State of the Art and Current Challenges on Electroactive Biomaterials and Strategies for Neural Tissue Regeneration.

The loss or failure of an organ/tissue stands as one of the healthcare system's most prevalent, devastating, and costly challenges. Strategies for neural tissue repair and regeneration have received significant attention due to their particularly strong impact on patients' well-being. Many research efforts are dedicated not only to control the disease symptoms but also to find solutions to repair the damaged tissues. Neural tissue engineering (TE) plays a key role in addressing this problem and significant efforts are being carried out to develop strategies for neural repair treatment. In the last years, active materials allowing to tune cell-materials interaction are being increasingly used, representing a recent paradigm in TE applications. Among the most important stimuli influencing cell behavior are the electrical and mechanical ones. In this way, materials with the ability to provide this kind of stimuli to the neural cells seem to be appropriate to support neural TE. In this scope, this review summarizes the different biomaterials types used for neural TE, highlighting the relevance of using active biomaterials and electrical stimulation. Furthermore, this review provides not only a compilation of the most relevant studies and results but also strategies for novel and more biomimetic approaches for neural TE.

Epidemiological Changes in Respiratory Viral Infections in Children: The Influence of the COVID-19 Pandemic.

BACKGROUND: Viruses are the major cause of acute respiratory infections in children, causing important morbimortality. Before the COVID-19 pandemic, in temperate regions, respiratory viruses displayed a typical seasonality in transmission. A disruption in this pattern was observed in several countries during the pandemic, with low prevalence during the typical season, and an interseasonal rise. We evaluated the effects of the COVID-19 pandemic in the epidemiology of non-COVID viral respiratory infections in children, in a tertiary care hospital in Portugal. METHODS: Between March 2020 and August 2022, nasopharyngeal samples from children with respiratory symptoms in the Emergency Department (ED) and the Pediatric Ward were tested for RSV, influenza and other respiratory viruses, by real-time reverse transcriptase PCR (RT-PCR). RESULTS: A seasonal variation was observed from 2018 to 2020, with prevalence increasing in winter (mainly RSV and influenza). In the winter of 2020/21, when measures to mitigate SARS-CoV-2 transmission were stricter, there was a disruption of the seasonal pattern, with unusually low numbers. In the summer of 2021, when measures were being relaxed, there was an atypical rise. In June 2021, RSV was first detected and peaked in October. Influenza (Influenza A H3) was detected for the first time in February 2022, peaking in March/April. CONCLUSIONS: These findings show a disruption of the seasonality of viral respiratory infections in children during the pandemic, with a virtual elimination during the months of usually higher prevalence, and a subsequent out-of-season increase, coinciding with variations in the measures implemented to control the SARS-CoV-2 transmission, and confirming their efficacy.

Electroactive Materials Surface Charge Impacts Neuron Viability and Maturation in 2D Cultures.

Since neurons were first cultured outside a living organism more than a century ago, a number of experimental techniques for their in vitro maintenance have been developed. These methods have been further adapted and refined to study specific neurobiological processes under controlled experimental conditions. Despite their limitations, the simplicity and visual accessibility of 2D cultures have enabled the study of the effects of trophic factors, adhesion molecules, and biophysical stimuli on neuron function and morphology. Nevertheless, the impact of fundamental properties of the surfaces to which neurons adhere when cultured in vitro has not been sufficiently considered. Here, we used an electroactive polymer with different electric poling states leading to different surface charges to evaluate the impact of the net electric surface charge on the behavior of primary neurons. Average negative and positive surface charges promote increased metabolic activity and enhance the maturation of primary neurons, demonstrating the relevance of considering the composition and electric charge of the culture surfaces. These findings further pave the way for the development of novel therapeutic strategies for the regeneration of neural tissues, particularly based on dynamic surface charge variation that can be induced in the electroactive films through mechanical solicitation.

Modulation of myoblast differentiation by electroactive scaffold morphology and biochemical stimuli.

