Unveiling the mechanism of action of 7α-acetoxy-6β-hydroxyroyleanone on an mrsa/visa strain: Membrane and cell wall interactions

UIDB/00100/2020 PTDC/MED-QUI/29036/2017 CEECIND/03414/2018 UIDB/04378/2020 PTDC/BIA-MIC/31645/2017 UID/DTP/04138/2019 UID/DTP/04567/2019 CBIOS/PRUID/BI1/2017 UIDB/04567/2020 UID/AMB/50017 UIDP/50017/2020 UIDB/50017/2020The number of cases of failure in the treatment of infections associated with resistant bacteria is on the rise, due to the decreasing efficacy of current antibiotics. Notably, 7α-Acetoxy-6β-hydroxyroyleanone (AHR), a diterpene isolated from different Plectranthus species, showed antibacterial activity, namely against Methicillin-resistant Staphylococcus aureus (MRSA) strains. The high antibacterial activity and low cytotoxicity render this natural compound an interesting alternative against resistant bacteria. The aim of this study is to understand the mechanism of action of AHR on MRSA, using the MRSA/Vancomycin-intermediate S. aureus (VISA) strain CIP 106760, and to study the AHR effect on lipid bilayers and on the cell wall. Although AHR interacted with lipid bilayers, it did not have a significant effect on membrane passive permeability. Alternatively, bacteria treated with this royleanone displayed cell wall disruption, without revealing cell lysis. In conclusion, the results gathered so far point to a yet undescribed mode of action that needs further investigation.publishersversionpublishe

Functional cell microcarriers: a new platform for cell separation and expansion

Publicado em "Journal of Tissue Engineering and Regenerative Medicine", vol. 7, supp. 1 (2013)The success of many stem cell applications in the biomedical field is highly dependent on the development of separation techniques for isolation and purification of cells with a very high yield and purity. Despite all the achievements made in the field over the past several years, new systems for effective cell separation are still needed. Previous work from our group demonstrated that functional chitosan films grafted with antibodies promote selective cell adhesion. 1 Herein we developed chitosan microparticles able to capture a specific cell types based in the concept of antibody coating for cell sorting. Our goal was to create new biomaterial surfaces capable of recruit a specific cell population within a mixture, reducing cell manipulation and time-consuming allowing at the same time cell expansion. Such system acts as a microcarrier for cell expansion of a specific cell target. Microcarrier culture system offers the advantage of providing a larger surface area for the growth of anchorage-dependent cells in a suspension culture system. Chitosan was chosen due to the excellent biocompatibility, gel forming properties, chemistry surface and low cell adhesion. This allows the modification with specific biochemical cues, for a controllable cell attachment. Here we develop functional biotinylated microparticles, such system allows tailoring microparticles to a variety of functional biomolecules. Here we tested the immobilization of antibodies to target specific cell types, CD31 for endothelial cells and CD90 for adipose stem cells. Primarily designed for an application in tissue engineering, two main challenges are accomplished with the herein presented microparticles: separation and scale-up expansion of specific cell type. The herein developed polymeric microparticles can also be used for directly deliver cells in vivo to repair and regenerate tissues

Functional chitosan microcarriers for selective cell attachment and expansion

The success of many stem cell applications in the biomedical field is highly dependent on the development of reliable techniques either for isolation or selection of specific cell populations with a very high yield and purity.1 In this work we propose the use of chitosan microparticles (μPs) to capture a specific cell type based in the concept of antibody-antigen binding. Our goal was to create new biomaterials capable of selecting within a heterotypic cell suspension, a specific sub-population, and supporting subsequent cell expansion. Such system simultaneously allows the selection and acts as a microcarrier for a specific target, thus reducing cell manipulation and time-consumption

World state of quality: a frontier approach to benchmark the performance of countries worldwide

