Publications by authors named "Paula Sampaio"

90 Publications

Advantages of an Automated Method Compared With Manual Methods for the Quantification of Intraepidermal Nerve Fiber in Skin Biopsy.

J Neuropathol Exp Neurol 2021 May 26. Epub 2021 May 26.

From the Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto (MFC, RM, RT, LM); Department of Neurology, Centro Hospitalar Universitário do Porto (CHUP) (MFC, IR, NV-C, APS, RM, RT, LM); Instituto de investigação e inovação em Saúde da Universidade do Porto (i3S) (MS, PS, LM), Porto, Portugal; Department of Medicine IV, Geriatrics, University Hospital, LMU Munich, Munich, Germany (FT).

Intraepidermal nerve fiber density (IENFD) measurements in skin biopsy are performed manually by 1-3 operators. To improve diagnostic accuracy and applicability in clinical practice, we developed an automated method for fast IENFD determination with low operator-dependency. Sixty skin biopsy specimens were stained with the axonal marker PGP9.5 and imaged using a widefield fluorescence microscope. IENFD was first determined manually by 3 independent observers. Subsequently, images were processed in their Z-max projection and the intradermal line was delineated automatically. IENFD was calculated automatically (fluorescent images automated counting [FIAC]) and compared with manual counting on the same fluorescence images (fluorescent images manual counting [FIMC]), and with classical manual counting (CMC) data. A FIMC showed lower variability among observers compared with CMC (interclass correlation [ICC] = 0.996 vs 0.950). FIMC and FIAC showed high reliability (ICC = 0.999). A moderate-to-high (ICC = 0.705) was observed between CMC and FIAC counting. The algorithm process took on average 15 seconds to perform FIAC counting, compared with 10 minutes for FIMC counting. This automated method rapidly and reliably detects small nerve fibers in skin biopsies with clear advantages over the classical manual technique.
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http://dx.doi.org/10.1093/jnen/nlab045DOI Listing
May 2021

Waste-derived volatile fatty acids as carbon source for added-value fermentation approaches.

FEMS Microbiol Lett 2021 05;368(9)

Industrial Biotechnology and Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, 15780 Athens, Greece.

The establishment of a sustainable circular bioeconomy requires the effective material recycling from biomass and biowaste beyond composting/fertilizer or anaerobic digestion/bioenergy. Recently, volatile fatty acids attracted much attention due to their potential application as carbon source for the microbial production of high added-value products. Their low-cost production from different types of wastes through dark fermentation is a key aspect, which will potentially lead to the sustainable production of fuels, materials or chemicals, while diminishing the waste volume. This article reviews the utilization of a volatile fatty acid platform for the microbial production of polyhydroxyalkanoates, single cell oil and omega-3 fatty acids, giving emphasis on the fermentation challenges for the efficient implementation of the bioprocess and how they were addressed. These challenges were addressed through a research project funded by the European Commission under the Horizon 2020 programme entitled 'VOLATILE-Biowaste derived volatile fatty acid platform for biopolymers, bioactive compounds and chemical building blocks'.
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http://dx.doi.org/10.1093/femsle/fnab054DOI Listing
May 2021

Protein-Based Films Functionalized with a Truncated Antimicrobial Peptide Sequence Display Broad Antimicrobial Activity.

ACS Biomater Sci Eng 2021 02 25;7(2):451-461. Epub 2021 Jan 25.

CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.

The increasing bacterial resistance to antibiotics is driving strong demand for new antimicrobial biomaterials. This work describes the fabrication of free-standing films exhibiting antimicrobial properties by combining, in the same polypeptide chain, an elastin-like recombinamer comprising 200 repetitions of the pentamer VPAVG (A200) and an 18-amino-acid truncated variant of the antimicrobial peptide BMAP-28, termed BMAP-18. The fusion protein BMAP-18A200 was overexpressed and conveniently purified by a simplified and scalable nonchromatographic process. Free-standing films of BMAP-18A200 demonstrated to be stable without requiring cross-linking agents and displayed high antimicrobial activity against skin pathogens including Gram-negative and Gram-positive bacteria as well as unicellular and filamentous fungi. The antimicrobial activity of the films was mediated by direct contact of cells with the film surface, resulting in compromised structural integrity of microbial cells. Furthermore, the BMAP-18A200 films showed no cytotoxicity on normal human cell lines (skin fibroblasts and keratinocytes). All of these results highlight the potential of these biotechnological multifunctional polymers as new drug-free materials to prevent and treat microbial infections.
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http://dx.doi.org/10.1021/acsbiomaterials.0c01262DOI Listing
February 2021

Development and Characterization of Monoolein-Based Liposomes of Carvacrol, Cinnamaldehyde, Citral, or Thymol with Anti- Activities.

Antimicrob Agents Chemother 2021 03 18;65(4). Epub 2021 Mar 18.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.

