Unraveling the role of cell metabolism in the molecular mechanisms of resistance in cancer. Encapsulation of glycolytic inhibitors in nanoparticles to increase their release in a lung cancer model.

A maioria dos tumores sólidos apresenta um metabolismo alterado, caracterizado pela elevada dependência da fermentação lática, mesmo em normóxia, sendo uma característica emergente das células tumorais. O aumento do fluxo glicolítico induz uma acidez no espaço extracelular, potenciando características mais agressivas das células tumorais, como a capacidade de migração e a resistência à terapia. Como o MCT1 e o MCT4 desempenham um papel na regulação do pH intracelular, exportando o lactato, apesentam uma sobreexpressão em tumores glicolíticos. Assim, o metabolismo alterado pode ser um alvo para novas terapias, nomeadamente a utilização de inibidores glicolíticos (GIs), inibindo o metabolismo celular e modificando o microambiente tumoral, de forma a afetar os mecanismos envolvidos na quimiorresistência. Deste modo, estudou-se o efeito de GIs (3-bromopiruvato (3BP), dicloroacetato (DCA) e 2-desoxiglicose (2DG)) nas propriedades das células tumorais e no fenótipo de multirresistência, usando como modelo linhas celulares derivadas de cancro de pulmão. Todos os compostos levaram à perda da viabilidade celular, sendo o efeito no metabolismo celular, na migração e na proliferação dependente do composto e da linha celular. Como o MCT1, o MCT4 e a chaperona CD147, estão envolvidos no efluxo de lactato e, no caso do 3BP, no influxo deste, analisou-se a sua expressão basal. Contudo, observou-se que a expressão basal do MCT1, do MCT4 e da CD147 não se correlacionava com a citotoxicidade dos GIs, demonstrando que outros fatores podem estar envolvidos no seu mecanismo de ação. De entre os GIs testados, o DCA apresentou um maior efeito inibitório sobre o metabolismo e a proliferação celular. O tratamento com DCA promoveu uma redução no consumo de glicose e na produção de ATP e de lactato nas linhas celulares A549 e NCI-H460. Apesar do efeito observado no metabolismo, foi apenas observado um pequeno efeito na inibição da migração. Apenas o 3BP foi capaz de induzir inibição da migração, e apenas na linha celular NCI-H460. Os resultados mostraram que as linhas celulares testadas apresentam baixa migração em condições basais e, consequentemente, os GIs não tiveram grande impacto nesta característica. Analisou-se o efeito do DCA na sensibilidade das células a quimioterápicos convencionais, como o Paclitaxel (PTX). Observou-se uma diminuição de 2,7 vezes e de 10 vezes no valor de IC50 do PTX nas linhas A549 e NCI-H460, respetivamente, mostrando que o DCA torna as células mais sensíveis ao PTX. Para aumentar a concentração intracelular de DCA, produziram-se nanopartículas de poli(ácido lático-co-glicólico) (PLGA NPs) contendo DCA. Para concentrações elevadas de DCA, a encapsulação aumentou a sua toxicidade em células tumorais. As PLGA NPs contendo DCA mostraram ser um sistema de entrega promissor para aumentar o efeito antitumoral do DCA. Esses resultados podem ajudar a encontrar uma nova estratégia de tratamento, através da terapia combinada, abrindo portas para novas abordagens de tratamento. Este estudo sugere que o metabolismo tumoral é um ator importante na tumorigénese e no fenótipo agressivo das células tumorais. Bloqueando os principais atores pode comprometer-se o mecanismo responsável pelo insucesso do tratamento e melhorar as opções terapêuticas utilizadas na prática clínica.Most solid tumors present an altered metabolism characterized by a high dependence on lactic acid fermentation, even in the presence of oxygen, which is an emergent hallmark of cancer cells. The glycolytic flux increase induces the acidification of the extracellular space and boosts the more aggressive characteristics of tumor cells, such as increased migration ability and resistance to therapy. Since MCT1 and MCT4 play a role in intracellular pH, by exporting the accumulating lactic acid, they are upregulated in glycolytic tumors. Therefore, the altered metabolism can be an excellent target for new therapies in the cancer field, namely through the use of glycolytic inhibitors (GIs), which can inhibit cell metabolism and modify tumor microenvironment, affecting mechanisms involved in chemotherapy resistance. Therefore, we studied the effect of GIs (3-bromopyruvate (3BP), dichloroacetate (DCA) and 2-deoxyglucose (2DG)) on cancer cell properties and on the multidrug resistance phenotype, using lung cancer cells. All compounds led to loss of cell viability, with the effect on cell metabolism, migration and proliferation being dependent on the drug and cell line assayed. As MCT1 and MCT4, as well as their chaperone CD147, are involved in lactate efflux and, in the case of 3BP, in its influx, we analyzed their basal expression. However, we observed that MCT1, MCT4 and CD147 basal expression was not correlated with the GI’s cytotoxicity, demonstrating that other factors should be involved in their mechanism of action. Among the GIs assayed, DCA was the most promising one, since it presented the highest inhibitory effect on cell metabolism and proliferation. DCA treatment led to a reduction in glucose consumption and in ATP and lactate production in A549 and NCI-H460 cell lines. In spite of the effect observed on metabolism, only a small effect was observed on the migratory capacity inhibition. In this case, only 3BP was able to induce some migration inhibition, and only in the NCI-H460 cell line. Our results showed that the cell lines assayed intrinsically exhibit a low migratory capacity in basal conditions and, consequently, the GIs did not have a major impact on this feature. We then analyzed DCA effect on the sensitivity of lung cancer cells to conventional chemotherapeutic agents namely Paclitaxel (PTX). A 2.7-fold and a 10-fold decrease in PTX IC50 value were observed in the A549 and NCI-H460 cell lines, respectively, showing that DCA sensitizes cells to PTX. To increase the intracellular DCA concentration, thereby potentiating its effect, DCA-loaded poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) were produced. It was found that for higher DCA concentrations, encapsulation increased its toxicity. Overall, DCA-loaded PLGA NPs showed to be a promising drug delivery system to enhance DCA anti-tumoral effect. These results may help finding a new treatment strategy, through combined therapy, which could open doors to new treatment approaches. This study suggests that tumor metabolism is an important player in tumorigenesis and in the aggressive cancer cell phenotype. Blocking the main actors involved in this relationship can disrupt the mechanism responsible for treatment failure and improve existing therapeutic options used in clinical practice

