Production of chitosan based films enriched with oregano essential oil for increased antibacterial activity

During the last years, there has been an increasing interest in developing bio-based active films to improve food safety, extend food shelf life and reduce the use of chemical preservatives. Chitosan, a deacetylated derivative of chitin, is a linear polysaccharide consisting of -(1 4) glucosamine and N-acetylglucosamine residues with potential to be used as a food packaging/coating material. This biopolymer can be used in a wide range of applications in the food industry due to several interesting properties such as its biodegradability, biocompatibility, non-toxicity, antimicrobial activity and versatile physical properties such as its film-forming capacity. Recently, different strategies have been explored to improve its natural properties for the development of food packaging/coating materials with enhanced antimicrobial activity. In particular, the incorporation in chitosan films of essential oils (EO) with acknowledged antibacterial properties, as an alternative of synthetic preservatives, is a matter of great interest since they are generally perceived by consumers as being “natural” food additives. Thus, the objective of this work was the production of chitosan films enriched with oregano EO to further improve the natural antimicrobial properties of chitosan. The obtained films which were then evaluated for its antibacterial activity

Pinus Susceptibility to Pitch Canker Triggers Specific Physiological Responses in Symptomatic Plants:An Integrated Approach

Fusarium circinatum, the causal agent of pine pitch canker (PPC), is an emergent and still understudied risk that threatens Pinus forests worldwide, with potential production and sustainability losses. In order to explore the response of pine species with distinct levels of susceptibility to PPC, we investigated changes in physiology, hormones, specific gene transcripts, and primary metabolism occurring in symptomatic Pinus pinea, Pinus pinaster, and Pinus radiata upon inoculation with F. circinatum. Pinus radiata and P. pinaster exhibiting high and intermediate susceptibility to PPC, respectively, suffered changes in plant water status and photosynthetic impairment. This was associated with sink metabolism induction, a general accumulation of amino acids and overexpression of pathogenesis-related genes. On the other hand, P. pinea exhibited the greatest resistance to PPC and stomatal opening, transpiration increase, and glycerol accumulation were observed in inoculated plants. A stronger induction of pyruvate decarboxylase transcripts and differential hormones regulation were also found for inoculated P. pinea in comparison with the susceptible Pinus species studied. The specific physiological changes reported herein are the first steps to understand the complex Pinus–Fusarium interaction and create tools for the selection of resistant genotypes thus contributing to disease mitigation

Production of chitosan based films enriched with oregano essential oil for increased antibacterial activity

During the last years, there has been an increasing interest in developing bio-based active films to improve food safety, extend food shelf life and reduce the use of chemical preservatives [1]. Chitosan, a deacetylated derivative of chitin, is a linear polysaccharide consisting of β-(I⇒t4) glucosarnine and N-acetylglucosamine residues with potential to be used as a food packaging/coating material. This biopolymer can be used in a wide range of applications in the food industry due to several interesting properties such as its biodegradability, biocompatibility, non-toxicity, antimicrobial activity and versatile physical properties such as its film-forming capacity [I]. Recently, different strategies have been explored to improve its natural properties for the development of food packaging/coating materials with enhanced antimicrobial activity. In particular, the incorporation of essential oils (EO) with recognized antibacterial properties, as an alternative of synthetic preservatives, is a matter of great interest since they are generally perceived by consumers as being "natural" food additives

Corrigendum: A Novel Small Molecule p53 Stabilizer for Brain Cell Differentiation

Brain tumor, as any type of cancer, is assumed to be sustained by a small subpopulation of stem-like cells with distinctive properties that allow them to survive conventional therapies and drive tumor recurrence. Thus, the identification of new molecules capable of controlling stemness properties may be key in developing effective therapeutic strategies for cancer by inducing stem-like cells differentiation. Spiropyrazoline oxindoles have previously been shown to induce apoptosis and cell cycle arrest, as well as upregulate p53 steady-state levels, while decreasing its main inhibitor MDM2 in the HCT116 human colorectal carcinoma cell line. In this study, we made modifications in this scaffold by including combinations of different substituents in the pyrazoline ring in order to obtain novel small molecules that could modulate p53 activity and act as differentiation inducer agents. The antiproliferative activity of the synthesized compounds was assessed using the isogenic pair of HCT116 cell lines differing in the presence or absence of the p53 gene. Among the tested spirooxindoles, spiropyrazoline oxindole 1a was selective against the cancer cell line expressing wild-type p53 and presented low cytotoxicity. This small molecule induced neural stem cell (NSC) differentiation through reduced SOX2 (marker of multipotency) and increased βIII-tubulin (marker of neural differentiation) which suggests a great potential as a non-toxic inducer of cell differentiation. More importantly, in glioma cancer cells (GL-261), compound 1a reduced stemness, by decreasing SOX2 protein levels, while also promoting chemotherapy sensitization. These results highlight the potential of p53 modulators for brain cell differentiation, with spirooxindole 1a representing a promising lead molecule for the development of new brain antitumor drugs.</p