There is an increasing need for novel drugs and new strategies for the therapy of invasive candidiasis. This study aimed to develop and characterize liposome-based nanoparticles of carvacrol, cinnamaldehyde, citral, and thymol with anti- activities. Dioctadecyldimethylammonium bromide- and monoolein-based liposomes in a 1:2 molar ratio were prepared using a lipid-film hydration method. Liposomes were assembled with equal volumes of liposomal stock dispersion and stock solutions of carvacrol, cinnamaldehyde, citral, or thymol in dimethyl sulfoxide. Cytotoxicity was tested on RAW 264.7 macrophages. antifungal activity of liposomes with phytocompounds was evaluated according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology using clinical isolates of , , , and Finally, the ability of macrophage cells to kill isolates after addition of phytocompounds and their nanoparticles was determined. Nanoparticles with 64 μg/ml of cinnamaldehyde, 256 μg/ml of citral, and 128 μg/ml of thymol had the best characteristics among the formulations tested. The highest encapsulation efficiencies were achieved with citral (78% to 83%) and carvacrol (66% to 71%) liposomes. Carvacrol and thymol in liposome-based nanoparticles were nontoxic regardless of the concentration. Moreover, carvacrol and thymol maintained their antifungal activity after encapsulation, and there was a significant reduction (∼41%) of yeast survival when macrophages were incubated with carvacrol or thymol liposomes. In conclusion, carvacrol and thymol liposomes possess high stability, low cytotoxicity, and antifungal activity that act synergistically with macrophages.
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http://dx.doi.org/10.1128/AAC.01628-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097438PMC
March 2021

Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections.

J Fungi (Basel) 2020 Nov 23;6(4). Epub 2020 Nov 23.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710 Braga, Portugal.

A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the for the identification of species, and the for the identification of species and . The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.
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http://dx.doi.org/10.3390/jof6040308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712097PMC
November 2020

Single Cell Oil Production by Oleaginous Yeasts Grown in Synthetic and Waste-Derived Volatile Fatty Acids.

Microorganisms 2020 Nov 17;8(11). Epub 2020 Nov 17.

CBMA-Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.

Four yeast isolates from the species-, , , and -previously selected by their oleaginous character and growth flexibility in different carbon sources, were tested for their capacity to convert volatile fatty acids into lipids, in the form of single cell oils. Growth, lipid yields, volatile fatty acids consumption, and long-chain fatty acid profiles were evaluated in media supplemented with seven different volatile fatty acids (acetic, butyric, propionic, isobutyric, valeric, isovaleric, and caproic), and also in a dark fermentation effluent filtrate. Yeasts and attained lipid productivities of more than 40% (/), mainly composed of oleic (>40%), palmitic (20%), and stearic (20%) acids, both in synthetic media and in the waste-derived effluent filtrate. These isolates may be potential candidates for single cell oil production in larger scale applications by using alternative carbon sources, combining economic and environmental benefits.
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http://dx.doi.org/10.3390/microorganisms8111809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698568PMC
November 2020

Oral colonization in healthy individuals: prevalence, genotypic diversity, stability along time and transmissibility.

J Oral Microbiol 2020 Sep 14;12(1):1820292. Epub 2020 Sep 14.

CBMA (Centre of Molecular and Environmental Biology) / Department of Biology / University of Minho, Braga, Portugal.

In this study, 181 healthy individuals, including 29 couples, were analysed regarding oral yeast colonization using a culture-based approach. Results showed that 39% of the individuals were yeast carriers, 89% being colonized with , 5% with , 3% with and 3% with . Sixty-two percent of the couples had at least one member colonized. Colonization and CFU counts were higher in the couples´ group. Eighty percent of the volunteers were colonized with strains with only one CAI genotype, while two but similar CAI genotypes inhabited the oral cavity of the remaining 20% individuals. The same CAI genotypes were found in 66.6% of the couples when both were colonized. Our results indicate that the intimacy among couples increases the probability of heavy cross-colonization, which is potentiated when one member of the couple is a smoker.
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http://dx.doi.org/10.1080/20002297.2020.1820292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534343PMC
September 2020

Author Correction: iLoF: An intelligent Lab on Fiber Approach for Human Cancer Single-Cell Type Identification.

Sci Rep 2020 Jun 26;10(1):10775. Epub 2020 Jun 26.

INESC TEC - INESC Technology and Science, Porto, Portugal.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-67916-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316816PMC
June 2020

The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction.

Elife 2020 03 20;9. Epub 2020 Mar 20.

Nerve Regeneration Group, Porto, Portugal.

Neurons have a membrane periodic skeleton (MPS) composed of actin rings interconnected by spectrin. Here, combining chemical and genetic gain- and loss-of-function assays, we show that in rat hippocampal neurons the MPS is an actomyosin network that controls axonal expansion and contraction. Using super-resolution microscopy, we analyzed the localization of axonal non-muscle myosin II (NMII). We show that active NMII light chains are colocalized with actin rings and organized in a circular periodic manner throughout the axon shaft. In contrast, NMII heavy chains are mostly positioned along the longitudinal axonal axis, being able to crosslink adjacent rings. NMII filaments can play contractile or scaffolding roles determined by their position relative to actin rings and activation state. We also show that MPS destabilization through NMII inactivation affects axonal electrophysiology, increasing action potential conduction velocity. In summary, our findings open new perspectives on axon diameter regulation, with important implications in neuronal biology.
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http://dx.doi.org/10.7554/eLife.55471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105375PMC
March 2020

Modified high-throughput Nile red fluorescence assay for the rapid screening of oleaginous yeasts using acetic acid as carbon source.

BMC Microbiol 2020 03 14;20(1):60. Epub 2020 Mar 14.

CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal.

Background: Over the last years oleaginous yeasts have been studied for several energetic, oleochemical, medical and pharmaceutical purposes. However, only a small number of yeasts are known and have been deeply exploited. The search for new isolates with high oleaginous capacity becomes imperative, as well as the use of alternative and ecological carbon sources for yeast growth.

