Publications by authors named "Inés Pinto"

32 Publications

Multiplexed Microfluidic Cartridge for At-Line Protein Monitoring in Mammalian Cell Culture Processes for Biopharmaceutical Production.

ACS Sens 2021 03 16;6(3):842-851. Epub 2021 Mar 16.

KTH Royal Institute of Technology, Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, 171 21 Solna, Sweden.

The biopharmaceutical market has been rapidly growing in recent years, creating a highly competitive arena where R&D is critical to strike a balance between clinical safety and profitability. Toward process optimization, the recent development and adoption of new process analytical technologies (PAT) highlight the dynamic complexity of mammalian/human cell culture processes, as well as the importance of fine-tuning and modeling key metabolites and proteins. In this context, simple, rapid, and cost-effective devices allowing routine at-line monitoring of specific proteins during process development and production are currently lacking. Here, we report the development of a versatile microfluidic protein analysis cartridge allowing the multiplexed bead-based immunodetection of specific proteins directly from complex mixtures with minimal hands-on time. Colorimetric quantification of Chinese hamster ovary (CHO) host cell proteins as key impurities, monoclonal antibodies as target biopharmaceuticals, and lactate dehydrogenase as a marker of cell viability was achieved with limits of detection in the 1-10 ng/mL range and analysis times as short as 30 min. The device was further demonstrated for the monitoring of a Rituximab-producing CHO cell bioreactor over the course of 8 days, providing comparable recoveries to standard enzyme-linked immunosorbent assay (ELISA) kits. The high sensitivity combined with robustness to matrix interference highlights the potential of the device to perform at-line measurements spanning from the bioreactor to the downstream processing.
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http://dx.doi.org/10.1021/acssensors.0c01884DOI Listing
March 2021

[Management of the Difficult Patient: The Example of Zamora].

Acta Med Port 2021 Feb 1;34(2):168. Epub 2021 Feb 1.

Departamento de Psiquiatria e Saúde Mental. Centro Hospitalar de Setúbal. Setúbal. Portugal.

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http://dx.doi.org/10.20344/amp.15330DOI Listing
February 2021

Reengineering Bone-Implant Interfaces for Improved Mechanotransduction and Clinical Outcomes.

Stem Cell Rev Rep 2020 12;16(6):1121-1138

Center for MicroElectroMechanical Systems (CMEMS-UMinho), University of Minho, Campus de Azurém, Guimarães, Portugal.

The number of patients undergoing joint replacement surgery has progressively increased worldwide due to world population ageing. In the Unites States, for example, the prevalence of hip and knee replacements has increased more than 6 and 10 times, respectively, since 1980. Despite advances in orthopaedic implant research, including the development of novel implantable biomaterials, failures are still observed due to inadequate biomechanical compliance at the bone-implant interface. This comprises static and dynamic mechanical mismatch between the bone and the implant surface. The importance and robustness of biomechanical cues for controlling osteogenic differentiation of mesenchymal stem cells (MSC) have been highlighted in recent studies. However, in the context of bone regenerative medicine, it remains elusive how mechanobiological signals controlling MSC osteogenic differentiation dynamics are modulated in their interaction with the bone and with implants. In this review, we highlight recent technological advances aiming to improve host bone-implant interactions based on the osteogenic and mechanoresponsive potential of MSC, in the context of joint replacement surgery. First, we discuss the extracellular and intracellular mechanical forces underlying proper receptivity and stimulation of physiological MSC differentiation and linked osteogenic activity. Second, we provide a critical overview on how this knowledge can be integrated towards the development of biomaterials for improved bone-implant interfaces. Third, we discuss cross-disciplinarily which contributes to the next generation design of novel pro-active orthopaedic implants and their implantation success. Graphical Abstract.
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http://dx.doi.org/10.1007/s12015-020-10022-9DOI Listing
December 2020

Sub-attomole detection of HIV-1 using padlock probes and rolling circle amplification combined with microfluidic affinity chromatography.

Biosens Bioelectron 2020 Oct 26;166:112442. Epub 2020 Jul 26.

Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden. Electronic address:

Despite significant progress in diagnostics and disease management during the past decades, human immunodeficiency virus (HIV) infections are still responsible for nearly 1 million deaths every year, mostly in resource-limited settings. Thus, novel, accurate and cost-effective tools for viral load monitoring become crucial to allow specific diagnostics and the effective monitoring of the associated antiviral therapies. Herein, we report an effective combination of a (1) padlock probe (PLP)-mediated rolling circle amplification (RCA) bioassay and an (2) agarose bead-based microfluidic device for the affinity chromatography-based capture and detection of RCA products (RCPs) pre-labelled simultaneously with biotin and an organic fluorophore. This method allowed the efficient capture of ~1 μm-sized RCPs followed by their quantification either as discrete signals or an average fluorescence signal, thus being compatible with both high-resolution imaging for maximum sensitivity as well as simpler optical detection setups. A limit of detection < 30 fM was obtained for HIV-1 synthetic target with just a single round of RCA, comparable to recently reported procedures requiring technically complex amplification strategies such as hyperbranching and/or enzymatic digestion/amplification. Furthermore, targeting a set of five conserved regions in the HIV-1 gag gene, the method could specifically detect HIV-1 in 293T cell culture supernatants, as well as a set of 11 HIV-1 NIH reference samples with four different subtypes. The reported method provides simplicity of operation, unique versatility of signal transduction (i.e. average or discrete signals), and potential coupling with previously reported miniaturized photodetectors. These combined features hold promise for bringing RCA-based molecular diagnostics closer to the point-of-care.
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http://dx.doi.org/10.1016/j.bios.2020.112442DOI Listing
October 2020

Evaluation of mental health stigma on medical education: an observational study with Portuguese medical students.

