthol may occur independently of TRPM8 activation. In the rodent urinary bladder, menthol facilitates the micturition reflex but inhibits muscarinic contractions of the detrusor smooth muscle. The site(s) of TRPM8 expression in the bladder are controversial. In this study we investigated the regulation of bladder contractility in vitro by menthol. Bladder strips from wild type and TRPM8 knockout male mice (25-30 g) were dissected free and mounted in organ baths. Isometric contractions to carbachol (1 nM-30 µM), CaCl2 (1 µM to 100 mM) and electrical field stimulation (EFS; 8, 16, 32 Hz) were measured. Strips from both groups contracted similarly in response to both carbachol and EFS. Menthol (300 µM) or nifedipine (1 µM) inhibited carbachol and EFS-induced contractions in both wild type and TRPM8 knockout bladder strips. Incubation with the sodium channel blocker tetrodotoxin (1 µM), replacement of extracellular sodium with the impermeant cation N-Methyl-D-Glucamine, incubation with a cocktail of potassium channel inhibitors (100 nM charybdotoxin, 1 µM apamin, 10 µM glibenclamide and 1 µM tetraethylammonium) or removal of the urothelium did not affect the inhibitory actions of menthol. Contraction to CaCl2 was markedly inhibited by either menthol or nifedipine. In cultured bladder smooth muscle cells, menthol or nifedipine abrogated the carbachol or KCl-induced increases in [Ca2+]i. Intravesical administration of menthol increased voiding frequency while decreasing peak voiding pressure. We conclude that menthol inhibits muscarinic bladder contractions through blockade of L-type calcium channels, independently of TRPM8 activation.

Cardioprotective mechanism of S-nitroso-N-acetylcysteine via S-nitrosated betadrenoceptor-2 in the LDLr-/- mice.

Previous studies from our group have demonstrated the protective effect of S-nitroso-N-acetylcysteine (SNAC) on the cardiovascular system in dyslipidemic LDLr-/- mice that develop atheroma and left ventricular hypertrophy after 15 days on a high fat diet. We have shown that SNAC treatment attenuates plaque development via the suppression of vascular oxidative stress and protects the heart from structural and functional myocardial alterations, such as heart arrhythmia, by reducing cardiomyocyte sensitivity to catecholamines. Here we investigate the ability of SNAC to modulate oxidative stress and cell survival in cardiomyocytes during remodeling and correlation with ß2-AR signaling in mediating this protection. Ventricular superoxide (O2⁻) and hydrogen peroxide (H2O2) generation was measured by HPLC methods to allow quantification of dihydroethidium (DHE) products. Ventricular histological sections were stained using terminal dUTP nick-end labeling (TUNEL) to identify nuclei with DNA degradation (apoptosis) and this was confirmed by Western blot for cleaved caspase-3 and caspase-7 protein expression. The findings show that O2⁻ and H2O2 production and also cell apoptosis were increased during left ventricular hypertrophy (LVH). SNAC treatment reduced oxidative stress during on cardiac remodeling, measured by decreased H2O2 and O2⁻ production (65% and 52%, respectively), and a decrease in the ratio of p-Ser1177 eNOS/total eNOS. Left ventricle (LV) from SNAC-treated mice revealed a 4-fold increase in ß2-AR expression associated with coupling change to Gi; ß2-ARs-S-nitrosation (ß2-AR-SNO) increased 61%, while apoptosis decreased by 70%. These results suggest that the cardio-protective effect of SNAC treatment is primarily through its anti-oxidant role and is associated with ß2-ARs overexpression and ß2-AR-SNO via an anti-apoptotic pathway.