The physico-chemical properties of the scaffold materials used for tissue regeneration strategies have a direct impact on cell shape, adhesion, proliferation, phenotypic and differentiation. Herewith, biophysical and biochemical cues have been widely used to design and develop biomaterial systems for specific tissue engineering strategies. In this context, the patterning of piezoelectric polymers that can provide electroactive stimuli represents a suitable strategy for skeletal muscle tissue engineering applications once it has been demonstrated that mechanoelectrical stimuli promote C2C12 myoblast differentiation. In this sense, this works reports on how C2C12 myoblast cells detect and react to physical and biochemical stimuli based on micropatterned poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)) electroactive scaffolds produced by soft lithography in the form of arrays of lines and hexagons (anisotropic and isotropic morphology, respectively) combined with differentiation medium. The scaffolds were evaluated for the proliferation and differentiation of C2C12 myoblast cell line and it is demonstrated that anisotropic microstructures promote muscle differentiation which is further reinforced with the introduction of biochemical stimulus. However, when the physical stimulus is not adequate to the tissue, e.g. isotropic microstructure, the biochemical stimulus has the opposite effect, hindering the differentiation process. Therefore, the proper morphological design of the scaffold combined with biochemical stimulus allows to enhance skeletal muscle differentiation and allows the development of advanced strategies for effective muscle tissue engineering.

Mental Health of Healthcare Professionals: Two Years of the COVID-19 Pandemic in Portugal.

The COVID-19 pandemic increased psychosocial risk factors among healthcare professionals (HCPs). Objective: To characterize Portuguese HCPs mental health (MH), estimate anxiety, depression, post-traumatic stress disorder (PTSD) and burnout symptoms, and identify risk/protective factors. A cross-sectional online survey and a longitudinal assessment were conducted in 2020 (T0) and 2021 (T1). Sociodemographic and occupational variables, COVID-19-related experiences and protective behavior data were collected from a non-probabilistic sample of HCPs in Portugal. Symptoms of anxiety, depression, PTSD, burnout and resilience were assessed using the Portuguese versions of the Generalized Anxiety Disorder Scale (GAD-7), the Patient Health Questionnaire (PHQ-9), the Post-traumatic Stress Disorder Checklist (PCL-5), the Shirom-Melamed Burnout Measure (MBSM) and the Connor-Davidson Resilience Scale (CD-RISC-10), respectively. Risk and protective factors were identified through simple and multiple logistic regression models. Overall, 2027 participants answered the survey in T0 and 1843 in T1. The percentage of moderate-to-severe symptoms decreased from T0 to T1; however, a considerable proportion of HCPs reported symptoms of distress in both years. Being a woman, working in a COVID-19-treatment frontline position and work-life balance increased the odds of distress. High resilience, good social/family support, and hobbies/lifestyle maintenance were found to be protective factors. Globally, our results show that performing as a HCP during the pandemic may result in long-term effects on MH.

Piezoelectric and Magnetically Responsive Biodegradable Composites with Tailored Porous Morphology for Biotechnological Applications.

The biomedical area in the scope of tissue regeneration pursues the development of advanced materials that can target biomimetic approaches and, ideally, have an active role in the environment they are placed in. This active role can be related to or driven by morphological, mechanical, electrical, or magnetic stimuli, among others. This work reports on the development of active biomaterials based on poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid), PHBV, a piezoelectric and biodegradable polymer, for tissue regeneration application by tailoring its morphology and functional response. PHBV films with different porosities were obtained using the solvent casting method, resorting to high-boiling-point solvents, as N,N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO), and the combination of chloroform (CF) and DMF for polymer dissolution. Furthermore, magnetoelectric biomaterials were obtained through the combination of the piezoelectric PHBV with magnetostrictive iron oxide (Fe3O4) nanoparticles. Independently of the morphology or filler content, all biomaterials proved to be suitable for biomedical applications.

New local ganirelix sustained release therapy for uterine leiomyoma. Evaluation in a preclinical organ model.

Currently, there is a limited number of treatment options available for patients with symptomatic leiomyomas, and surgical removal is by far the most frequent procedure. Previous studies found that GnRH agonists and antagonists acting through GnRH receptors led to cell death and decreased extracellular synthesis in cultured leiomyoma cells. In this study, we encapsulated the GnRH antagonist ganirelix in PLGA microspheres contained in an alginate scaffold that also supports a leiomyoma ex vivo tissue explant. Microspheres maintained ganirelix concentration stably during six days of culture, inducing significant cell death in 50-55% of tumor cells. Although no changes were observed in the expression of extracellular matrix genes, a decreased expression of the Nuclear Factor of Activated T cells 5, a transcription factor involved in osmotic stress and tumor size. Interestingly, all tumors analyzed experienced apoptosis independently of the original driver mutation. These data indicate that local therapy of ganirelix would induce tumor reduction in a wide range of uterine leiomyomas.

