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Since the last decade of the last century, it is known that many old grapevine varieties are descendants of other varieties through natural crossing. Portugal has an important program for the conservation of representative samples of intra-varietal variability of all autochthonous varieties, managed by the Portuguese Association for Grapevine Diversity (PORVID), which makes looking for genotypes with dubious identification an important activity from a perspective of its valorisation. This communication presents the results of the molecular analysis of 5,000 samples (accessions) from the PORVID’s collection, using nine microsatellite loci currently recommended by the International Organization of Vine and Wine (OIV) for genetic grapevine identification. The results obtained confirmed the molecular identity of 4,220 samples corresponding to 214 varieties present in the official list of Portuguese varieties. In 780 samples, 95 profiles with a plural number of accessions revealed not to be listed in the Vitis International Variety Catalogue (VIVC) database, corresponding to possible varieties either descendent from natural crossing from at least one known parental variety, or from undetermined origin. Furthermore, the need for a comprehensive strategy aimed at uncovering other hidden varieties is discussed to prevent their imminent loss, deepen understanding of their origin, and add economic value and sustainability to the vine and wine sector.

Current needs for increased drug delivery carrier efficacy and specificity in cancer necessitate the adoption of intelligent materials that respond to environmental stimuli. Therefore, we developed and optimized pH-triggered drug delivery nanoassemblies that exhibit an increased release of doxorubicin (DOX) in acidic conditions typical of cancer tissues and endosomal vesicles (pH 5.5) while exhibiting significantly lower release under normal physiological conditions (pH 7.5), indicating the potential to reduce cytotoxicity in healthy cells. The hybrid (polymeric/lipid) composition of the lyotropic non-lamellar liquid crystalline (LNLCs) nanoassemblies demonstrated high encapsulation efficiency of the drug (>90%) and high drug loading content (>7%) with colloidal stability lasting at least 4 weeks. Confocal microscopy revealed cancer cellular uptake and DOX-loaded LNLCs accumulation near the nucleus of human hepatocellular carcinoma cells, with a large number of cells appearing to be in apoptosis. DOX-loaded LNLCs have also shown higher citotoxicity in cancer cell lines (MDA-MB 231 and HepG2 cell lines after 24 h and in NCI-H1299 cell line after 48 h) when compared to free drug. After 24 h, free DOX was found to have higher cytotoxicity than DOX-loaded LNLCs and empty LNLCs in the normal cell line. Overall, the results demonstrate that DOX-loaded LNLCs have the potential to be explored in cancer therapy. [ABSTRACT FROM AUTHOR]

Grapevine is regarded as a highly profitable culture, being well spread worldwide and mostly directed to the wine-producing industry. Practices to maintain the vineyard in healthy conditions are tenuous and are exacerbated due to abiotic and biotic stresses, where fungal grapevine trunk diseases (GTDs) play a major role. The abolishment of chemical treatments and the intensification of several management practices led to an uprise in GTD outbreaks. Symptomatology of GTDs is very similar among diseases, leading to underdevelopment of the vines and death in extreme scenarios. Disease progression is widely affected by biotic and abiotic factors, and the prevalence of the pathogens varies with country and region. In this review, the state-of-the-art regarding identification and detection of GTDs is vastly analyzed. Methods and protocols used for the identification of GTDs, which are currently rather limited, are highlighted. The main conclusion is the utter need for the development of new technologies to easily and precisely detect the presence of the pathogens related to GTDs, allowing to readily take phytosanitary measures and/or proceed to plant removal in order to establish better vineyard management practices. Moreover, new practices and methods of detection, identification, and quantification of infectious material would allow imposing greater control on nurseries and plant exportation, limiting the movement of infected vines and thus avoiding the propagation of fungal inoculum throughout wine regions. [ABSTRACT FROM AUTHOR]

