microRNAs as biomarkers of cancer drug resistance

Cancer drug resistance (CDR) is a central problem in therapeutic failure. Several mechanisms can underlie CDR, including increased expression and activity of efflux ABC transporters and epigenetic phenomena. A topic that is not usually addressed is the mechanism underlying the loss of resistance to therapy once the challenge to these cells is withdrawn. A KCR cell line (doxorubicin-resistant and expressing ABCB1) was used to induce loss of resistance by withdrawing doxorubicin in culture medium, assess ABCB1 expression and activity and determine a signature of microRNAs expression. The activity of ABCB1 was analysed by fluorescence microscopy and flow cytometry through fluorescence (DiOC2) substrate retention assays. Expression of 1008 microRNAs was assessed before and after doxorubicin withdrawal. After 16 weeks of doxorubicin withdrawal we observed a decrease of ABCB1 activity and expression. Moreover, we determined a signature of 23 microRNAs, 13 under-expressed and 10 over expressed, as a tool to assess loss of resistance. Through pathway enrichment analysis, “Pathways in cancer”, “Proteoglycans in cancer” and “ECM-receptor interaction” were identified as relevant pathways in loss of CDR. Taken together, the data reported reinforces the assumption that ABCB1 may play a major role in the kinetics of CDR and their levels of expression are in the dependence of the circuitry of cell miRNAs

Next-generation sequencing as a promising approach for assessing the entomological origin of honey

Honey is a food widely consumed worldwide and much appreciated for its nutritional and organoleptic properties as well as for its beneficial health effects. However, honey is also considered one of the foods most prone to be adulterated either by the admixing of honey with lower quality, by the addition of sugars, or by mislabeling of botanical and geographical origins, among other possible frauds.1 Therefore, typically, honey authentication has focused mainly on the development of techniques targeting these types of frauds. Recently, increased attention has been paid to honey’s entomological origin since it also relates with geographical origin whose label non-compliances are difficult to detect. Moreover, in the current context where native honeybees are increasingly threatened by introgression, due to the use of exotic queens, preservation of honeybee subspecies in their native ranges, to which they are better adapted, is perceived as of high importance. In this sense, valorisation of the honey produced by native subspecies has been suggested as a possible approach to generate higher income for beekeepers, contributing to the development of rural regions and of sustainable beekeeping based on conservation strategiesThe authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020), to Fenapícola and Capemel for supplying the Portuguese honeys, to Dr. Antonio Nanetti (CREA-AA) for the Italian honeys and to António Pajuelo (Pajuelo Consultores Apícolas S.L.) for the Spanish honeys. D. Henriques is supported by the project BeeHappy (POCI-01-0145-FEDER-029871) funded by FEDER, COMPETE 2020-POCI and FCT and A. Quaresma by the PhD scholarship funded by the FCT (DFA/BD/5155/2020)info:eu-repo/semantics/publishedVersio

Development of a new approach based on NADH dehydrogenase subunit 1(ND1) marker and high resolution melting (HRM) analysis towards the authentication of the geographical origin of honey

In the present study, an approach for verifying the geographical origin of honey based on its entomological origin is proposed. The method was developed based on the polymerase chain reaction (PCR) amplification of a new markercontaining different single nucleotide polymorphisms characteristic of honeybees of different mitochondrial (mtDNA) lineages, therefore generating different fluorescent curves on HRM analysis. The method was successfully applied to honeys from Portugal and Italy, expected to be from lineages A and C, respectively, demonstrating their origin compliance.The authors are grateful to Programa Nacional Apícola 2020-2022 for funding the project “AUTENT+ Desenvolvimento de abordagens inovadoras com vista à valorização e exploração do potencial de mercado do mel Português”, to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO UIDB/00690/2020, to Fenapícola and Capemel for supplying the Portuguese honeys and to Dr. Antonio Nanetti (CREA-AA) for the Italian honeys. D. Henriques is supported by the project Bee Happy (POCI-01-0145-FEDER-029871) funded by FEDER, COMPETE 2020-POCIand FCT and A.R. Lopes by the PhD scholarship funded by the FCT SFRH/BD/143627/2019.info:eu-repo/semantics/publishedVersio