Porto Biomed J 2020 Jul-Aug;5(4):e074. Epub 2020 Jul 17.

Centro Hospitalar Universitário de S. João - Alameda Prof. Hernâni Monteiro, Porto, Portugal.

Background: The Portuguese mental health care plan emphasizes that health care professionals can be a source of stigma against people with mental illness enhancing self-stigma and leading to a decrease in the search for help and adherence to treatment.

Methods: In this exploratory study, we surveyed 111 first and last year students from the Faculty of Medicine of the University of Porto, Portugal, using the Portuguese version of the Attribution Questionnaire AQ-27 to assess the attitudes toward mental illness.

Results: The students showed a significant difference in the segregation dimension, and in some items related with pity and coercion in the end of the course. These results express a positive will to integrate people with mental illness in community, a decrease of pity and a valorization of the pharmacological treatment in this kind of disease. The previous personal experience of psychiatric problems decreases the level of segregation and psychological problems increase the motivation to help.

Conclusion: Final-year students express more positive and less discriminatory attitudes toward people with severe mental illness than first-year students. This is likely due to education and contact opportunities promoted throughout the medical school, as well as due to the experience of having gone to a psychology or psychiatric consultation. Knowledge of stigma levels of future medical doctors is therefore crucial for the prevention of attitudes that could condition the provision of medical care.
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http://dx.doi.org/10.1097/j.pbj.0000000000000074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386544PMC
July 2020

Pushing myelination - developmental regulation of myosin expression drives oligodendrocyte morphological differentiation.

J Cell Sci 2020 08 5;133(15). Epub 2020 Aug 5.

International Iberian Nanotechnology Laboratory (INL), 4715-330 Braga, Portugal

Oligodendrocytes are the central nervous system myelin-forming cells providing axonal electrical insulation and higher-order neuronal circuitry. The mechanical forces driving the differentiation of oligodendrocyte precursor cells into myelinating oligodendrocytes are largely unknown, but likely require the spatiotemporal regulation of the architecture and dynamics of the actin and actomyosin cytoskeletons. In this study, we analyzed the expression pattern of myosin motors during oligodendrocyte development. We report that oligodendrocyte differentiation is regulated by the synchronized expression and non-uniform distribution of several members of the myosin network, particularly non-muscle myosins 2B and 2C, which potentially operate as nanomechanical modulators of cell tension and myelin membrane expansion at different cell stages.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.232264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426197PMC
August 2020

Whole-Genome Approach to Understanding the Mechanism of Action of a Histatin 5-Derived Peptide.

Antimicrob Agents Chemother 2020 02 21;64(3). Epub 2020 Feb 21.

Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas, USA

The incidence of opportunistic fungal infections that threaten immunocompromised patients, along with the limited arsenal of antifungal drugs, calls for renewed efforts to develop novel antifungal therapies. Antimicrobial peptides have garnered interest as potential therapeutics. Among naturally occurring peptides, histatin 5 is a well-characterized 24-amino-acid peptide with strong antifungal activity. Our lab has identified a smaller histatin derivative, KM29, with stronger activity against multiple spp., prompting us to investigate its fungicidal mechanism. A genetic screen was developed to test the genomewide deletion collection for mutants with increased or decreased peptide sensitivity. The goal was to identify genes that would reveal insights into the mechanism of action of KM29, to be assessed in Several biological processes yielded increased sensitivity, with endosomal transport and vacuolar function appearing at high frequencies. Among the pathways involved in increased resistance, mitochondrial function showed the highest normalized genome frequency; hence, we focused on characterizing this pathway. KM29 localizes to mitochondria, and the killing activity depends on a functional electron transport chain. In addition, KM29 triggered reactive oxygen species (ROS) production, which was responsible for some cell death but insufficient to account for the complete killing activity. In agreement with this finding, we found that KM29 induced mitochondrial fragmentation and a mild loss of mitochondrial membrane potential. Furthermore, respiratory mutants exhibited severely diminished KM29 uptake. We confirmed this behavior in a respiratory mutant. Taking our findings together, this work delineates the mitochondrial functions associated with KM29 fungicidal activity and provides additional pathways for further characterization in spp.
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http://dx.doi.org/10.1128/AAC.01698-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038261PMC
February 2020

NRARP displays either pro- or anti-tumoral roles in T-cell acute lymphoblastic leukemia depending on Notch and Wnt signaling.

Oncogene 2020 01 4;39(5):975-986. Epub 2019 Oct 4.

Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina Universidade de Lisboa, Lisboa, Portugal.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a dismal prognosis in patients with resistant or relapsed disease. Although NOTCH is a known driver in T-ALL, its clinical inhibition has significant limitations. Our previous studies suggested that NRARP, a negative regulator of Notch signaling, could have a suppressive role in T-ALL. Here, we report that NRARP levels are significantly increased in primary T-ALL cells suggesting that NRARP is not sufficient to block NOTCH oncogenic signals. Interestingly, although NRARP overexpression blocks NOTCH1 signaling and delays the proliferation of T-ALL cells that display high levels of Notch1 signaling, it promotes the expansion of T-ALL cells with lower levels of Notch1 activity. We found that NRARP interacts with lymphoid enhancer-binding factor 1 (LEF1) and potentiates Wnt signaling in T-ALL cells with low levels of Notch. Together these results indicate that NRARP plays a dual role in T-ALL pathogenesis, regulating both Notch and Wnt pathways, with opposite functional effects depending on Notch activity. Consistent with this hypothesis, mice transplanted with T-cells co-expressing NOTCH1 and NRARP develop leukemia later than mice transplanted with T-NOTCH1 cells. Importantly, mice transplanted with T-cells overexpressing NRARP alone developed leukemia with similar kinetics to those transplanted with T-NOTCH1 cells. Our findings uncover a role for NRARP in T-ALL pathogenesis and indicate that Notch inhibition may be detrimental for patients with low levels of Notch signaling, which would likely benefit from the use of Wnt signaling inhibitors. Importantly, our findings may extend to other cancers where Notch and Wnt play a role.
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http://dx.doi.org/10.1038/s41388-019-1042-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989401PMC
January 2020

Protrusion membrane pearling emerges during 3D cell division.

Phys Biol 2019 10 10;16(6):066009. Epub 2019 Oct 10.

Nanobioengineering Group, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 15-21, 08028 Barcelona, Spain. Department of Electronics and Biomedical Engineering, University of Barcelona, C/Martí i Franqués 1, 08028 Barcelona, Spain. Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Av. Monforte de Lemos, 3-5, 28029 Madrid, Spain. Author to whom any correspodence should be addressed. Current address: 3B's Research Group, I3Bs-Research Institute on Biomaterials, Biodegradables and Biomimetics of University of Minho, AvePark-Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco, Guimarães, Portugal.

Cell division is accompanied by dramatic changes in shape that ultimately lead to the physical separation of one cell into two. In 2D microenvironments, cells round up and remain adhered onto the substrate by thin retraction fibers during division. In contrast, in 3D environments, cells divide exhibiting long protrusions that guide the orientation of the division axis. However, the mechanism of cell division in three dimensions still remains poorly understood. Here we report the spontaneous formation of transient quasiperiodic membrane pearling on extended mitotic protrusions during 3D cell division. Protrusion membrane pearling may be initiated by the non-uniform distribution of focal adhesions and consequent stationary instability of the protrusive membrane. Overall, membrane pearling emergence may provide insights into a novel modality of 3D cell division with potential physiological relevance.
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http://dx.doi.org/10.1088/1478-3975/ab4549DOI Listing
October 2019

Optimizing the Performance of Chromatographic Separations Using Microfluidics: Multiplexed and Quantitative Screening of Ligands and Target Molecules.

Biotechnol J 2019 Oct 23;14(10):e1800593. Epub 2019 Jul 23.

IBB - Institute for Bioengineering and Biosciences Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais 1, 1049-001, Lisbon, Portugal.

The optimization of chromatography ligands for the purification of biopharmaceuticals is highly demanded to meet the needs of the pharmaceutical industry. In the case of monoclonal antibodies (mAbs), synthetic ligands comprising multiple types of interactions (multimodal) provide process and economic advantages compared to protein-based affinity ligands. However, optimizing the operation window of these ligands requires the development of effective high-throughput screening platforms. Here, a novel microfluidics-based methodology to perform rapid and multiplexed screening of various multimodal ligands relative to their ability to bind different target molecules is demonstrated. The microfluidic structure comprises three individual chambers (≈8 nL each) packed with different types of chromatography beads in series with the feed flow. An artificial mixture composed of immunoglobulin G (IgG) and bovine serum albumin, labeled with different thiol-reactive neutral fluorescent dyes, is used as a model to quantitatively optimize the performance (yield and purity) of the separation. This approach can potentially be used as a predictive analytical tool in the context of mAb purification, allowing low consumption of molecules and providing results in <3 min. Furthermore, this versatile approach can potentially be extended not only with respect to the number of different resins and target molecules, but also for parallel analysis of multiple conditions.
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http://dx.doi.org/10.1002/biot.201800593DOI Listing
October 2019

Silica bead-based microfluidic device with integrated photodiodes for the rapid capture and detection of rolling circle amplification products in the femtomolar range.

Biosens Bioelectron 2019 Mar 18;128:68-75. Epub 2018 Dec 18.

Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, SE-171 65 Solna, Sweden. Electronic address:

The rapid and sensitive detection of specific nucleic acid sequences at the point-of-care (PoC) is becoming increasingly in demand for a variety of emergent biomedical applications ranging from infectious disease diagnostics to the screening of antimicrobial resistance. To meet such demand, considerable efforts have been invested towards the development of portable and integrated analytical devices combining microfluidics with miniaturized signal transducers. Here, we demonstrate the combination of rolling circle amplification (RCA)-based nucleic acid amplification with an on-chip size-selective trapping of amplicons on silica beads (~8 nL capture chamber) coupled with a thin-film photodiode (200 × 200 µm area) fluorescence readout. Parameters such as the flow rate of the amplicon solution and trapping time were optimized as well as the photodiode measurement settings, providing minimum detection limits below 0.5 fM of targeted nucleic acids and requiring only 5 μL of pre-amplified sample. Finally, we evaluated the analytical performance of our approach by benchmarking it against a commercial instrument for RCA product (RCP) quantification and further investigated the effect of the number of RCA cycles and elongation times (ranging from 10 to 120 min). Moreover, we provide a demonstration of the application for diagnostic purposes by detecting RNA from influenza and Ebola viruses, thus highlighting its suitability for integrated PoC systems.
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http://dx.doi.org/10.1016/j.bios.2018.12.004DOI Listing
March 2019

In vitro evaluation of novel reverse transcriptase inhibitors TAF (tenofovir alafenamide) and OBP-601 (2,3-didehydro-3-deoxy-4-ethynylthymidine) against multi-drug resistant primary isolates of HIV-2.

