Combinatory approach for developing silk fibroin-based scaffolds seeded with human adipose-derived stem cells for a cartilage tissue engineering applications

Several processing technologies have been combined to create scaffolds for different tissue engineering (TE) applications. Hydrogels have been extensively used for cartilage TE applications, presenting several structural similarities to the natural extracellular matrix of cartilage tissue environment[1]. From the different biodegradable materials proposed as matrices for cartilage scaffolding[2], silk fibroin (SF) presents high versatility, processability and tailored mechanical properties, which make this protein attractive for the development of innovative matrices for cartilage TE purposes[3]. In a previous study, we proposed fast formed SF hydrogels produced through a horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) crosslinking reaction, taking advantage of the presence of tyrosine groups[4]. In this work, macro-/micro-porous SF scaffolds derived from enzymatically cross- linked SF hydrogels by a HRP/H2O2 complex were produced in combination with salt-leaching and freeze-drying methodologies. The scaffolds morphology, mechanical properties and chemical characterization were assessed by mean of different characterization techniques (SEM, micro-CT, Instron, FTIR and XRD). The scaffolds structural integrity was evaluated by swelling ratio and degradation profile studies. The in vitro ability to support the adhesion, proliferation and differentiation into the chondrogenic lineage was tested using human adipose-derived stem cells (hASCs) cultured over 28 days in basal and chondrogenic conditions. Cell behaviour in the presence of the SF scaffolds was evaluated through different quantitative (GAGs/DNA and RT-PCR) and qualitative (live/dead, SEM, histology and immunocytochemistry) assays. The in vivo biocompatibility of the SF-based scaffolds was also assessed by subcutaneous implantation in mice for 2 and 4 weeks and analysed by means of hematoxylin & eosin (H&E) staining and immunohistochemical analysis of CD31 angiogenic marker. The results showed highly porous and interconnected SF structures that allowed cell adhesion and infiltration into the scaffolds. In vitro cell viability and proliferation were also observed over the 28 days of culturing in basal conditions and a significant increase of GAGs content was detected on constructs cultured in chondrogenic differentiation medium. In vivo results showed that the implanted scaffolds allowed tissue ingrowthâ s and blood vessels formation/infiltration. The obtained results demonstrated that the innovative approach of combining enzymatically cross-linked SF hydrogels with the salt- leaching and freeze-drying methodologies allowed to produce more versatile scaffold architectures with appropriate mechanical properties and large swelling ability. The positive influence over in vitro chondrogenic differentiation and in vivo response, revealed by the new tissue formation and angiogenesis within the porous scaffolds, validates the proposed macro-/micro-porous SF scaffolds for being used in cartilage TE applications. Moreover, the versatility of these combinatory approach can allow for further applications in other musculoskeletal TE strategies.Investigator FCT program IF/00423/2012 and IF/00411/2013 are also greatly acknowledged. Financial support from FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) and Fundo Social Europeu através do Programa Operacional do Capital Humano (FSE/POCH), PD/59/2013, PD/BD/113806/2015.info:eu-repo/semantics/publishedVersio

Human-derived NLS enhance the gene transfer efficiency of chitosan

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA. In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5. These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.FCT: PTDC/BTM/ORG/28121/2017; PD/BD/52424/2013; SFRH/BD/76873/2011;PIRG-GA-2009-249314info:eu-repo/semantics/publishedVersio

Satellite-based estimation of soil organic carbon in Portuguese grasslands

Soil organic carbon (SOC) sequestration is one of the main ecosystem services provided by well-managed grasslands. In the Mediterranean region, sown biodiverse pastures (SBP) rich in legumes are a nature-based, innovative, and economically competitive livestock production system. As a co-benefit of increased yield, they also contribute to carbon sequestration through SOC accumulation. However, SOC monitoring in SBP require time-consuming and costly field work. Methods: In this study, we propose an expedited and cost-effective indirect method to estimate SOC content. In this study, we developed models for estimating SOC concentration by combining remote sensing (RS) and machine learning (ML) approaches. We used field-measured data collected from nine different farms during four production years (between 2017 and 2021). We utilized RS data from both Sentinel-1 and Sentinel-2, including reflectance bands and vegetation indices. We also used other covariates such as climatic, soil, and terrain variables, for a total of 49 inputs. To reduce multicollinearity problems between the different variables, we performed feature selection using the sequential feature selection approach. We then estimated SOC content using both the complete dataset and the selected features. Multiple ML methods were tested and compared, including multiple linear regression (MLR), random forests (RF), extreme gradient boosting (XGB), and artificial neural networks (ANN). We used a random cross-validation approach (with 10 folds). To find the hyperparameters that led to the best performance, we used a Bayesian optimization approach. Results: Results showed that the XGB method led to higher estimation accuracy than the other methods, and the estimation performance was not significantly influenced by the feature selection approach. For XGB, the average root mean square error (RMSE), measured on the test set among all folds, was 2.78 g kg−1 (r2 equal to 0.68) without feature selection, and 2.77 g kg−1 (r2 equal to 0.68) with feature selection (average SOC content is 13 g kg−1). The models were applied to obtain SOC content maps for all farms. Discussion: This work demonstrated that combining RS and ML can help obtain quick estimations of SOC content to assist with SBP managementinfo:eu-repo/semantics/publishedVersio