Purpose - The World State of Quality (WSQ) Project aims to evaluate, analyse, rank and categorise countries according to their performance in quality as a multidimensional concept. The Project involves the computation of an overall score for each country, obtained as a weighted average of ranking positions of 16 metrics, with weights determined by a panel of experts. Methodology-This work proposes an alternative strategy for that procedure, using a Benefit-of-the-Doubt (BoD) Composite Indicator approach under the framework of Data Envelopment Analysis (DEA). This strategy avoids the need of using subjective weights and normalising data by rank positions, using a more objective procedure to obtain the countries’ ranking. A new overall score of the World State of Quality is proposed, which allows the categorisation of countries’ performance. The novel insights resulting from the use of this methodology are discussed, including the identification of strengths and weaknesses of the various countries, and the peers that can be used for facilitating continuous improvements policies. Findings - The results show that the BoD approach and the original method used by the WSQ Project present comparable results. Countries’ strengths and weaknesses and their suitable peers and targets for benchmarking are presented with illustrative examples. Originality/value – A novel frontier approach for countries’ benchmarking regarding their performance in quality is proposed, incorporating new insights into the current method.FCT - Fundação para a Ciência e a Tecnologia(2021.05244)The authors acknowledge the financial support provided by FCT- Fundação para a Ciência e a Tecnologia (Portuguese National Funding Agency for Science, Research and Technology) through PhD research grants and SFRH/BD/131285/2017. This work has been supported by FCT – Fundação para a Ciência e Tecnologia within the R&D Units Project Scope: UIDB/00319/2020

The effect of chitosan on the in vitro biological performance of chitosan-poly(butylene succinate) blends

Chitosan blends with synthetic biodegradable polymers have been proposed for various biomedical applications due to their versatile mechanical properties and easier processing. However, details regarding the main surface characteristics that may benefit from the blending of these two types of materials are still missing. Hence, this work aims at investigating the surface properties of chitosan-based blends, illustrating the way these properties determine the material-proteins interactions and ultimately the behavior of osteoblast-like cells. The surface characteristics of modified and nonmodified blends were assessed using complimentary techniques such as optical microscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR-ATR), X-ray photoelectron spectroscopy (XPS), contact angle measurements and surface energy calculations. The adsorption of human serum albumin (HSA) and human plasma fibronectin (HFN) onto the different surfaces was quantified by association of an indirect method with a colorimetric assay. It was found that the presence of chitosan on the surface promoted the adsorption of proteins. Moreover, a preferential adsorption of albumin over fibronectin was registered. The in vitro biological performance of the studied materials was further investigated by a direct contact assay with an osteoblastic-like cell line (SaOs-2). A synergistic effect of the two components of the blend was observed. While the synthetic polyester promoted the adhesion of SaOs-2, the presence of chitosan significantly enhanced the osteoblastic activity of these cells. This work further confirmed the interest in designing polymeric blends with natural polymers as a successful strategy to enhance the biological performance of a biomaterial

Novel and revisited approaches in nanoparticle systems for buccal drug delivery

The buccal route is considered patient friendly due to its non-invasive nature and ease of administration. Such delivery route has been used as an alternative for the delivery of drugs that undergo first-pass metabolism or are susceptible to pH and enzymatic degradation, such as occurs in the gastrointestinal tract. However, the drug concentration absorbed in the buccal mucosa is often low to obtain an acceptable therapeutic effect, mainly due to the saliva turnover, tongue and masticatory movements, phonation, enzymatic degradation and lack of epithelium permeation. Therefore, the encapsulation of drugs into nanoparticles is an important strategy to avoid such problems and improve their buccal delivery. Different materials from lipids to natural or synthetic polymers and others have been used to protect and deliver drugs in a sustained, controlled or targeted manner, and enhance their uptake through the buccal mucosa improving their bioavailability and therapeutic outcome. Overall, the main aim of this review is to perform an overview about the nanotechnological approaches developed so far to improve the buccal delivery of drugs. Herein, several types of nanoparticles and delivery strategies are addressed, and a special focus on pipeline products is also given.info:eu-repo/semantics/acceptedVersio