Antiviral Res 2019 01 2;161:85-89. Epub 2018 Nov 2.

Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003, Lisboa, Portugal; Centro de Investigação Interdisciplinar Egas Moniz (CiiEM), Instituto Universitário Egas Moniz (IUEM), Campus Universitário, Quinta da Granja Monte de Caparica, 2829 - 511, Caparica, Portugal. Electronic address:

New antiretroviral drugs are needed to treat HIV-2 infected patients failing therapy. Herein, we evaluate the activity of novel reverse transcriptase inhibitors tenofovir alafenamide (TAF) and OBP-601(2,3-didehydro-3-deoxy-4-ethynylthymidine) against primary isolates from HIV-2 infected patients experiencing virologic failure. TAF and OBP-601 were tested against twelve primary isolates obtained from nine drug-experienced patients failing therapy and three drug naïve patients using a single-round infectivity assay in TZM-bl cells. The RT-coding region of pol was sequenced and the GRADE algorithm was used to identify resistance profiles and mutations. TAF and OBP-601 inhibited the replication of almost all isolates at a median EC of 0.27 nM and 6.83 nM, respectively. Two isolates showed moderate-level resistance to OBP-601 or TAF and two other isolates showed high-level resistance to OBP-601 or to both drugs. With one exception, all resistant viruses had canonical nucleoside reverse transcriptase inhibitors (NRTIs)-associated resistance mutations (K65R, N69S, V111I, Y115F, Q151M and M184V). Our results show that TAF has potent activity against most multi-drug resistant HIV-2 isolates and should be considered for the treatment of HIV-2 infected patients failing therapy.
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http://dx.doi.org/10.1016/j.antiviral.2018.10.018DOI Listing
January 2019

Nanoparticles provide long-term stability of bevacizumab preserving its antiangiogenic activity.

Acta Biomater 2018 09 21;78:285-295. Epub 2018 Jul 21.

I3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal; INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal; CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Rua Central de Gandra 1317, 4585-116 Gandra, Portugal; School of Pharmacy, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK. Electronic address:

Bevacizumab is one of the most common monoclonal antibodies used to treat cancer due to its antiangiogenic role. However, the frequent parenteral administrations are not attractive for the patient adhesion to the therapy. Nanoencapsulation of bevacizumab might be a useful alternative to increase administration intervals, due to controlled release properties. To achieve a long-term bevacizumab stability into PLGA nanoparticles, we developed an optimized and validated lyophilization protocol. The co-encapsulation of trehalose and bevacizumab into PLGA nanoparticles, associated to their lyophilization with external 10% (w/v) of trehalose, allowed maintenance of the physical-chemical characteristics of nanoparticles and bevacizumab secondary and tertiary structure. More relevant, the antiangiogenic activity of bevacizumab was kept over 6 months of storage while formulated with this protocol. No significant differences were found upon 6 months of storage at 4 °C and 25 °C/60% HR, and minor differences were observed for storage at 40 °C/75% HR, bringing to our knowledge, the first successfully report for monoclonal antibody storage at room temperature, without losing its structural and functional features. Our results served as starting point to understand the monoclonal antibody-based nanoparticle behavior over time, creating an innovative approach for a long-term monoclonal antibody stability.

Statement Of Significance: Nanoencapsulation of monoclonal antibodies has boost the interest of researchers as an alternative to the current antibody-based therapy, changing the route of administrations through controlled release of monoclonal antibodies. Despite good results have been achieved with nanoencapsulation process, no strategy has still found concerning a long-term stability of nanoparticles and monoclonal antibodies. In this study, the aim was to find out a validated and optimized method that allows a long-term stability of nanoparticles and antibodies. Over 6 months of storage, an optimized nanosystem was considered stable for both nanoparticles and antibody structure, at 4 °C and 25 °C, resulting the first successfully report for monoclonal antibody storage at room temperature.
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http://dx.doi.org/10.1016/j.actbio.2018.07.040DOI Listing
September 2018

Multiplexed microfluidic fluorescence immunoassay with photodiode array signal acquisition for sub-minute and point-of-need detection of mycotoxins.

Lab Chip 2018 05;18(11):1569-1580

Instituto de Engenharia de Sistemas e Computadores - Microsistemas e Nanotecnologias (INESC MN) and IN - Institute of Nanoscience and Nanotechnology, Lisbon, Portugal.

Portable, rapid, cost effective and simple analytical tools are in increasing demand to facilitate the routine monitoring of target chemical/biological compounds at the point-of-need. Such devices are highly relevant within the context of food safety, particularly concerning the screening of highly toxic and strictly regulated mycotoxins. To achieve ultrarapid detection of mycotoxins, namely aflatoxin B1, ochratoxin A and deoxynivalenol, at the point-of-need, a novel multiplexed bead-based microfluidic competitive immunosensor, coupled with an array of a-Si:H thin-film photodiodes for integrated fluorescence signal acquisition, is reported. Simultaneously measuring the initial binding rate for each analyte of the sample under analysis against an internal reference, this device provided limits of detection below 1 ng mL-1 for all mycotoxins in a single-step assay and within 1 minute after mixing the sample under analysis with a fluorescent conjugate. The compatibility of the device with the analysis of mycotoxins spiked in corn samples was further demonstrated after performing a sample preparation procedure based on aqueous two-phase extraction. The short times of analysis and sensitivities in the low ng mL-1 range make these devices potentially competitive with the lateral flow devices that are currently the standard for this application. Furthermore, this device architecture and concept is amenable of being expanded to other analytes in food safety, biomedical and other applications.
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http://dx.doi.org/10.1039/c8lc00259bDOI Listing
May 2018

Mechanical plasticity during oligodendrocyte differentiation and myelination.