Aprendizagem através de role-playing games: uma abordagem para a educação ativa / Learning through role-playing games: an approach for active learning and teaching

This study evaluates the use of role-playing games (RPGs) as a methodological approach for teaching cellular biology, assessing student satisfaction, learning outcomes, and retention of acquired knowledge. First-year undergraduate medical students at two Brazilian public universities attended either an RPG-based class (RPG group) or a lecture (lecture-based group) on topics related to cellular biology. Pre- and post-RPG-based class questionnaires were compared to scores in regular exams and in an unannounced test one year later to assess students' attitudes and learning. From the 230 students that attended the RPG classes, 78.4% responded that the RPG-based classes were an effective tool for learning; 55.4% thought that such classes were better than lectures but did not replace them; and 81% responded that they would use this method. The lecture-based group achieved a higher grade in 1 of 14 regular exam questions. In the medium-term evaluation (one year later), the RPG group scored higher in 2 of 12 questions. RPG classes are thus quantitatively as effective as formal lectures, are well accepted by students, and may serve as educational tools, giving students the chance to learn actively and potentially retain the acquired knowledge more efficiently.

Avaliamos o uso dos role-plying games (RPGs) como uma metodologia de ensino e aprendizagem em Biologia Celular. Alunos do primeiro ano de Medicina participaram de aula expositiva tradicional (grupo referência) ou de aula com RPG (grupo RPG). Comparações foram feitas pela análise de questionários pré- e pós-aula com RPG, das notas nas provas regulares, e das notas obtidas em um teste aplicado um ano após a conclusão da disciplina. Dos 230 alunos que participaram das aulas com RPG, 78,4% responderam que o RPG é uma ferramenta eficaz na aprendizagem; 55,4% acharam que essas aulas são melhores que as aulas tradicionais, mas não as substituem; e 81% responderam que usariam essa metodologia. Os alunos do grupo referência tiveram nota maior em uma de 14 questões das provas regulares; os alunos do grupo RPG tiveram notas maiores em duas de 12 questões aplicadas um ano depois. Portanto, aulas com RPG foram tão eficientes quanto as aulas tradicionais (quantitativamente), são bem aceitas pelos estudantes, e podem ser uma metodologia que lhes dá a chance de aprender ativamente e, potencialmente, levar a uma melhor retenção do conhecimento aprendido.

Gene expression in distinct regions of rat tendons in response to jump training combined with anabolic androgenic steroid administration.

The aim of this study was to evaluate the expression of key genes responsible for tendon remodeling of the proximal and distal regions of calcaneal tendon (CT), intermediate and distal region of superficial flexor tendon (SFT) and proximal, intermediate and distal region of deep flexor tendon (DFT) submitted to 7 weeks of jumping water load exercise in combination with AAS administration. Wistar male rats were grouped as follows: sedentary (S), trained (jumping water load exercise) (T), sedentary animals treated with AAS (5 mg/kg, twice a week) and animals treated with AAS and trained (AAST). mRNA levels of COL1A1, COL3A1, TIMP-1, TIMP-2, MMP-2, IGF-IEa, GAPDH, CTGF and TGF-ß-1 were evaluated by quantitative PCR. Our main results indicated that mRNA levels alter in different regions in each tendon of sedentary animals. The training did not alter the expression of COL1A1, COL3A, IGF-IEa and MMP-2 genes, while AAS administration or its combination with training reduced their expression. This study indicated that exercise did not alter the expression of collagen and related growth factors in different regions of rat tendon. Moreover, the pattern of gene expression was distinct in the different tendon regions of sedentary animals. Although, the RNA yield levels of CT, SFT and DFT were not distinct in each region, these regions possess not only the structural and biochemical difference, but also divergence in the expression of key genes involved in tendon adaptation.

On the road to understanding of the osteoblast adhesion: cytoskeleton organization is rearranged by distinct signaling pathways.