A fast and highly specific detection of COVID-19 infections is essential in managing the virus dissemination networks. The most relevant technologies developed for SARS-CoV-2 detection, along with their advantages and limitations, will be presented and fully explored. Additionally, some of the newest and emerging COVID-19 diagnosis tools, such as biosensing platforms, will also be introduced. Considering the extreme relevance that all these technologies assume in pandemic control, it is of the utmost relevance to have an intrinsic knowledge of the parameters that need to be taken into consideration before choosing the most adequate test for a particular situation. Moreover, the new variants of the virus and their potential impact on the detection method's effectiveness will be discussed. In order to better manage the pandemic, it is essential to maintain continuous research into the SARS-CoV-2 genome and updated genomic surveillance at the global level. This will allow for timely detection of new mutations and viral variants, which may affect the performance of COVID-19 detection tests. [ABSTRACT FROM AUTHOR]

The wine sector is a billion euro business and therefore subjected to multiple attempts of fraudulent practices. This requires the development of rapid and reliable methods to detect such situations. Several methodologies have been developed based on the chemical profiles of the wines, but they are limited due to the environmental conditions that cannot be controlled. The use of DNA-based detection systems are an emergent research field that have been extended to a wide variety of food prod- ucts and are still the most reliable methods for varietal identification. However these methods are not suitable for geographical determination. Soil related fingerprints have a primary role considering that there is a relationship between the elemental compo- sition of wine and the composition of the provenance soil. WineBioCode is a project aiming to define the best strategy for wine authenticity based on a multidisciplinary approach. Two DNA-based strategies have been developed based on Real-time PCR and a label free optical biosensor platform. Both platforms enabled successful identification of specific DNA-targets when applied to Vitis vinifera L., and can be applied throughout the grape-wine chain. The methods are complementary and can be used in dif- ferent situations, according to the requirements. The geographical evaluation has been assessed by the strontium 87Sr/86Sr isotope ratio determination involving soil evaluation in the vineyards followed by its assay in the wine samples. The results are being integrated in order to establish the best procedure to be undertaken for wine fingerprinting, including varietal composition and geographical origin, therefore fulfilling the requirements of the geographical denominations in wine certification.

The wine sector is one of the most economically important agro-food businesses. The wine market value is largely associated to terroir, in some cases resulting in highly expensive wines that attract fraudulent practices. The existent wine traceability system has some limitations that can be overcome with the development of new technological approaches that can tackle this problem with several means. This review aims to call attention to the problem and to present several strategies that can assure a more reliable and authentic wine system, identifying existent technologies developed for the sector, which can be incorporated into the current traceability system. [ABSTRACT FROM AUTHOR]

The need to support food labelling has driven to the development of PCR‐based techniques suitable for food analysis. DNA‐based markers have been successfully employed for varietal tracing in Protected Designation of Origin (PDO) olive oils. In this study, we report a fast, sensitive, and easy‐to‐use strategy for PDO olive varietal identification. To achieve this aim four different DNA extraction methods were tested and compared, based on initial volume, extraction time, the gDNA concentration, and quality ratios. The optimized DNA extraction protocol from extra virgin olive oils, based on CTAB–hexane–chloroform extraction, proved to be the most effective. High‐resolution melting (HRM) DNA assay was developed based on nuclear microsatellites (gSSR) and plastid DNA (cpDNA) aiming an accurate identification of the olive varieties present in the olive oil samples. After PCR reproducibility evaluation, six molecular markers: three SSRs and three cpDNA loci were chosen based on their discrimination power. The SSR‐HRM strategy assays were designed to target UDO99‐011, UDO99‐039, UDO99‐024, and ssrOeUA‐DCA16 loci. All SSR‐PCR products generated from these primers were analyzed by capillary electrophoresis (CE) for HRM data validation. The SSR coupled with HRM melting curve analysis generated 14 HRM profiles sufficient to genotype all varieties, highlighting their potential use for varietal discrimination. The locus ssrOeUA‐DCA16 generated a specific melting curve that allow a high‐throughput discrimination of the Picual and Cobrançosa varieties in olive oil samples. Further, the UDO99‐024 was also tested by SSR‐HRM assay in commercial olive oil samples with promising results. Considering time, cost, and performance SSR‐HRM proved to be a reliable method suitable for varietal tracing of olive oils. Practical Application: Olive oil authenticity is a form of protecting producers and consumers against fraudulent practices. Herein, we present a DNA barcode suitable for the identification of olive varieties, allowing an accurate identification of the olive varieties in olive oil samples using SSR‐HRM assay. Its applicability in commercial olive oil samples is viable. This methodology can be used as a tool for Extra Virgin Olive Oil (EVOO) adulterations detection. [ABSTRACT FROM AUTHOR]