Disentangling protostellar evolutionary stages in clustered environments using Spitzer-IRS spectra and comprehensive SED modeling

When studying the evolutionary stages of protostars that form in clusters, the role of any intracluster medium cannot be neglected. High foreground extinction can lead to situations where young stellar objects (YSOs) appear to be in earlier evolutionary stages than they actually are, particularly when using simple criteria like spectral indices. To address this issue, we have assembled detailed SED characterizations of a sample of 56 Spitzer-identified candidate YSOs in the clusters NGC 2264 and IC 348. For these, we use spectra obtained with the Infrared Spectrograph onboard the Spitzer Space Telescope and ancillary multi-wavelength photometry. The primary aim is twofold: 1) to discuss the role of spectral features, particularly those due to ices and silicates, in determining a YSO's evolutionary stage, and 2) to perform comprehensive modeling of spectral energy distributions (SEDs) enhanced by the IRS data. The SEDs consist of ancillary optical-to-submillimeter multi-wavelength data as well as an accurate description of the 9.7 micron silicate feature and of the mid-infrared continuum derived from line-free parts of the IRS spectra. We find that using this approach, we can distinguish genuine protostars in the cluster from T Tauri stars masquerading as protostars due to external foreground extinction. Our results underline the importance of photometric data in the far-infrared/submillimeter wavelength range, at sufficiently high angular resolution to more accurately classify cluster members. Such observations are becoming possible now with the advent of the Herschel Space Observatory.Comment: Accepted for publication in Ap

mRNA transcription profile of potato (Solanum tuberosum L.) exposed to ultrasound during different stages of in vitro plantlet development

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PHYSICAL CONNECTION BETWEEN BVRF SEGMENTS BASED ON LEVELING ASSOCIATED WITH GRAVIMETRY

