Venenos e panaceias do vinho tinto

É o propósito dos autores apresentar nesta comunicação uma reflexão crítica, com base nos dados disponíveis até à data, sobre os fundamentos dos debates recentes sobre segurança alimentar no vinho, bem como sobre o papel do vinho na saúde, usando como caso de estudo dados conjuntos sobre a casta Vinhão, quanto à presença da toxina ocratoxina A e antocianas. Aspectos como a avaliação e gestão do risco quanto à ocratoxina A, em termos de estabelecimento de limites máximos recomendados, e comunicação do risco ao consumidor serão aqui debatidos dos pontos de vista científicos, políticos e económicos, tendo a saúde do consumidor em vista final. Os mesmos pontos são focados na visão inversa, dos compostos com efeito benéficos para a saúde, quanto às antocianas. Um balanço final é apresentado

Aspergillus ibericus : a new species of section nigri characterised by MALDI-TOF MS

Strains from the new described species Aspergillus ibericus were characterised using MALDI-TOF MS and the results were compared with other related species of section Nigri.Fundação para a Ciência e a Tecnologia (FCT

Ochratoxin A and filamentous fungi in red wine grapes from Santa Catarina, Brazil

The quality of wines has been evaluated traditionally according to sensorial properties. Recently, safety issues have been raised, such as pesticide residues and mycotoxins, with the introduction of new agricultural practices and the development of analytical methods with higher sensitivity. Ochratoxin A (OTA) is such a mycotoxin, produced by some Aspergillus and Penicillium species and is one of the most recent safety issues for wine. The mycobiota of, and the occurrence of OTA in Southern Brazilian grapes are not known. The presence of these contaminants was assessed by collecting 30 samples of grapes, from 16 vineyards, from the two most important wine subregions in the State of Santa Catarina, Brazil. The mycobiota was evaluated by plating 10 grapes from each sample in Dichloran Rose Bengal Chloramphenicol Agar and Sabouraud Dextrose Agar, supplemented with chloramphenicol. Production of OTA by black Aspergillus strains was estimated after growing in Czapeck Yeast Agar. OTA was analysed in 9 grape samples by chromatography with immunoaffinity clean-up, as stipulated by the European regulation. Three hundred and eighty seven strains were isolated. The dominant genera were Cladosporium (found in 86.7% of plated berries), Alternaria (80.0%), Botrytis (70.0%), Aspergillus (66.7%), and Penicillium (63.3%). Sixteen A. niger aggregate strains (26 % of total Aspergillus strains) were isolated, and OTA was not detected from any of these strains. No A. carbonarius was isolated. OTA was found in 6 grape samples, with a range of values from 0.16 μg/Kg to 0.77 μg/Kg. In conclusion, no OTA producing black Aspergillus strains were found in grapes, although some grape samples contain the mycotoxin. The fungal source of OTA requires further investigation

Bioprocess intensification for the continuous expansion of 3D human induced pluripotent stem cell aggregates in bioreactors

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. Firstly, we demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4% oxygen) and perfusion at 1.3 day-1 dilution rate to improve hiPSC growth as 3D aggregates in xeno-free medium (Cellartis® DEF-CS™ 500 Xeno-Free Culture Medium). This strategy allowed for increased cell specific growth rate, maximum volumetric cell concentrations (4.7x106 cell/mL) and expansion factors (approximately 19), resulting in an overall improvement of 2.6-fold in cell yields. Extensive cell characterization, including whole proteomic analysis was performed to confirm that the pluripotent phenotype was maintained during culture. Secondly, we have tested different chemical and mechanical strategies for hiPSC aggregate dissociation, revealing similar viable cell recovery yields (approximately 50%). However, only the mechanical dissociation strategies enabled the re-aggregation of hiPSC in stirred conditions, with the mechanical dissociation using a 70 μm pore size nylon mesh allowing a higher expansion factor after dissociation. Finally, a scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using the mechanical dissociation for aggregate disruption/passaging. A total expansion factor of 1100 in viable cells was obtained in 11 days of culture after 3 sequential passages in bioreactors, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability. To our knowledge, this is the highest expansion factor reported for hiPSC for such a short culture time frame. The strategy described herein for continuous expansion of hiPSC provides important insights towards up-scaling the production of hiPSC. Integrative biomanufacturing processes using this continuous strategy are now being pursued for hiPSC expansion and differentiation towards cardiac lineages in order to recreate cardiac models for drug discovery, toxicity testing and disease modeling. Acknowledgments: This work was supported by Fundação para a Ciência e Tecnologia (FCT)-funded projects CARDIOSTEM (MITP-TB/ECE/0013/2013) and CardioRegen (HMSP-ICT/0039/2013); and iNOVA4Health UID/Multi/04462/2013, a program supported by FCT/Ministério da Educação e Ciência, through national funds and cofounded by FEDER under the PT2020 Partnership Agreement. BA. was supported by FCT Grant SFRH/BD/52475/2013. The work was also funded by a Vinnova Grant, registration number 2014-00310 (Cell therapies via large scale expansion of pluripotent stem cells)

Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4% oxygen) and perfusion at 1.3 day-1 dilution rate to improve hiPSC growth as 3D aggregates in xeno-free medium (Cellartis® DEF-CS™ 500 Xeno-Free Culture Medium). This strategy allowed for increased cell specific growth rate, maximum volumetric cell concentrations (4.7x106 cell/mL) and expansion factors (approximately 19), resulting in an overall improvement of 2.6-fold in cell yields. Extensive cell characterization, including whole proteomic analysis was performed to confirm that the pluripotent phenotype was maintained during culture. Furthermore, a scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation protocols for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11 days of culture (Figure 1), while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors. To our knowledge, this is the highest expansion factor reported for hiPSC for such a short culture time frame. The strategy described herein for continuous expansion of hiPSC provides important insights towards up-scale production of hiPSC. This will strengthen the utility of hiPSC in cell therapy, drug discovery, toxicity testing and disease modeling. Please click Additional Files below to see the full abstract

Microencapsulation Technology: A Powerful Tool for Integrating Expansion and Cryopreservation of Human Embryonic Stem Cells

The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors

ZmOrphan94 Transcription Factor Downregulates ZmPEPC1 Gene Expression in Maize Bundle Sheath Cells

Spatial separation of the photosynthetic reactions is a key feature of C4 metabolism. In most C4 plants, this separation requires compartmentation of photosynthetic enzymes between mesophyll (M) and bundle sheath (BS) cells. The upstream region of the gene encoding the maize PHOSPHOENOLPYRUVATE CARBOXYLASE 1 (ZmPEPC1) has been shown sufficient to drive M-specific ZmPEPC1 gene expression. Although this region has been well characterized, to date, only few trans-factors involved in the ZmPEPC1 gene regulation were identified. Here, using a yeast one-hybrid approach, we have identified three novel maize transcription factors ZmHB87, ZmCPP8, and ZmOrphan94 as binding to the ZmPEPC1 upstream region. Bimolecular fluorescence complementation assays in maize M protoplasts unveiled that ZmOrphan94 forms homodimers and interacts with ZmCPP8 and with two other ZmPEPC1 regulators previously reported, ZmbHLH80 and ZmbHLH90. Trans-activation assays in maize M protoplasts unveiled that ZmHB87 does not have a clear transcriptional activity, whereas ZmCPP8 and ZmOrphan94 act as activator and repressor, respectively. Moreover, we observed that ZmOrphan94 reduces the trans-activation activity of both activators ZmCPP8 and ZmbHLH90. Using the electromobility shift assay, we showed that ZmOrphan94 binds to several cis-elements present in the ZmPEPC1 upstream region and one of these cis-elements overlaps with the ZmbHLH90 binding site. Gene expression analysis revealed that ZmOrphan94 is preferentially expressed in the BS cells, suggesting that ZmOrphan94 is part of a transcriptional regulatory network downregulating ZmPEPC1 transcript level in the BS cells. Based on both this and our previous work, we propose a model underpinning the importance of a regulatory mechanism within BS cells that contributes to the M-specific ZmPEPC1 gene expression

Synergistic Binding of bHLH Transcription Factors to the Promoter of the Maize NADP-ME Gene Used in C4 Photosynthesis Is Based on an Ancient Code Found in the Ancestral C3 State.

C4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialized form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world's most productive crops. The C4 cycle is accomplished through cell-type-specific accumulation of enzymes but cis-elements and transcription factors controlling C4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we tested whether mechanisms impacting on transcription in C4 plants evolved from ancestral components found in C3 species. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C4NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C3 and C4 Panicoid grass species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C3 state. We conclude that C4 photosynthesis has co-opted an ancient C3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C4 photosynthesis

Influence of passage number on the impact of the secretome of adipose tissue stem cells on neural survival, neurodifferentiation and axonal growth

Mesenchymal stem cells (MSCs), and within them adipose tissue derived stem cells (ASCs), have been shown to have therapeutic effects on central nervous system (CNS) cell populations. Such effects have been mostly attributed to soluble factors, as well as vesicles, present in their secretome. Yet, little is known about the impact that MSC passaging might have in the secretion therapeutic profile. Our aim was to show how human ASCs (hASCs) passage number influences the effect of their secretome in neuronal survival, differentiation and axonal growth. For this purpose, post-natal rat hippocampal primary cultures, human neural progenitor cell (hNPCs) cultures and dorsal root ganglia (DRGs) explants were incubated with secretome, collected as conditioned media (CM), obtained from hASCs in P3, P6, P9 and P12. Results showed no differences when comparing percentages of MAP-2 positive cells (a mature neuronal marker) in neuronal cultures or hNPCs, after incubation with hASCs secretome from different passages. The same was observed regarding DRG neurite outgrowth. In order to characterize the secretomes obtained from different passages, a proteomic analysis was performed, revealing that its composition did not vary significantly with passage number P3 to P12. Results allowed us to identify several key proteins, such as pigment epithelium derived factor (PEDF), DJ-1, interleucin-6 (IL-6) and galectin, all of which have already proven to play neuroprotective and neurodifferentiating roles. Proteins that promote neurite outgrowth were also found present, such as semaphorin 7A and glypican-1. We conclude that cellular passaging does not influence significantly hASCs's secretome properties especially their ability to support post-natal neuronal survival, induce neurodifferentiation and promote axonal growth.Prémios Santa Casa Neurociências - Prize Melo e Castro for Spinal Cord Injury Research (MC-17-2013 and MC-04-2017), Canada Research Chair in Biomedical Engineering (LAB), Northern Portugal Regional Operational Programme (NORTE 2020),, European Regional Development Fund (FEDER), Competitiveness Factors Operational Programme (COMPETE), National Mass Spectrometry Network (RNEM)info:eu-repo/semantics/publishedVersio