Detecção e Quantificação de Aeroalergenos Polínicos de Gramíneas e Olea spp. em Amostras de Ar Exterior e sua Correlação com as Contagens Polínicas

Detecção e Quantificação de Aeroalergenos Polínicos de Gramíneas e Olea spp. em Amostras de Ar Exterior e sua Correlação com as Contagens Polínicas C.M Antunes1,5 R Ferro1*, R Ribeiro1*, E Caeiro4, L. Lopes8, C. Nunes6 R Brandão2,3 & M. Morais-Almeida7 e o HIALINE working group9 1Dep. De Química, Universidade de Évora, Portugal; 2Mediterranean Inst. Crop and Environment Sciences, Univ.Evora, Portugal; 3Depto. Biologia, Universidade de Évora, Portugal; 4Soc.Portuguesa Alergol.Imunologia Clínica , Portugal 5Center for NeuroSciences and Cell Biology-University of Coimbra, Portugal; 6Centro de Imunoalergologia do Algarve, Portugal 7Hospital CUF Descobertas 8Hospital Stª Luzia, Elvas, Portugal 9 M. Thibaudon, France, M. Smith, United Kingdom, C. Galan, Spain R. Albertini, Italy, L. Grewling, Poland, G. Reese, Germany, A. Rantio-Lehtimäki, Finland, S. Jäger and U. Berger, Austria, M. Sofiev, Finland, I. Sauliene, Lithuania, L. Cecchi, Italy Introducão: As Doenças Alérgicas Respiratórias desencadeadas após uma exposição ao pólen atmosférico, como a asma e a rinite alérgica, representam actualmente um grave problema de saúde pública. Em Portugal, o pólen de Poaceae (gramíneas) e de Olea europaea constitui uma das principais fontes de aeroalergenos polínicos atmosféricos e, como tal, uma das principais causas da alergia respiratória. Por essa razão, é importante o desenvolvimento de novas estratégias para a respectiva prevenção e tratamento. O objectivo deste estudo, desenvolvido no âmbito do projecto europeu Hialine, consistiu em avaliar a correlação entre o doseamento, por imunoensaios, de 2 importantes aeroalergenos – Phl p5 e Ole e1 – e as contagens polínicas do pólen de gramíneas e Olea europaea.. Metodologia: Numa plataforma meteorologica situada no centro da cidade de Évora (sul de Portugal), foram recolhidas amostras do ar ambiente a 800L/min com um colector Chemvol high-volume cascade impactor, equipado com filtros para os níveis PM>10µm e 10 µm>PM>2.5µm. As amostras dos filtros em polyurethane foram extraídas com 0.1M NH4HCO3, pH 8.1, suplementado com 0.1% BSA. Os aeroalergenos Phleum p 5 e Ole e 1 foram determinados por ELISA´s específicas. O polen atmosférico de Poaceae e Olea europaea foi monitorizado em simultâneo com um Burkard Seven Day Recording Volumetric Spore Trap* , entre 30 de Abril e 8 de Julho de 2009. Ambos os colectores estiveram colocados lado a lado, com o nível de entrada de ar à mesma altura. Resultados: Durante a estação polínica de 2009, foram registados elevados valores de gramíneas entre os dias 2 de Maio e 1 de Junho. Foi observado também que o conteúdo das amostras de ar dos aeroalergenos Phl p5 ou Ole e1 estavam directamente correlacionados com as contagens polínicas de Poaceae e Oleaceae, respectivamente. Conclusões: Estes resultados sugerem que a quantificação directa de aeroalergenos pode contribuir, juntamente com as contagens polínicas atmosféricas, para definir o risco alergénico com maior precisão. Agradecimentos: Este estudo está integrado no projecto europeu HIALINE (Executive Agency for Health and Consumers under grant agreement No 2008 11 07

Bilateral Amygdala Radio-Frequency Ablation for Refractory Aggressive Behavior Alters Local Cortical Thickness to a Pattern Found in Non-refractory Patients