Stroke due to Percheron artery occlusion: description of a consecutive case series from Southern Portugal

The artery of Percheron (AOP) is an abnormal variant of the arterial supply of the thalamus. Stroke caused by AOP occlusion is seldom reported. AOP leads to bilateral thalamic and rostral midbrain infarct presenting with unspecific manifestations. There are few descriptions of case series of stroke caused by AOP. We sought to review the clinicoradiological characteristics of AOP infarction from Algarve, Southern Portugal. Eight consecutive cases were retrospectively identified by searching the electronic clinical charts, as well as the stroke Unit database (2015–2020). Sociodemographic (age and gender) and clinicoradiological characteristics (etiological classification, admission severity, manifestations, and short- and long-term prognoses) were retrieved. The corresponding frequency of AOP infarction was 0.17% (95% confidence interval: 0.05–0.28). The mean age was 67.1 (range: 60–80) years. The range of stroke severity evaluated assessed by the National Institute of Health Stroke Scale ranged from 5 to 23 (median ¼ 7.5). None of the patients receive acute ischemic stroke reperfusion treatment. AOP patterns were isolated bilateral paramedian thalamic (n ¼ 2), bilateral paramedian and anterior thalamic (n ¼ 2), and bilateral paramedian thalamic with rostral midbrain (n ¼ 4). Two patients (20%) died on the short term (30 days). At hospital discharge, six patients had functional disability of 2 on the modified Rankin scale. In the follow-up at 6 months, half (n ¼ 3) of the survivors had persistent hypersomnia and two had vascular dementia. Stroke from AOP presents with variable clinical and radiological presentations and patients do not receive alteplase. The shortterm survivor and the long-term functional independency can be compromised after AOS infarct.info:eu-repo/semantics/publishedVersio

Effects of residues on the degradation of PHA produced from mixed microbial cultures and processed in extrusion

Thermal degradation upon melting is one of the major drawbacks reported for polyhydroxyalkanoates (PHA). However, the role of residues originating from the fermentation and the extraction steps on the thermal stability of this class of biopolymers still needs to be clarified. In the particular case of PHA produced from mixed microbial cultures (MMC), this topic is even less documented in the literature. Here, two polyhydroxy(butyrate-co-valerate) (PHBV) produced from MMC enriched in PHA accumulating organisms and fed with cheese whey were studied. A micro extrusion line is used to produce filaments and assess the processability and the degradation of processed PHBV. The prototype micro extrusion line allows for studying grams of materials. The two PHBV contain 18 mol% HV. PHBV was recovered with 11 wt% residues, and further submitted to a purification procedure resulting in a second biopolyester containing less than 2 wt% impurities. The thermorheological characterization of the two PHBV is first presented, together with their semicrystalline properties. Then the processing windows of the two biopolyesters are presented. Finally, the properties of extruded filaments are reported and the thermomechanical degradation of PHBV is extensively studied. The structure was assessed by wide angle X-ray diffraction, mechanical and rheological properties are reported, thermal properties are studied with differential scanning calorimetry and thermogravimetric analysis, whereas Fourier Transform Infrared spectroscopy was used to assess the impact of the extrusion on PHBV chemical structure. All results obtained with the two PHBV are compared to assess the effects of residues on both PHBV processability and degradation

Proof-of-Concept Study of Multifunctional Hybrid Nanoparticle System Combined with NIR Laser Irradiation for the Treatment of Melanoma