Glia 2018 01 21;66(1):5-14. Epub 2017 Sep 21.

International Iberian Nanotechnology Laboratory - INL, Braga, Portugal.

In the central nervous system, oligodendrocyte precursor cells are exclusive in their potential to differentiate into myelinating oligodendrocytes. Oligodendrocyte precursor cells migrate within the parenchyma and extend cell membrane protrusions that ultimately evolve into myelinating sheaths able to wrap neuronal axons and significantly increase their electrical conductivity. The subcellular force generating mechanisms driving morphological and functional transformations during oligodendrocyte differentiation and myelination remain elusive. In this review, we highlight the mechanical processes governing oligodendrocyte plasticity in a dynamic interaction with the extracellular matrix.
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http://dx.doi.org/10.1002/glia.23206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743057PMC
January 2018

Polyester-Based Nanoparticles for the Encapsulation of Monoclonal Antibodies.

Methods Mol Biol 2018 ;1674:239-253

i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 208, 4200-393, Porto, Portugal.

Aliphatic polyesters have been widely explored for biomedical applications (e.g., drug delivery systems, biomedical devices, and tissue engineering). Recently, polyesters have been used in nanoparticle formulations for the controlled release of monoclonal antibodies (mAbs) for the enhanced efficacy of antibody-based therapy. Polyester-based nanoparticles for mAb delivery provide decreased antibody dosage, increased antibody stability and protection and longer therapeutic action, ultimately translating to an increased therapeutic index. Additionally, nanoencapsulation holds the potential for the selective cellular recognition and internalization of mAbs, in the disease context when intracellular organelles and molecules (e.g., enzymes, transcription factors and oncogenic proteins) are the preferred target. We present here a detailed method to prepare mAb-loaded polyester-based nanoparticles and the various techniques to characterize the resulting nanoparticles and mAb structure. Finally, we highlight different biological approaches to assess the in vitro bioactivity of the antibody upon nanoparticle release.
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http://dx.doi.org/10.1007/978-1-4939-7312-5_20DOI Listing
May 2018

A new paradigm for antiangiogenic therapy through controlled release of bevacizumab from PLGA nanoparticles.

Sci Rep 2017 06 16;7(1):3736. Epub 2017 Jun 16.

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 208, 4200-393, Porto, Portugal.

Monoclonal antibodies have deserved a remarkable interest for more than 40 years as a vital tool for the treatment of various diseases. Still, there is a raising interest to develop advanced monoclonal antibody delivery systems able to tailor pharmacokinetics. Bevacizumab is a humanized immunoglobulin IgG1 used in antiangiogenic therapies due to its capacity to inhibit the interaction between vascular endothelial growth factor and its receptor. However, bevacizumab-based antiangiogenic therapy is not always effective due to poor treatment compliance associated to multiples administrations and drug resistance. In this work, we show a promising strategy of encapsulating bevacizumab to protect and deliver it, in a controlled manner, increasing the time between administrations and formulation shelf-life. Nanoencapsulation of bevacizumab represents a significant advance for selective antiangiogenic therapies since extracellular, cell surface and intracellular targets can be reached. The present study shows that bevacizumab-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles does not impair its native-like structure after encapsulation and fully retain the bioactivity, making this nanosystem a new paradigm for the improvement of angiogenic therapy.
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http://dx.doi.org/10.1038/s41598-017-03959-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473878PMC
June 2017

Anxiety, Family Functioning and Neuroendocrine Biomarkers in Obese Children.

Acta Med Port 2017 Apr 28;30(4):273-280. Epub 2017 Apr 28.

Department of Neurosciences and Mental Health. Faculty of Medicine. University of Porto. Porto. Portugal. Institute for Research and Innovation in Health (i3S). University of Porto. Porto. Portugal.

Introduction: This observational study explores potential links between obese children's cortisol, and parental mental state, family functioning, and the children's symptoms of anxiety and depression.

Material And Methods: A non-random sample of 104 obese children (55 boys), mean age 10.9 years (standard deviation 1.76), was recruited from a childhood obesity clinic. Obesity was defined as body mass index above the 95th age- and gender-specific percentiles. Neuroendocrine biomarkers were measured. Symptoms of anxiety and depression were assessed with self and parent-reported questionnaires (Anxiety, Depression and Stress Scales; Child Behaviour Checklist). Family functioning was assessed with parent-reported questionnaires (Family Adaptation and Cohesion Scales-III).

Results: A significant, negative correlation (rs = -0.779; p = 0.003) between girls' cortisol and their parents' anxiety symptoms was found, limited to high functioning families. Boys scored significantly higher than girls on parent-reported internalizing symptoms but not on self-report. No association was found between cortisol in children and parental depressive symptoms.

Discussion: Whether the association between cortisol levels in obese children and parental mental health is effectively restricted to girls from high functioning families or is due to study limitations, requires further research. The lack of associations between cortisol in children and parental depressive symptoms, suggests a specific association between cortisol and parental anxiety symptoms.

