Dynamic gastrointestinal system as a tool to evaluate the behaviour of carbohydrates after ingestion: from macro to nano scale

GLUPOR 12 - 12nd International Meeting of the Portuguese Carbohydrate Chemistry GroupIn the recent years, much effort has been dedicated to the development of in vitro gastrointestinal systems that closely mimic the physiological processes occurring during human digestion , i.e ., systems that provide accurate results in short time , serving as a tool for rapid screening of foods or delivery systems with different compositions and structures [1] . Static gastrointestinal systems are extensively used , however , most of the times , their simplified gastrointestinal conditions do not accurately simulate the complex physicochemical and physiological processes that occur within the human gastrointestinal tract. A dynamic gastrointestinal system , composed of stomach, duodenum, jejunum and ileum and that simulates the main events that occur during human digestion has been used by our group to evaluate the behaviour of food structures (from macro to nano scale ) under digestion . This dynamic gastrointestinal system can be used for example to predict the glycemic index of food (e .g ., rice) , predicting the blood glucose response after their ingestion and allowing the se lection of the appropriate diet for people that suffer from glucose intol erance. Also , the knowledge of the behaviour of nanostructures (e .g ., carbohydrate- based nanostructures) as well as the fate of the bioactive compounds encapsulated within them in the gastrointestinal tract is of utmost importance for optimizing the bioactivity of encapsulated compounds and to ensure that these structures are safe for human consumption . In fact, the development of novel delivery systems for food applications through the use of nanotechnology has been extensive ly explored [2] . Although the encapsulation of bioactive compounds in bio-based nanostructures have been reported as promising mean of protecting the valuable bioactive compounds and providing new functiona li ties (e.g. increase of bioavailab ility ), the use of very small particle sizes may alter the biological fate of the ingested materials and bioactive compounds , wh i ch could potentially have adverse effects on human health [3]Foundation for Science and Technology (FCT) for her fellowship (SFRH/BPD/1011811/2014). This work was supported by Portuguese Foundation for Science and Technology (FCT) under the scope of the Project PTDC/AGR-TEC/52151/2014 and of the strategic funding of UID/BIO/044691/2013 unit, and COMPETE 2020 (POCl-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 -Programa Operacional Regional do Norte. The authors would also like to thank the investement project n° 017931, co-funded by Fundo Europeu de Desenvolvimento Regional (FEDER) through Programa Operacional Competitividade e lnternacionalização (COMPETE 2020)info:eu-repo/semantics/publishedVersio

Bio-based nanocarriers incorporating curcumin bioaccessibility and cell viability evaluation

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] For decades, curcumin (Cur), a natural polyphenol product derived from turmeric (Curcuma longa) has been considered one of the most promising bioactive compounds due to its health benefits such as anti-inflammatory, antioxidant and anticarcinogenic properties. However, Cur application as functional compound in food products has been limited due to light, heat, and oxidation sensitive and mainly, to poor aqueous solubility which limit its bioavailability [1]. To increase Cur bioaccessibility and consequently, increase bioavailability, several carriers have been investigated, particularly nanocarriers. Among the various nanocarriers described in the literature, lipid-based nanocarriers may offer a promising tool to increase the stability, efficacy and safety of lipophilic compounds, namely Cur [2]. Moreover, the understanding of Cur-loaded nanocarriers’ behaviour under gastrointestinal (GI) conditions is fundamental to produce safe and customized nanocarriers with optimized bioactivity for oral consumption. The aim of this study was to comparatively analyze the impact of two different lipid nanocarriers incorporating Cur - solid lipid nanoparticles (SLN) and nanoemulsions (NE) – on bioaccessibility and Caco-2 cells viability. [...]info:eu-repo/semantics/publishedVersio

In vitro digestion and storage stability of riboflavin-loaded WPI nanostructures towards foods fortification

The consumption of fortified foods incorporating bioactive compounds as a way to promote a healthier lifestyle has gain particular interest in research community and food industry. However, due to their chemical instabilities, bioactive compounds bioavailability can be compromised during post-processing, storage, and digestion. Their encapsulation/association in nanostructures offers a good strategy to enhance bioactive compounds bioavailability. Whey protein isolate (WPI) nanostructures were developed to associate riboflavin (Rb), aiming at its incorporation in foods, and their storage stability and digestion behavior were evaluated. Rb bioaccessibility was determined through spectrofluorimetry by quantifying Rb concentration in the soluble fraction after digestion, that was performed using INFOGEST static in vitro gastrointestinal model. Also, storage stability was evaluated by assessing nanostructures size and polydispersity (PdI) through dynamic light scattering, over 45 days at 4 °C and 25 °C. Rb-loaded WPI nanostructures showed no statistically significant differences in terms of size (ca. 120 nm) and PdI (0.2) during storage period, at both temperatures tested. Rb showed a bioaccessibility of 56 % when associated in WPI nanostructures, enhancing Rb bioaccessibility. These results contribute to improve the knowledge on the use of WPI nanostructures as effective encapsulating systems to augment hydrophilic bioactive compounds bioaccessibility, towards food fortification.info:eu-repo/semantics/publishedVersio