Considering the efforts to establish Global Reference Systems linked to the geopotential space, new alternatives are sought to address the problems found in the classic national vertical networks. The Brazilian Vertical Reference Frame (BVRF) was materialized in two different segments with independent datums (Imbituba and Santana tide gauges) due to the terrain difficulties for conventional leveling. The 2018 BVRF realization, in the geopotential space, still remains without interoperability between its segments. We analyze alternatives for physical connection based on the new precepts of the International Association of Geodesy (IAG) involving the geopotential space. Some proposed solutions for physical connection based on GPS leveling associated with gravimetry are presented. These solutions were developed with the aim of evidencing the discrepancy between the two BVRF segments, now carried out in terms of geopotential numbers and normal heights. The results indicate differences ranging from about 45 cm to 140 cm between the two segments depending on the strategy employed. Comparisons with previous determinations based on indirect strategies and involving previous BVRF realizations are made

Análise populacional e funcional dos polimorfismos genéticos CYP2D6*4 e CYP2C19*17 do citocromo P450 e C3435T do gene MDR1

A variabilidade da resposta aos fármacos deve-se, em grande parte, a fatores genéticos. Polimorfismos em genes que codificam enzimas metabolizadoras de xenobióticos, como as enzimas CYP2D6 e CYP2C19 do citocromo P450 (CYP450), e em genes codificantes de proteínas que promovem o seu efluxo, como o gene MDR1, codificante da glicoproteína-P (Pgp), podem afetar a eficácia de um tratamento farmacológico ou a sua toxicidade, determinando assim a variabilidade interindividual na resistência a fármacos. Como tal, e porque os genes que codificam estas proteínas são extremamente polimórficos torna-se necessária uma análise genética e funcional aprofundada. Com o intuito de prever a incidência de algumas variações genéticas na população Portuguesa, procedeu-se ao estudo dos polimorfismos CYP2D6*4 e CYP2C19*17, do CYP450, e C3435T do gene MDR1. Estes polimorfismos foram pesquisados quer por PCR-RFLP quer por High Resolution Melting (HRM) em amostras de DNA genómico extraído de dadores voluntários do Instituto Superior de Ciências da Saúde - Norte. Os resultados obtidos permitiram identificar diferentes genótipos, tendo-se verificado uma frequência de 7,4% para o polimorfismo CYP2C19*17, de 7,7% para o polimorfismo CYP2D6*4 e, por último, de 13,0% para o polimorfismo C3435T. De forma a complementar o estudo populacional, o polimorfismo C3435T do gene MDR1 foi também genotipado em linhas celulares tumorais de adenocarcinoma do intestino, HCT-15, HT29-MTX e Caco-2, com o objetivo de determinar a influência da presença do polimorfismo na funcionalidade da Pgp. Assim, foram realizados ensaios de resistência à doxorrubicina, um fármaco antitumoral substrato da Pgp, que permitissem avaliar uma possível relação entre a presença/ausência dos polimorfismos e diferentes perfis de metabolização. Os resultados obtidos demonstraram que a linha celular HCT-15 de genótipo TT apresentou uma maior resistência ao fármaco do que as restantes linhas de genótipo CT. Foi também observado que a linha HCT-15 apresentava uma maior expressão da Pgp, e que esta era induzida após exposição ao fármaco, o que poderá estar associado à presença do polimorfismo C3435T. Contudo, a existência de um perfil genético e metabólico diferente nas linhas celulares utilizadas impossibilita a determinação do papel específico do polimorfismo C3435T na funcionalidade do gene MDR1 por esta abordagem. Assim, com o intuito de relacionar a presença do polimorfismo com o efluxo da doxorrubicina, efetuou-se a expressão heteróloga do gene MDR1 na presença e na ausência do polimorfismo na estirpe Saccharomyces cerevisiae AD1-8 desprovida dos principais transportadores de fármacos. Os resultados obtidos demonstraram que a proteína apresentava funcionalidade neste sistema heterólogo, demonstrando assim que a levedura S. cerevisiae constitui um bom modelo para estudos funcionais de polimorfismos do gene MDR1 humano

Angiotensin ii therapy in refractory septic shock: which patient can benefit most? A narrative review

Angiotensina II; Shock sèptic; VasopressorsAngiotensin II; Septic shock; VasopressorsAngiotensina II; Shock séptico; VasopresoresPatients with septic shock who experience refractory hypotension despite adequate fluid resuscitation and high-dose noradrenaline have high mortality rates. To improve outcomes, evidence-based guidelines recommend starting a second vasopressor, such as vasopressin, if noradrenaline doses exceed 0.5 µg/kg/min. Recently, promising results have been observed in treating refractory hypotension with angiotensin II, which has been shown to increase mean arterial pressure and has been associated with improved outcomes. This narrative review aims to provide an overview of the pathophysiology of the renin-angiotensin system and the role of endogenous angiotensin II in vasodilatory shock with a focus on how angiotensin II treatment impacts clinical outcomes and on identifying the population that may benefit most from its use

Tuberculin skin testing versus interferon-gamma release assay among users of a public health unit in Northeast Portugal

he screening of groups with a high risk for developing tuberculosis (TB) is a priority in order to control this disease. Since there is no gold standard for the diagnosis of latent TB infection (LTBI), both the tuberculin skin test (TST) and the interferon-gamma release assays (IGRA) have been used for this purpose. The aim of this study was to determine the proportion of LTBI by using the TST and the IGRA tests, and to assess the risk factors related with discordant results between tests across several risk groups advised for screening in Northeast Portugal. Data were collected from the database of patients with suspected LTBI and advised for the screening in a public health unit (January 2014 to December 2015). The proportion of LTBI was computed using both tests. Logistic regression models assessed risk factors for a positive test and for discordant results between tests. The adjusted odds ratio (OR) and respective 95% confidence interval (95% CI) were obtained. Out of 367 patients included in the analysis, 79.8% had a positive TST and 46.0% of them had a positive IGRA. In comparison with contacts of active TB cases, healthcare workers and inmates presented higher odds of TST positivity (OR 4.38, 95% CI 1.59–12.09 and OR 4.74, 95% CI 1.45–15.49, respectively), but immunocompromised people presented lower odds of TST positivity (OR 0.14; 95% CI 0.06–0.31). Instead, healthcare workers (OR 0.44, 95% CI 0.24–0.80) and immunocompromised people (OR 0.24, 95% CI 0.10–0.56) presented lower odds of a positive IGRA. There were 42.0% concordant positive results, 16.1% concordant negative results, and 41.9% discordant results, with healthcare workers presenting higher odds of discordant results (OR 3.34, 95% CI 1.84–6.05). The proportion of LTBI estimated by TST and IGRA among people advised for screening in our setting is high, highlighting the need of preventive strategies. Among healthcare workers, TST results should be read with caution as the higher proportion of discordant results with a positive TST suggests the impact of the booster reaction in this group.info:eu-repo/semantics/publishedVersio

Contributions of IFN-gamma and granulysin to the clearance of Plasmodium yoelii blood stage

P. vivax-infected Retics (iRetics) express human leukocyte antigen class I (HLA-I), are recognized by CD8+ T cells and killed by granulysin (GNLY) and granzymes. However, how Plasmodium infection induces MHC-I expression on Retics is unknown. In addition, whether GNLY helps control Plasmodium infection in vivo has not been studied. Here, we examine these questions using rodent infection with the P. yoelii 17XNL strain, which has tropism for Retics. Infection with P. yoelii caused extramedullary erythropoiesis, reticulocytosis and expansion of CD8+CD44+CD62L- IFN-gamma-producing T cells that form immune synapses with iRetics. We now provide evidence that MHC-I expression by iRetic is dependent on IFN-gamma-induced transcription of IRF-1, MHC-I and beta2-microglobulin (beta2-m) in erythroblasts. Consistently, CTLs from infected wild type (WT) mice formed immune synapses with iRetics in an IFN-gamma- and MHC-I-dependent manner. When challenged with P. yoelii 17XNL, WT mice cleared parasitemia and survived, while IFN-gamma KO mice remained parasitemic and all died. beta2-m KO mice that do not express MHC-I and have virtually no CD8+ T cells had prolonged parasitemia, and 80% survived. Because mice do not express GNLY, GNLY-transgenic mice can be used to assess the in vivo importance of GNLY. Parasite clearance was accelerated in GNLY-transgenic mice and depletion of CD8+ T cells ablated the GNLY-mediated resistance to P. yoelii. Altogether, our results indicate that in addition to previously described mechanisms, IFN-gamma promotes host resistance to the Retic-tropic P. yoelii 17XNL strain by promoting MHC-I expression on iRetics that become targets for CD8+ cytotoxic T lymphocytes and GNLY