Conclusion: These results highlight the importance of taking into account family functioning, parental mental state and gender, when investigating neuroendocrine biomarkers in obese children associated with symptoms of anxiety and depression.
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http://dx.doi.org/10.20344/amp.7919DOI Listing
April 2017

The Iron-Dependent Regulation of the Candida albicans Oxidative Stress Response by the CCAAT-Binding Factor.

PLoS One 2017 25;12(1):e0170649. Epub 2017 Jan 25.

Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas, United States of America.

Candida albicans is the most frequently encountered fungal pathogen in humans, capable of causing mucocutaneous and systemic infections in immunocompromised individuals. C. albicans virulence is influenced by multiple factors. Importantly, iron acquisition and avoidance of the immune oxidative burst are two critical barriers for survival in the host. Prior studies using whole genome microarray expression data indicated that the CCAAT-binding factor is involved in the regulation of iron uptake/utilization and the oxidative stress response. This study examines directly the role of the CCAAT-binding factor in regulating the expression of oxidative stress genes in response to iron availability. The CCAAT-binding factor is a heterooligomeric transcription factor previously shown to regulate genes involved in respiration and iron uptake/utilization in C. albicans. Since these pathways directly influence the level of free radicals, it seemed plausible the CCAAT-binding factor regulates genes necessary for the oxidative stress response. In this study, we show the CCAAT-binding factor is involved in regulating some oxidative stress genes in response to iron availability, including CAT1, SOD4, GRX5, and TRX1. We also show that CAT1 expression and catalase activity correlate with the survival of C. albicans to oxidative stress, providing a connection between iron obtainability and the oxidative stress response. We further explore the role of the various CCAAT-binding factor subunits in the formation of distinct protein complexes that modulate the transcription of CAT1 in response to iron. We find that Hap31 and Hap32 can compensate for each other in the formation of an active transcriptional complex; however, they play distinct roles in the oxidative stress response during iron limitation. Moreover, Hap43 was found to be solely responsible for the repression observed under iron deprivation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170649PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5266298PMC
August 2017

Robust gap repair in the contractile ring ensures timely completion of cytokinesis.

J Cell Biol 2016 Dec 13;215(6):789-799. Epub 2016 Dec 13.

Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal

Cytokinesis in animal cells requires the constriction of an actomyosin contractile ring, whose architecture and mechanism remain poorly understood. We use laser microsurgery to explore the biophysical properties of constricting rings in Caenorhabditis elegans embryos. Laser cutting causes rings to snap open. However, instead of disintegrating, ring topology recovers and constriction proceeds. In response to severing, a finite gap forms and is repaired by recruitment of new material in an actin polymerization-dependent manner. An open ring is able to constrict, and rings repair from successive cuts. After gap repair, an increase in constriction velocity allows cytokinesis to complete at the same time as controls. Our analysis demonstrates that tension in the ring increases while net cortical tension at the site of ingression decreases throughout constriction and suggests that cytokinesis is accomplished by contractile modules that assemble and contract autonomously, enabling local repair of the actomyosin network. Consequently, cytokinesis is a highly robust process impervious to discontinuities in contractile ring structure.
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http://dx.doi.org/10.1083/jcb.201605080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5166501PMC
December 2016

Attachment Strategies and Neuroendocrine Biomarkers in Obese Children.

Acta Med Port 2016 May 31;29(5):332-9. Epub 2016 May 31.

Department of Neurosciences and Mental Health. Faculdade de Medicina da Universidade do Porto. Porto. Institute for Research and Innovation inHealth (i3S). University of Porto. Porto.

Introduction: Quality of the parent-infant relationship influences the mechanisms of development of the child's physiological stress regulation. This study explored associations between attachment strategies and both cortisol and thyroid stimulating hormone, hypothesized to be respectively a potential mediator and a potential intervening variable of the mother-child relationship in obese children.

Material And Methods: A sample of 83 obese children (46 boys), aged 10.9 (1.8) years was recruited from a child obesity clinic. Obesity was defined by body mass index percentile adjusted for age and sex. Metabolic biomarkers were measured by routine methods. Attachment strategies were assessed with self and parent-report questionnaires. Family functioning was assessed with parent-reported questionnaires (FACES-III). Multivariate linear regression analyses were performed.

Results: Type A, avoidant attachment strategies, had significant positive association with thyroid stimulating hormone levels and negative association with cortisol levels (R2 = 0.352). Type B, secure attachment strategies, had significant positive associations with both hypothyroidism and body mass index percentile (R2 = 0.541). 'Insecure attachment' (types A and C combined) strategies showed some evidence of positive association with thyroid stimulating hormone (R2 = 0.250).

Discussion: These findings suggest that there may be commonalities in the regulation of hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid axes. Processes involved in development of the type A attachment strategy appear to be associated with effects on the regulatory mechanisms of the hypothalamic-pituitary-adrenal axis.

Conclusions: In obese children, different attachment strategies are associated with diverse metabolic profiles. How this may contribute to developing differentiated treatment approaches remains to be explored.
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http://dx.doi.org/10.20344/amp.6826DOI Listing
May 2016

High-Throughput Nanoliter-Scale Analysis and Optimization of Multimodal Chromatography for the Capture of Monoclonal Antibodies.

Anal Chem 2016 08 28;88(16):7959-67. Epub 2016 Jul 28.

Instituto de Engenharia de Sistemas e Computadores-Microsistemas e Nanotecnologias, and Institute of Nanoscience and Nanotechnology, 1000-029 Lisbon, Portugal.