Enhancing curcumin bioaccessibility through different nanoformulations

5th International Conference on Food Digestion[Excerpt] Introduction: Curcumin, a natural polyphenolic phytochemical, is known for its wide range of biological activities, however it has an extremely low water solubility as well as a low bioavailability, which limit its application as a bioactive ingredient in food. The use of delivery systems at nanoscale such as nanoemulsions (NE) and solid lipid nanoparticles (SLN) has been reported as a promising mean of improving the lipophilic bioactive compounds’ bioavailability and their physical and chemical stability. However, the knowledge of the behaviour of different nanoformulations as well as the fate of bioactive compounds encapsulated within them in the gastrointestinal (GI) tract is of utmost importance to either assess their safety for human consumption and to produce tailored delivery systems (i.e. with optimized bioactivity). The aim of this work was the evaluation of the behaviour of two different bio-based nanoformulations (NE and SLN) incorporating curcumin when submitted to an in vitro digestion and the assessment of their cytotoxicity. [...]Ana C. Pinheiro and Joana T. Martins acknowledge the Foundation for Science and Technology (FCT) for their fellowships (SFRH/BPD/101181/2014 and SFRH/BPD/89992/2012). This work was supported by Portuguese Foundation for Science and Technology (FCT) under the scope of the Project PTDC/AGR-TEC/5215/2014, of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Advances in nutraceutical delivery systems: From formulation design for bioavailability enhancement to efficacy and safety evaluation

Background: Aiming at the enhancement of food products' nutritional and health value, the incorporation of nutraceuticals has attracted increasing interest in the last years. However, they often exhibit low water solubility and stability, limiting their direct incorporation into food products. Also, they show very low bioavailability due to limited bioaccessibility, poor absorption and/or chemical transformation within the gastrointestinal tract. This renders their health benefits extremely difficult to be realized by the consumers. Scope and Approach In the present review the recent innovations regarding the formulation and design of bio-based micro and nano-delivery systems to encapsulate nutraceuticals is discussed; it also gives an overview of the challenges associated to their development; and highlights some strategies to enhance nutraceuticals' bioavailability. An insight about delivery systems' potential toxicity (in particular at nano-scale) is also provided. Key Findings and Conclusions Recent developments in the design of bio-based delivery systems offer the possibility of stabilizing and enhancing nutraceuticals' functionality within food products. In fact, different strategies can be used to enhance nutraceuticals' bioavailability: i) nano-delivery systems, besides showing a huge potential for the protection of valuable nutraceuticals during food processing/digestion, can be used to increase their bioavailability; ii) absorption enhancement technologies have been successfully used to increase nutraceuticals' membrane permeation; and iii) excipient foods have been shown to improve nutraceuticals' biological activity. However, the application of these enabling technologies to food is hindered by very pertinent issues that can be summarized in the effective preservation/maximization of the nutraceuticals' bioactivity and safety, once inside the human body.Ana C. Pinheiro and Joana T. Martins acknowledge the Foundation for Science and Technology (FCT) for their fellowships (SFRH/BPD/ 101181/2014 and SFRH/BPD/89992/2012). This work was supported by Portuguese Foundation for Science and Technology (FCT) under the scope of the Project PTDC/AGR-TEC/5215/2014, of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-010145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

An in vitro fermentation model to study the impact of bacteriophages targeting shiga toxin-encoding escherichia coli on the colonic microbiota

Lytic bacteriophages are considered safe for human consumption as biocontrol agents against foodborne pathogens, in particular in ready-to-eat foodstuffs. Phages could, however, evolve to infect different hosts when passing through the gastrointestinal tract (GIT). This underlines the importance of understanding the impact of phages towards colonic microbiota, particularly towards bacterial families usually found in the colon such as the Enterobacteriaceae. Here we propose in vitro batch fermentation as model for initial safety screening of lytic phages targeting Shiga toxin-producing Escherichia coli (STEC). As inoculum we used faecal material of three healthy donors. To assess phage safety, we monitored fermentation parameters, including short chain fatty acid production and gas production/intake by colonic microbiota. We performed shotgun metagenomic analysis to evaluate the outcome of phage interference with colonic microbiota composition and functional potential. During the 24h incubation, concentrations of phage and its host were also evaluated. We found the phage used in this study, named E. coli phage vB_EcoS_Ace (Ace), to be safe towards human colonic microbiota, independently of the donors faecal content used. This suggests that individuality of donor faecal microbiota did not interfere with phage effect on the fermentations. However, the model revealed that the attenuated STEC strain used as phage host perturbed the faecal microbiota as based on metagenomic analysis, with potential differences in metabolic output. We conclude that the in vitro batch fermentation model used in this study is a reliable safety screening for lytic phages intended to be used as biocontrol agents.info:eu-repo/semantics/publishedVersio