High resolution melting analysis of a COI mini-barcode as a simple approach for the entomological authentication of honey

Honey is highly valued for its taste, aroma, content in bioactive compounds and for being a natural food. In the European Union (EU), market demand for honey is higher than the domestic production and therefore a substantial amount of honey is imported. According to a 2014 European Parliament report on fraud in the food chain, honey was ranked as the 6th food product most prone to adulterateration.1 Up until now, honey authenticity addressed mainly sugars addition and botanical origin. However, an increased attention has recently been paid to honey entomological origin as it also relates to its geographical origin since honeybees carrying mitochondrial DNA (mtDNA) of distinct ancestries can be found across Europe. While in Portugal the predominant mtDNA of the autochthonous subspecies Apis mellifera iberiensis belongs to the A-lineage, when moving towards the northeastern part of the Iberian Peninsula this lineage is gradually replaced by the M-lineage. The native distribution of the M-lineage A. m. mellifera expands from the Pyrenees to Scandinavia and from the British Isles to the Ural Mountains while the C-lineage A. m. ligustica and A. m. carnica are naturally found in the Apennine and Balkan peninsulas, respectivelyThis work was funded by the project-2022, financed by IFAP. The authors are also grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020), to Fenapícola and Capemel for supplying the Portuguese honeys, to Dr. Antonio Nanetti (CREA-AA) for the Italian honeys and to António Pajuelo (Pajuelo Consultores Apícolas S.L.) for the Spanish honeys. D. Henriques is supported by the project BeeHappy (POCI-01-0145-FEDER-029871) funded by FEDER, COMPETE 2020-POCI and FCT. A. R. Lopes and A. Quaresma acknowledge the PhD scholarship funded by the FCT (SFRH/BD/143627/2019 and DFA/BD/5155/2020, respectively).info:eu-repo/semantics/publishedVersio

Kinetics of Expression in Cancer Drug Resistance

This research was funded by Fundação de Ciência e Tecnologia (FCT), grant number UID-BIM-00009-2020 and GHTM-UID/Multi/04413/2013. The APC was funded by Fundação de Ciência e Tecnologia (FCT), grant number UID-BIM-00009-2020.Cancer drug resistance (CDR) is a major problem in therapeutic failure. Over 90% of patients with metastatic cancer present CDR. Several mechanisms underlie CDR, including the increased expression of efflux ABC transporters and epigenetic phenomena. Nevertheless, a topic that is not usually addressed is the mechanism underlying the loss of CDR once the challenge to these cells is withdrawn. A KCR cell line (doxorubicin-resistant, expressing ABCB1) was used to induce loss of resistance by withdrawing doxorubicin in culture medium. ABCB1 activity was analysed by fluorescence microscopy and flow cytometry through substrate (DiOC2) retention assays. The expression of 1008 microRNAs was assessed before and after doxorubicin withdrawal. After 16 weeks of doxorubicin withdrawal, a decrease of ABCB1 activity and expression occurred. Moreover, we determined a signature of 23 microRNAs, 13 underexpressed and 10 overexpressed, as a tool to assess loss of resistance. Through pathway enrichment analysis, “Pathways in cancer”, “Proteoglycans in cancer” and “ECM-receptor interaction” were identified as relevant in the loss of CDR. Taken together, the data reinforce the assumption that ABCB1 plays a major role in the kinetics of CDR, and their levels of expression are in the dependence of the circuitry of cell miRNAspublishersversionpublishe