Results: In the present study a high-throughput screening comprising 366 distinct yeast isolates was performed by applying an optimised protocol based on two approaches: (I) yeast cultivation on solid medium using acetic acid as carbon source, (II) neutral lipid estimation by fluorimetry using the lipophilic dye Nile red.

Conclusions: Results showed that, with the proposed methodology, the oleaginous potential of yeasts with broad taxonomic diversity and variety of growth characteristics was discriminated. Furthermore, this work clearly demonstrated the association of the oleaginous yeast character to the strain level, contrarily to the species-level linkage, as usually stated.
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http://dx.doi.org/10.1186/s12866-020-01742-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071767PMC
March 2020

iLoF: An intelligent Lab on Fiber Approach for Human Cancer Single-Cell Type Identification.

Sci Rep 2020 02 21;10(1):3171. Epub 2020 Feb 21.

INESC TEC - INESC Technology and Science, Porto, Portugal.

With the advent of personalized medicine, there is a movement to develop "smaller" and "smarter" microdevices that are able to distinguish similar cancer subtypes. Tumor cells display major differences when compared to their natural counterparts, due to alterations in fundamental cellular processes such as glycosylation. Glycans are involved in tumor cell biology and they have been considered to be suitable cancer biomarkers. Thus, more selective cancer screening assays can be developed through the detection of specific altered glycans on the surface of circulating cancer cells. Currently, this is only possible through time-consuming assays. In this work, we propose the "intelligent" Lab on Fiber (iLoF) device, that has a high-resolution, and which is a fast and portable method for tumor single-cell type identification and isolation. We apply an Artificial Intelligence approach to the back-scattered signal arising from a trapped cell by a micro-lensed optical fiber. As a proof of concept, we show that iLoF is able to discriminate two human cancer cell models sharing the same genetic background but displaying a different surface glycosylation profile with an accuracy above 90% and a speed rate of 2.3 seconds. We envision the incorporation of the iLoF in an easy-to-operate microchip for cancer identification, which would allow further biological characterization of the captured circulating live cells.
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http://dx.doi.org/10.1038/s41598-020-59661-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035380PMC
February 2020

Relevance of Macrophage Extracellular Traps in Killing.

Front Immunol 2019 4;10:2767. Epub 2019 Dec 4.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.

causes systemic life-threatening infections, particularly in immunocompromised individuals, such as patients in intensive care units, patients undergoing chemotherapy, and post-surgical and neutropenic patients. The proliferation of invading cells is mainly limited by the action of the human innate immune system, in which phagocytic cells play a fundamental role. This function is, however, limited in neutropenic patients, who rely mainly on the protective immunity mediated by macrophages. Macrophages have been shown to release extracellular DNA fibers, known as macrophage extracellular traps (METs), which can entrap and kill various microbes by a process called ETosis. In this study, we observed that, upon contact with , macrophages became active in phagocyting and engulfing yeast cells. ETosis was induced in 6% of macrophages within the first 30 min of contact, and this percentage increased with the multiplicity of infection until a plateau was reached. After 2.5 h incubation, the presence of extracellular macrophage DNA was observed in approximately half of the cells. This study suggests that the formation of METs occurs before pyroptosis (first 6-8 h) and macrophage cell death (up to 24 h), and thus, METs could be included in models describing -macrophage interactions. We also observed that macrophage ETosis and phagocytosis can occur simultaneously and that, in the first hours of infection, both processes are similarly important in controlling the proliferation of yeast cells, this being critical in neutropenic patients. Finally, it can also be concluded that, since can degrade DNA, the structural component of METs, yeast extracellular DNase activity can be considered as an important virulence factor.
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http://dx.doi.org/10.3389/fimmu.2019.02767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904331PMC
October 2020

Higher IL-6 peri-tumoural expression is associated with gastro-intestinal neuroendocrine tumour progression.

Pathology 2019 Oct 26;51(6):593-599. Epub 2019 Aug 26.

Endocrine, Cardiovascular and Metabolic Research, Multidisciplinary Unit for Biomedical Research (UMIB), Department of Anatomy of Instituto de Ciências Biomédicas Abel Salazar, University of Porto (ICBAS/UP), Porto, Portugal. Electronic address:

An association of well-differentiated gastroenteropancreatic neuroendocrine tumours (WD GEP NETs) with metabolic syndrome (MetS) was recently described. Yet no molecular mechanisms linking the two conditions are known. This study's aim was to identify putative molecular signatures linking WD GEP NETs and MetS to gain further insight into potential mechanisms for this association. Patients with WD GEP NETs (n=39), pancreatic (panNET) and gastro-intestinal (GI-NET), were clinically evaluated for presence of MetS. WD GEP NETs immunohistochemistry staining for Forkhead box protein M1 (FOXM1), insulin growth factor 1 receptor (IGF1R), Ki-67 and interleukin 6 (IL-6) was performed and quantified by computerised morphometric analysis. FOXM1, Ki-67, IGF1R or IL-6 expression in WD GEP NETs was not influenced by the presence of MetS. IL-6 peritumoural expression was higher in GI-NETs of patients with low HDL cholesterol (0.018±0.005% vs 0.030±0.005%, p=0.02). In GI-NETs, a higher IL-6 expression was also associated with disease progression (0.026±0.004% vs 0.016±0.002%, p=0.03). In WD GEP-NETs, MetS did not influence FOXM1, IGF1R and IL-6 expression. In GI-NETs, IL-6 expression was influenced by the MetS feature low HDL, and positively associated with disease progression. These data suggest that local and systemic inflammatory status can potentially modulate GI-NET behaviour.
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http://dx.doi.org/10.1016/j.pathol.2019.07.001DOI Listing
October 2019

Variation in primary health care services after implementation of quality improvement policy in Brazil.

