Recent Decisions

Comments on recent decisions by James M. Corcoran, Manuel A. Sequeira, D. D. Robertson, Joseph B. Joyce, A. J. Deutsch, Lawrence J. Dolan, Otto K. Hilbert, and J. Robert Geiman

Uma visão crítica sobre a utilização de cortinas de ar em estabelecimentos comerciais climatizados

Tendo por base um conjunto de ensaios realizados numa instalação experimental especialmente concebida para o efeito, é avaliada a influência de alguns parâmetros geométricos e dinâmicos sobre a eficácia da vedação térmica alcançada com a utilização de aparelhos de cortina de ar. A informação recolhida permitiu estabelecer diversas recomendações sobre a selecção, instalação e operação deste tipo de aparelho, as quais, na prática, nem sempre são seguidas, conforme se pode antever dos dados preliminares recolhidos em vistorias técnicas realizadas a estabelecimentos comerciais que utilizam este tipo de solução

A modular approach for efficient production of multi-HA Influenza VLP-based vaccines

Safer and broadly protective vaccines are needed to cope with the continuous evolution of circulating influenza virus strains. Promising approaches based on the expression of multiple hemagglutinins (HA) (alone or in combination with neuraminidase and matrix M1 proteins), in a single vector or virus-like particle (VLP) have been proposed. However, expression of multiple genes in the same vector can be an issue due to tandem repetition of promoter sequences leading to its instability. By combining stable with transient expression we can rationally distribute the number of genes to be expressed by each system and thus mitigate this risk. Therefore, we developed a modular system using stable and baculovirus-mediated expression of HA in insect High Five cells for production of multi-HA influenza enveloped VLPs. First, a stable pool of High Five cells expressing two HA was established by random integration and intracellular HA expression confirmed by immunofluorescence microscopy. This cell pool was then infected at CCI of 2 or 3×106 cells/mL with M1-encoding baculovirus to evaluate the incorporation of stable expressed HA in the M1 core, thus generating Influenza VLPs. Similar levels of Influenza VLPs could be detected in culture medium by hemagglutination assay regardless of the CCI used. Aiming to increase HA production, infections at a higher CCI were attempted by implementing a feeding strategy designed based on the exhaustion of key nutrients, analyzed by 1H-NMR spectroscopy. Noteworthy, the shake flask cultures that were supplemented and infected at a CCI of 4×106 cells/mL showed a 8-fold increase in HA levels when compared to above tested conditions. The robustness of our modular system was then challenged by infecting the stable High Five cell pool with a baculovirus encoding M1 plus three HA proteins. Results obtained at CCI of 4×106 cells/mL with supplementation showed a 4-fold increase in HA levels when compared to standard infection conditions (CCI of 2 and 3×106 cells/mL). Finally, to demonstrate the scalability of the strategy herein designed, cultures in fully controlled 2L stirred tank bioreactors were performed, and a 1.5-fold improvement in HA levels was obtained when compared to shake flask cultures. Overall, this work demonstrates the suitability of combining a stable insect cell line with baculovirus-mediated expression as a faster platform for production of multi-HA Influenza VLPs surpassing standard methods such as coinfections or the use of larger, unstable vectors. Acknowledgements This work was supported by EU-funded project EDUFLUVAC (FP7-HEALTH-2013-INNOVATION)

Insect cells platforms for fast production of Pseudo-Typed VLPs for drug and vaccine development