NMR Reveals Functionally Relevant Thermally Induced Structural Changes within the Native Ensemble of G-CSF.

Structure-function relationships in proteins refer to a trade-off between stability and bioactivity, molded by evolution of the molecule. Identifying which protein amino acid residues jeopardize global or local stability for the benefit of bioactivity would reveal residues pivotal to this structure-function trade-off. Here, we use 15N-1H heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy to probe the microenvironment and dynamics of residues in granulocyte colony-stimulating factor (G-CSF) through thermal perturbation. From this analysis, we identified four residues (G4, A6, T133, and Q134) that we classed as significant to global stability, given that they all experienced large environmental and dynamic changes and were closely correlated to each other in their NMR characteristics. Additionally, we observe that roughly four structural clusters are subject to localized conformational changes or partial unfolding prior to global unfolding at higher temperature. Combining NMR observables with structure relaxation methods reveals that these structural clusters concentrate around loop AB (binding site III inclusive). This loop has been previously implicated in conformational changes that result in an aggregation prone state of G-CSF. Residues H43, V48, and S63 appear to be pivotal to an opening motion of loop AB, a change that is possibly also important for function. Hence, we present here an approach to profiling residues in order to highlight their potential roles in the two vital characteristics of proteins: stability and bioactivity.

Stem Cell Growth and Differentiation in Organ Culture: New Insights for Uterine Fibroid Treatment.

Organ culture allows for the understanding of normal and tumor cell biology, and tissues generally remain viable for 5-7 days. Strikingly, we determined that myometrial and MED12 mutant leiomyoma cells repopulated cell-depleted tissue slices after 20 days of culture. Using immunofluorescence and quantitative PCR of stem cell and undifferentiated cell markers, we observed clusters of CD49b+ cells in tumor slices. CD49b+ cells, however, were sparsely detected in the myometrial slices. Almost all LM cells strongly expressed Ki67, while only a few myometrial cells were stained for this proliferation marker. The CD73 marker was expressed only in tumor cells, whereas the mesenchymal stem cell receptor KIT was detected only in normal cells. HMGA2 and CD24 showed broader expression patterns and higher signal intensity in leiomyoma than in myometrial cells. In this study, we propose that activating CD49b+ stem cells in myometrium leads to asymmetrical division, giving rise to transit-amplifying KIT+ cells that differentiate to smooth muscle cells. On the contrary, activated leiomyoma CD49b+ cells symmetrically divide to form clusters of stem cells that divide and differentiate to smooth muscle cells without losing proliferation ability. In conclusion, normal and mutant stem cells can proliferate and differentiate in long-term organ culture, constituting a helpful platform for novel therapeutic discovery.

Hidden Multivalency in Phosphatase Recruitment by a Disordered AKAP Scaffold.

Disordered scaffold proteins provide multivalent landing pads that, via a series of embedded Short Linear Motifs (SLiMs), bring together the components of a complex to orchestrate precise spatial and temporal regulation of cellular processes. One such protein is AKAP5 (previously AKAP79), which contains SLiMs that anchor PKA and Calcineurin, and recruit substrate (the TRPV1 receptor). Calcineurin is anchored to AKAP5 by a well-characterised PxIxIT SLiM. Here we show, using a combination of biochemical and biophysical approaches, that the Calcineurin PxIxIT-binding groove also recognises several hitherto unknown lower-affinity SLiMs in addition to the PxIxIT motif. We demonstrate that the assembly is in reality a complex system with conserved SLiMs spanning a wide affinity range. The capture is analogous to that seen for many DNA-binding proteins that have a weak non-specific affinity for DNA outside the canonical binding site, but different in that it involves (i) two proteins, and (ii) hydrophobic rather than electrostatic interactions. It is also compatible with the requirement for both stable anchoring of the enzyme and responsive downstream signalling. We conclude that the AKAP5 C-terminus is enriched in lower-affinity/mini-SLiMs that, together with the canonical SLiM, maintain a structurally disordered but tightly regulated signalosome.

Carbapenemase-Producing Enterobacteriaceae (CPE) Newborn Colonization in a Portuguese Neonatal Intensive Care Unit (NICU): Epidemiology and Infection Prevention and Control Measures.

