Impact of glycerol feeding profiles on STEAP1 biosynthesis by Komagataella pastoris using a methanol-inducible promoter.

Currently, the lack of reliable strategies for the diagnosis and treatment of cancer makes the identification and characterization of new therapeutic targets a pressing matter. Several studies have proposed the Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) as a promising therapeutic target for prostate cancer. Although structural and functional studies may provide deeper insights on the role of STEAP1 in cancer, such techniques require high amounts of purified protein through biotechnological processes. Based on the results presented, this work proposes the application, for the first time, of a fed-batch profile to improve STEAP1 biosynthesis in mini-bioreactor Komagataella pastoris X-33 Mut+ methanol-induced cultures, by evaluating three glycerol feeding profiles-constant, exponential, and gradient-during the pre-induction phase. Interestingly, different glycerol feeding profiles produced differently processed STEAP1. This platform was optimized using a combination of chemical chaperones for ensuring the structural stabilization and appropriate processing of the target protein. The supplementation of culture medium with 6 % (v/v) DMSO and 1 M proline onto a gradient glycerol/constant methanol feeding promoted increased biosynthesis levels of STEAP1 and minimized aggregation events. Deglycosylation assays with peptide N-glycosidase F showed that glycerol constant feed is associated with an N-glycosylated pattern of STEAP1. The biological activity of recombinant STEAP1 was also validated, once the protein enhanced the proliferation of LNCaP and PC3 cancer cells, in comparison with non-tumoral cell cultures. This methodology could be a crucial starting point for large-scale production of active and stable conformation of recombinant human STEAP1. Thus, it could open up new strategies to unveil the structural rearrangement of STEAP1 and to better understand the biological role of the protein in cancer onset and progression.

Glycolysis Inhibition as a Strategy for Hepatocellular Carcinoma Treatment?

Hepatocellular carcinoma (HCC) is the most frequently detected primary malignant liver tumor, representing a worldwide public health problem due to its high morbidity and mortality rates. The HCC is commonly detected in advanced stage, precluding the use of treatments with curative intent. For this reason, it is crucial to find effective therapies for HCC. Cancer cells have a high dependence of glycolysis for ATP production, especially under hypoxic environment. Such dependence provides a reliable possible strategy to specifically target cancer cells based on the inhibition of glycolysis. HCC, such as other cancer types, presents a clinically well-known upregulation of several glycolytic key enzymes and proteins, including glucose transporters particularly glucose transporter 1 (GLUT1). Such enzymes and proteins constitute potential targets for therapy. Indeed, for some of these targets, several inhibitors were already reported, such as 2-Deoxyglucose, Imatinib or Flavonoids. Although the inhibition of glycolysis presents a great potential for an anticancer therapy, the development of glycolytic inhibitors as a new class of anticancer agents needs to be more explored. Herein, we propose to summarize, discuss and present an overview on the different approaches to inhibit the glycolytic metabolism in cancer cells, which may be very effective in the treatment of HCC.

Targeting STEAP1 Protein in Human Cancer: Current Trends and Future Challenges.

Cancer is a global health issue that impairs the life quality of patients and origins thousands of deaths annually worldwide. Six-transmembrane epithelial antigen of the prostate (STEAP1) was identified to be overexpressed in several types of cancers, namely in prostate cancer (PCa). Considering its secondary structure, associated with its location in the cell membrane, has been suggested a role in intercellular communication between tumour cells. Taking into account its high specificity and overexpression in human cancers, STEAP1 is nowadays a promising candidate to be imposed as a therapeutic target. Several strategies have been developed during the last few years for targeting STEAP1, including antibody-drug conjugates, monoclonal antibodies (mAbs), DNA vaccines and small noncoding RNAs (ncRNAs). This review presents the current knowledge about STEAP1 protein expression in human tissues, its biochemical properties and targeting strategies with the purpose to evaluate its potential as therapeutic agent for cancer.