Pre-osteoblast adhesion attracts increasing interest in both medicine and dentistry. However, how this physiological event alters osteoblast phenotype is poorly understood. We therefore attempted to address this question by investigating key biochemical mechanism that governs pre-osteoblast adhesion on polystyrene surface. Importantly, we found that cofilin activity was strongly modulated by PP2A (Ser/Thr phosphatase), while cell-cycle was arrested. Accordingly, we observed that the profile of cofilin phosphorylation (at Ser03) was similar to phospho-PP2A (at Tyr307). Also, it is plausible to suggest during pre-osteoblast adhesion that PP2A phosphorylation at Y307 was executed by phospho-Src (Y416). In addition, it was observed that MAPKp38, but not MAPK-erk, played a key role on pre-osteoblast adhesion by phosphorylating MAPKAPK-2 and ATF-2 (also called CRE-BP1). Also, the up-modulation of RhoA reported here suggests its involvement at the beginning of osteoblast attachment, while Akt remained active during all periods. Altogether, our results clearly showed that osteoblast adhesion is under an intricate network of signaling molecules, which are responsible to guide their interaction with substrate mainly via cytoskeleton rearrangement.

Traque-oclusão intra-útero: técnica cirúrgica assistida à corticoterapia promove aumento nos receptores de VEGF (Vascular Endothelial Growth Factor) em pulmões de fetos de ratas / In utero tracheal occlusion: surgical approach associated to steroid treatment increase VEGF (Vascular Endothelial Growth Factor) receptors in lungs of fetal rats

A hérnia diafragmática congênita (HDC) causa hipoplasia e hipertensãopulmonar e em geral leva a alta morbidade e mortalidade neonatal. Traqueo-oclusão fetal (TO) e corticoterapia pré-natal são alternativas para acelerar o crescimento pulmonar fetal e diminuir a hipoplasia na HDC. A produção de VEGF (Vascular Endothelial Growth Factor) está relacionada com a maturidade pulmonar e sofre alterações na HDC ainda não elucidadas.Materiais e métodos: Seis grupos de 12 fetos de ratos Spreague-Dawley foram comparados: TO, Sham, Controle, TO+Dex, Sham+Dex e Controle+Dex. No dia 18,5º foi realizada TO come sem corticoterapia utilizando dexametasona. No 21,5º dia gestacional os pesos corporal e...

Congenital diaphragmatic hernia (CDH) presents with hypoplastic lungs and usually leads to pulmonary hypertension and high neonatal mortality. Fetal tracheal occlusion (TO) and prenatal corticotherapy are alternatives to accelerate fetal pulmonary growth and decrease hypoplasia in CDH. VEGF (Vascular Endothelial Growth Factor)production and surfactant production by type II pneumocytes are related with pulmonary maturity and are altered in CDH, but little has been described about VEGF receptors. Our objective wasto quantify the receptors of VEGF (VEGFR) and type II pneumocytes, verifying the effects of TO and corticotherapy on normal lungs of fetal rats...

Fluorescence and confocal laser scannig microscopy of H&E staned sections for the study of elastic fiber in skin and some skin disorders

Elastic fibers are important components of the skin and contribute to its biomechanical properties. Primary changes in elastic fiber structure are responsible for some diseases while modifications of the elastic fibers results from disorders in other extracellular matix components. We have previously suggested that examination of H&E stained sections by fluorence microscopy allows for good imaging of elastic fiber structure and distribution. Since these paramenters are important for the chacacterization of elastic fibers and for diagnosing some skin diseases, we show in this paper that dermal elastic fibers are consistently imaged by H&E plus fluorescence microscopy, by the analysis of normal and diseased skin samples, and that the use of confocal laser scanning microscopy greatly increases the image qualitity, which in turn will permit image analysis to be carried out. We are clearly convinced that the proposed procedure is a revelant tool in elastic fiber research and the present tecnique is of interest to a large group of investigators using H&E as a routine method.

Aspectos biológicos e moleculares das lectinas / Biological and molecular aspects of lectins

Alguns aspectos da estrutura e do modo de açäo das lectinas säo brevemente revisados. Especial ênfase é dada as funçöes biológicas dessas moléculas, bem como às suas aplicaçöes como ferramentas em diversas áreas