The yeast Saccharomyces cerevisiae is sensitive to colorectal cancer routine treatment EGFR antibody Cetuximab

Cetuximab/Erbitux® (Merk Sereno), a drug used in routine treatment of colorectal cancer and other malignant pathologies, is a monoclonal antibody against the Epidermal Growth Factor Receptor (EGFR). A frequent problem affecting the clinical use of Cetuximab is the lack of effectiveness deriving from frequent mutations in K-ras (1-3). A set of mutations in K-ras gene, KRAS c.35G>A (G12D), KRAS c.38G>A (G13D) in exon 2, and KRAS c.183A>T (Q61H) in exon 3, all implicated in the development of colorectal cancer, have been recognized as impeding Cetuximab’s EGFR inhibitory action in human (1). Ras human genes have recognized counterparts in yeast, RAS1 and RAS2. The corresponding proteins belong to the PKA/cAMP MAPK pathway are involved in cell proliferation, in differentiation into hyphae and spores, in response to nitrogen starvation, and in carbon source regulation (4, 5). In opposition to Ras, yeasts do not have a recognized ortholog of EGFR. Nevertheless, yeast is sensitive to Imatinib, another drug that targets specifically EGFR in human cells (6). We generated recombinant yeast strains expressing human wild-type (wt) and mutated open reading frames (ORFs) of K-ras to use in the optimization of phenotypic tests appropriate for the assessment of cell sensitivity to Cetuximab. We observed that Saccharomyces cerevisiae, is sensitive to the treatment with this drug at identical concentration as human cell cultures. Moreover, the complementation of yeast deletions in RAS1 and/or RAS2 with wt or the above mentioned mutated forms of human K-RAS did not alter the response of the cells to the treatment. This suggests that the sensitivity of S. cerevisiae to Cetuximab is independent of the Ras/cAMP pathway. These results further indicate the existence of a paralog of EGFR protein in yeast cell surface. In view of these results, research focused on identifying the EGFR yeast counterpart, downstream effectors and target genes, and determining the correspondent Cetuximab/Erbitux® mode of action.Este trabalho é financiado por Fundos FEDER através do Programa Operacional Factores de Competitividade – COMPETE e por Fundos Nacionais através da FCT – Fundação para a Ciência e a Tecnologia no âmbito do projecto PEst-C/BIA/UI4050/201

Gellan-gum coated gold nanorods: a new tool for biomedical applications

Publicado em "Frontiers in Bioengineering and Biotechnology. Conference Abstract: 10th World Biomaterials Congress"Introduction: Gold nanorods (AuNRs) have been widely studied, in the scope of cancer research and biomedical applications [1]. Their optical properties, easy synthesis and high surface area make AuNRs an outstanding tool for a plethora of applications, such as drug delivery, imaging and tissue engineering [2]. However, before biomedical usage, it is necessary to modify AuNRs surface chemistry, to improve their biocompatibility and stability under biological conditions [3]. One possible approach is the use of biocompatible natural-based polymers that enhance AuNRs performance while allowing the controlled release of drugs/bioactive agents. Herein, we report the successful preparation of a core-shell nanostructure using low-acyl gellan gum (GG) [4], [5] for the coating of AuNRs. Methods: AuNRs were prepared following the seed-mediated growth method [6]. Then, particles were coated with a successive deposition of anionic and cationic polyelectrolytes (poly(acrylic acid) and poly(allylamine hydrochloride), respectively). The pre-coated nanorods were added to a low-acyl gellan gum (GG) solution, previously heated at 90ºC to allow dissolution, and the mixture was stirred overnight at room temperature. The GG-coated AuNRs (AuNR-GG) were characterized by UVvisible spectrometry, zeta potential measurements and transmission electron microscopy (TEM). AuNRs cytotoxicity was accessed in vitro after 1, 3, 7 and 14 days of SaOS-2 cell culture, using an MTS assay. Nanoparticles internalization was confirmed by TEM. In vivo biocompatibility tests were also performed by delivering a solution of AuNRs-GG via tail injection in mice. Results and Discussion: AuNRs were successfully synthesized and coated with a GG shell of approximately 7 nm, as shown in Figure 1. The presence of the GG around AuNRs clearly improved particles stability at different salt and pH conditions, as observed by UV-vis spectroscopy. The in vitro studies using SaOS-2 showed that AuNRs-GG are noncytotoxic. TEM analyses have confirmed that nanoparticles are uptaken by cells and aggregate within cytoplasmic vesicles as depicted in Figure 2. Additionally, in vivo tests suggest that AuNRs are harmless for mice after 24 hours. Conclusion: In this study, AuNRs were individually coated with a gellan gum (GG) shell, resulting in nanoparticles with enhanced stability under different salt concentrations and range of pH’s. Thence, one can conclude that the GG present around the nanoparticles acts as a stabilizer, improving AuNRs stability and biocompatibility. AuNRs-GG have shown noteworthy features and a high potential for further use on biomedical applications including intracellular drug delivery and imaging.QREN (ON.2 – NORTE-01-0124-FEDER-000018) co-financed by North Portugal Regional Operational Program (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF)The Portuguese Foundation for Science and Technology (FCT) - SFRH/BD/102710/201

Speech-Mediated Retention in Dyslexics

Dyslexics (6 to 9 yr. old) and a control group of 12 normal readers (second-graders) were tested on a memory task with rhyming and non-rhyming items. The most important finding is that both groups were penalized by rhyme, indicating speech coding. It was observed that the dyslexics were not less penalized by rhyme than were controls. The present results are compared with those of other recent studies.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

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

Development & performance assessment of a new ATMP for cartilage tissue engineering