miR-335 targets LRRK2 and mitigates inflammation in Parkinson’s disease

Copyright © 2021 Oliveira, Dionísio, Gaspar, Correia Guedes, Coelho, Rosa, Ferreira, Amaral and Rodrigues. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Parkinson's disease (PD) is mainly driven by dopaminergic neuronal degeneration in the substantia nigra pars compacta accompanied by chronic neuroinflammation. Despite being mainly sporadic, approximately 10% of all cases are defined as heritable forms of PD, with mutations in the leucine-rich repeat kinase (LRRK2) gene being the most frequent known cause of familial PD. MicroRNAs (miRNAs or miRs), including miR-335, are frequently deregulated in neurodegenerative diseases, such as PD. Here, we aimed to dissect the protective role of miR-335 during inflammation and/or neurodegenerative events in experimental models of PD. Our results showed that miR-335 is significantly downregulated in different PD-mimicking conditions, including BV2 microglia cells stimulated with lipopolysaccharide (LPS) and/or overexpressing wild-type LRRK2. Importantly, these results were confirmed in serum of mice injected with 1-methyl-1-4-phenyl-1,2,3,6-tetrahydripyridine hydrochloride (MPTP), and further validated in patients with idiopathic PD (iPD) and those harboring mutations in LRRK2 (LRRK2-PD), thus corroborating potential clinical relevance. Mechanistically, miR-335 directly targeted LRRK2 mRNA. In the BV2 and N9 microglia cell lines, miR-335 strongly counteracted LPS-induced proinflammatory gene expression, and downregulated receptor interacting protein 1 (RIP1) and RIP3, two important players of necroptotic and inflammatory signaling pathways. Further, miR-335 inhibited LPS-mediated ERK1/2 activation. LRRK2-Wt-induced proinflammatory gene expression was also significantly reduced by miR-335 overexpression. Finally, in SH-SY5Y neuroblastoma cells, miR-335 decreased the expression of pro-inflammatory genes triggered by α-synuclein. In conclusion, we revealed novel roles for miR-335 in both microglia and neuronal cells that strongly halt the effects of classical inflammatory stimuli or LRRK2-Wt overexpression, thus attenuating chronic neuroinflammation.This research was funded in part by UIDB/04138/2020 from Fundação para a Ciência e Tecnologia (FCT), Portugal. SO received a Ph.D. fellowship (PD/BD/128332/2017) from FCT.info:eu-repo/semantics/publishedVersio

A Novel Small Molecule p53 Stabilizer for Brain Cell Differentiation

Brain tumor, as any type of cancer, is assumed to be sustained by a small subpopulation of stem-like cells with distinctive properties that allow them to survive conventional therapies and drive tumor recurrence. Thus, the identification of new molecules capable of controlling stemness properties may be key in developing effective therapeutic strategies for cancer by inducing stem-like cells differentiation. Spiropyrazoline oxindoles have previously been shown to induce apoptosis and cell cycle arrest, as well as upregulate p53 steady-state levels, while decreasing its main inhibitor MDM2 in the HCT116 human colorectal carcinoma cell line. In this study, we made modifications in this scaffold by including combinations of different substituents in the pyrazoline ring in order to obtain novel small molecules that could modulate p53 activity and act as differentiation inducer agents. The antiproliferative activity of the synthesized compounds was assessed using the isogenic pair of HCT116 cell lines differing in the presence or absence of the p53 gene. Among the tested spirooxindoles, spiropyrazoline oxindole 1a was selective against the cancer cell line expressing wild-type p53 and presented low cytotoxicity. This small molecule induced neural stem cell (NSC) differentiation through reduced SOX2 (marker of multipotency) and increased βIII-tubulin (marker of neural differentiation) which suggests a great potential as a non-toxic inducer of cell differentiation. More importantly, in glioma cancer cells (GL-261), compound 1a reduced stemness, by decreasing SOX2 protein levels, while also promoting chemotherapy sensitization. These results highlight the potential of p53 modulators for brain cell differentiation, with spirooxindole 1a representing a promising lead molecule for the development of new brain antitumor drugs