Multimodal ligands are synthetic molecules comprising multiple types of interactions that have been increasingly used for the capture of different biopharmaceutical compounds within complex biological mixtures. For monoclonal antibodies (mAbs) in particular, these ligands have shown the possibility of direct capture from cell culture supernatants in native conditions, as well as enhanced selectivity and affinity compared to traditional single-mode ligands. However, performing the capture of a target mAb using multimodal chromatography comes with the need for extensive optimization of the operating conditions, due to the multitude of interactions that can be promoted in parallel. In this work, a high-throughput microfluidic platform was developed for the optimization of chromatographic conditions regarding the capture of an anti-interleukin 8 mAb, using a multimodal ligand (2-benzamido-4-mercaptobutanoic acid), under a wide range of buffer pH and conductivities. The interaction of the ligand with the fluorescently labeled target mAb was also analyzed with respect to the individual contribution of the hydrophobic (phenyl) and electrostatic (carboxyl) moieties using fluorescence microscopy. The results were further validated at the macroscale using prepacked columns in standard chromatography assays, and recovery yield values of 94.6% ± 5.2% and 97.7% ± 1.5% were obtained under optimal conditions for the miniaturized and conventional approaches, respectively. In summary, this study highlights that a microfluidic-based approach is a powerful analytical tool to expedite the optimization process while using reduced reagent volumes (<50 μL), less resin (∼70 nL), and delivering results in less than 1 min per assay condition.
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http://dx.doi.org/10.1021/acs.analchem.6b00781DOI Listing
August 2016

Nano- and micro-based systems for immunotolerance induction in multiple sclerosis.

Hum Vaccin Immunother 2016 07 18;12(7):1886-90. Epub 2016 Feb 18.

a International Iberian Nanotechnology Laboratory , Braga , Portugal.

It is estimated that more than 2.5 million individuals worldwide have multiple sclerosis (MS). MS is an autoimmune neurodegenerative disease resulting from the destruction of the myelin sheath that enwraps axons driven by an immune cell attack to the central nervous system. Current therapeutic programs for MS focus in immunosuppression and more recently in the use of immunomodulatory molecules. These therapeutic approaches provide significant improvements in the management of the disease, but are frequently associated with an increased susceptibility of opportunistic infection. In this commentary, we highlight the application of nano and micro-technologies as emerging and innovative solutions for MS therapy with the potential to restore immune homeostasis via antigen-specific interactions. Furthermore, we propose and discuss the usage of a minimally invasive approach, namely microneedle patches, as a new therapeutic route. Microneedle patches for the delivery of specific antigens to restore immunotolerance in the context of multiple sclerosis.
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http://dx.doi.org/10.1080/21645515.2016.1138190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4964821PMC
July 2016

Epithelia migration: a spatiotemporal interplay between contraction and adhesion.

Cell Adh Migr 2015 15;9(5):340-4. Epub 2015 Jul 15.

b International Iberian Nanotechnology Laboratory ; Braga , Portugal.

Epithelial tissues represent 60% of the cells that form the human body and where more than 90% of all cancers derived. Epithelia transformation and migration involve altered cell contractile mechanics powered by an actomyosin-based cytoskeleton and influenced by cell-cell and cell-extracellular matrix interactions. A balance between contractile and adhesive forces regulates a large number of cellular and tissue properties crucial for epithelia migration and tumorigenesis. In this review, the forces driving normal epithelia transformation into highly motile and invasive cells and tissues will be discussed.
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http://dx.doi.org/10.1080/19336918.2015.1008329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4955367PMC
August 2016

The Exchange Relationship between Work-Family Enrichment and Affective Commitment: the Moderating Role of Gender.

Span J Psychol 2015 Jun 3;18:E35. Epub 2015 Jun 3.

Universidade de Lisboa (Portugal).

Workers' perception that their job experience enriches their family life has been considered a mechanism that explains their positive attitudes toward the organization where they work. However, because women and men live their work and family differently, gender may condition this relationship between the work-family enrichment and workers' attitudes. With a sample of 1885 workers from one Portuguese bank, with 802 women, the current study investigated the relationship between work-family enrichment and organizational affective commitment as well as the role of sex as a moderator of this relationship. The hypotheses were tested by using regression analysis. The results indicated that the perception held by workers that their work enriches their family is positively correlated with their affective commitment toward the organization. Furthermore, the data revealed that this relationship is stronger for women than for men. Study results have implications for management, particularly for human resource management, enhancing their knowledge about the relationship of work-family enrichment and workers' affective commitment toward organization.
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http://dx.doi.org/10.1017/sjp.2015.38DOI Listing
June 2015

Analytical protocols for separation and electron microscopy of nanoparticles interacting with bacterial cells.

Anal Chem 2015 15;87(9):4641-8. Epub 2015 Apr 15.

‡International Iberian Nanotechnology Laboratory, Braga 4715-330, Portugal.

An important step toward understanding interactions between nanoparticles (NPs) and bacteria is the ability to directly observe NPs interacting with bacterial cells. NP-bacteria mixtures typical in nanomedicine, however, are not yet amendable for direct imaging in solution. Instead, evidence of NP-cell interactions must be preserved in derivative (usually dried) samples to be subsequently revealed in high-resolution images, for example, via scanning electron microscopy (SEM). Here, this concept is realized for a mixed suspension of model NPs and Staphylococcus aureus bacteria. First, protocols for analyzing the relative colloidal stabilities of NPs and bacteria are developed and validated based on systematic centrifugation and comparison of colony forming unit (CFU) counting and optical density (OD) measurements. Rate-dependence of centrifugation efficiency for each component suggests differential sedimentation at a specific predicted rate as an effective method for removing free NPs after co-incubation; the remaining fraction comprises bacteria with any associated NPs and can be examined, for example, by SEM, for evidence of NP-bacteria interactions. These analytical protocols, validated by systematic control experiments and high-resolution SEM imaging, should be generally applicable for investigating NP-bacteria interactions.
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http://dx.doi.org/10.1021/ac503835aDOI Listing
November 2015

Histones and genome integrity.

Front Biosci (Landmark Ed) 2012 Jan 1;17:984-95. Epub 2012 Jan 1.

Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701, USA.

Chromosomes undergo extensive structural rearrangements during the cell cycle, from the most open chromatin state required for DNA replication to the highest level of compaction and condensation essential for mitotic segregation of sister chromatids. It is now widely accepted that chromatin is a highly dynamic structure that participates in all DNA-related functions, including transcription, DNA replication, repair, and mitosis; hence, histones have emerged as key players in these cellular processes. We review here the studies that implicate histones in functions that affect the chromosome cycle, defined as the cellular processes involved in the maintenance, replication, and segregation of chromosomal DNA. Disruption of the chromosome cycle affects the integrity of the cellular genome, leading to aneuploidy, polyploidy or cell death. Histone stoichiometry, mutations that affect the structure of the nucleosome core particle, and mutations that affect the structure and/or modifications of the histone tails, all have a direct impact on the fidelity of chromosome transmission and the integrity of the genome.
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http://dx.doi.org/10.2741/3969DOI Listing
January 2012

Modular coherence of protein dynamics in yeast cell polarity system.

Proc Natl Acad Sci U S A 2011 May 18;108(18):7647-52. Epub 2011 Apr 18.

Stowers Institute for Medical Research, Kansas City, MO 64110, USA.

In this study, we investigated on a systems level how complex protein interactions underlying cell polarity in yeast determine the dynamic association of proteins with the polar cortical domain (PCD) where they localize and perform morphogenetic functions. We constructed a network of physical interactions among >100 proteins localized to the PCD. This network was further divided into five robust modules correlating with distinct subprocesses associated with cell polarity. Based on this reconstructed network, we proposed a simple model that approximates a PCD protein's molecular residence time as the sum of the characteristic time constants of the functional modules with which it interacts, weighted by the number of edges forming these interactions. Regression analyses showed excellent fitting of the model with experimentally measured residence times for a large subset of the PCD proteins. The model is able to predict residence times using small training sets. Our analysis also revealed a scaffold protein that imposes a local constraint of dynamics for certain interacting proteins.
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http://dx.doi.org/10.1073/pnas.1017567108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3088571PMC
May 2011

Estimated age of acquisition norms for 834 Portuguese nouns and their relation with other psycholinguistic variables.

Behav Res Methods 2007 Aug;39(3):439-44

University of Lisbon, Lisbon, Portugal.

The main objective of this study is to report rated age of acquisition (AoA) norms for 834 nouns in Portuguese (European). AoA ratings were collected on a 7 point scale, generally following Gilhooly and Logie (1980) procedure with an 8 extra point for "don't know the word" answers. Results were analyzed considering AoA ratings and their standard deviations and considering the relationship between AoA ratings and other psycholinguistic variables (imageability, familiarity, written word frequency, concreteness, number of syllables and number of words). AoA ratings and their standard deviations were significantly and positively correlated, with early acquired word ratings showing higher agreement. Correlation and multiple regression analyses confirmed the major contribution of imageability and familiarity to AoA ratings obtained in other languages. The full database of AoA ratings and other psycholinguistic variables may be downloaded from www.psychonomic.org/archive or www.fpce.ul.pt/pessoal/ulfpfred/aoa.htm.
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http://dx.doi.org/10.3758/bf03193013DOI Listing
August 2007

[Therapeutic community model in short psychiatric hospitalization. Descriptive study on the dynamic psychiatric inpatient unit of the Italian hospital of Buenos Aires].

Vertex 2006 Jan-Feb;17(65):55-64

Hospital Italiano de Buenos Aires, Argentina.

Objective: the aim of this paper is to communicate a project of short term psychiatric hospitalization, based on a therapeutic community model, considering qualitative and quantitative aspects in the present socio - cultural context.

Introduction: this psychiatric hospitalization model that embraces psychodynamic and pharmacological interventions is focused in the intensity of interactions between members of the therapeutic community and integrated to the administrative structure of a general hospital; this will be the key to consider patient's return to the community and to move forward over the prejudices that inpatients suffer.

Material And Methods: quantitative, prospective, observational and transversal study on a Dynamic Psychiatric Inpatient Unit. 605 patients were included.

Results: mean length of stay was 16.34 days; principal causes of admission were depression (19.4%), suicide ideas (17.7%), suicide attempt (17.6%), substance abuse or dependence (14.3%), psychosis (13.8%), behavioral and psychological symptoms of dementia (6%). There were 75 readmissions. 14.88% patients were physically restrained. Principal Axis I diagnosis were depression (32.1%), substance dependence (13.2%), bipolar disorder (10.2%), dementia (7.6%), schizophrenia (7.5%), and psychotic disorder (5.8). Axis II diagnosis were borderline personality disorder (27.3%), narcissistic personality disorder (8.9%), histrionic personality disorder (5.3%).

Discussion: this kind of approach shows a structural model that allows possible and persistent favorable changes for psychiatric inpatients.
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August 2006