Fam Pract 2020 02;37(1):69-80

School of Public Health, University of California, Berkeley, CA, USA.

Background: Brazil is the most populous country with a public, universal and free health care system. The National Program for Access and Quality Improvement in Primary Care (PMAQ) was created to improve the quality of primary health care (PHC).

Objective: To evaluated whether progress generally has been made within Brazil's PHC since PMAQ implementation, and if changes occurred uniformly in the country, while also identifying municipal characteristics that may have influenced the improvement.

Methods: This is an observational study using data from PMAQ external evaluation (2012 and 2014), a 1200-item survey used to evaluate Brazilian PHC quality. After confirming the groupings of items using factor analysis, we created 23 composed indexes (CIs) related to infrastructure and work process.

Results: On average, the large majority of CIs showed improvements between 2012 and 2014. Region and city size moderated changes in the PHC indices differently. Overall, there were better improvements in infrastructure in the Northeast compared with other country regions, and in smaller cities (10 000-20 000 people). Infrastructure indices appear to have improved equitably across the country. Work process improvements varied with city size and region.

Conclusion: Despite similar support of PMAQ across the country, improvements are not predictable nor homogeneous. Non-uniform improvements were seen in Brazil's PHC. Though we do not directly evaluate the effectiveness of the PMAQ (financial reward) method, these initial findings suggest that it is a potentially useful tool to improve health systems, but additional support may be needed in regions that lag behind in quality improvements.
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http://dx.doi.org/10.1093/fampra/cmz040DOI Listing
February 2020

Optical fiber-based sensing method for nanoparticle detection through supervised back-scattering analysis: a potential contributor for biomedicine.

Int J Nanomedicine 2019 2;14:2349-2369. Epub 2019 Apr 2.

INESC Technology and Science, Porto, Portugal,

Background: In view of the growing importance of nanotechnologies, the detection/identification of nanoparticles type has been considered of utmost importance. Although the characterization of synthetic/organic nanoparticles is currently considered a priority (eg, drug delivery devices, nanotextiles, theranostic nanoparticles), there are many examples of "naturally" generated nanostructures - for example, extracellular vesicles (EVs), lipoproteins, and virus - that provide useful information about human physiology or clinical conditions. For example, the detection of tumor-related exosomes, a specific type of EVs, in circulating fluids has been contributing to the diagnosis of cancer in an early stage. However, scientists have struggled to find a simple, fast, and low-cost method to accurately detect/identify these nanoparticles, since the majority of them have diameters between 100 and 150 nm, thus being far below the diffraction limit.

Methods: This study investigated if, by projecting the information provided from short-term portions of the back-scattered laser light signal collected by a polymeric lensed optical fiber tip dipped into a solution of synthetic nanoparticles into a lower features dimensional space, a discriminant function is able to correctly detect the presence of 100 nm synthetic nanoparticles in distilled water, in different concentration values.

Results And Discussion: This technique ensured an optimal performance (100% accuracy) in detecting nanoparticles for a concentration above or equal to 3.89 µg/mL (8.74E+10 particles/mL), and a performance of 90% for concentrations below this value and higher than 1.22E-03 µg/mL (2.74E+07 particles/mL), values that are compatible with human plasmatic levels of tumor-derived and other types of EVs, as well as lipoproteins currently used as potential biomarkers of cardiovascular diseases.

Conclusion: The proposed technique is able to detect synthetic nanoparticles whose dimensions are similar to EVs and other "clinically" relevant nanostructures, and in concentrations equivalent to the majority of cell-derived, platelet-derived EVs and lipoproteins physiological levels. This study can, therefore, provide valuable insights towards the future development of a device for EVs and other biological nanoparticles detection with innovative characteristics.
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http://dx.doi.org/10.2147/IJN.S174358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452810PMC
May 2019

Coherent-hybrid STED: high contrast sub-diffraction imaging using a bi-vortex depletion beam.

Opt Express 2019 Mar 5;27(6):8092-8111. Epub 2019 Mar 5.

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal.

Stimulated emission depletion (STED) fluorescence microscopy squeezes an excited spot well below the wavelength scale using a doughnut-shaped depletion beam. To generate a doughnut, a scale-free vortex phase modulation (2D-STED) is often used because it provides maximal transverse confinement and radial-aberration immunity (RAI) to the central dip. However, RAI also means blindness to a defocus term, making the axial origin of fluorescence photons uncertain within the wavelength scale provided by the confocal detection pinhole. Here, to reduce the uncertainty, we perturb the 2D-STED phase mask so as to change the sign of the axial concavity near focus, creating a dilated dip. By providing laser depletion power, the dip can be compressed back in three dimensions to retrieve lateral resolution, now at a significantly higher contrast. We test this coherent-hybrid STED (CH-STED) mode in imaging of complex biological structures, such as the dividing cell. The proposed strategy creates an orthogonal direction in the STED parametric space that uniquely allows independent tuning of resolution and contrast using a single depletion beam in a conventional (circular polarization-based) STED setup.
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http://dx.doi.org/10.1364/OE.27.008092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420153PMC
March 2019

Ploidy Determination in the Pathogenic Fungus spp.