3D printing techniques and their applications to organ-on-a-chip platforms: a systematic review

Three-dimensional (3D) in vitro models, such as organ-on-a-chip platforms, are an emerging and effective technology that allows the replication of the function of tissues and organs, bridging the gap amid the conventional models based on planar cell cultures or animals and the complex human system. Hence, they have been increasingly used for biomedical research, such as drug discovery and personalized healthcare. A promising strategy for their fabrication is 3D printing, a layer-by-layer fabrication process that allows the construction of complex 3D structures. In contrast, 3D bioprinting, an evolving biofabrication method, focuses on the accurate deposition of hydrogel bioinks loaded with cells to construct tissue-engineered structures. The purpose of the present work is to conduct a systematic review (SR) of the published literature, according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses, providing a source of information on the evolution of organ-on-a-chip platforms obtained resorting to 3D printing and bioprinting techniques. In the literature search, PubMed, Scopus, and ScienceDirect databases were used, and two authors independently performed the search, study selection, and data extraction. The goal of this SR is to highlight the importance and advantages of using 3D printing techniques in obtaining organ-on-a-chip platforms, and also to identify potential gaps and future perspectives in this research field. Additionally, challenges in integrating sensors in organs-on-chip platforms are briefly investigated and discussed.The authors are grateful for the funding of FCT through the projects NORTE-01-0145- FEDER-029394, NORTE-01-0145-FEDER-030171 funded by COMPETE2020, NORTE2020, PORTUGAL2020, and FEDER. This work was also supported by Fundação para a Ciência e a Tecnologia (FCT) under the strategic grants UIDB/04077/2020, UIDB/00319/2020, UIDB/04436/2020 and UIDB/00532/2020. This work was also funded by AMED-CREST Grant Number JP20gm1310001h0002.Violeta Carvalho acknowledges the PhD scholarship UI/BD/151028/2021 attributed by FCT. Inês Gonçalves acknowledges the PhD scholarship BD/08646/2020 attributed by FCT

Organ-on-a-chip platforms for drug screening and delivery in tumor cells: a systematic review

The development of cancer models that rectify the simplicity of monolayer or static cell cultures physiologic microenvironment and, at the same time, replicate the human system more accurately than animal models has been a challenge in biomedical research. Organ-on-a-chip (OoC) devices are a solution that has been explored over the last decade. The combination of microfluidics and cell culture allows the design of a dynamic microenvironment suitable for the evaluation of treatments’ efficacy and effects, closer to the response observed in patients. This systematic review sums the studies from the last decade, where OoC with cancer cell cultures were used for drug screening assays. The studies were selected from three databases and analyzed following the research guidelines for systematic reviews proposed by PRISMA. In the selected studies, several types of cancer cells were evaluated, and the majority of treatments tested were standard chemotherapeutic drugs. Some studies reported higher drug resistance of the cultures on the OoC devices than on 2D cultures, which indicates the better resemblance to in vivo conditions of the former. Several studies also included the replication of the microvasculature or the combination of different cell cultures. The presence of vasculature can influence positively or negatively the drug efficacy since it contributes to a greater diffusion of the drug and also oxygen and nutrients. Co-cultures with liver cells contributed to the evaluation of the systemic toxicity of some drugs metabolites. Nevertheless, few studies used patient cells for the drug screening assays.This work has been supported by the projects NORTE-01-0145-FEDER-030171 (project reference PTDC/EME-SIS/30171/2017), NORTE-01-0145-FEDER-029394 (project reference PTDC/EMDEMD/29394/2017), through the COMPETE2020, the Lisb@2020, the Programa Operacional Regional do Norte–Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (FEDER) and by Fundação para a Ciência e Tecnologia (FCT), and through FEDER and FCT, project references EXPL/EMD-EMD/0650/2021 and PTDC/EEI-EEE/2846/2021. The authors also acknowledge the partial financial support within the R&D Units Project Scope: UIDB/00319/2020, UIDB/04077/2020, UIDB/00690/2020, UIDB/04436/2020. This work was also funded by AMED-CREST Grant Number JP20gm1310001h0002. Raquel O. Rodrigues (R.O.R.) thanks FCT for her contract funding provided through 2020.03975.CEECIND