Aggressive behaviors comprise verbal and/or physical aggression directed toward oneself, others, or objects and are highly prevalent among psychiatric patients, especially patients diagnosed with autism spectrum disorder and severe intellectual disabilities. Some of these patients are considered refractory to treatment, and functional neurosurgery targeting the amygdala can result in widespread plastic brain changes that might reflect ceasing of some abnormal brain function, offering symptom alleviation. This study investigated cortical thickness changes in refractory aggressive behavior patients that were treated with bilateral amygdala ablation and compared to control patients presenting non-refractory aggressive behavior [three refractory and seven non-refractory patients, all males diagnosed with autism spectrum disorder (ASD) and intellectual disabilities]. The Overt Aggression Scale (OAS) was used to quantify behavior and magnetic resonance imaging was performed to investigate cortical thickness. Before surgery, both groups presented similar total OAS score, however refractory patients presented higher physical aggression against others. After surgery the refractory group showed 88% average reduction of aggressive behavior. Imaging analysis showed that while refractory patients present an overall reduction in cortical thickness compared to non-refractory patients across both timepoints, the local pattern of thickness difference found in areas of the neurocircuitry of aggressive behavior present before surgery is diminished and no longer detected after surgery. These results corroborate the hypotheses on induction of widespread neuronal plasticity following functional neurosurgical procedures resulting in modifications in brain morphology and improvement in behavior. Further studies are necessary to determine the underlying cause of these morphological changes and to better understand and improve treatment options

Uncovering the Cyanobacterial Chemical Diversity: The Search for Novel Anticancer Compounds

This article belongs to the Proceedings of The 7th Iberian Congress on Cyanotoxins/3rd Iberoamerican Congress on Cyanotoxins[Abstract] Cancer has a tremendous negative socio-economic impact on our society. Thus, the discovery of new and more effective anticancer drugs is of utmost importance. To address this societal challenge, the main goal of the CYANCAN project was the discovery of anticancer compounds from cyanobacteria. These photosynthetic bacteria are considered among the most promising groups capable of producing metabolites with pharmaceutical applications. A valuable and underexplored natural resource that can underpin the discovery of promising compounds can be found in the Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC) at CIIMAR (Interdisciplinary Centre of Marine and Environmental Research), comprising more than 700 different cyanobacterial strains. Herein, we present the recent advances implemented for finding robust anticancer lead compounds from LEGE-CC cyanobacteria. For this purpose, we developed a natural product library. Sixty cyanobacterial strains, representative of LEGE-CC biodiversity, were chromatographed to yield 480 fractions that were tested for their cytotoxic activity against 2D and 3D models of human colon carcinoma (HCT 116). The conjugation of monolayer assays and 3D cancer spheroids led to the selection of 11 active fractions, of which the chemical space was studied using an untargeted metabolomics approach. The putative annotation and identification of several compounds led to the selection of two marine strains for compound isolation: Leptothoe sp. and Lusitaniella coriacea. The isolation of the compounds was guided through bioactivity assays and mass spectrometry. These molecules were purified from the crudes by employing several chromatography methods, and the chemical structures were determined by means of NMR (nuclear magnetic resonance) and MS/MS (tandem mass spectrometry) experiments. Thus, a novel macrolide-type compound was isolated from Leptothoe sp., which presented a highly cytotoxic activity against our cancer cell models. Its effects on cancer vascularization and metastasis were studied using the zebrafish model. Moreover, from L. coriacea, five unprecedented salicyl-capped thiazol(in)e NRPs-PKs (nonribosomal peptides-polyketides) compounds were isolated. These compounds showed the potential to act as reversers of P-glycoprotein efflux activity.CIIMAR strategic funds UIDB/04423/2020 and UIDP/04423/2020; CYANCAN PTDC/MED-QUI/30944/2017, co-financed by NORTE 2020, Portugal 2020, and the European Union through the ERDF. EMERTOX: EU Horizon 2020 R&I programme under the Marie Skłodowska-Curie grant agreement No 778,069. Ribeiro, T: FCT grant SFRH/BD/139131/2018. Castelo-Branco, R: FCT grant SFRH/BD/136367/2018Centro Interdisciplinar de Investigação Marinha e Ambiental (Porto); UIDB/04423/2020Portugal. Fundação para a Ciência e a Tecnologia; PTDC/MED-QUI/30944/2017Portugal. Fundação para a Ciência e a Tecnologia; SFRH/BD/139131/2018Portugal. Fundação para a Ciência e a Tecnologia; SFRH/BD/136367/201

Genetic diversity and structure of two species of Enyalius (Squamata: Leiosauridae) from neotropical biodiversity hotspots