Front Microbiol 2019 25;10:284. Epub 2019 Feb 25.

Life and Health Sciences Research Institute (ICVS), School of Medicine, Braga, Portugal.

The pathogenic clade of the genus comprises the etiological agents of sporotrichosis, a worldwide emergent disease. Despite the growing understanding of their successful pathogen traits, there is little information on genome sizes and ploidy within the genus. Therefore, in this work, we evaluated the ploidy of four species of the genus, specifically , , , and . Through cell cycle analysis of the yeast-phase cells, we showed that the DNA content of G/G cells was similar to the genome size determined by whole genome sequencing. Moreover, ploidy of , , and that was determined by allele composition using next-generation sequencing (NGS) data is consistent with monomorphic positions at each allele. These data show that the analyzed strains of are haploid, or at least aneuploid, thereby laying the foundation for the development of a molecular toolbox for spp.
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http://dx.doi.org/10.3389/fmicb.2019.00284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397882PMC
February 2019

The intertwined effect of lack of emotional warmth and child abuse and neglect on common mental disorders in adolescence.

Child Abuse Negl 2018 09 17;83:74-82. Epub 2018 Jul 17.

Institute of Nutrition Josué de Castro, Rio de Janeiro Federal University Centro de Ciências da Saúde, Bloco J - Av. Carlos Chagas Filho, 373 - Cidade Universitária, Rio de Janeiro RJ, 21941-902, Brazil. Electronic address:

Adolescence is a vulnerable period for mental health problems. Although child abuse and neglect (CAN) are known risk factors for some of them, it is not clear if the negative consequences on mental health also occur in families where CAN and a warm parent-child relationship coexist. The aim of this study is to explore this gap and investigate the effects of different types of CAN according to levels of warmth in the parent-child relationship on common mental disorders (CMD) in adolescence. This is a cross-sectional study encompassing 487 adolescents attending the ninth grade at 2 public and 4 private schools in Rio de Janeiro, Brazil. CAN was measured by the Child Trauma Questionnaire (CTQ), CMD by the General Health Questionnaire (GHQ-12) and the level of warmth in the parent-child relationship were recalled using short Egna Minnen Betraffände Uppfostran (s-EMBU-23). The adjusted separate effects of different types of CAN and a low level of warmth in the parent-child relationship, and both in tandem, were estimated using multivariate linear regression models. Results indicated that emotional abuse and neglect, physical abuse and neglect, and a low level of warmth in the parent-child relationship are important risk factors for CMD in adolescence. Nevertheless, in families where CAN coexist with a warm and affectionate parent-child relationship, the negative effects of CAN on mental health are attenuated. Evidence indicates that actions to prevent or interrupt CAN and improving parental practices could be effective strategies to reduce CMD in adolescence.
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http://dx.doi.org/10.1016/j.chiabu.2018.07.008DOI Listing
September 2018

The Role of Transcription Factor in Response to Carbon Adaptation.

Front Microbiol 2018 29;9:1127. Epub 2018 May 29.

Department of Biology, CBMA, School of Sciences, University of Minho, Braga, Portugal.

is the main causative agent of candidiasis and one of the most frequent causes of nosocomial infections worldwide. In order to establish an infection, this pathogen supports effective stress responses to counter host defenses and adapts to changes in the availability of important nutrients, such as alternative carbon sources. These stress responses have clear implications on the composition and structure of cell wall. Therefore, we studied the impact of lactate, a physiologically relevant carbon source, on the activity of transcriptional factor. is involved in the cell wall integrity pathway and plays an important role in regulating the flow of carbohydrates into cell wall biosynthesis pathways. The role of in response to lactate adaptation was assessed in respect to several virulence factors, such as the ability to grow under cell wall damaging agents, filament, adhere or form biofilm, as well as to immune recognition. The data showed that growth of cells in the presence of lactate induces the secretion of tartaric acid, which has the potential to modulate the TCA cycle on both the yeast and the host cells. In addition, we found that adaptation of cells to lactate reduces their internalization by immune cells and consequent % of killing, which could be correlated with a lower exposure of the cell wall β-glucans. In addition, absence of has a minor impact on internalization, compared with the wild-type and complemented strains, but it reduces the higher efficiency of lactate grown cells at damaging phagocytic cells and induces a high amount of IL-10, rendering these cells more tolerable to the immune system. The data suggests that mediates cell wall remodeling during carbon adaptation, impacting their interaction with immune cells.
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http://dx.doi.org/10.3389/fmicb.2018.01127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986929PMC
May 2018

Jmy regulates oligodendrocyte differentiation via modulation of actin cytoskeleton dynamics.

Glia 2018 09 6;66(9):1826-1844. Epub 2018 May 6.

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.