The major limitation of cereal production in acidic soils is aluminium (Al) phytotoxicity which inhibits root growth. Recent evidence indicates that different genotypes within the same species have evolved different mechanisms to cope with this stress. With these facts in mind, root responses of two highly Al tolerant Portuguese bread wheat genotypes--Barbela 7/72/92 and Viloso mole--were investigated along with check genotype Anahuac (Al sensitive), using different physiological and histochemical assays. All the assays confirmed that Barbela 7/72/92 is much more tolerant to Al phytotoxicity than Viloso Mole. Our results demonstrate that the greater tolerance to Al phytotoxicity in Barbela 7/72/92 than in Viloso Mole relies on numerous factors, including higher levels of organic acid (OAs) efflux, particularly citrate efflux. This might be associated with the lower accumulation of Al in the root tips, restricting the Al-induced lipid peroxidation and the consequent plasma membrane integrity loss, thus allowing better root regrowth under Al stress conditions. Furthermore, the presence of root hairs in Barbela 7/72/92 might also help to circumvent Al toxicity by facilitating a more efficient uptake of water and nutrients, particularly under Al stress on acid soils. In conclusion, our findings confirmed that Portuguese bread wheat genotype Barbela 7/72/92 represents an alternative source of Al tolerance in bread wheat and could potentially be used to improve the wheat productivity in acidic soils. [ABSTRACT FROM AUTHOR]

Objective: To prepare stable and easy to handle formulation of solid lipid nanoparticles (SLNs) by freeze-drying with or without cryoprotectants, as appropriate. Materials and methods: SLNs were freeze-dried without cryoprotectants or with cryoprotectants in quantities selected by freeze-thaw test (sucrose, glucose) or literature search (trehalose, maltose). Appearance, re-dispersability and size distribution of re-dispersed samples were evaluated. Results: SLN could be freeze-dried using 10% sucrose, trehalose or maltose. Trehalose was effective in protecting one of presented formulations that was already very stable on its own; its efficiency in protecting other two formulations was limited. Discussion: Our results are in line with various reports of successful freeze-drying of SLN, but considering the stability of original dispersions, no improvement was achieved. Conclusion: We confirmed that trehalose is among the most suitable cryoprotectant for SLN, however it did not improve shelf-life of the most stable formulation. [ABSTRACT FROM AUTHOR]

In bread wheat, besides malate, the importance of citrate efflux for Al tolerance has also been reported. For better understanding the Al tolerance mechanism in bread wheat, here, we performed both a molecular characterization of the citrate transporter gene TaMATE1 and an investigation on the upstream variations in citrate and malate transporter genes. TaMATE1 belong to multidrug transporter protein family, which are located on the long arm of homoeologous group 4 chromosomes (TaMATE1-4A, TaMATE1-4B TaMATE1-4D). TaMATE1 homoeologues transcript expression study exhibited the preponderance of homoeologue TaMATE1-4B followed by TaMATE1-4D whereas homoeologue TaMATE1-4A seemed to be silenced. TaMATE1, particularly homoeologue TaMATE1-4B and TaALMT1 transcripts were much more expressed in the root apices than in shoots of Al tolerant genotype Barbela 7/72/92 under both control and Al stress conditions. In addition, in both tissues of Barbela 7/72/92, higher basal levels of these gene transcripts were observed than in Anahuac (Al sensitive). Noticeably, the presence of a transposon in the upstream of TaMATE1-4B in Barbela 7/72/92 seems to be responsible for its higher transcript expression where it may confer citrate efflux. Thus, promoter variations (transposon in TaMATE1-4B upstream and type VI promoter in TaALMT1) associated with higher basal transcript expression of TaMATE1-4B and TaALMT1 clearly show how different mechanisms for Al tolerance operate simultaneously in a single genotype. In conclusion, our results demonstrate that Barbela 7/72/92 has favorable alleles for these organic acids transporter genes which could be utilized through genomic assisted selection to develop improved cultivars for acidic soils. [ABSTRACT FROM AUTHOR]

Hexaploid tritordeum (HHAABB; 2n = 42) results from the cross between Hordeum chilense (HH; 2n = 14) and cultivated durum wheat ( Triticum turgidum ssp. durum (AABB; 2n = 28). Morphologically, tritordeum resembles the wheat parent, showing promise for agriculture and wheat breeding. Start Codon Targeted (SCoT) polymorphism is a recently developed technique that generates gene-targeted markers. Thus, we considered it interesting to evaluate its potential for the DNA fingerprinting of newly synthesized hexaploid tritordeums and their respective parents. In this study, 60 SCoT primers were tested, and 18 and 19 of them revealed SCoT polymorphisms in the newly synthesized tritordeum lines HT27 and HT22, respectively, and their parents. An analysis of the presence/absence of bands among tritordeums and their parents revealed three types of polymorphic markers: (i) shared by tritordeums and one of their parents, (ii) exclusively amplified in tritordeums, and (iii) exclusively amplified in the parents. No polymorphism was detected among individuals of each parental species. Three SCoT markers were exclusively amplified in tritordeums of lines HT22 and HT27, being considered as polyploidization-induced rearrangements. About 70 % of the SCoT markers of H. chilense origin were not transmitted to the allopolyploids of both lines, and most of the SCoTs scored in the newly synthesized allopolyploids originated from wheat, reinforcing the potential use of tritordeum as an alternative crop. [ABSTRACT FROM AUTHOR]

ABSTRACT Cationic solid lipid nanoparticles (cSLN) are colloidal carriers for genes or drugs, particularly lipophilic drugs. Several reports exist on their high efficiency, but only a few studies report the effect of cSLNs on living cells. In the present work, internalization, cell viability (alamar blue assay) and genotoxic potential (alkaline comet assay) of three cSLN formulations (A-C) were evaluated in HepG2 and Caco-2 cells. cSLN showed an average hydrodynamic diameter (z-ave) of 141-222 nm, zeta-potential of 55.0-72.5 mV and polidispersity indices (PdI) of 0.336-0.421. Dispersion in physiological buffers increased z-ave and PdI. 0.01 mg ml-1 cSLN unaffected cell viability, but 1.0 mg ml-1 significantly decreased it, being cSLN-C (Compritol-based) the most toxic and HepG2 the most affected. DNA damage was not significantly increased by 0.1 mg ml-1 cSLN but damage was observed at 1.0 mg ml-1 cSLN-C. Thus, no genotoxicity is to be expected at concentrations that do not reduce cell viability. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: Aluminium (Al) toxicity is considered to be one of the major constraints affecting crop productivity on acid soils. Being a trait governed by multiple genes, the identification and characterization of novel transcription factors (TFs) regulating the expression of entire response networks is a very promising approach. Therefore, the aim of the present study was to clone, localize, and characterize the TaSTOP1 gene, which belongs to the zinc finger family (Cys2His2 type) transcription factor, at molecular level in bread wheat. Results: TaSTOP1 loci were cloned and localized on the long arm of homoeologous group 3 chromosomes [3AL (TaSTOP1-A), 3BL (TaSTOP1-B) and 3DL (TaSTOP1-D)] in bread wheat. TaSTOP1 showed four potential zinc finger domains and the homoeologue TaSTOP1-A exhibited transactivation activity in yeast. Expression profiling of TaSTOP1 transcripts identified the predominance of homoeologue TaSTOP1-A followed by TaSTOP1-D over TaSTOP1-B in root and only predominance of TaSTOP1-A in shoot tissues of two diverse bread wheat genotypes. Al and proton (H+) stress appeared to slightly modulate the transcript of TaSTOP1 homoeologues expression in both genotypes of bread wheat. Conclusions: Physical localization of TaSTOP1 results indicated the presence of a single copy of TaSTOP1 on homoeologous group 3 chromosomes in bread wheat. The three homoeologues of TaSTOP1 have similar genomic structures, but showed biased transcript expression and different response to Al and proton (H+) toxicity. These results indicate that TaSTOP1 homoeologues may differentially contribute under Al or proton (H+) toxicity in bread wheat. Moreover, it seems that TaSTOP1-A transactivation potential is constitutive and may not depend on the presence/ absence of Al at least in yeast. Finally, the localization of TaSTOP1 on long arm of homoeologous group 3 chromosomes and the previously reported major loci associated with Al resistance at chromosome 3BL, through QTL and genome wide association mapping studies suggests that TaSTOP1 could be a potential candidate gene for genomic assisted breeding for Al tolerance in bread wheat. [ABSTRACT FROM AUTHOR]

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DNA analysis with molecular markers has opened a way to understand complex organism's genome. It is presently being widely applied across different fields, where food takes a preeminent position. Constant outbreaks of foodborne illnesses are increasing consumer's attention towards more detailed information related to what they are consuming. This overview reports on the areas where food traceability has been considered, and the problems that still remain to be bypassed in order to be widely applied. An outline of the most broadly used PCR-based methods for food traceability is described. Applications in the area of detection of genetically modified organisms, protected denomination of origin, allergenic and intolerance reactions are detailed in order to understand the dimension of the performed studies. [ABSTRACT FROM AUTHOR]

Nanotechnology is an emerging field of research that has been widely applied in different scientific and engineering areas. The agro-food sector is not an exception, which considers its applicability in several areas of major interest for both consumers and producers. This review considers major concepts related to nanostructures and nano-based instruments used in the food sector, as well as their applications in agro-food products. Food safety through the use of nanosensors for pathogen detection, smart packaging, and valorisation of food products by nanoencapsulation/nanodelivery of food ingredients (e.g. flavours) are examples of important areas of nanotechnology. Consumers' apprehension regarding food stability and safety issues is also considered. [ABSTRACT FROM AUTHOR]

Abstract: Olive tree is mainly vegetatively propagated; therefore a small level of polymorphism is expected among clones of the same cultivar. In order to access the level of intra-varietal genetic variability within a collection of 120 clones of the Portuguese olive ‘Cobrançosa’ DNA fingerprinting was performed. Ten random amplified polymorphism DNA (RAPD) primers amplified 150 fragments, of which 75 were polymorphic, while 10 inter-simple sequence repeats (ISSR) primers amplified 179 fragments of which 98 were polymorphic. ‘Cobrançosa’ clones were compared and analysed using Jaccard''s coefficient with unweighted pair group mathematical averages, using the RAPD data, the ISSR data and both markers simultaneously. The data indicates a wide intra-varietal genetic variability among the clones. It was possible to cluster some clones according to geographical provenance. Analysis of molecular variance revealed greater variation within clones than among different geographical origins. Significant genetic diversity among the ‘Cobrançosa’ olive cultivar, even though the clones came from a limited geographical area, was also detected. [Copyright &y& Elsevier]

The interest in developing reliable wine authenticity schemes is a hot-topic, especially for wines with recognized added-value. In order to accomplish this goal, two dimensions need to be considered: the grapevine variety determination and the geographical provenance. The aim of this study was to develop a multidisciplinary approach applicable to wines from the sub region Melgaço and Monção of the demarcated Vinho Verde region and from the demarcated Douro region. The proposed scheme consists on the use of DNA-based assays to detect Single Nucleotide Polymorphisms (SNPs) on three genes of the anthocyanin pathway (UFGT, F3H and LDOX) coupled with High-resolution melting (HRM) analysis aiming the varietal identification. The Alvarinho wines revealed to have the same haplotype using this marker set, demonstrating its applicability for genetic identification. In addition, to assess their geographical provenance, a multi-elemental approach using Sr and Pb isotopic ratios of wine, soil and bedrock samples was used. The isotopic data suggest a relation between Sr and Pb uptake by vine roots and soil's texture and clay content, rather than with the whole rock's isotopic ratios, but also highlights the potential of a discriminating method based on the combination of selected isotopic signatures. [ABSTRACT FROM AUTHOR]