Publicado em "Frontiers in Bioengineering and Biotechnology. Conference Abstract: 10th World Biomaterials Congress"NORTE-07-0202-FEDER-023189 / ARTICULATE - Desenvolvimento de novos produtos e terapias regenerativas para o tratamento de patologias articulares / AD

Comparison of some theoretical models for fittings of the temperature dependence of the fundamental energy gap in GaAs

In this work we report on a comparison of some theoretical models usually used to fit the dependence on temperature of the fundamental energy gap of semiconductor materials. We used in our investigations the theoretical models of Viña, Pässler-p and Pässler-ρ to fit several sets of experimental data, available in the literature for the energy gap of GaAs in the temperature range from 12 to 974 K. Performing several fittings for different values of the upper limit of the analyzed temperature range (Tmax), we were able to follow in a systematic way the evolution of the fitting parameters up to the limit of high temperatures and make a comparison between the zero-point values obtained from the different models by extrapolating the linear dependence of the gaps at high T to T = 0 K and that determined by the dependence of the gap on isotope mass. Using experimental data measured by absorption spectroscopy, we observed the non-linear behavior of Eg(T) of GaAs for T > ΘD.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)CNP

End-on PEGylation of heparin: Effect on anticoagulant activity and complexation with protamine

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2023.125957.Heparin is the most common anticoagulant used in clinical practice but shows some downsides such as short half- life (for the high molecular weight heparin) and secondary effects. On the other hand, its low molecular weight analogue cannot be neutralized with protamine, and therefore cannot be used in some treatments. To address these issues, we conjugated polyethylene glycol (PEG) to heparin reducing end (end-on) via oxime ligation and studied the interactions of the conjugate (Hep-b-PEG) with antithrombin III (AT) and protamine. Isothermal titration calorimetry showed that Hep-b-PEG maintains the affinity to AT. Dynamic light scattering demonstrated that the Hep-b-PEG formed colloidal stable nanocomplexes with protamine instead of large multi-molecular aggregates, associated with heparin side effects. The in vitro (human plasma) and in vivo experiments (Sprague Dawley rats) evidenced an extended half-life and higher anticoagulant activity of the conjugate when compared to unmodified heparin.  The authors thank INNO Laboratório Veterinário for the measurements of in vivo coagulation times, Raul Pacheco for discussions about the ITC results, Teresa Oliveira for her help in the in vivo experiments and Ramón Rail for his help in the 3D structures of Fig. 1, Fig. 4. We thank funding provided by the Portuguese Foundation for Science and Technology (PTDC/QUI-POL/28117/2017 and CEECIND/00814/2017). África González-Fernández thanks Xunta de Galicia (Grupo de Referencia competitiva, GRC-ED431C 2020/02) 2020-2023

Die Ikonographie der Maitreyafigur mit gekreuzten Füßen und des Tusita-Himmels, dargestellt anhand des Sutra von der Visualisierung der Wiedergeburt des Bodhisattva Maitreya im himmlischen Paradies Tusita

Basing much of my study on analysis and classification of 127 figures of Maitreya, I interpreted the iconography of this Bodhisattva and accompanied with my translation of the Maitreya Sutra "the visualization of the rebirth of the Bodhisattva Maitreya in the heavenly paradise Tusita". I took the contemplating figure of Maitreya with crossed legs in the grottos of Yungang, China, as the focus of my analysis and interpreted it as a "practicing visualization", based on the aforementioned sutra and on commentary about this sutra by the Silla monk Yonhyo. Yonhyo explained that adherents of Maitreya had to practice the visualization which was described in the sutra in order to attain future opportunities seeing Maitreya either in Tusita heaven or in Jambdvipa. And I also interpreted the crown of the contemplating figure, which is Korean National Treasure No.78. The crown of the contemplating figure No.78 described the palace of Maitreya in Tusita. This figure of Maitreya is practicing the visualization of heavenly paradise Tusita

Bioactive fibrous membrane containing endogenous nerve growth factor promotes cellular and functional recuperation of impaired cavernous nerve: An in vivo animal study

Introduction And Objective Prostate cancer is a frequent disease and the radical prostatectomy one of the most used treatment when the cancer is localized. This procedure commonly causes erectile dysfunction (ED) by injure the cavernous nerve (CN) during the surgery.With this research we investigate the ability of a bioactive fibrous membrane (FM) to regenerate the damaged CN in a rat model of CN injury. Methods Male Spragueâ Dawley (SD) rats were distributed by 4 experimental groups: sham (only a lower midline abdominal incision), negative control (lower midline abdominal incision with pelvic dissection and CN crush but no FM implantation), electrospun fibrous membrane [eFM] (with CN crush and FM implantation) and electrospun fibrous membrane biofunctionalized with NGF from rat urine [eFM-uNGF] (with CN crush). Function was evaluated five weeks later after an injection of apomorphine by glans observation (visual scoring) and intracavernous pressure (ICP) measurements. Neurogenic genes expression and Histological and Immunohistochemical analysis of CN and cavernous tissue was performed. Results in this model of bilateral CN crush the treatment with implanted bioactive fibrous membrane induces CN regeneration and restoration of erectile function (p <0.001) (figure), showing a significant increased number of smooth muscle cells and nNOS and eNOS contents. Additionally, the bioactive fibrous membrane promotes a CN protective effect and increase the nerve regeneration capacity by increasing the number of myelinated axons and nNOS-positive cells, recovering from the CN fibrosis observed in rats not treated. Conclusions This personalized regenerative strategy could help better recovery of erectile function after CN injuries, and it may constitute an effective novel option to prostate cancer patients suffering from ED after being subject to radical prostatectomy