During central nervous system development, oligodendrocytes form structurally and functionally distinct actin-rich protrusions that contact and wrap around axons to assemble myelin sheaths. Establishment of axonal contact is a limiting step in myelination that relies on the oligodendrocyte's ability to locally coordinate cytoskeletal rearrangements with myelin production, under the control of a transcriptional differentiation program. The molecules that provide fine-tuning of actin dynamics during oligodendrocyte differentiation and axon ensheathment remain largely unidentified. We performed transcriptomics analysis of soma and protrusion fractions from rat brain oligodendrocyte progenitors and found a subcellular enrichment of mRNAs in newly-formed protrusions. Approximately 30% of protrusion-enriched transcripts encode proteins related to cytoskeleton dynamics, including the junction mediating and regulatory protein Jmy, a multifunctional regulator of actin polymerization. Here, we show that expression of Jmy is upregulated during myelination and is required for the assembly of actin filaments and protrusion formation during oligodendrocyte differentiation. Quantitative morphodynamics analysis of live oligodendrocytes showed that differentiation is driven by a stereotypical actin network-dependent "cellular shaping" program. Disruption of actin dynamics via knockdown of Jmy leads to a program fail resulting in oligodendrocytes that do not acquire an arborized morphology and are less efficient in contacting neurites and forming myelin wraps in co-cultures with neurons. Our findings provide new mechanistic insight into the relationship between cell shape dynamics and differentiation in development.
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http://dx.doi.org/10.1002/glia.23342DOI Listing
September 2018

High variability within Candida albicans transcription factor RLM1: Isolates from vulvovaginal infections show a clear bias toward high molecular weight alleles.

Med Mycol 2018 Jul;56(5):649-651

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.

Previous studies have correlated the severity of recurrent vulvovaginal Candida infections (VVC) and balanitis in patients from China with the presence of some dominant genotypes at the ORF RLM1. Here we tested VVC vs non-VVC isolates from Portugal, Brazil and Greece and, although the same genotypes were identified in VVC isolates, they were present in only five out of 150 strains. However, this analysis showed that VVC isolates presented a higher percentage of genotypes with similar high molecular weight alleles, in comparison with strains isolated from other biological sources.
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http://dx.doi.org/10.1093/mmy/myx079DOI Listing
July 2018

Genomic and transcriptomic analysis of Saccharomyces cerevisiae isolates with focus in succinic acid production.

FEMS Yeast Res 2017 09;17(6)

CBMA (Centre of Molecular and Environmental Biology) / Department of Biology / University of Minho, 4710-057 Braga, Portugal.

Succinic acid is a platform chemical that plays an important role as precursor for the synthesis of many valuable bio-based chemicals. Its microbial production from renewable resources has seen great developments, specially exploring the use of yeasts to overcome the limitations of using bacteria. The objective of the present work was to screen for succinate-producing isolates, using a yeast collection with different origins and characteristics. Four strains were chosen, two as promising succinic acid producers, in comparison with two low producers. Genome of these isolates was analysed, and differences were found mainly in genes SDH1, SDH3, MDH1 and the transcription factor HAP4, regarding the number of single nucleotide polymorphisms and the gene copy-number profile. Real-time PCR was used to study gene expression of 10 selected genes involved in the metabolic pathway of succinic acid production. Results show that for the non-producing strain, higher expression of genes SDH1, SDH2, ADH1, ADH3, IDH1 and HAP4 was detected, together with lower expression of ADR1 transcription factor, in comparison with the best producer strain. This is the first study showing the capacity of natural yeast isolates to produce high amounts of succinic acid, together with the understanding of the key factors associated, giving clues for strain improvement.
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http://dx.doi.org/10.1093/femsyr/fox057DOI Listing
September 2017

Poncet's disease after the intravesical instillation of Bacillus Calmette-Guérin (BCG): a case report.

BMC Res Notes 2017 Aug 18;10(1):416. Epub 2017 Aug 18.

Section of Clinical Oncology, Division of Oncology, Department of Clinical Care, Centro Universitário do Pará, 3775 Almirante Barroso Avenue, Souza, Belém, Pará, 66613-903, Brazil.

Background: Poncet's disease is a rare syndrome characterized by articular impairment in a form of rare tuberculid. One of the theories of its cause involves an autoimmune response induced by the intravesical administration of the Calmette-Guerin Bacillus or the treatment of bladder carcinoma. Furthermore, there may be an appearance of oligoarticular or polyarticular arthritis, beginning 1-3 months after the start of therapy. Few physicians know the disease and the literature related to that syndrome is scarce and restricted to case reports, which contributes to its under diagnosis.

Case Presentation: Female patient, 64 years old, Caucasian, in whom was noticed firstly dark urine, without haematuria or dysuria. Later felt also colic pain in the hypogastric region. Microscopically, the conclusive diagnosis was a high grade non-invasive papillary urothelial carcinoma. Thereupon, the treatment of the tumour began with transurethral resection technique and intravesical instillation of Calmette-Guérin Bacillus as adjuvant treatment. Eight months after the beginning of treatment, the lingering presence of the carcinoma was identified. Nevertheless, arthritis was identified through radiographs, after an increase in the clavicle capitation, right knee and left ankle in bone scintigraphy. Coinciding with the joint manifestations, the patient developed fever and purulent urethral discharge (culture was negative). Therefore, trying to investigate the cause of the arthritis, Purified Protein Derivate was taken, with reactive results. An increase of acute phase reactants was found, with other tests resulting normal: blood chemistry, Complete Blood Count, immunology and serology. Human Leukocyte Antigen typing by polymerase chain reaction revealed the presence of A24/AX, B44, B27, BW4/BW4, DQ7 and DQ5. Consequently, Poncet's disease was the diagnostic conclusion. The treatment with intravesical Calmette-Guérin Bacillus was immediately discontinued. The patient received corticosteroids associated with etoricoxib and isoniazid for 4 months, achieving disappearance of the inflammatory joint signs in 3 months. After 6 months, no joint pain recurrence or other manifestations suggesting active disease had been seen.

Conclusions: Therefore, such diagnosis should be considered when confronted with an osteoarticular clinical picture in patients treated with intravesical Calmette-Guérin Bacillus, especially patients with HLA-B27 (+) and B7 (+), as Poncet's disease is a reactive arthritis.
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http://dx.doi.org/10.1186/s13104-017-2606-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563016PMC
August 2017

Comparative bonding ability to dentin of a universal adhesive system and monomer conversion as functions of extended light curing times and storage.

J Mech Behav Biomed Mater 2017 11 27;75:41-49. Epub 2017 Jun 27.

Department of Dentistry, School of Health Sciences, Universidade de Brasília (UnB), SQNW 109 Bloco A, Apt 422, Ed. Real Evolution, Noroeste, Brasília CEP 70686-405, DF, Brazil. Electronic address:

Objectives: The purpose of this in vitro study was to evaluate the bonding ability and monomer conversion of a universal adhesive system applied to dentin as functions of different curing times and storage. The results were compared among a variety of commercial adhesives.

Materials And Methods: Flat superficial dentin surfaces were exposed on human molars and assigned into one of the following adhesives (n = 15): total-etch Adper Single Bond 2 (SB) and Optibond Solo Plus (OS), self-etch Optibond All in One (OA) and Clearfil SE Bond (CSE), and Scotchbond Universal Adhesive in self-etch mode (SU). The adhesives were applied following the manufacturers' instructions and cured for 10, 20, or 40s. Specimens were processed for the microtensile bond strength (µTBS) test in accordance with the non-trimming technique and tested after 24h and 2 years. The fractured specimens were classified under scanning electron microscopy (SEM). Infrared (IR) spectra were obtained and monomer conversion (%) was calculated by comparing the aliphatic-to-aromatic IR absorption peak ratio before and after polymerization (n=5). Data were analyzed by 2-way ANOVA/Tukey's tests (α = 0.05).

Results: At 24-h evaluation, OA and CSE presented similar bond strength means irrespective of the curing time, whereas SB and SU exhibited significantly higher means when cured for 40s as did OS when cured for 20 or 40s (p < 0.05). At 2-year evaluation, only OA exhibited significantly higher bond strength when cured for 20 and 40s (p < 0.05). When the evaluation times were compared, OA also exhibited the same bonding ability when cured for longer periods of time (20 and 40s). All of the adhesives tested exhibited significantly lower monomer conversion when photoactivated according to the manufacturers' instructions (10s).

Conclusions: Higher monomer conversions obtained with longer light exposure allow only higher immediate bond strength for most of the adhesives tested. After 2-year storage, only the self-etching adhesive Optibond All-In-One exhibited the same bonding ability when cured for longer periods of time.
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http://dx.doi.org/10.1016/j.jmbbm.2017.06.033DOI Listing
November 2017

Microfluidic-based platform to mimic the in vivo peripheral administration of neurotropic nanoparticles.

Nanomedicine (Lond) 2016 Dec 10;11(24):3205-3221. Epub 2016 Nov 10.

INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal.

Aim: Propose a nanoparticle for neuron-targeted retrograde gene delivery and describe a microfluidic-based culture system to provide insight into vector performance and safety.

Methods: Using compartmentalized neuron cultures we dissected nanoparticle bioactivity upon delivery taking advantage of (quantitative) bioimaging tools.

Results: Targeted and nontargeted nanoparticles were internalized at axon terminals and retrogradely transported to cell bodies at similar average velocities but the former have shown an axonal flux 2.7-times superior to nontargeted nanoparticles, suggesting an improved cargo-transportation efficiency. The peripheral administration of nanoparticles to axon terminals is nontoxic as compared with their direct administration to the cell body or whole neuron.

Conclusion: A neuron-targeted nanoparticle system was put forward. Microfluidic-based neuron cultures are proposed as a powerful tool to investigate nanoparticle bio-performance.
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http://dx.doi.org/10.2217/nnm-2016-0247DOI Listing
December 2016

[Dimensional structure of the Brazilian version of the s-EMBU instrument for measuring parental educational practices in adolescents].

Cad Saude Publica 2016 Aug;32(8):e00179915

Instituto de Medicina Social, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brasil.

The aim of this study was to assess the dimensional structure of the s-EMBU, used to measure parental educational practices in adolescents. The sample included 487 students from Greater Metropolitan Rio de Janeiro, Brazil, in 2013. Mean age was 14 years, and 47% were girls. The original dimensional structure was assessed by confirmatory factor analysis (CFA). The study also applied exploratory structural equation modeling (ESEM). CFA adjustment was unsatisfactory. In light of the ESEM, the most parsimonious solution concerned the three-factor model (RMSEA = 0.03, CFI = 0.97, and TLI = 0.96), but various items in the overprotection dimension showed higher factor loads in the rejection dimension. The items in the rejection and emotional warmth dimensions proved more appropriate. In Brazil, s-EMBU partially captures the proposed dimensions for measuring parental educational practices in adolescents. Emotional warmth had its dimensional structure confirmed, but rejection and especially overprotection require further refinement.
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http://dx.doi.org/10.1590/0102-311X00179915DOI Listing
August 2016

New integrative computational approaches unveil the Saccharomyces cerevisiae pheno-metabolomic fermentative profile and allow strain selection for winemaking.