Supplementary Materials - available online at https://www.mdpi.com/article/10.3390/biom11040511/s1The global impact of cancer emphasizes the importance of developing innovative, effective and minimally invasive therapies. In the context of superficial cancers, the development of a multifunctional nanoparticle-based system and its in vitro and in vivo safety and efficacy characterization are, herein, proposed as a proof-of-concept. This multifunctional system consists of gold nanoparticles coated with hyaluronic and oleic acids, and functionalized with epidermal growth factor for greater specificity towards cutaneous melanoma cells. This nanoparticle system is activated by a near-infrared laser. The characterization of this nanoparticle system included several phases, with in vitro assays being firstly performed to assess the safety of gold nanoparticles without laser irradiation. Then, hairless immunocompromised mice were selected for a xenograft model upon inoculation of A375 human melanoma cells. Treatment with near-infrared laser irradiation for five minutes combined with in situ administration of the nanoparticles showed a tumor volume reduction of approximately 80% and, in some cases, led to the formation of several necrotic foci, observed histologically. No significant skin erythema at the irradiation zone was verified, nor other harmful effects on the excised organs. In conclusion, these assays suggest that this system is safe and shows promising results for the treatment of superficial melanoma.The authors would like to thank to Fundação para a Ciência e Tecnologia (FCT) for the essential financial support under the project’s references PTDC/BBB-BMC/0611/2012, UIDB/00645/2020, UIDB/04138/2020 and UIDP/04138/2020 as well as for the PhD fellowships SFRH/BD/148044/2019 and SFRH/BD/147306/2019.info:eu-repo/semantics/publishedVersio

Sodium alginate/polycaprolactone co-axial wet-spun microfibers modified with N-carboxymethyl chitosan and the peptide AAPV for Staphylococcus aureus and human neutrophil elastase inhibition in potential chronic wound scenarios

In chronic wound (CW) scenarios, Staphylococcus aureus-induced infections are very prevalent. This leads to abnormal inflammatory processes, in which proteolytic enzymes, such as human neutrophil elastase (HNE), become highly expressed. Alanine-Alanine-Proline-Valine (AAPV) is an antimicrobial tetrapeptide capable of suppressing the HNE activity, restoring its expression to standard rates. Here, we proposed the incorporation of the peptide AAPV within an innovative co-axial drug delivery system, in which the peptide liberation was controlled by N-carboxymethyl chitosan (NCMC) solubilization, a pH-sensitive antimicrobial polymer effective against Staphylococcus aureus. The microfibers' core was composed of polycaprolactone (PCL), a mechanically resilient polymer, and AAPV, while the shell was made of the highly hydrated and absorbent sodium alginate (SA) and NCMC, responsive to neutral-basic pH (characteristic of CW). NCMC was loaded at twice its minimum bactericidal concentration (6.144 mg/mL) against S. aureus, while AAPV was loaded at its maximum inhibitory concentration against HNE (50 μg/mL), and the production of fibers with a core-shell structure, in which all components could be detected (directly or indirectly), was confirmed. Core-shell fibers were characterized as flexible and mechanically resilient, and structurally stable after 28-days of immersion in physiological-like environments. Time-kill kinetics evaluations revealed the effective action of NCMC against S. aureus, while elastase inhibitory activity examinations proved the ability of AAPV to reduce HNE levels. Cell biology testing confirmed the safety of the engineered fiber system for human tissue contact, with fibroblast-like cells and human keratinocytes maintaining their morphology while in contact with the produced fibers. Data confirmed the engineered drug delivery platform as potentially effective for applications in CW care.Authors acknowledge the Portuguese Foundation for Science and Technology (FCT), FEDER funds by means of Portugal 2020 Competitive Factors Operational Program (POCI) and the Portuguese Government (OE) for funding the project PEPTEX with reference PTDC/CTMTEX/28074/2017 (POCI-01-0145-FEDER-028074). Authors also acknowledge project UIDP/00264/2020 of 2C2T and UID/QUI/00686/2020 of CQ, funded by national funds through FCT/MCTES. C.S.M. and H.P.F. also acknowledge FCT for PhD funding via scholarship 2020.08547.BD and for auxiliary researcher contract via 2021.02720.CEEIND, respectively