ASPECTOS IMUNOLÓGICOS E CLÍNICOS DA LEISHMANIOSE TEGUMENTAR AMERICANA: uma revisão

Leishmanioses são doenças infecciosas, zoonóticas, causadas por protozoários do gênero Leishmania que, ainda hoje, se constituem em importantes problemas de saúde pública mundial. Caracterizam-se como um complexo de doenças com importante espectro clínico e diversidade epidemiológica e de acordo com a espécie de Leishmania inoculada pelo vetor, o indivíduo infectado apresentará quadros diferentes, variando desde o aparecimento de lesões cutâneas, até a visceralização da infecção. A resposta imune nos seres humanos, devido a sua complexidade, ainda não está tão bem caracterizada como em camundongos. essa resposta envolve citocinas, moléculas coestimulatórias e ativação de linfócitos t auxiliares. De maneira geral, é aceito que a diferença entre resistência e susceptibilidade à infecção está relacionada à expansão de células t auxi-liares (th) dos tipos 1 e 2 (th1 e th2). Pacientes com a forma cutânea localizada desenvolvem ativação de linfócitos th1, na região da lesão, enquanto, aqueles com a forma cutâneo-mucosa apresentam ativação mista de linfócitos th1 e th2. Já os pacientes com a forma cutâneo-difusa exibem quase exclusivamente a ativação de linfócitos th2. o estudo da interação entre parasitas e hospedeiros, mediante a avaliação da biologia parasitária, a saliva do inseto- vetor, e mecanismos imunológicos da resposta do hospedeiro vertebrado têm dado uma nova luz ao entendimento dos mecanimos que regem essa relação. re-visamos aqui alguns desses aspectos.descritores: Leishmaniose tegumentar Americana; Clínica; Epidemiologia; ImunologiaAbstract:  Leishmaniasis are zoonot-ic infectious disease caused by protozoa of the genus Leishmania that still today constitute important public health problems worldwide. Characterized as a complex spectrum of diseases with signifcant clinical and epidemiological and diversity ac-cording to the species of Leishmania inoculated by the vector and infected individuals will present different conditions, rang-ing from the development of cutaneous lesions to visceral infection. the immune response in humans, due to its complexity, is still not as well characterized as in mice. this response involves cytokines, co-stimulatory molecules and activation of helper t lymphocytes. in general, it is assumed that the difference between resistance and susceptibility to infection is related to the expansion of t helper cells (th) type 1 and type 2 (th2 and th1). Patients diagnosed with localized skin develop activa-tion of th1 lymphocytes in the region of the lesion, while those with the present form mucocutaneous activation mixed th1 and th2 lymphocytes. Patients with diffuse cutaneous-form exhibit almost exclusively the activation of th2 lymphocytes.the study of the interaction between parasites and hosts, through the assessment, parasite biology, insect saliva-vector and vertebrate host response has given birth to a new understanding of the engine that governs this relationship. We review here some of these aspects.descriptors: American Tegumentar Leishmaniasis; Clinics; Epidemiology; Immunolog

H. pylori phages: from genome release to hope for use as therapy

The increasing antibiotic-resistant Helicobacter pylori infections worldwide and the ineffectiveness of treatments led the World Health Organization to designate clarithromycin-resistant H. pylori as a high-priority bacterium for antibiotic research and development. (Bacterio)phages, viruses that infect bacteria, showing effectiveness in the treatment of pathogenic bacteria, could be a promising alternative strategy in the fight against H. pylori infections. Material and methods In this work, a collection of 74 Portuguese H. pylori-clinical strains was used to screen for the presence of phage genes, using a new PCR-based method. Selected strains were subsequently sequenced and prophage isolation was attempted using UV radiation. Three phages were isolated, one of which was further characterized genetically and biologically. Results PCR-based detection indicated the presence of target phage sequences in 14 strains, and the induction strategies resulted in the release of a new phage. It presents a genome length of 31,162 bp with a G+C content of 37.1 %. This podovirus showed capability to form phage plaques in five strains, was stable under an in vitro gastric digestion model, and was able to maintain a H. pylori population at low levels for up to 24h post-infection. Conclusion The new PCR screening method proved to be very effective in the selection of strains carrying prophages, resulting in the isolation of a new H. pylori phage. This phage presented very promising characteristics in terms of stability and efficacy, being therefore a small step towards the future use of phage therapy in the fight against H. pylori infections.info:eu-repo/semantics/publishedVersio