Non-small-cell lung cancer (NSCLC) is a major cause of death worldwide. Alterations in such genes as EGFR and ALK are considered important biomarkers in NSCLC due to the existence of targeted therapies with specific tyrosine kinase inhibitors (TKIs). However, specific resistance-related mutations can occur during TKI treatment, which often result in therapy inefficacy. Liquid biopsies arise as a reliable tool for the early detection of these types of alterations, allowing a non-invasive follow-up of the patients. Furthermore, they can be essential for cancer screening, initial diagnosis and to check surgery success. Despite the great advantages of liquid biopsies in NSCLC and the high input that next-generation sequencing (NGS) approaches can provide in this field, its use in oncology is still limited. With improvement of assay sensitivity and the establishment of clinical guidelines for liquid biopsy analysis, it is expected that they will be used in routine procedures. This review focuses on the usefulness of liquid biopsies of NSCLC patients as a means to detect alterations in EGFR and ALK genes and in disease management, highlighting the impact of NGS methods. [ABSTRACT FROM AUTHOR]

Olive is an evergreen tree belonging to the Oleaceae family and producing edible fruits. Olive production is limited by a huge number of diseases, among them, anthracnose, caused by the ascomycete fungus Colletotrichum spp, which is considered to be the most damaging one in terms of economic loss. Although several methods have been developed to detect the pathogen that causes the disease, its quantification is still limited among infected food products. The interest in developing quantification methods has increased, so risk assessment can be performed in fresh or processed food/beverage samples. This work presents the state-of-the-art of the methods used in pathogen identification, considering several hosts and/or food products, with particular emphasis given to olive anthracnose disease. Additionally, the reasons why pathogen quantification methods are necessary for crop control and food products are also explored. So far, the most reliable detection and quantification methods described are based on DNA amplification, more particularly, real-time PCR. Through a deep knowledge of the C. acutatum infection patterns, improvements may be made to enhance anthracnose disease detection, control and avoidance strategies. • Olive anthracnose (Colletotrichum spp.) has a huge impact on olive oil production. • Colletotrichum detection is accurately achieved by PCR-based methods. • PCR-based methods are independent of environmental conditions. • Quantification of inoculum in processed matrices is still difficult. • Comparative genomics with infections pathway factors is an alternative approach. [ABSTRACT FROM AUTHOR]

Olive anthracnose caused by the hemibiotrophic fungal pathogen Colletotrichum acutatum is a serious threat to the olive sector. Olive oil and fruit production is severely constrained by Colletotrichum spp. infection, being C. acutatum the most distributed pathogen in Portuguese olive orchards. To understand the impact of C. acutatum on phenylpropanoids biosynthesis, the enzyme activity, phenolic compounds, ortho-diphenols, and flavonoids content were determined and correlated with the expression of gene encoding key enzymes within phenylpropanoids metabolism in susceptible and tolerant olive fruits, during maturation and when infected with C. acutatum. Differences between cultivars was observed, the tolerant olive cv. Picual presented a higher basal value and a stable phenolic content throughout the infection process, supporting its high C. acutatum tolerance, whereas in the susceptible olive cv. Galega these secondary metabolites were significantly increased only after the elicitation with C. acutatum. [ABSTRACT FROM AUTHOR]

Abstract: Aluminium (Al) toxicity is particularly evident under acid soil conditions limiting crop production, namely in wheat (Triticum aestivum). Although largely studied, the effects of Al on nutrition imbalances and putative correlations with Al tolerance degrees still remain inconclusive. We investigated the uptake of Al and transport to shoots in one wheat line and one cultivar differing in their tolerance to Al: ‘Barbela 7/72’ (Al-tolerant) and ‘Anahuac’ (Al-sensitive). Wheat seedlings were exposed for a short period to 185μM AlCl3 and then grown in the absence of the metal. In general, Al inhibited root growth, mostly in the sensitive cultivar ‘Anahuac’, while water content decreased and organic matter increased in ‘Barbela 7/72’ line. Similar but less evident effects were found in leaves of both genotypes. The profiles of macro- (Ca, K, P, Mg) and micronutrients (Mn, Fe, Zn, B, Cu) accumulation in response to aluminium were analyzed in both genotypes: Al levels increased, mostly in ‘Anahuac’ cultivar. Overall, P, Mg and K levels decreased and Si and Ca increased in roots of both genotypes, while other nutrients had a more inconsistent behaviour. Histological data showed that ‘Barbela 7/72’ initiated endodermis differentiation more efficiently than ‘Anahuac’, at the hairy root zone. These data show that: (a) Al negatively interferes with P, Mg and K in wheat and (b) Al interactions with some nutrients depend on the level of plant tolerance. Finally, data also provide support to the hypothesis that endodermis differentiation may be stimulated limiting Al accumulation/allocation in roots of the tolerant wheat landrace. [Copyright &y& Elsevier]

Abstract CAB51, a compact antibody against human epithelial growth receptor 2 (HER2, ErbB2), has been linked to cationic Solid Lipid Nanoparticles (SLN) via streptavidin-biotin interaction and their targeting potential evaluated against breast cancer cells. The amount of streptavidin and biotinylated antibody was optimised by monitoring the mean complex size (intensity weighed average diameter), polydispersity index and immediate stability in phosphate buffer saline (PBS). The effect on MCF-7 and BT-474 cells was evaluated at concentrations of 0.01 mg/mL and 0.1 mg/mL (counted as solid lipid). Streptavidin adsorption onto SLN surface had no influence on cell viability. Linking the antibody showed a synergistic effect on cell viability at lowest concentration tested (0.01 mg/mL) which was lower than that observed after exposure to SLN alone or antibody alone. At the higher tested concentration (0.1 mg/mL), the observed toxicity was entirely governed by the inherent toxicity of the SLN themselves. Streptavidin adsorption had no effect on accumulation in cells, while the antibody-containing complexes showed clearly increased internalisation in both cell lines. In HER2/neu positive BT-474 higher internalisation was observed than in HER2/neu negative MCF-7. Graphical Abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Olive oil and wine production have a worldwide economic impact. Their market reliability is under great concern because of the increasing number of fraud and adulteration attempts. The need for a traceability system in all its extension is crucial particularly for the cases of olive oils and wines with certified labels, in which only a limited number of olives and grapevine varieties, respectively, are allowed in a restricted well-defined geographical area. Molecular markers have been vastly applied to the food sector, and in particular High-Resolution DNA Melting technology has been successfully applied for olive oil and wine authentication, as part of the traceability system. In this review, the applications of HRM and their usefulness for this sector considering, Safety , Security and Authenticity will be reviewed. A broad overview of the HRM technique will be presented, focusing on the aspects that are crucial for its success, in particular the new generation of fluorescent dsDNA dyes used for amplicon detection and quantification, and the data analysis. A brief outlook on the olive oil and wine authenticity procedures, based on new DNA technology advances, and in which way this may influence the future establishment of a traceability system will be discussed. [ABSTRACT FROM AUTHOR]

The ability to detect and quantify small amounts of DNA in biological complex samples is a hot research area. Up until recently most of the work performed in this area used label-dependent protocols that increases its complexity and overall costs. The aim the work was to develop a label-free technology suitable for DNA detection and quantification using real complex DNA samples. The applicability of this system was tested using synthetic ssDNA targets that guaranteed the systems specificity, in the sense that only complementary sequences hybridized with the probe. When using real samples extracted from Vitis vinifera L. the system was able to successfully detect and quantify the DNA present without any of the time consuming and costly amplification steps. The detection and quantification limits of the proposed system were 60 ± 20 nM and 201 ± 20 nM, respectively for Target 1 concentrations between 31 and 350 nM. This method can easily be applied to other species and purposes, allowing the direct detection of DNA in a label-free environment with high accuracy and specificity. [ABSTRACT FROM AUTHOR]

Abstract: Aluminium (Al) irreversibly inhibits root growth in sensitive, but not in some tolerant genotypes. To better understand tolerance mechanisms, seedlings from tolerant (‘Barbela 7/72’ line) and sensitive (‘Anahuac’) Triticum aestivum L. genotypes were exposed to AlCl3 185μM for: (a) 24h followed by 48h without Al (recovery); (b) 72h of continuous exposure. Three root zones were analyzed (meristematic (MZ), elongation (EZ) and hairy (HZ)) for callose deposition, reserves (starch and lipids) accumulation, endodermis differentiation and tissue architecture. Putative Al-induced genotoxic or cytostatic/mytogenic effects were assessed by flow cytometry in root apices. Tolerant plants accumulated less Al, presented less root damage and a less generalized callose distribution than sensitive ones. Starch and lipid reserves remained constant in tolerant roots but drastically decreased in sensitive ones. Al induced different profiles of endodermis differentiation: differentiation was promoted in EZ and HZ, respectively, in sensitive and tolerant genotypes. No ploidy changes or clastogenicity were observed. However, differences in cell cycle blockage profiles were detected, being less severe in tolerant roots. After Al removal, only the ‘Barbela 7/72’ line reversed Al-induced effects to values closer to the control, mostly with respect to callose deposition and cell cycle progression. We demonstrate for the first time that: (a) cell cycle progression is differently regulated by Al-tolerant and Al-sensitive genotypes; (b) Al induces callose deposition >3cm above root apex (in HZ); (c) callose deposition is a transient Al-induced effect in tolerant plants; and (d) in HZ, endodermis differentiation is also stimulated only in tolerant plants, probably functioning in tolerant genotypes as a protective mechanism in addition to callose. [Copyright &y& Elsevier]

• Colletotrichum acutatum real-time qPCR assay. • Colletotrichum acutatum detection and quantification in olive drupes. • Colletotrichum acutatum single-copy gene: klap 1. Olive anthracnose is caused by fungal species within the Colletotrichum acutatum, C. gloeosporioides and C. boninense complexes. Anthracnose causes severe pre- and post-harvest olive drupe fall. This study aimed to design a species-specific qPCR assay, based on klap1 gene, suitable for C. acutatum s.s. quantification in cv. Galega Vulgar fruit samples. The developed qPCR assay presented a detection limit of 10.14 fg/reaction, and a linear cycle threshold of R2 = 0.996. C. acutatum inoculum was detected in pulverized olive fruits, and in early infection stages, before symptom appearance, 16 h after inoculation (Ct values = 28.29 ± 1.1). In olive samples, the derived melting curve was specific presenting a single dissociation peak (T melting = 88.7 °C). The designed assay was effectively applied in C. acutatum detection and quantification using infected olive samples, with a LOD of 0.59 ng and a LOQ of 1.8 ng, allowing its application to orchard management. [ABSTRACT FROM AUTHOR]

• Real-time PCR assay applied to food control. • Colletotrichum acutatum detection in olive drupes. • Colletotrichum acutatum detection in olive oils. The detection of latent Colletotrichum spp infection in olive drupes is crucial, to avoid contamination in the olive oil production chain. In order to detect the presence of C. acutatum in complex olive matrices a real-time PCR assay was developed, using olive drupe and oil samples from C. acutatum susceptible and tolerant olive cultivars (Galega Vulgar, Cobrançosa and Picual) with different infection levels. A C. acutatum specific sequence, belonging to the Internal Transcribed Spacers region, was used to design the real-time PCR detection assay, resulting in an 490 bp amplicon with a consistent melting temperature (T m = 87.8 °C). The assay allowed a rapid and high-sensitive C. acutatum detection mean, being able to detect the infection in a latent phase, for the first time, in olive drupes, 16 hai, and in olive oils containing 20% of infected olives. This novel method can be used to monitor C. acutatum presence in olive orchards. [ABSTRACT FROM AUTHOR]