Diversidade e estrutura genética de duas espécies de Enyalius (Squamata: Leiosauridae) em hotspots neotropicais de biodiversidade. Os lagartos do gênero Enyalius são endêmicos da América do Sul, sendo predominantemente encontrados no Cerrado e em fragmentos da Floresta Atlântica. Este é um gênero pouco estudado, e não foram encontrados dados relacionados à diversidade e à estrutura genética das espécies do gênero. Neste trabalho, investigamos a diversidade genética de populações de E. bilineatus (N = 20) e E. perditus (N = 28), usando um fragmento de 234-pb do citocromo b, e comparamos as sequências geradas com outras publicadas. Dezenove haplótipos distintos foram encontrados (11 de E. perditus e oito de E. bilineatus), sendo oito deles novos registros. Os valores de diversidade haplotípica foram muito similares para as duas espécies (0.684 para E. perditus e 0.647 para E. bilineatus). A distância genética entre as duas espécies foi de 20.3%, e as distâncias intraespecíficas foram 2.0% para E. perditus e 5.6% para E. bilineatus. Nossos dados sugerem que as populações de E. bilineatus são altamente divergentes e que a espécie deve apresentar diversidade críptica. Este é o primeiro estudo medindo a diversidade genética de espécies do gênero Enyalius oriundas de regiões consideradas hotspots da biodiversidade neotropical e apresenta dados relevantes para um melhor entendimento das relações inter e intrapopulacionais, assim como a distribuição das linhagens genéticas desse gênero endêmico.Genetic diversity and structure of two species of Enyalius (Squamata: Leiosauridae) from neotropical biodiversity hotspots. Enyalius, a lizard genus endemic to South America, is mostly distributed in the remains of the Atlantic Forest and in the Cerrado. The genus has been the topic of a few studies but none has quantified the genetic diversity and structure within and among populations of Enyalius. The genetic diversity and structure of populations of E. bilineatus (N = 20) and E. perditus (N = 28) are examined using a 234-bp fragment of the cytochrome b gene and compared with the sequences reported in other published data. Nineteen distinct haplotypes (eleven for E. perditus and eight for E. bilineatus) were found, eight of which were recorded for the first time. The haplotype diversities are highly similar for both species (0.684 for E. perditus and 0.647 for E. bilineatus). The genetic distance between the two species is 20.3% and the distance within species is 2.0% and 5.6% for E. perditus and E. bilineatus, respectively. Our data suggest that populations of E. bilineatus are genetically divergent and may reveal cryptic diversity. This is the first study to quantify the genetic diversity of species of Enyalius from Neotropical biodiversity hotspots. These data facilitate a better understanding of both within and among population variation, and highlight the distribution of genetic lineages of an endemic and poorly studied genus

Genome-wide diversity and differentiation in New World populations of the human malaria parasite Plasmodium vivax.

BACKGROUND: The Americas were the last continent colonized by humans carrying malaria parasites. Plasmodium falciparum from the New World shows very little genetic diversity and greater linkage disequilibrium, compared with its African counterparts, and is clearly subdivided into local, highly divergent populations. However, limited available data have revealed extensive genetic diversity in American populations of another major human malaria parasite, P. vivax. METHODS: We used an improved sample preparation strategy and next-generation sequencing to characterize 9 high-quality P. vivax genome sequences from northwestern Brazil. These new data were compared with publicly available sequences from recently sampled clinical P. vivax isolates from Brazil (BRA, total n = 11 sequences), Peru (PER, n = 23), Colombia (COL, n = 31), and Mexico (MEX, n = 19). PRINCIPAL FINDINGS/CONCLUSIONS: We found that New World populations of P. vivax are as diverse (nucleotide diversity π between 5.2 × 10-4 and 6.2 × 10-4) as P. vivax populations from Southeast Asia, where malaria transmission is substantially more intense. They display several non-synonymous nucleotide substitutions (some of them previously undescribed) in genes known or suspected to be involved in antimalarial drug resistance, such as dhfr, dhps, mdr1, mrp1, and mrp-2, but not in the chloroquine resistance transporter ortholog (crt-o) gene. Moreover, P. vivax in the Americas is much less geographically substructured than local P. falciparum populations, with relatively little between-population genome-wide differentiation (pairwise FST values ranging between 0.025 and 0.092). Finally, P. vivax populations show a rapid decline in linkage disequilibrium with increasing distance between pairs of polymorphic sites, consistent with very frequent outcrossing. We hypothesize that the high diversity of present-day P. vivax lineages in the Americas originated from successive migratory waves and subsequent admixture between parasite lineages from geographically diverse sites. Further genome-wide analyses are required to test the demographic scenario suggested by our data