Infections due to carbapenemase-producing Enterobacterales (CPE) are increasing worldwide and are especially concerning in a neonatal intensive care unit (NICU). Risk factors for CPE gut colonization in neonates need to be clarified. In this work, we describe the epidemiological and clinical features of CPE-colonized newborns and the infection control measures in a Portuguese NICU. We performed a prospective, observational, longitudinal, cohort study for surveillance of CPE colonization. Maternal and neonatal features of colonized newborns and surveillance strategy were described. A statistical analysis was performed with SPSS23.0, and significance was indicated by p-value ≤ 0.05. Between March and November 2019, CPE was isolated in 5.8% of 173 admitted neonates. Carbapenemase-producing Klebsiella pneumoniae were the most frequently isolated. There was no associated infection. Birth weight, gestational age, length of stay, and days of central line were the identified risk factors for CPE colonization (bivariate analysis with Student's t-test or Mann-Whitney U test, according to normality). No independent risk factors for CPE colonization were identified in the logistic regression analysis. CPE colonization risk factors are still to be determined accurately in the neonatal population. Active surveillance and continuous infection control measures restrained the current cluster of colonized newborns and helped to prevent infection and future outbreaks.

Dual agonist-antagonist effect of ulipristal acetate in human endometrium and myometrium.

The aim of this study was to assess the molecular effect of ulipristal acetate (UPA) on gene expression in myometrium and endometrium of patients with symptomatic fibroids. Tissues isolated from four women treated preoperatively with UPA (5 mg) were compared to those from untreated controls using NanoString platform to assess the expression of 75 candidate genes modulated by UPA and ovarian steroids. Deregulated genes were then validated by real-time PCR. In myometrium, UPA exerted an antagonistic effect similar to that observed in fibroids. In UPA-treated endometrium, six genes were identified as highly and significantly upregulated, including matricellular genes CCN1 (54-fold, P = 0.0018) and CCN2 (11-fold, P = 0.00044), Krüppel-like factor 4 (>3-fold, P = 0.0036), and mast cell markers including tryptases TPSAB1/TPSB2 (31-fold, P = 0.023) and carboxypeptidase A (CPA3, 17-fold, P = 0.05). In endometrium, UPA induced the expression of genes involved in fibrogenesis and mast cell function-some of them being widely involved in hepatic injury, which could explain the marked fibrosis and inflammatory cell infiltration observed in explanted livers from patients under UPA treatment.

NMR Reporter Assays for the Quantification of Weak-Affinity Receptor-Ligand Interactions.

Biophysical methods are widely employed in academia and the pharmaceutical industry to detect and quantify weak molecular interactions. Such methods find broad application in fragment-based drug discovery (FBDD). In an FBDD campaign, a suitable affinity determination method is key to advancing a project beyond the initial screening phase. Protein-observed (PO) nuclear magnetic resonance (NMR) finds widespread use due to its ability to sensitively detect very weak interactions at residue-level resolution. When there are issues precluding the use of PO-NMR, ligand-observed (LO) NMR reporter assays can be a useful alternative. Such assays can measure affinities in a similar range to PO-NMR while offering some distinct advantages, especially with regard to protein consumption and compound throughput. In this paper, we take a closer look at setting up such assays for routine use, with the aim of getting high-quality, accurate data and good throughput. We assess some of the key characteristics of these assays in the mathematical framework established for fluorescence polarization assays with which the readers may be more familiar. We also provide guidance on setting up such assays and compare their performance with other affinity determination methods that are commonly used in drug discovery.

Patterned Piezoelectric Scaffolds for Osteogenic Differentiation.

The morphological clues of scaffolds can determine cell behavior and, therefore, the patterning of electroactive polymers can be a suitable strategy for bone tissue engineering. In this way, this work reports on the influence of poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)) electroactive micropatterned scaffolds on the proliferation and differentiation of bone cells. For that, micropatterned P(VDF-TrFE) scaffolds were produced by lithography in the form of arrays of lines and hexagons and then tested for cell proliferation and differentiation of pre-osteoblast cell line. Results show that more anisotropic surface microstructures promote bone differentiation without the need of further biochemical stimulation. Thus, the combination of specific patterns with the inherent electroactivity of materials provides a promising platform for bone regeneration.

Organotypic culture as a research and preclinical model to study uterine leiomyomas.