Discovery of MDM2-p53 and MDM4-p53 protein-protein interactions small molecule dual inhibitors

MDM2 and MDM4 are key negative regulators of p53, an important protein involved in several cell processes (e. g. cell cycle and apoptosis). Not surprisingly, the p53 tumor suppressor function is inactivated in tumors over -expressing these two proteins. Therefore, both MDM2 and MDM4 are considered important therapeutic targets for an effective reactivation of the p53 function. Herein, we present our studies on the development of spi-ropyrazoline oxindole small molecules able to inhibit MDM2/4-p53 protein-protein interactions (PPIs). Twenty-seven potential spiropyrazoline oxindole dual inhibitors were prepared based on in silico structural optimization studies of a hit compound with MDM2 and MDM4 proteins. The antiproliferative activity of the target com-pounds was evaluated in cancer cell lines harboring wild-type p53 and overexpressing MDM2 and/or MDM4. The most active compounds in SJSA-1 cells, 2q and 3b, induce cell death via apoptosis and control cell growth by targeting the G0/G1 cell cycle checkpoint in a concentration-dependent manner. The ability of the five most active spiropyrazoline oxindoles in dissociating p53 from MDM2 and MDM4 was analyzed by an immu-noenzymatic assay. Three compounds inhibited MDM2/4-p53 PPIs with IC50 values in the nM range, while one compound inhibited more selectively the MDM2-p53 PPI over the MDM4-p53 PPI. Collectively, these results show: i) 3b may serve as a valuable lead for obtaining selective MDM2-p53 PPI inhibitors and more efficient anti-osteosarcoma agents; ii) 2a, 2q and 3f may serve as valuable leads for obtaining dual MDM2/4 inhibitors and more effective p53 activators

Preclinical validation of a new hybrid molecule loaded in liposomes for melanoma management

The aggressiveness of melanoma and lack of effective therapies incite the discovery of novel strategies. Recently, a new dual acting hybrid molecule (HM), combining a triazene and a ʟ-tyrosine analogue, was synthesized. HM was designed to specifically be activated by tyrosinase, the enzyme involved in melanin biosynthesis and overexpressed in melanoma. HM displayed remarkable superior antiproliferative activity towards various cancer cell lines compared with temozolomide (TMZ), a triazene drug in clinical use, that acts through DNA alkylation. In B16-F10 cells, HM induced a cell cycle arrest at phase G0/G1 with a 2.8-fold decrease in cell proliferation index. Also, compared to control cells, HM led to a concentration-dependent reduction in tyrosinase activity and increase in caspase 3/7 activity. To maximize the therapeutic performance of HM in vivo, its incorporation in long blood circulating liposomes, containing poly(ethylene glycol) (PEG) at their surface, was performed for passively targeting tumour sites. HM liposomes (LIP HM) exhibited high stability in biological fluids. Preclinical studies demonstrated its safety for systemic administration and in a subcutaneous murine melanoma model, significantly reduced tumour progression. In a metastatic murine melanoma model, a superior antitumour effect was also observed for mice receiving LIP HM, with markedly reduction of lung metastases compared to positive control group (TMZ). Biodistribution studies using 111In-labelled LIP HM demonstrated its ability for passively targeting tumour sites, thus correlating with the high therapeutic effect observed in the two experimental murine melanoma models. Overall, our proposed nanotherapeutic strategy was validated as an effective and safe alternative against melanoma.</p

Initial modelled outputs at field scale

This report comprises Deliverable 6.16 in the project, which contributes to the third objective as it presents field-scale evaluation of innovations, in order to adapt and evaluate agroforestry designs and practices for locations where agroforestry is currently not-widely practised or declining. The modelling of outputs at field scale to support best agroforestry practices is an ongoing activity during the AGFORWARD project. This report highlights some of the outputs which has been produced in the form of three papers (either submitted or about to be submitted to a peer-reviewed journal) or in four presentations at the Third European Agroforestry Conference in May 2016N/