Animal species authentication in dairy products

Milk is one of the most important nutritious foods, widely consumed worldwide, either in its natural form or via dairy products. Currently, several economic, health and ethical issues emphasize the need for a more frequent and rigorous quality control of dairy products and the importance of detecting adulterations in these products. For this reason, several conventional and advanced techniques have been proposed, aiming at detecting and quantifying eventual adulterations, preferentially in a rapid, cost-effective, easy to implement, sensitive and specific way. They have relied mostly on electrophoretic, chromatographic and immunoenzymatic techniques. More recently, mass spectrometry, spectroscopic methods (near infrared (NIR), mid infrared (MIR), nuclear magnetic resonance (NMR) and front face fluorescence coupled to chemometrics), DNA analysis (real-time PCR, high-resolution melting analysis, next generation sequencing and droplet digital PCR) and biosensors have been advanced as innovative tools for dairy product authentication. Milk substitution from high-valued species with lower-cost bovine milk is one of the most frequent adulteration practices. Therefore, this review intends to describe the most relevant developments regarding the current and advanced analytical methodologies applied to species authentication of milk and dairy products.This work was funded by national funds (FCT, Fundação para a Ciência e Tecnologia) through the strategic funding of CIMO (UIDB/00690/2020) and LAQV-REQUIMTE (UIDB/50006/2020, UIDP/50006/2020). This study was also supported by the European Union through European Regional Development Fund (NORTE-01-0145-FEDER-000052) and SYSTEMIC (Knowledge Hub on Food and Nutrition Security, ERA-Net Cofund ERA-HDHL no. 696295). I.Mafra thanks FCT for funding through the Individual Call to Scientific Employment Stimulus (2021.03670.CEECIND).M. Honrado is grateful to FCT grant 2021.08119.BD, financed by POPH-QREN (subsidized by FSE andMCTES).info:eu-repo/semantics/publishedVersio

Development of a new approach based on real - time PCR coupled with high resolution melting (HRM) analysis towards the entomological authentication of honey

This work has received funding from the Programa Nacional Apícola 2020-2022 under the project “AUTENT+ Desenvolvimento de abordagens inovadoras com vista à valorização e exploração do potencial de mercado do mel Português”. The authors are also grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020). D. Henriques is supported by the project BeeHappy (POCI-01-0145-FEDER-029871) funded by FEDER (Fundo Europeu de Desenvolvimento Regional) through the program COMPETE 2020—POCI (Programa Operacional para a Competividade e Internacionalização), and by Portuguese funds through FCT and A.R. Lopes by the PhD scholarship funded by the FCT (SFRH/BD/143627/2019).Honey is a natural product widely consumed around the globe, not only for its taste and nutritional value, but also for its health benefits. Being a product of high dietary relevance and increasing demand, it has also become a target of economically motivated adulteration. According to the 2014 European Parliament report on the food crisis, fraud in the food chain and the control thereof, honey is among the 10 food products most prone of being adulterated [1]. Up until now, honey authenticity was mainly focused on the issues of sugars addition and botanical and geographical origin. However, recently an increased attention has been paid to the entomological origin of honey. To this aim, different approaches have been proposed to differentiate honey produced by different Apis mellifera subspecies, including those from distinct mitochondrial (mt) DNA lineages [2]. This work aimed to develop a novel real-time PCR method coupled with HRM analysis that allows for the simultaneous differentiation of honeybee from maternal lineages A, M and C, for further application in honey authentication. In this sense, data previously obtained from the mitogenomes of a total of 112 honeybees of different lineages were considered for the development of new DNA markers. Considering the aim of further application in honey, new primer sets were designed to amplify short fragments that included different single nucleotide polymorphisms (SNPs) allowing for HRM application. Three primer sets were proposed, amsCOI-F/amsCOI-R targeting the Cytochrome oxidase I (COI) gene, amsND1-F-amsND1-R targeting the NADH-ubiquinone oxidoreductase chain 1 (ND1) gene and amsCox3-F/amsCox3-R targeting the Cytochrome oxidase subunit III (Cox3) gene. Each primer set was first tested using qualitative PCR using DNA extracted from honeybees of A, M and C mtDNA lineages. After optimizing the real-time PCR conditions, each primer set was tested using a series of mtDNA extracted from honeybees. While amsCOI-F/amsCOI-R allowed only for the separation of the honeybees in two clusters, with lineage C and M clustering together, both the amsCox3-F/amsCox3-R and amsND1-F/amsND1-R set of primers allowed to differentiate the three lineages in separate clusters, with high level of confidence. As future work, the methodology will be assayed in commercial honey samples.info:eu-repo/semantics/publishedVersio