Organotypic cultures of tissue slices have been successfully established in lung, prostate, colon, gastric and breast cancer among other malignancies, but until now an ex vivo model based on tissue slices has not been established for uterine leiomyoma. In the present study, we describe a method for culturing tumour slides onto an alginate scaffold. Morphological integrity of tissue slices was maintained for up to 7 days of culture, with cells expressing desmin, estrogen and progesterone receptors. Driver mutations were present in the ex vivo slices at all-time points analyzed. Cultivated tumour slices responded to ovarian hormones stimulation upregulating the expression of genes involved in leiomyoma pathogenesis. This tissue model preserves extracellular matrix, cellular diversity and genetic background simulating more in-vivo-like situations. As a novelty, this platform allows encapsulation of microspheres containing drugs that can be tested on the ex vivo tumour slices. After optimizing drug release rates, microspheres would then be directly tested in animal models through local injection.

Tailoring Electrospun Poly(l-lactic acid) Nanofibers as Substrates for Microfluidic Applications.

Novel microfluidic substrates based on electrospun poly(l-lactic acid) (PLLA) membranes were developed to increase the limited range of commercially available paper substrates, commonly used for the fabrication of microfluidic paper-based analytical devices. PLLA's advantageous properties include biodegradability, biocompatibility, ease of being processed in various tailored morphologies, and cost effectiveness, among others. Oriented and nonoriented electrospun PLLA membranes were fabricated using electrospinning and the influence of fiber orientation, addition of hydrophilic additives, and plasma treatments on the morphology, physicochemical properties, and capillary flow rates were evaluated and compared with the commercial Whatman paper. In addition, a proof-of-concept application based on the colorimetric detection of glucose in printed PLLA and paper-based microfluidic systems was also performed. The results show the potential of PLLA substrates for the fabrication of portable, disposable, eco-friendly, and cost-effective microfluidic systems with controllable properties that can be tailored according to specific biotechnological application requirements, being a suitable alternative to conventional paper-based substrates.

Mitocondrial COI and 16S rDNA sequences support morphological identification and biogeography of deep-sea red crabs of the genus Chaceon (Crustacea, Decapoda, Geryonidae) in the Eastern Central and South Atlantic Ocean.

The geographical spreading of new fishing activities and the increasingly deeper locations of these activities have shown the worldwide distribution of gerionid crabs and new descriptions of Chaceon taxa. However, incomplete penetrance, variable expressivity, and phenotypic overlap make the morphometric identification of these species difficult. In this study, partial sequences of the cytochrome c oxidase subunit 1 (COI) and 16S mitochondrial ribosomal RNA (16S rRNA) genes have been analyzed in Chaceon species from the Eastern Central and South Atlantic and compared with sequences of species from Western Atlantic. Our results corroborate the proposed morphological species and highlight the significant separation of the Eastern Atlantic species and those from Atlantic coasts of South America for both markers (97% Bayesian posterior probability, BPP / 83% Bootstrap replicates, BT). Interestingly, Chaceon sanctaehelenae shows a closer relationship with the species of the American coast than with those from the Eastern Atlantic. On the other hand, while COI marker clearly separates Chaceon atopus and Chaceon erytheiae species (99 BPP / 91% BT), these species share haplotypes for the 16S rRNA marker, pointing to a recent speciation process. Moreover, a close relationship was observed between Chaceon maritae and Chaceon affinis (94% BPP / 77% BT). The topologies of the trees obtained indicate that the ancestor of this genus was closer related to those species from South America than to those from the Eastern Atlantic.

Tuning Myoblast and Preosteoblast Cell Adhesion Site, Orientation, and Elongation through Electroactive Micropatterned Scaffolds.

Electroactive polymers are being increasingly used in tissue engineering applications. Together with the electromechanical clues, morphological ones have been demonstrated to determine cell proliferation and differentiation. This work reports on the micropatterning of poly(vinylidene fluoride-co-trifluoroethylene), P(VDF-TrFE) scaffolds, and their interaction with myoblast and preosteoblasts cell lines, selected based on their different functional morphology. The scaffolds were obtained by soft lithography and obtained in the form of arrays of lines, intermittent lines, hexagons, linear zigzags, and curved zigzags with dimensions of 25, 75, and 150 µm. Moreover, the scaffolds were tested in cell adhesion assays of myoblasts and preosteoblasts cell lines. The results show that more linear surface topographies and dense morphology have a large potential in the regeneration of musculoskeletal tissue, while nonpatterned scaffolds or more anisotropic surface microstructures present largest potential to promote the growth and regeneration of bone tissue. In this way, cell adhesion site, orientation, and elongation can be controlled by choosing properly the topography and morphology of the scaffolds, indicating their suitability and potential for further proliferation and differentiation assays.