Food Chem 2016 Nov 13;211:509-20. Epub 2016 May 13.

CBMA - Molecular and Environmental Research Centre, Department of Biology, University of Minho, Braga, Portugal.

During must fermentation by Saccharomyces cerevisiae strains thousands of volatile aroma compounds are formed. The objective of the present work was to adapt computational approaches to analyze pheno-metabolomic diversity of a S. cerevisiae strain collection with different origins. Phenotypic and genetic characterization together with individual must fermentations were performed, and metabolites relevant to aromatic profiles were determined. Experimental results were projected onto a common coordinates system, revealing 17 statistical-relevant multi-dimensional modules, combining sets of most-correlated features of noteworthy biological importance. The present method allowed, as a breakthrough, to combine genetic, phenotypic and metabolomic data, which has not been possible so far due to difficulties in comparing different types of data. Therefore, the proposed computational approach revealed as successful to shed light into the holistic characterization of S. cerevisiae pheno-metabolome in must fermentative conditions. This will allow the identification of combined relevant features with application in selection of good winemaking strains.
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http://dx.doi.org/10.1016/j.foodchem.2016.05.080DOI Listing
November 2016

Protective effect of antigen delivery using monoolein-based liposomes in experimental hematogenously disseminated candidiasis.

Acta Biomater 2016 07 2;39:133-145. Epub 2016 May 2.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710-057 Braga, Portugal. Electronic address:

Unlabelled: We evaluated the potential of a liposomal antigen delivery system (ADS) containing Candida albicans cell wall surface proteins (CWSP) in mediating protection against systemic candidiasis. Treatment of bone-marrow-derived dendritic cells with CWSP-loaded dioctadecyldimethylammonium bromide:monoolein (DODAB:MO) liposomes enhanced and prolonged their activation comparatively to free antigen, indicating that liposome-entrapped CWSP were released more sustainable. Therefore, we immunized mice with CWSP either in a free form or loaded into two different DODAB:MO liposome formulations, respectively designated as ADS1 and ADS2, prior to intravenous C. albicans infection. Immunization with ADS1, but not with ADS2, conferred significant protection to infected mice, comparatively to immunization with CWSP or empty liposomes as control. ADS1-immunized mice presented significantly higher serum levels of C. albicans-specific antibodies that enhanced phagocytosis of this fungus. In these mice, a mixed cytokine production profile was observed encompassing IFN-γ, IL-4, IL-17A and IL-10. Nevertheless, only production of IL-4, IL-17 and IL-10 was higher than in controls. In this study we demonstrated that DODAB:MO liposomes enhance the immunogenicity of C. albicans antigens and host protection in a murine model of systemic candidiasis. Therefore, this liposomal adjuvant could be a promising candidate to assess in vaccination against this pathogenic fungus.

Statement Of Significance: This work describes the immunomodulation capacity of the previously validated antigen delivery system (ADS) composed by dioctadecyldimethylammonium bromide (DODAB) and monoolein (MO) lipids incorporating the cell wall surface proteins (CWSP) from C. albicans. Here, we not only present the ability of this system in facilitating antigen uptake by DCs in vitro, but also that this system induces higher levels of pro-inflammatory cytokines and opsonizing specific IgG antibodies in serum of mice immunized subcutaneously. We show that the ADS are efficient nanocarrier and modulate the immune response against intravenous C. albicans infection favoring mouse protection. In sum, we show that the incorporation of C. albicans antigens in DODAB:MO nanocarries are a promising vaccine strategy against C. albicans fungal infection.
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http://dx.doi.org/10.1016/j.actbio.2016.05.001DOI Listing
July 2016

Effect of three months of soft contact lens wear on conjunctival cytology.

Clin Exp Optom 2016 Jul 5;99(4):336-41. Epub 2016 May 5.

Centre of Physics, University of Minho, Braga, Portugal.

Background: The purpose of this study was to investigate the effect of three months of soft contact lens wear on conjunctival goblet cell density and epithelial cell morphology.

Methods: This was a longitudinal clinical trial. Conjunctival impression cytology was performed on the superior palpebral conjunctiva in fifty-four eyes of twenty-seven neophyte contact lens wearers before and after three months of contact lens wear. Goblet cell density was determined by optical microscopy and epithelial cell morphology was classified according to the Tseng classification. Changes in goblet cell density as well as epithelial cell grading were determined. The effects of lens material and wearing modality on cytological changes were also investigated.

Results: Goblet cell density reduced significantly by 85 ± 151 cells/mm(2) (p < 0.001) after three months of contact lens wear. Reduction in goblet cell density was associated with lens materials; it was higher in conventional hydrogel lenses in comparison to silicone-hydrogel lenses (p = 0.008). The highest reduction in goblet cell density was found with Nelfilcon A lens wear (p = 0.002) and the lowest with Comfilcon A lens wear (p = 0.414). There was no statistically significant difference in grading of epithelial metaplasia before and after three months of contact lens wear (p = 0.075). Age was not correlated with the reduction in goblet cell density (r = -0.196, p = 0.160) but it was associated with the change in epithelial cell morphology (p = 0.036).

Conclusion: Three months of soft contact lens wear statistically significantly reduced goblet cell density; however, no significant changes were found in the grading of epithelial metaplasia. Contact lenses with lower oxygen permeability, higher Young modulus and higher thickness highly affected the conjunctival cytology.
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http://dx.doi.org/10.1111/cxo.12373DOI Listing
July 2016
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