DNA-based methods as a powerful tool for the entomological authentication of honey

Honey is a food widely consumed worldwide and much appreciated for its nutritional, organoleptic and health properties. However, it is also considered one of the food products most prone to be adulterated in the EU. Up until now, honey authenticity addressed mainly the issue of sugars addition and botanical origin. Still, increased attention has recently been paid to honey entomological origin as it also relates to its geographical origin since honeybees carrying mitochondrial DNA (mtDNA) of distinct ancestries can be found across Europe. While in Portugal mtDNA of the autochthonous subspecies Apis mellifera iberiensis belongs to the African (A) lineage, in the northeastern part of Iberia African mitotypes are replaced by mitotypes of western European (M-lineage) ancestry. The native distribution of the M-lineage A. m. mellifera expands from the Pyrenees to Scandinavia and from the British Isles to the Ural Mountains while the C-lineage A. m. ligustica and A. m. carnica subspecies are naturally found in the Apennine and Balkan peninsulas, respectively [1]. Also, certain honeys holding the protected designation of origin (PDO) label should be produced by autochthonous A. mellifera subspecies, as mentioned in their EU geographical indications register.PAN 2020-2022, financed by IFAP. To the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020), to Fenapícola and Capemel for supplying the Portuguese honeys, to Dr. Antonio Nanetti (CREA-AA) for the Italian honeys and to António Pajuelo (Pajuelo Consultores Apícolas S.L.) for the Spanish honeys. D. Henriques is supported by the project BeeHappy (POCI-01-0145-FEDER-029871) funded by FEDER, COMPETE 2020-POCI and FCT. M. Honrado, A.R. Lopes, A. Quaresma acknowledge the PhD scholarship funded by the FCT (2021.08119.BD, SFRH/BD/143627/2019 and DFA/BD/5155/2020). projeto AUTENT+ "Desenvolvimento de abordagens inovadoras com vista à valorização e exploração do potencial de mercado do mel Português", PAN 2020-2022, financiado pelo IFAP.info:eu-repo/semantics/publishedVersio

A sequenciação de nova geração como uma abordagem promissora para a identificação da origem entomológica do mel

O mel é um alimento muito consumido e apreciado em todo o mundo pelas suas propriedades nutricionais e organoléticas, bem como pelos seus efeitos benéficos para a saúde. No entanto, é também considerado um dos alimentos mais suscetíveis de ser adulterado, quer pela mistura de mel de qualidade inferior, quer pela adição de açúcares, ou pela rotulagem incorreta da origem botânica e/ou geográfica, entre outras possíveis fraudes. Nos últimos anos, tem sido dada uma atenção crescente à origem entomológica do mel, uma vez que esta também está relacionada com a origem geográfica. No âmbito do projeto PRIMA “MEDIBEES” (https://medibees.org/), a sequenciação de nova geração (NGS) será utilizada com vista ao desenvolvimento de ferramentas moleculares que permitam identificar a origem entomológica de amostras de mel provenientes dos 8 países mediterrânicos do consórcio, de forma a diferenciar e valorizar méis produzidos por abelhas autóctones destes países. Com este objetivo, inicialmente procedeu-se à construção da base de dados das sequências de DNA mitocondrial das abelhas de modo a incluir 10 subespécies mediterrânicas das 4 linhagens maternas (A, M, C e O). Para tal, procedeu-se à extração de DNA e à respetiva sequenciação dos genomas completos, na plataforma Illumina Novaseq 6000, de um total de 1095 abelhas destes países. Posteriormente, utilizou-se o programa mitoZ 3.6 para fazer a montagem do genoma mitocondrial de cada uma das amostras, resultando na seleção de 283 sequências mitocondriais com boa montagem. Em seguida, foi utilizado o software MEGA 11, para realizar o alinhamento destas sequências. A informação obtida será posteriormente utilizada para a seleção de regiões com variantes (SNPs) informativos que possam ser usadas para o desenho de primers adequados e desenvolvimento de ferramentas para a identificação de méis produzidos por abelhas de diferentes linhagens mitocondriais e respetivas subespécies.Financiado pelos projetos “PRIMA, MEDIBEES: Monitoring the Mediterranean honeybee subspecies and their resilience to climate change for the improvement of sustainable agro-ecosystems.”. Os autores agradecem também à Fundação para a Ciência e Tecnologia (FCT, Portugal) pelo apoio financeiro através dos fundos nacionais FCT/MCTES (PIDDAC) ao CIMO (UIDB/00690/2020 e UIDP/00690/2020) e à SusTEC (LA/P/0007/2020), M. Honrado e Carlos Garcia agradecem a bolsa de doutoramento financiada pela FCT (2021.08119.BD e 2021.06948.BD, respetivamente)info:eu-repo/semantics/publishedVersio