Biologia floral e sistema reprodutivo da erva-baleeira (Varronia curassavica Jacq. ) / Flowering phenology and reproductive system of the Cordia (Varronia curassavica Jacq. )

RESUMOO conhecimento do sistema reprodutivo é fundamental para a conservação e manejo de uma espécie. O objetivo deste trabalho foi descrever a fenologia da floração, a antese, registrar os insetos visitantes no período de floração, determinar as características morfométricas das flores e o sistema reprodutivo da erva-baleeira, em um ambiente de Cerrado do Norte de Minas Gerais. Entre maio a dezembro de 2012 foi caracterizado o comportamento fenológico da floração. Na análise da fenologia floral foi determinado: o crescimento da inflorescência, o número de flores e frutos por inflorescências. Utilizou-se seis acessos que tiveram dez inflorescências marcadas em cada acesso, totalizando 60 inflorescências. A antese foi determinada utilizando quatro inflorescências em duas plantas. Os visitantes florais foram observados in loco e capturados em três dias consecutivos de coleta. As características morfométricas foram determinadas com paquímetro utilizando 20 flores, sendo cinco flores de quatro acessos. Para determinar o sistema reprodutivo utilizou-se a razão pólen:óvulo (P:O), utilizando 50 flores, sendo 10 flores de cinco acessos em pré-antese. Nas condições de Montes Claros, o crescimento das inflorescências de erva-baleeira ocorreu entre meados de agosto e início de outubro, totalizando 45 dias. O florescimento foi observado entre meados de setembro e final de outubro, enquanto a frutificação ocorreu de meados de outubro a início de dezembro, sendo que ambos ocorreram de forma irregular. A antese floral de erva-baleeira, neste estudo, ocorre entre 7:00 e 11:00 horas. Os insetos visitantes pertencem as ordens Coleoptera, Hemiptera, Diptera e Hymenoptera. As flores apresentaram o diâmetro de 2,13 ± 0,05 (mm), o comprimento de 3,29 ± 0,08 (mm), diâmetro do ovário de 0,70 ± 0,02 (mm), o comprimento do ovário de 2,48 ± 0,12 (mm), o diâmetro da antera de 0,67 ± 0,01(mm) e o comprimento da antera de 0,93 ±0,02 (mm), quatro óvulos e cinco anteras por flor. A razão P:O foi de 576,542, indicando que a espécie é alógama facultativa.

ABSTRACTThe knowledge of the reproductive system is essential for the conservation and management of the species. This study aimed on several procedures, as follows: to describe the phenology of flowering, the anthesis; to record the visiting insects during flowering and to determine the morphometric characteristics of the flowers and the reproductive system of the "erva-baleeira", in an environment of Northern Cerrado in the State of Minas Gerais. From May to December 2012, the flowering phenology of six access was characterized. In the analyzes of floral phenology it were determined the growth of the inflorescence and the number of flowers and fruits per inflorescence by using six access which had ten inflorescences marked in each access l, totalizing sixty inflorescences. The anthensis was assessed using four inflorescences in two plants. The flower`s visitors were observed live and captured in three consecutive days of sampling. The morphometric characteristics were determined with a caliper using 20 flowers, with five flowers from four access. In order to determine the reproductive system it was employed a ratiopollen: ovule (P: O) with 50 flowers and 10 of them belonging to five accesses in the pre-anthesis. Under the conditions of Montes Claros, the growth of inflorescences from Cordia occurred between mid-August and early October, totalizing 45 days. The flowering was observed between mid-September and late October, and the fruiting occurred from mid-October to early December. Both phases happened irregularly. The anthesis of the Cordia, in this study, occurred between 7:00 and 11:00 o`clock.. The visiting insects identified were from the orders of Coleoptera, Hemiptera, Diptera and Hymenoptera. The flowers exhibited a diameter of 2.13 ± 0.05 (mm), length of 3.29 ± 0.08 (mm), diameter of 0.70 ± 0.02 Ovarian (mm), the length of the ovary was 2.48 ± 0.12 (mm), the diameter of the anther 0.67 ± 0.01 (mm) and the length anther was 0.93 ± 0.02 (mm), with five anthers and four ovules per flower. The reason P: O was 576.542, indicating that the species is facultative allogamous.