Expression systems capable of delivering high concentrations of membrane proteins in their native structure are essential in the vaccine field as well as in drug discovery. In this work, we took advantage of insect cell expression and site-specific gene integration based on flipase-mediated cassette exchange (FMCE) technology to generate cell platforms for efficient production of membrane proteins on the surface of a protein scaffold, namely enveloped virus-like particles (VLPs). The expression of membrane proteins concomitantly with capsid proteins of enveloped viruses (e.g. HIV Gag or influenza M1) will enable their capturing in lipid rafts of the cellular plasma membrane and their display on the surface of budding VLPs, thus providing a native conformation for downstream assays. Parental insect Sf-9 and High Five cells were randomly tagged with GFP-fused Gag or M1 proteins and FACS enriched with cells tagged in genomic “hot-spots” supporting high expression. A linker including a Flp recognition target (FRT) site was used to allow posterior removal of the marker gene from the particle through cassette exchange. By confocal microscopy we could observe that Gag localizes preferentially at the plasma membrane whereas M1 disperses within the cell. Upon promoting Flp-mediated recombination in the tagging populations, cassette exchange was well succeeded, allowing to recover cells tagged in loci supporting FMCE. We are currently evaluating the capability of both core proteins as scaffolds to display GPCRs (e.g. beta-2 adrenergic receptor) and Influenza HA proteins. For the latter, we will present recent results on the feasibility of combining stable and baculovirus-mediated expression of HA in insect High Five cells for production of multi-HA influenza enveloped VLPs towards the development of an “universal” vaccine. This strategy surpasses standard methods for production of multivalent Influenza VLPs such as coinfections or the use of larger, unstable vectors. Overall, modular insect cells platforms are being generated to be readily adaptable for production of a broad range of VLP-based vaccines as well as receptor display particles for drug screening or antibody discovery. Acknowledgments: Funding from European Commission (Project EDUFLUVAC; Grant nr. 602640) and Fundação para a Ciência e a Tecnologia through the project EXPL/BBB-BIO/1541/2013 and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/2012

Synthesis of Bis(3-indolyl)methanes Mediated by Potassium tert-Butoxide

The authors thank the Fundação para a Ciênciae Tecnologia for the fellowship PD/BD/142876/2018.This work was supported by the Associate Laboratory for Green Chemistry– LAQV which is financed by national funds from FCT/MCTESUIDB/50006/2020,UIDP/50006/2020(LAQV).The National NMR Facility is supported by FCT,ROTEIRO/0031/2013-PINFRA/22161/2016,co-financed by FEDER through COMPETE2020,POCI,andPORLandFCT through PIDDAC)and CERMAX(022162).LBM also thanks to FCT/MCTES for the CEEC-Individual Program Contract(CEECIND/03810/2017)The indole moiety is an important N-heterocycle found in natural products, and a key structural component of many value-added chemicals including pharmaceuticals. In particular, bis(3-indolyl)methanes (BIMs) are an important subgroup of indoles, composed of two indole units. Herein, we report the development of a simple method to access BIMs derivatives in yields of up to 77 % by exploiting a tBuOK-mediated coupling reaction of indoles and benzyl alcohols.publishersversionpublishe

Seasonal sensory evaluation of low commercial value or unexploited fish species from the Portuguese coast

Overfishing is increasing over time, and according to FAO (Food and Agriculture Organization), about one third of the world’s fish stocks are now overfished. Thus, diversifying the target species is essential for fisheries sustainability contributing to improve resource-effcient processes. Non-target species can be valuable resources for the development of new food products. However, those species are scarcely studied, and it is of high importance to trace their seasonal sensory profile as a first step towards their valorisation. Therefore, in this study, seasonal influence on sensory properties of five low commercial value or unexploited fish species, namely Trachurus picturatus (blue jack mackerel), Spondyliosoma cantharus (black seabream), Trigla lyra (piper gurnard), Serranus cabrilla (comber) and Capros aper (boarfish), was assessed in order to identify the most favourable season for catching each species. Fish samples were assessed by a panel of 16 semi-trained assessors for sensory attributes previously identified. The evaluation takes place every 2 months. Statistical differences were reported between attributes and seasons for all species, except for T. lyra, which did not present any di erence in its sensory attributes throughout the year.info:eu-repo/semantics/publishedVersio

Adding Value to Bycatch Fish Species Captured in the Portuguese Coast—Development of New Food Products