Estrutura populacional e estado de conservação das subespécies de Apis mellifera no Oriente Próximo e Médio

A abelha melífera, Apis mellifera, é composta por 31 subespécies que se encontram distribuídas na Ásia, África e Europa. O objetivo deste trabalho é desvendar a estrutura populacional e verificar o estado de conservação de três subespécies do Médio Oriente, as quais têm sido pouco estudadas. Para isso, foi extraído o DNA a partir de tóraxes inteiros de machos de 329 amostras de A. m. lamarckii (Egito, 68 amostras), A. m. syriaca (Jordânia, 238 amostras) e A. m. jemenitica (Omã e Emirados Árabes Unidos, 23 amostras). Foram adicionadas 21 amostras de A. m. ligustica, que é uma subespécie amplamente utilizada pelos apicultores no mundo inteiro e por isso fonte de introgressão genética. O genoma completo das 329 amostras foi sequenciado na plataforma Illumina NovaSeq 600 tendo como objetivo uma cobertura de 20X. Os 329 genomas foram mapeados usando o genoma de referência Amel_HAv3.1 e foi implementada uma pipeline que garante a qualidade dos dados. No final, obteve-se um total de 4.030.485 de SNPs que foram usados na reconstrução da estrutura populacional com o ADMIXTURE e PCA. As amostras egípcias mostraram que apesar de terem alguma introgressão de A. m. ligustica, essa não é relevante e é variável (Q-values entre 1E-05 e 0.44), com a maior parte (97%) das amostras apresentando um valor médio de 0.07 ± 0.06 (Q-values, meia ± DP). A. m. syriaca apresenta uma estrutura complexa, tendo sido observados dois grupos distintos pelo PCA e três pelo ADMIXTURE. Relativamente seu ao estado de conservação, foram detetados 76 indivíduos com uma proporção considerável (Q-values entre 0.15 e 0.47) de introgressão com A. m. ligustica. No caso de A. m. jemenitica, foram observados dois cenários diferentes. Em Omã, todas as amostras estudadas mostraram ser puras. Por outro lado, apenas sete amostras dos Emirados Árabes Unidos foram classificadas como tal, enquanto as restantes mostraram proporções de introgressão semelhantes às do Egito. Estes resultados evidenciam o estado precário de integridade genética que estas subespécies apresentam nos locais estudados. No entanto, a existência de indivíduos que podem ser considerados puros para suas respetivas subespécies pode servir como ponto de partida para o desenvolvimento de planos de conservação.Este trabalho foi financiado pelos projetos “PRIMA, MEDIBEES: Monitoring the Mediterranean honeybee subspecies and their resilience to climate change for the improvement of sustainable agro-ecosystems.”. Os autores agradecem também à Fundação para a Ciência e Tecnologia (FCT, Portugal) pelo apoio financeiro através dos fundos nacionais FCT/MCTES (PIDDAC) ao CIMO (UIDB/00690/2020 e UIDP/00690/2020) e à SusTEC (LA/P/0007/2020). Carlos Garcia e M. Honrado agradecem a bolsa de doutoramento financiada pela FCT (2021.06948.BD, 2021.08119.BD, respetivamente).info:eu-repo/semantics/publishedVersio