Reguladores vegetais e substratos no enraizamento de estacas de erva-baleeira (Varronia curassavica Jacq. ) / Growth regulators and substrates on rooting of Varronia curassavica Jacq

A erva-baleeira, espécie medicinal anti-inflamatória, pode ser propagada por sementes, no entanto, o desenvolvimento de métodos eficientes para a propagação vegetativa possibilitará a uniformidade nas populações e a clonagem de plantas de interesse quanto aos aspectos agronômico e de composição química. O presente trabalho objetivou avaliar os efeitos do ácido naftaleno acético (NAA) e do ácido indolbutírico (IBA) e de diferentes substratos no enraizamento de estacas semi-lenhosas de erva-baleeira. Os experimentos foram conduzidos em casa de vegetação em Montes Claros em delineamento inteiramente casualizados. Para o experimento teste da influência das auxinas, as estacas foram submetidas a tratamentos com NAA e IBA nas concentrações de 0, 500, 1000, 2000, 3000 e 4000 mg L-1, e para o experimento dos substratos, cinco substratos foram avaliados (vermiculita expandida + casca de arroz parcialmente carbonizada (proporção de 1:1); vermiculita expandida; substrato orgânico comercial para hortaliça (Tropstrato HT ®); areia; solo + compostagem (proporção de 1:1)). Após 90 dias, para ambos os experimentos, foram avaliadas porcentagem de estacas enraizadas, de estacas mortas, de estacas vivas com calos, de estacas mortas com calos, número de raízes formadas por estaca e comprimento das três maiores raízes (cm). O regulador vegetal ideal para o enraizamento da espécie é o IBA, na concentração de 2.000 mg L-1, sendo que o NAA não é recomendado para a indução radicial em estacas erva-baleeira. O substrato vermiculita com casca de arroz carbonizada na proporção de 1:1 proporcionou maior porcentagem de enraizamento nas estacas da erva-baleira que o substrato solo + compostagem (1:1), os demais substratos não diferenciaram entre si.

The Varronia curassavica Jacq., black sage, is an anti-inflammatory medicinal species that can be propagated by seed; however, the development of efficient methods for vegetative propagation will enable uniformity in the populations and cloning of plants of interest regarding the agronomic aspects and chemical composition. The aim of this study was to assess the effects of the naphthalene acetic acid (NAA) and indole butyric acid (IBA) and different substrates on the rooting of semi-hardwood cuttings of black sage. The experiments were conducted in a greenhouse at Montes Claros, in the State of Minas Gerais, Brazil, as a randomized complete design. For the experiment to test the influence of auxin, the cuttings were subjected to treatments with NAA and IBA at the concentrations of 0, 500, 1000, 2000, 3000 and 4000 mg L-1, and for the experiment with substrates, five substrates were evaluated (expanded vermiculite + carbonized rice husk (1:1); expanded vermiculite; commercial organic substrate for vegetable (Tropstrato ® HT); sand; soil + compost (1:1)). After 90 days, in both experiments, we evaluated the rooting percentage of cuttings, dead cuttings, live cuttings with callus, dead cuttings with callus, number of roots per cutting and length of the three longest roots (cm). The best plant growth regulator for the rooting of the species is the IBA at a concentration of 2.000 mg L-1, and the NAA is not recommended for the induction of roots in cuttings of black sage. Vermiculite with carbonized rice husk in the ratio of 1:1 showed greater percentage of rooting in cuttings of black sage than soil + composting (1:1); other substrates did not differ among themselves.

Allergic asthma exhaled breath metabolome: a challenge for comprehensive two-dimensional gas chromatography.

Allergic asthma represents an important public health issue, most common in the paediatric population, characterized by airway inflammation that may lead to changes in volatiles secreted via the lungs. Thus, exhaled breath has potential to be a matrix with relevant metabolomic information to characterize this disease. Progress in biochemistry, health sciences and related areas depends on instrumental advances, and a high throughput and sensitive equipment such as comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-ToFMS) was considered. GC×GC-ToFMS application in the analysis of the exhaled breath of 32 children with allergic asthma, from which 10 had also allergic rhinitis, and 27 control children allowed the identification of several hundreds of compounds belonging to different chemical families. Multivariate analysis, using Partial Least Squares-Discriminant Analysis in tandem with Monte Carlo Cross Validation was performed to assess the predictive power and to help the interpretation of recovered compounds possibly linked to oxidative stress, inflammation processes or other cellular processes that may characterize asthma. The results suggest that the model is robust, considering the high classification rate, sensitivity, and specificity. A pattern of six compounds belonging to the alkanes characterized the asthmatic population: nonane, 2,2,4,6,6-pentamethylheptane, decane, 3,6-dimethyldecane, dodecane, and tetradecane. To explore future clinical applications, and considering the future role of molecular-based methodologies, a compound set was established to rapid access of information from exhaled breath, reducing the time of data processing, and thus, becoming more expedite method for the clinical purposes.

GDNF contributes to oestrogen-mediated protection of midbrain dopaminergic neurones.

Parkinson's disease (PD) is characterised by the preferential loss of dopaminergic neurones from the substantia nigra (SN) that leads to the hallmark motor disturbances. Animal and human studies suggest a beneficial effect of oestrogen to the nigrostriatal system, and the regulation of neurotrophic factor expression by oestrogens has been suggested as a possible mechanism contributing to that neuroprotective effect. The present study was designed to investigate whether the neuroprotection exerted by 17ß-oestradiol on nigrostriatal dopaminergic neurones is mediated through the regulation of glial cell line-derived neurotrophic factor (GDNF) expression. Using an in vivo rat model of PD, we were able to confirm the relevance of 17ß-oestradiol in defending dopaminergic neurones against 6-hydroxydopamine (6-OHDA) toxicity. 17ß-oestradiol, released by micro-osmotic pumps, implanted 10 days before intrastriatal 6-OHDA injection, prevented the loss of dopaminergic neurones induced by 6-OHDA. 17ß-oestradiol treatment also promoted an increase in GDNF protein levels both in the SN and striatum. To explore the relevance of GDNF increases to 17ß-oestradiol neuroprotection, we analysed, in SN neurone-glia cultures, the effect of GDNF antibody neutralisation and RNA interference-mediated GDNF knockdown. The results showed that both GDNF neutralisation and GDNF silencing abolished the dopaminergic protection provided by 17ß-oestradiol against 6-OHDA toxicity. Taken together, these results strongly identify GDNF as an important player in 17ß-oestradiol-mediated dopaminergic neuroprotection.

Profiling allergic asthma volatile metabolic patterns using a headspace-solid phase microextraction/gas chromatography based methodology.

Allergic asthma represents an important public health issue with significant growth over the years, especially in the paediatric population. Exhaled breath is a non-invasive, easily performed and rapid method for obtaining samples from the lower respiratory tract. In the present manuscript, the metabolic volatile profiles of allergic asthma and control children were evaluated by headspace solid-phase microextraction combined with gas chromatography-quadrupole mass spectrometry (HS-SPME/GC-qMS). The lack of studies in breath of allergic asthmatic children by HS-SPME led to the development of an experimental design to optimize SPME parameters. To fulfil this objective, three important HS-SPME experimental parameters that influence the extraction efficiency, namely fibre coating, temperature and time extractions were considered. The selected conditions that promoted higher extraction efficiency corresponding to the higher GC peak areas and number of compounds were: DVB/CAR/PDMS coating fibre, 22 °C and 60 min as the extraction temperature and time, respectively. The suitability of two containers, 1L Tedlar® bags and BIOVOC®, for breath collection and intra-individual variability were also investigated. The developed methodology was then applied to the analysis of children exhaled breath with allergic asthma (35), from which 13 had also allergic rhinitis, and healthy control children (15), allowing to identify 44 volatiles distributed over the chemical families of alkanes (linear and ramified) ketones, aromatic hydrocarbons, aldehydes, acids, among others. Multivariate studies were performed by Partial Least Squares-Discriminant Analysis (PLS-DA) using a set of 28 selected metabolites and discrimination between allergic asthma and control children was attained with a classification rate of 88%. The allergic asthma paediatric population was characterized mainly by the compounds linked to oxidative stress, such as alkanes and aldehydes. Furthermore, more detailed information was achieved combining the volatile metabolic data, suggested by PLS-DA model, and clinical data.

Detection of rancid defect in virgin olive oil by the electronic nose.

A sensor array of 32 conducting polymer sensors has been used to detect the rancid defect in virgin olive oils. A training set, composed of admixtures of a Portuguese virgin olive oil with different percentages (0-100%) of a rancid standard oil, was used for the selection of the best sensors classifying correctly the samples. Information on volatile compounds responsible for rancidity and the sensory evaluation of samples by assessors were used for explaining the mathematical selection of sensors. A tentative calibration, using unsupervised procedures (PCA and MDS) and a nonlinear regression, was carried out, with the training set, and later confirmed with a test set with which rancid commercial samples of different varieties were used to spike a Greek extra virgin olive oil at low levels of rancidity (0.5-6%).

[Agents of nursing in São Paulo health practices: from the bacteriological model to the health program (1889-1983)]. / Os agentes de enfermagem nas práticas sanitárias paulistas: do modelo bacteriológico à programação em saúde (1889-1983).

The historical roots of the nursing profession in public health in the state of São Paulo from 1900-1980 have been identified. Nurses have effectively participated to implement a new public health program--"Programação em Saúde", having the assignment to train, coordinate, supervise/manage other nurses who lacked specialized training. The nurse's understanding of her role in the health process was limited to individual performance with emphasis on technical ability, without understanding the purpose of her actions in the overall model.

Solucoes alternativas para hidratacao oral em pediatria. II. - Variacoes na concentracao de sodio e sacarose observadas na preparacao de uma solucao hidratante caseira. / Alternative solutions for oral hydration in Pediattrics. II - Variations in the concentration of sodium and sucrose observed in the preparation of a homemade hydrating solution

A quatro grupos de adultos foi pedido que preparassem uma solucao hidratante caseira utilizando a tampinha da garrafa de refrigerantes ("tampinha") para medida de sal e acucar refinados e a mamadeira para agua.O grupo I foi constituido de 13 tecnicos de laboratorio e dois medicos, que prepararam a solucao em duplicatas, o grupo II constituido de 30 estudantes de Enfermagem; o grupo III constituido de 30 maes registradas num Posto de Saude que prepararam a solucao na propria instituicao, mediante receita e orientacao; o grupo IV de outras 30 maes do mesmo Posto que prepararam a solucao em suas proprias casas apos orientacao e demonstracao.Os resultados mostraram que os valores medios das amostras do grupo I correspondem aproximadamente aos que seriam esperados utilizando-se as medidas preconizadas. Os devios padroes para o sodio e osmolalidade determinada pela sacarose foram maiores nos grupos II, III e IV em relacao ao grupo I indicando maior dispersao dos valores. A maior amplitude entre os valores extremos de concentracao de sodio e sacarose foi observada no grupo II. O valor mais elevado de sodio foi observado no grupo IV (76 mEq/L) e o menor no grupo II (6,5 mEq/L). Em conclusao podemos dizer que, embora a amplitude dos valores observados nos grupos de maes seja maior que naquele grupo habituado a trabalhar com medidas mais exatas, os extremos obtidos nao atingem niveis de erros muito elevados que impecam o emprego da "tampinha" como utensilio de medida do sal e acucar na preparacao da solucao hidratante caseira