We live in a world of limited biological resources and ecosystems, which are essential to feed people. Consequently, diversifying target species and considering full exploitation are essential for fishery sustainability. The present study focuses on the valorization of three low commercial value fish species (blue jack mackerel, Trachurus picturatus; black seabream, Spondyliosoma cantharus; and piper gurnard, Trigla lyra) and of two unexploited species (comber, Serranus cabrilla and boarfish, Capros aper) through the development of marine-based food products with added value. A preliminary inquiry with 155 consumers from Região de Lisboa e Vale do Tejo (Center of Portugal) was conducted to assess fish consumption, the applicability of fish product innovation, and the importance of valorizing discarded fish. Five products (black seabream ceviche, smoked blue jack mackerel pâté, dehydrated piper gurnard, fried boarfish, and comber pastries) were developed and investigated for their sensory characteristics and consumer liking by hedonic tests to 90 consumers. The most important descriptors were identified for each product (texture, flavor, color, and appearance). Comber pastries had the highest purchase intention (88%), followed by black seabream ceviche (85%) and blue jack mackerel pâté (76%). Sensory evaluations showed a clear tendency of consumers to accept reformulated products, with the introduction of the low-value and unexploited species under study.info:eu-repo/semantics/publishedVersio

Effectiveness and long-term retention of anti-tumour necrosis factor treatment in juvenile and adult patients with juvenile idiopathic arthritis: data from Reuma.pt

Methods. We prospectively collected patient and disease characteristics from patients with JIA who started biological therapy. Adverse events were collected during the follow-up period. Predictors of response at 1 year and drug retention rates were assessed at 4 years of treatment for the first biologic agent.Results. A total of 812 JIA patients [65% females, mean age at JIA onset 6.9 years (s.d. 4.7)], 227 received biologic therapy; 205 patients (90.3%) were treated with an anti-TNF as the first biologic. All the parameters used to evaluate disease activity, namely number of active joints, ESR and Childhood HAQ/HAQ, decreased significantly at 6 months and 1 year of treatment. The mean reduction in Juvenile Disease Activity Score 10 (JADAS10) after 1 year of treatment was 10.4 (s.d. 7.4). According to the definition of improvement using the JADAS10 score, 83.3% respond to biologic therapy after 1 year. Fourteen patients discontinued biologic therapies due to adverse events. Retention rates were 92.9% at 1 year, 85.5% at 2 years, 78.4% at 3 years and 68.1% at 4 years of treatment. Among all JIA subtypes, only concomitant therapy with corticosteroids was found to be univariately associated with withdrawal of biologic treatment (P = 0.016).Conclusion. Biologic therapies seem effective and safe in patients with JIA. In addition, the retention rates for the first biologic agent are high throughout 4 years

Topography-driven isolation, speciation and a global increase of endemism with elevation

Aim: Higher-elevation areas on islands and continental mountains tend to be separated by longer distances, predicting higher endemism at higher elevations; our study is the first to test the generality of the predicted pattern. We also compare it empirically with contrasting expectations from hypotheses invoking higher speciation with area, temperature and species richness. Location: Thirty-two insular and 18 continental elevational gradients from around the world. Methods: We compiled entire floras with elevation-specific occurrence information, and calculated the proportion of native species that are endemic (‘percent endemism’) in 100-m bands, for each of the 50 elevational gradients. Using generalized linear models, we tested the relationships between percent endemism and elevation, isolation, temperature, area and species richness. Results: Percent endemism consistently increased monotonically with elevation, globally. This was independent of richness–elevation relationships, which had varying shapes but decreased with elevation at high elevations. The endemism–elevation relationships were consistent with isolation-related predictions, but inconsistent with hypotheses related to area, richness and temperature. Main conclusions: Higher per-species speciation rates caused by increasing isolation with elevation are the most plausible and parsimonious explanation for the globally consistent pattern of higher endemism at higher elevations that we identify. We suggest that topography-driven isolation increases speciation rates in mountainous areas, across all elevations and increasingly towards the equator. If so, it represents a mechanism that may contribute to generating latitudinal diversity gradients in a way that is consistent with both present-day and palaeontological evidence

Preferential Localization of Human Origins of DNA Replication at the 5′-Ends of Expressed Genes and at Evolutionarily Conserved DNA Sequences

Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7–1.5 kb obtained from the breast cancer cell line MCF–7 hybridized to a custom tiling array containing 50–60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5′ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors