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Traumatic Spinal Cord Injury (SCI) leads to severe motor and sensory functional impairments that affect personal and social behaviors. Medical advancements have improved supportive therapeutic measures for SCI patients, but no effective neuroregenerative therapeutic options exist to date. Deficits in motor function are the most visible consequence of SCI. However, other complications, as spasticity, produce a significant impact on SCI patient's welfare. Baclofen, a GABA agonist, is the most effective drug for spasticity treatment. Interestingly, emerging data reveals that Baclofen can also play a role on neuroprotection and regeneration after SCI.The goal of this study was to understand the potential of Baclofen as a treatment to promote recovery after SCI.We used a pre-clinical SCI mouse model with the administration of Baclofen 1 mg/Kg at different time-points after injury.Behavior analysis (locomotor and bladder function) were performed during nine weeks of the in vivo experiment. Afterwards, spinal cords were collected and processed for histological and molecular analysis.Our data showed that Baclofen leads to locomotor improvements in mice when its administered acutely after SCI. Moreover, Baclofen administration also led to improved bladder function control in all experimental groups. Interestingly, acute Baclofen administration modulates microglia activation state and levels of circulating chemokines and cytokines, suggesting a putative role of Baclofen in the modulation of the immune response.Although further studies must be performed to understand the mechanisms that underlie the functional improvements produced by Baclofen, our data shed light into the pharmacological potential of Baclofen to promote recovery after SCI.Our outcomes revealed that Baclofen, a well-known drug used for spasticity management, improves the motor performance after SCI in a pre-clinical animal model. Our data opens new avenues for pharmacological strategies design to promote SCI recovery.

Over 90% of chronic pain (CP) patients receive opioids-based treatments, which led to a public health crisis with lasting impacts on social and economic wellbeing based on opioid addiction. Opioids act through activation of μ (MOR), δ (DOR), and κ (KOR) opioid receptors, which are broadly and differentially distributed throughout the brain. Chronic opioid consumption leads to brain changes such as alterations on neurotransmission, dendritic branching, and spine density, as well as an increase in apoptosis. To overcome opioid-related issues, extensive efforts have been made to search for an alternative treatment. Bioactive molecules secreted by stem cells, collectively known as secretome, have shown a positive impact in different pain models. However, there is a lack of studies on the role of secretome in modulating opioid receptors. By using cerebral organoids (CeO), a self-organized, functional, and multicellular 3D structure that resemble the brain, we were able to identify MOR, DOR, and KOR at different stages of maturation. Treatment with secretome increased MOR expression highlighting a possible role in pain signaling mechanisms. Opioid treatments did not impact the expression of neuronal maturation markers but together with secretome, they increased astrogliogenesis. Opioid-treated organoids presented higher dopamine secretion recapitulating an important physiological event after opioid exposure. This work demonstrates that CeO is an important model system for the study of opioid signaling with potential implications to the understanding of basic mechanisms related to pain physiology.

Mesenchymal stem cells (MSCs) hold promising therapeutic potential in several clinical applications, mainly due to their paracrine activity. The implementation of future secretome-based therapeutic strategies requires the use of easily accessible MSCs sources that provide high numbers of cells with homogenous characteristics. MSCs obtained from induced pluripotent stem cells (iMSCs) have been put forward as an advantageous alternative to the gold-standard tissue sources, such as bone marrow (BM-MSCs). In this study, we aimed at comparing the secretome of BM-MSCs and iMSCs over long-term culture. For that, we performed a broad characterization of both sources regarding their identity, proteomic secretome analysis, as well as replicative senescence and associated phenotypes, including its effects on MSCs secretome composition and immunomodulatory action. Our results evidence a rejuvenated phenotype of iMSCs, which is translated into a superior proliferative capacity before the induction of replicative senescence. Despite this significant difference between iMSCs and BM-MSCs proliferation, both untargeted and targeted proteomic analysis revealed a similar secretome composition for both sources in pre-senescent and senescent states. These results suggest that shifting from the use of BM-MSCs to a more advantageous source, like iMSCs, may yield similar therapeutic effects as identified over the past years for this gold-standard MSC source.

Electroactive smart materials play an important role for tissue regenerative applications. Poly(vinylidene fluoride) (PVDF) is a specific subtype of piezoelectric electroactive material that generates electrical potential upon mechanical stimulation. This work focuses on the application of piezoelectric PVDF films for neural differentiation. Human neural precursor cells (hNPCs) are cultured on piezoelectric poled and non-poled -PVDF films with or without a pre-coating step of poly-d-lysine and laminin (PDL/L). Subsequently, hNPCs differentiation into the neuronal lineage is assessed (MAP2+ and DCX+) under static or dynamic (piezoelectric stimulation) culture conditions. The results demonstrate that poled and coated -PVDF films induce neuronal differentiation under static culture conditions which is further enhanced with mechanical stimulation. In silico calculations of the electrostatic potential of different domains of laminin, highlight the high polarity of those domains, which shows a clear preference to interact with the varying surface electric field of the piezoelectric material under mechanical stimulation. These interactions might explain the higher neuronal differentiation induced by poled -PVDF films pre-coated with PDL/L under dynamic conditions. Our results suggest that electromechanical stimuli, such as the ones induced by piezoelectric -PVDF films, are suitable to promote neuronal differentiation and hold great promise for the development of neuroregenerative therapies., Fundação para a Ciência e a Tecnologia, Grant/Award Numbers: POCI-01-0145-FEDER-029206, POCI-01-0145-FEDER-031392, PTDC/MED-NEU/31417/2017, UIDB/50026/2020, UIDP/50026/2020, NORTE-01-0145-FEDER-029968, POCI-01-0145-FEDER-029751, POCI-01-0145-FEDER-032619, UID/FIS/04650/2020, PTDC/EMD-EMD/28159/2017, PTDC/BTM-MAT/28237/2017, PTDC/QUI-OUT/32243/2017, LA/P/0058/2020, UIDB/04539/2020, UIDP/04539/2020, POCI-01-0145-FEDER-031356, DSAIPA/DS/0118/2020; Santa Casa da Misericórdia de Lisboa, Grant/Award Numbers: MC-04-17, MC-18-21; Basque Government Industry Departments; Spanish State Research Agency; European Regional Development Fund, Grant/Award Number: PID2019-106099RB-C43/AEI; Northern Portugal Regional Operational Program, Grant/Award Numbers: NORTE-01-0145-FEDER-000023, NORTE-01-0145-FEDER-000013; Portuguese Platform of Bioimaging, Grant/Award Number: PPBI-POCI-01-0145-FEDER-022122; ICVS Scientific Microscopy Platform; Portuguese Foundation for Science and Technology, Grant/Award Number: PD/BDE/143150/2019, info:eu-repo/semantics/publishedVersion

Funding Acknowledgements Type of funding sources: None. Introduction Electrocardiogram (ECG) is the most performed cardiovascular tool in medicine(1). First described by Einthoven at the beginning of the 20th century, it is now a useful, cheap and safe exam, accessible to millions of people on devices as light as smartwatches. Several studies have demonstrated the variability in the ECG interpretation. A recent systematic review described a low median accuracy of 54%, across different training levels (from students to experimented Cardiologists), and suggested that continuous training with courses, software and databases significantly improves that accuracy(2). With the easy access to this exam, along with the high prevalence of arrhythmias, Primary Care Physicians play an important role in this ECG era. Nevertheless, some articles reported that they too make frequent errors in interpretation and feel "uncomfortable" dealing with the 120 diagnostic ECG patterns described(3). Purpose To develop a website designed to aid Primary Care Physicians in diagnosing and managing patients with most ECG diagnostic patterns. Methods Literature regarding ECG pattern diagnosis and management was reviewed. The research combined European and North American guidelines, medical textbooks and websites, reviews and original articles available on Pubmed. The website information was assembled and written by Primary Care Physicians, then reviewed by Cardiologists that work in the Arrhythmology area. Results It was created an open, free access, nonprofitable, continuously updated website, written in Portuguese, that has 4 main components: 1. A Homepage with a search toolbar; 2. An "Index Page", with all the ECG diagnostic statements organized as a book index (FIGURE1); 3. A "Toolkit" link, with tools that are useful in ECG management such as CHA2DDS2-VASc and HASBLED scores, doses and contraindications of drugs, etc (FIGURE2).; 4. When searching for an ECG pattern, the user can access a summarized text with its definition, causes, exams that might be required (e.g. echocardiogram), and management (FIGURE1). The management section organizes procedures from the most to the less urgent. A version of the website is available and fully functional for testing purposes. The website will be continuously updated with images of all ECG diagnostic patterns from a local hospital database and specific information for pediatrics, pregnant women, and athletes. Conclusion Nowadays, Primary Care Physicians face a fascinating era of increasing amounts of information, fast discoveries and possibilities that demand permanent attention and update. The ECG is a complex but valuable and accessible tool with more than 100 different diagnostic statements. With this website, the aim is to empower Primary Care Physicians so they can confidently interpret ECG, and also manage patients according to the best medical recommendations available, with an updated tool fully designed for them.

Phase transition temperatures of three ionic liquids (ILs) with ammonium cations of different alkyl chain length and nitrate common anion were determined in this paper by differential scanning calorimetry. Thermal stability of these ILs in air and nitrogen atmosphere and the maximum operation temperatures of the compounds were also studied using a thermogravimetric analyser. Furthermore, thermophysical properties as density, viscosity, thermal conductivity, and heat capacity against temperature of these pure ionic liquids were determined and compared with the corresponding values for water saturated samples. For this purpose, different techniques were employed: for density and viscosity a rotational automated viscodensimeter; for thermal conductivity measurements a thermal conductimeter that follows the transient hot-wire method; and for heat capacity measurements a microdifferential scanning calorimeter. Different behaviours were observed depending on the analysed thermophysical properties. In case of dried samples, density and thermal conductivity decrease with alkyl chain; for saturated samples, same tendency is observed for density but is the opposite in case of thermal conductivity. Isobaric specific heat and viscosity increase with alkyl chain length in case of dried samples; for saturated samples same tendency is observed for the viscosity, whereas the tendency is the contrary in case of isobaric specific heat.

After the introduction of large-bore catheters with the Seldinger technique into the vena cava superior via the internal jugular veins in 1979, the advantages of this puncturing technique versus the puncture of the femoral or subclavian veins were seen. However, complications and side effects of the puncture of the internal jugular vein, such as faulty punctures, bleeding, hematothorax, thrombosis and faults in catheter material and infections were observed, too. Infections, thrombosis, and stenosis are among the most frequent side effects associated with blood-contacting catheters. These side effects are usually related to surface properties and the material of these catheters. Surface treatment processes, such as ion implantation and ion beam assisted deposition, and microdomain structured surfaces, could be used to mitigate such complications. The complication rate was first retrospective about 28 %. A second retrospective study of surface treated catheters with silver versus untreated catheters showed 75 % decline in the infection and thrombosis rate in the surface treated catheters. However, this cannot be confirmed with more available data of these patients. One reason may be that in the surfaces treated catheters only the outer surface was coated with silver and the possibilities of contamination by handling during treatments. New materials and technologies which include the outer and inner surface are necessary to reduce the tremendous discomfort of patients and the high costs of the catheter-related infections.

Spinal cord injury (SCI) affects an estimated three million persons worldwide, with ∼180,000 new cases reported each year leading to severe motor and sensory functional impairments that affect personal and social behaviors. To date, no effective treatment has been made available to promote neurological recovery after SCI. Deficits in motor function is the most visible consequence of SCI; however, other secondary complications produce a significant impact on the welfare of patients with SCI. Spasticity is a neurological impairment that affects the control of muscle tone as a consequence of an insult, trauma, or injury to the central nervous system, such as SCI. The management of spasticity can be achieved through the combination of both nonpharmacological and pharmacological approaches. Baclofen is the most effective drug for spasticity treatment, and it can be administered both orally and intrathecally, depending on spasticity location and severity. Interestingly, recent data are revealing that baclofen can also play a role in neuroprotection after SCI. This new function of baclofen in the SCI scope is promising for the prospect of developing new pharmacological strategies to promote functional recovery in patients with SCI.

Parkinson's disease is the second most prevalent neurodegenerative disorder worldwide. Clinically, it is characterized by severe motor complications caused by progressive degeneration of dopaminergic neurons. Current treatment is focused on mitigating the symptoms through the administration of levodopa, rather than on preventing dopaminergic neuronal damage. Therefore, the use and development of neuroprotective/disease-modifying strategies is an absolute need that can lead to promising gains on translational research of Parkinson's disease. For instance, N-acetylcysteine, a natural compound with strong antioxidant effects, has been shown to modulate oxidative stress, preventing dopamine-induced cell death. Despite the evidence of neuroprotective and modulatory effects of this drug, as far as we know, it does not induce per se any regenerative process. Therefore, it would be of interest to combine the latter with innovative therapies that induce dopaminergic neurons repair or even differentiation, as stem cell-based strategies. Stem cells secretome has been proposed as a promising therapeutic approach for Parkinson's disease, given its ability to modulate cell viability/preservation of dopaminergic neurons. Such approach represents a shift in the paradigm, showing that cell-transplantation free therapies based on the use of stem cells secretome may represent a potential alternative for regenerative medicine of Parkinson's disease. Thus, in this review, we address the current understanding of the potential combination of stem cell free-based strategies and neuroprotective/disease-modifying strategies as a new paradigm for the treatment of central nervous system neurodegenerative diseases, like Parkinson's disease.

Background: Manual segmentation of the Foveal Avascular Zone (FAZ) has a high level of variability. Research into retinas needs coherent segmentation sets with low variability. Methods: Retinal optical coherence tomography angiography (OCTA) images from type-1 diabetes mellitus (DM1), type-2 diabetes mellitus (DM2) and healthy patients were included. Superficial (SCP) and deep (DCP) capillary plexus FAZs were manually segmented by different observers. After comparing the results, a new criterion was established to reduce variability in the segmentations. The FAZ area and acircularity were also studied. Results: The new segmentation criterion produces smaller areas (closer to the real FAZ) with lower variability than the different criteria of the explorers in both plexuses for the three groups. This was particularly noticeable for the DM2 group with damaged retinas. The acircularity values were also slightly reduced with the final criterion in all groups. The FAZ areas with lower values showed slightly higher acircularity values. We also have a consistent and coherent set of segmentations with which to continue our research. Conclusions: Manual segmentations of FAZ are generally carried out with little attention to the consistency of the measurements. A novel criterion for segmenting the FAZ allows segmentations made by different observers to be more similar.

Background aims The capacity of the secretome from bone marrow-derived mesenchymal stem cells (BMSCs) to prevent dopaminergic neuron degeneration caused by overexpression of alpha-synuclein (α-syn) was explored using two Caenorhabditis elegans models of Parkinson's disease (PD). Methods First, a more predictive model of PD that overexpresses α-syn in dopamine neurons was subjected to chronic treatment with secretome. This strain displays progressive dopaminergic neurodegeneration that is age-dependent. Following chronic treatment with secretome, the number of intact dopaminergic neurons was determined. Following these initial experiments, a C. elegans strain that overexpresses α-syn in body wall muscle cells was used to determine the impact of hBMSC secretome on α-syn inclusions. Lastly, in silico analysis of the components that constitute the secretome was performed. Results The human BMSC (hBMSC) secretome induced a neuroprotective effect, leading to reduced dopaminergic neurodegeneration. Moreover, in animals submitted to chronic treatment with secretome, the number of α-syn inclusions was reduced, indicating that the secretome of MSCs was possibly contributing to the degradation of those structures. In silico analysis identified possible suppressors of α-syn proteotoxicity, including growth factors and players in the neuronal protein quality control mechanisms. Conclusions The present findings indicate that hBMSC secretome has the potential to be used as a disease-modifying strategy in future PD regenerative medicine approaches.

Spinal cord injury (SCI) leads to severe functional deficits. Currently, there are no available pharmacological treatments to promote neurological recovery in SCI patients. Recent work from our group has shown that a baclofen treatment can promote functional recovery after a compression SCI in mice [1]. Here, we provide transcriptomic (RNA-seq) data from adult mouse spinal cords collected 7 days after a compression SCI and baclofen (vs vehicle) administration. The Illumina NovaSeq 6000 platform was used to generate the raw transcriptomic data. In addition, we also present bioinformatic analyses including differential gene expression analysis, enrichment analyses for various functional annotations (gene ontology, KEGG and BioCarta pathways or InterPro domains) and transcription factor targets. The raw RNA-seq data has been uploaded to the NCBI Sequence Read Archive (SRA) database (Bioproject ID PRJNA886048). The data generated from the bioinformatic analyses is contained within the article This work was supported by the Wings for Life Spinal Cord Research Foundation (grant reference WFL-ES-03/19), the Portuguese Foundation for Science and Technology (FCT) EXPL/MED-FAR/1529/2021 to N. de Sousa and through the Scientific Employment Stimulus to N. Silva and S. Monteiro (CEECIND/04794/2017 and CEECIND/01902/2017) and Grant PID2020-115121GB-I00 funded by MCIN/AEI/10.13039/501100011033 to A. Barreiro-Iglesias SI

Numerical Analysis is a broad field, and coming to grips with all of it may seem like a daunting task. This text provides a thorough and comprehensive exposition of all the topics contained in a classical graduate sequence in numerical analysis. With an emphasis on theory and connections with linear algebra and analysis, the book shows all the rigor of numerical analysis. Its high level and exhaustive coverage will prepare students for research in the field and become a valuable reference as they continue their career. Students will appreciate the simple notation, clear assumptions and arguments, as well as the many examples and classroom-tested exercises ranging from simple verification to qualifying exam-level problems. In addition to the many examples with hand calculations, readers will also be able to translate theory into practical computational codes by running sample MATLAB codes as they try out new concepts.

Background: Our goal in the construction of the transman phalloplasty is not only to decrease the patient’s level of gender dysphoria, obviate the use of an external prosthesis, give the patient the ability to urinate in the standing position and orgasm but also to decrease urinary complications. Material and Methods: A retrospective review of transmen patients from June 2016 to June 2018 was performed on patients undergoing a two- stage mucosa only prelaminated neourethra radial forearm flap phalloplasty. The surgical technique is detailed in addition to patient demographics including co-morbidities, flap complications, and urinary sequelae.

The regional heterogeneity of microglia was first described a century ago by Pio del Rio Hortega. Currently, new information on microglia heterogeneity throughout central nervous system (CNS) regions is being revealed by high-throughput techniques. It remains unclear whether these spatial specificities translate into different microglial behaviors in vitro. We cultured microglia isolated from the cortex and spinal cord and analyzed the effect of the CNS spatial source on behavior in vitro by applying the same experimental protocol and culture conditions. We analyzed the microglial cell numbers, function, and morphology and found a distinctive in vitro phenotype. We found that microglia were present in higher numbers in the spinal-cord-derived glial cultures, presenting different expressions of inflammatory genes and a lower phagocytosis rate under basal conditions or after activation with LPS and IFN-γ. Morphologically, the cortical microglial cells were more complex and presented longer ramifications, which were also observed in vivo in CX3CR1+/GFP transgenic reporter mice. Collectively, our data demonstrated that microglial behavior in vitro is defined according to specific spatial characteristics acquired by the tissue. Thus, our study highlights the importance of microglia as a source of CNS for in vitro studies.

Endoplasmic reticulum (ER) stress is shown to promote nucleus pulposus (NP) cell apoptosis and intervertebral disc degeneration. However, little is known about ER stress regulation by the hypoxic disc microenvironment and its contribution to extracellular matrix homeostasis. NP cells were cultured under hypoxia (1% partial pressure of oxygen) to assess ER stress status, and gain-of-function and loss-of-function approaches were used to assess the role of hypoxia-inducible factor (HIF)-1α in this pathway. In addition, the contribution of ER stress induction on the NP cell secretome was assessed by a nontargeted quantitative proteomic analysis by sequential windowed data independent acquisition of the total high-resolution mass spectra–mass spectrometry. NP cells exhibited a lower ER stress burden under hypoxia. Knockdown of HIF-1α increased C/EBP homologous protein, protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) levels, whereas HIF-1α stabilization decreased the expression of ER stress markers Ddit3, Hsp5a, Atf6, and Eif2a. Interestingly, ER stress inducers tunicamycin and thapsigargin induced HIF-1α activity under hypoxia while promoting the unfolded protein response. NP cell secretome analysis demonstrated an impact of ER stress induction on extracellular matrix secretion, with decreases in collagens and cell adhesion–related proteins. Moreover, analysis of transcriptomic data of NP tissues from aged mice and degenerated human discs showed higher levels of unfolded protein response markers and decreased levels of matrix components. Our study shows, for the first time, that hypoxia and HIF-1α attenuate ER stress responses in NP cells, and ER stress promotes inefficient extracellular matrix secretion under hypoxia.

Spinal cord injury (SCI) leads to dramatic impairments of motor, sensory, and autonomic functions of affected individuals. Following the primary injury, there is an increased release of glutamate that leads to excitotoxicity and further neuronal death. Therefore, modulating glutamate excitotoxicity seems to be a promising target to promote neuroprotection during the acute phase of the injury. In this study, we evaluated the therapeutic effect of a FDA approved antiepileptic drug (levetiracetam-LEV), known for binding to the synaptic vesicle protein SV2A in the brain and spinal cord. LEV therapy was tested in two models of SCI—one affecting the cervical and other the thoracic level of the spinal cord. The treatment was effective on both SCI models. Treated animals presented significant improvements on gross and fine motor functions. The histological assessment revealed a significant decrease of cavity size, as well as higher neuronal and oligodendrocyte survival on treated animals. Molecular analysis revealed that LEV acts by stabilizing the astrocytes allowing an effective uptake of the excess glutamate from the extracellular space. Overall, our results demonstrate that Levetiracetam may be a promising drug for acute management of SCI.

Background: Hidradenitis suppurativa is a painful and disfiguring disease, which requires surgical management in severe cases. Improvements in management are necessary to decrease disease morbidity and improve outcomes. Aim: To determine the efficacy of applying amniotic tissue matrix to surgically-excised hidradenitis suppurativa wounds. Materials & Methods: A 5-year retrospective chart review of patients who underwent surgical treatment of perineal, perianal, and inguinal hidradenitis suppurativa at the University of Miami Hospital was completed. The study group consisted of 5 patients who had amniotic tissue matrix applied to their surgical wounds postoperatively. The control group, 18 patients, did not receive the amniotic tissue matrix application. Results: The average disease surface area in the study group was larger at 1278 ± 723 cm2 compared to 700 ± 555 cm2 for the control group. Duration of intensive care unit admission in the study group was shorter at 4.2 ± 4.3 days compared to 6.7 ± 8.8 days for the control group. Duration of inpatient admission was longer in the study group, 28.2 ± 12.4 days, compared to 24.6 ± 11.9 days for the control group. Postoperative hypergranulation tissue occurred more frequently in the control group (52.6%) than in the study group (20%). Conclusion: While further evaluation in a larger cohort of patients is necessary, application of amniotic tissue matrix to surgically-excised hidradenitis wounds appears to offer an adjunctive improvement to the treatment of hidradenitis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with short life expectancy and no effective therapy. We previously identified upregulated miR-124 in NSC-34-motor neurons (MNs) expressing human SOD1-G93A (mSOD1) and established its implication in mSOD1 MN degeneration and glial cell activation. When anti-miR-124-treated mSOD1 MN (preconditioned) secretome was incubated in spinal cord organotypic cultures from symptomatic mSOD1 mice, the dysregulated homeostatic balance was circumvented. To decipher the therapeutic potential of such preconditioned secretome, we intrathecally injected it in mSOD1 mice at the early stage of the disease (12-week-old). Preconditioned secretome prevented motor impairment and was effective in counteracting muscle atrophy, glial reactivity/dysfunction, and the neurodegeneration of the symptomatic mSOD1 mice. Deficits in corticospinal function and gait abnormalities were precluded, and the loss of gastrocnemius muscle fiber area was avoided. At the molecular level, the preconditioned secretome enhanced NeuN mRNA/protein expression levels and the PSD-95/TREM2/IL-10/arginase 1/MBP/PLP genes, thus avoiding the neuronal/glial cell dysregulation that characterizes ALS mice. It also prevented upregulated GFAP/Cx43/S100B/vimentin and inflammatory-associated miRNAs, specifically miR-146a/miR-155/miR-21, which are displayed by symptomatic animals. Collectively, our study highlights the intrathecal administration of the secretome from anti-miR-124-treated mSOD1 MNs as a therapeutic strategy for halting/delaying disease progression in an ALS mouse model., This research was funded by Santa Casa da Misericórdia de Lisboa: ELA-2015-002 (to DB); Fundação para a Ciência e a Tecnologia (FCT): PTDC/MED-NEU/31395/2017 (to D.B.), UIDB/UIDP/04138/2020, and UID/DTP/04138/2019-2020 (to iMed.ULisboa); Programa Operacional Regional de Lisboa and the Programa Operacional Competitividade e Internacionalização LISBOA-01-0145-FEDER-031395 (to D.B.); La Caixa Foundation and Francisco Luzón Foundation through project HR21-00931 (to D.B.); and an individual fellowship from FCT: SFRH/BD/129586/2017 (to M.B.). This work was also funded by the ICVS Scientific Microscopy Platform, a member of the national infrastructure of PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122).

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): MCIN/AEI/ 10.13039/501100011033 and “ERDF A way of making Europe” by the European Union Background Ca2+ ion is pivotal in excitation-contraction coupling. The dysregulation of Ca2+ fluxes is at the core of inherited and acquired arrhythmias. In recent years zebrafish have become an attractive vertebrate model to study heart development, cardiotoxicity, and heart diseases. We recently reported that the transgenic zebrafish line Tg(myl7:Twitch-4) expressing the ratiometric Ca2+ indicator Twitch-4 in the heart, allowed imaging Ca2+ transients (CaT) simultaneously with heart contraction in zebrafish larvae, thus maintaining the physiological mechano-electrical feedback. Purpose This study aimed to detect subtle disturbances in Ca2+ cycling, contractility, and hemodynamics induced by arrhythmogenic drugs in zebrafish larvae. Methods We have used the zebrafish transgenic line Tg(myl7:Twitch-4) and a custom software to acquire and analyze fluorescence emission ratio images of the heart. Larvae of 3- and 5-days post-fertilization (dpf) were incubated with arrhythmogenic drugs, and their effects on the systolic and diastolic Ca2+ and the CaT amplitude in the atrium and ventricle were determined. As the ventricular diameter was assessed in the same fluorescence images, a simultaneous, real-time, measurement of contraction correlated with Ca2+ was obtained. The systolic and diastolic areas, measured independently, were used to estimate hemodynamic parameters. Results Dofetilide caused time-dependent bradycardia and ultimately a 2:1 arrhythmia in 3 and 5 dpf larvae, with an increase in the CaT amplitude. the ejection fraction and the stroke volume increased, but the cardiac output decreased due to the low heart rate (2:1 arrhythmia). The T-type Ca2+ channel antagonist ML218 induced bradycardia and decreased systolic and diastolic Ca2+ levels in 3 dpf larvae, but the CaT amplitude did not change. Interestingly, some larvae (3 out of 14) displayed a progressive lengthening of the time between the start of atrial and ventricular CaT, analogous to increased PR interval of the electrocardiogram, until a ventricular systole failed. This is reminiscent of a second-degree atrioventricular block in humans. At higher ML218 concentration, a 2:1 arrhythmia was observed and increased ventricular CaT amplitude in all larvae. Conclusions We characterized Ca2+ changes induced by arrhythmogenic drugs and correlated them with contractility and hemodynamic parameters. The zebrafish line Tg(myl7:Twitch-4) allowed investigating drug-induced arrhythmias in this animal model with unprecedented detail. This transgenic line will serve to screen cardiotoxicity during drug development and to study conduction and rhythm defects in zebrafish, and their correlation with human disease.

The crosstalk between glial cells and neurons represents an exceptional feature for maintaining the normal function of the central nervous system (CNS). Increasing evidence has revealed the importance of glial progenitor cells in adult neurogenesis, reestablishment of cellular pools, neuroregeneration, and axonal (re)myelination. Several types of glial progenitors have been described, as well as their potentialities for recovering the CNS from certain traumas or pathologies. Among these precursors, glial-restricted precursor cells (GRPs) are considered the earliest glial progenitors and exhibit tripotency for both Type I/II astrocytes and oligodendrocytes. GRPs have been derived from embryos and embryonic stem cells in animal models and have maintained their capacity for self-renewal. Despite the relatively limited knowledge regarding the isolation, characterization, and function of these progenitors, GRPs are promising candidates for transplantation therapy and reestablishment/repair of CNS functions in neurodegenerative and neuropsychiatric disorders, as well as in traumatic injuries. Herein, we review the definition, isolation, characterization and potentialities of GRPs as cell-based therapies in different neurological conditions. We briefly discuss the implications of using GRPs in CNS regenerative medicine and their possible application in a clinical setting. MAIN POINTS: GRPs are progenitors present in the CNS with differentiation potential restricted to the glial lineage. These cells have been employed in the treatment of a myriad of neurodegenerative and traumatic pathologies, accompanied by promising results, herein reviewed.

Background Alterations in the immune system are a complication of spinal cord injury (SCI) and have been linked to an excessive sympathetic outflow to lymphoid organs. Still unknown is whether these peripheral immune changes also contribute for the deleterious inflammatory response mounted at the injured spinal cord. Methods We analyzed different molecular outputs of the splenic sympathetic signaling for the first 24 h after a thoracic compression SCI. We also analyzed the effect of ablating the splenic sympathetic signaling to the innate immune and inflammatory response at the spleen and spinal cord 24 h after injury. Results We found that norepinephrine (NE) levels were already raised at this time-point. Low doses of NE stimulation of splenocytes in vitro mainly affected the neutrophils’ population promoting an increase in both frequency and numbers. Interestingly, the interruption of the sympathetic communication to the spleen, by ablating the splenic nerve, resulted in reduced frequencies and numbers of neutrophils both at the spleen and spinal cord 1 day post-injury. Conclusion Collectively, our data demonstrates that the splenic sympathetic signaling is involved in the infiltration of neutrophils after spinal cord injury. Our findings give new mechanistic insights into the dysfunctional regulation of the inflammatory response mounted at the injured spinal cord.

Background Asthma is heterogeneous disease with different phenotypes, endotypes and severities. Definition of these subgroups requires the identification of biomarkers in biological samples, and serum proteomics is a useful and minimally invasive method for this purpose. Therefore, the aim of this study was to detect serum proteins whose abundance is distinctively associated with different asthma phenotypes (allergic vs nonallergic) or severities. Methods For each group of donors (32 healthy controls, 43 allergic rhinitis patients and 192 asthmatics with different phenotypes and severities), we generated two pools of sera that were analysed by a shotgun MS approach based on combinatorial peptide ligand libraries and iTRAQ-LC-MS/MS. Results MS analyses identified 18 proteins with a differential abundance. Functional/network study of these proteins identified key processes for asthma pathogenesis, such as complement activation, extracellular matrix organization, platelet activation and degranulation, or post-translational protein phosphorylation. Furthermore, our results highlighted an enrichment of the "Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs)" route in allergic asthma and the lectin pathway of complement activation in nonallergic asthma. Thus, several proteins (eg IGFALS, HSPG2, FCN2 or MASP1) displayed a differential abundance between the different groups of donors. Particularly, our results revealed IGFALS as a useful biomarker for moderate-severe allergic asthma. Conclusion Our data suggest a set of serum biomarkers, especially IGFALS, capable of differentiating allergic from nonallergic asthma. These proteins reveal different pathophysiological mechanisms and may be useful in the future for diagnosis, prognosis or targeted therapy purposes.

Presence of bacteria in wounds can delay healing. Addition of a regularly instilled topical solution over the wound during negative‐pressure wound therapy (NPWT) may reduce bioburden levels compared with standard NPWT alone. We performed a prospective, randomised, multi‐centre, post‐market trial to compare effects of NPWT with instillation and dwell of polyhexamethylene biguanide solution vs NPWT without instillation therapy in wounds requiring operative debridement. Results showed a significantly greater mean decrease in total bacterial counts from time of initial surgical debridement to first dressing change in NPWT plus instillation (n = 69) subjects compared with standard NPWT (n = 63) subjects (−0.18 vs 0.6 log10 CFU/g, respectively). There was no significant difference between the groups in the primary endpoint of required inpatient operating room debridements after initial debridement. Time to readiness for wound closure/coverage, proportion of wounds closed, and incidence of wound complications were similar. NPWT subjects had 3.1 times the risk of re‐hospitalisation compared with NPWT plus instillation subjects. This study provides a basis for exploring research options to understand the impact of NPWT with instillation on wound healing.

Animal models of human diseases are crucial experimental tools to investigate the mechanisms involved in disease pathogenesis and to develop new therapies. In spite of the numerous animal models currently available that reproduce several neuropathological features of Parkinson disease (PD), it is challenging to have one that consistently recapitulates human PD conditions in both motor behaviors and biochemical pathological outcomes. Given that, we have implemented a new paradigm to expose rats to a chronic low dose of paraquat (PQ), using osmotic minipumps and characterized the developed pathologic features over time. The PQ exposure paradigm used lead to a rodent model of PD depicting progressive nigrostriatal dopaminergic neurodegeneration, characterized by a 41% significant loss of dopaminergic neuron in the substantia nigra pars compacta (SNpc), a significant decrease of 18% and 40% of dopamine levels in striatum at week 5 and 8, respectively, and a significant 1.5-fold decrease in motor performance. We observed a significant increase of microglia activation state, sustained levels of α-synucleinopathy and increased oxidative stress markers in the SNpc. In summary, this is an explorative study that allowed to characterize an improved PQ-based rat model that recapitulates cardinal features of PD and may represent an attractive tool to investigate several mechanisms underlying the various aspects of PD pathogenesis as well as for the validation of the efficacy of new therapeutic approaches that targets different mechanisms involved in PD neurodegeneration.

In two dimensions, we propose and analyze an a posteriori error estimator for finite element approximations of the stationary Navier--Stokes equations with singular sources on Lipschitz, but not ne...

We study the regularity of the solution to an obstacle problem for a class of integro–differential operators. The differential part is a second order elliptic operator, whereas the nonlocal part is given by the integral fractional Laplacian. The obtained smoothness is then used to design and analyze a finite element scheme.

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The severity and long-term consequences of brain damage in traumatic brain injured (TBI) patients urgently calls for better neuroprotective/neuroreparative strategies for this devastating disorder. Mesenchymal stromal cells (MSCs) hold great promise and have been shown to confer neuroprotection in experimental TBI, mainly through paracrine mechanisms via secreted bioactive factors (i.e. secretome), which indicates significant potential for a cell-free neuroprotective approach. The secretome is composed of cytokines, chemokines, growth factors, proteins, lipids, nucleic acids, metabolites, and extracellular vesicles; it may offer advantages over MSCs in terms of delivery, safety, and variability of therapeutic response for brain injury. Immunomodulation by molecular factors secreted by MSCs is considered to be a key mechanism involved in their multi-potential therapeutic effects. Regulated neuroinflammation is required for healthy remodeling of central nervous system during development and adulthood. Moreover, immune cells and their secreted factors can also contribute to tissue repair and neurological recovery following acute brain injury. However, a chronic and maladaptive neuroinflammatory response can exacerbate TBI and contribute to progressive neurodegeneration and long-term neurological impairments. Here, we review the evidence for MSC-derived secretome as a therapy for TBI. Our framework incorporates a detailed analysis of in vitro and in vivo studies investigating the effects of the secretome on clinically relevant neurological and histopathological outcomes. We also describe the activation of immune cells after TBI and the immunomodulatory properties exerted by mediators released in the secretome. We then describe how ageing modifies central and systemic immune responses to TBI and discuss challenges and opportunities of developing secretome based neuroprotective therapies for elderly TBI populations. Finally, strategies aimed at modulating the secretome in order to boost its efficacy for TBI will also be discussed.

The space traffic management is becoming more and more complex with the growing number of maneuvering satellites in particular in low Earth Orbit (LEO) due to the race for large constellations of communication satellites. One of the major challenges for CNES [1] and the EU SST [2] in particular is the analysis of the collision risk when a conjunction that involves one of these satellites is scheduled. Preliminary analysis showed that about 20% of the close approach conjunctions with active satellites involve a secondary object with some maneuver capability, while this figure was rather below 10% a couple of years ago. This evolution is partially due to the deployment of more than 800 Starlink satellites over year 2020. In some cases, satellite operators can share their ephemerides and coordinate their actions to minimize the collision risk. However, in most cases, the behavior of the secondary object is unknown. The purpose of this work is to identify patterns of life of the secondary object to reduce the risk of collision. A conjunction is mainly characterized by a time of closest approach (TCA), a miss distance, a relative velocity, and uncertainty in position at TCA which allows to compute a probability of collision. Recent studies discussed the realism of uncertainties and proposed solutions to reassess the collision risk [3, 4, 5]. These works impact the decision making process that precedes a collision avoidance maneuver. However, if a maneuver is performed by the secondary satellite between the prediction date and the TCA, the risk assessment can turn out to be wrong. More importantly, some dangerous events can also be missed or detected just a few hours before TCA. In general, the whole process of orbit fitting and trajectory forecast becomes inaccurate when the secondary objects performs regular frequent station-keeping maneuvers. To tackle this issue, secondary objects with maneuver capability are treated separately from the passive objects. First, a list of such objects is maintained on a daily basis using satellite databases. Secondly, the two-line elements (TLE) data of these objects are analyzed to determine how often station keeping maneuvers are realized. This step is based on filtered differences on the orbital elements [6] and yields a first time interval where each maneuver occurred, as well as an estimate of the maneuver Delta-V. Then, an estimate of the thrust capability and thrust direction of the secondary satellite is determined using an iterative method based on forward and backward orbit propagation of the special perturbation (SP) orbital data from the US Air Force. The uncertainty on the object position comes mainly from the uncertainty related to the time and duration of the maneuvers, and the Gaussian assumption is not valid anymore. Hence, we define an exclusion volume at each date whose bounds are defined by the extreme maneuver scenarios that would be possible for the secondary satellite. In order to avoid a collision, the primary satellite should keep out of this exclusion volume, at any epoch. In this paper, we describe each of these methods in details and present simulation results for a limited set of primary objects in LEO. [1] M Moury and L Newman. Middle man concept for in-orbit collision risk mitigation: Caesar and cara examples. In SpaceOps 2014 Conference, page 1637, 2014. [2] P Faucher, R Peldszus, and A Gravier. Operational space surveillance and tracking in europe. Journal of Space Safety Engineering, 7(3):420425, 2020. [3] M D Hejduk and L C Johnson. Approaches to evaluating probability of collision uncertainty. NASA Technical Reports (Presentation), 2016. [4] S Laurens, P Seimandi, J Couetdic, and JC Dolado. Covariance matrix uncertainty analysis and correction. In 68th International Astronautical Congress, 2017. [5] A B Poore, J M Aristoff, Joshua T Horwood, R Armellin, William T Cerven, Y Cheng, C M Cox, R S Erwin, J H Frisbee, M D Hejduk, et al. Covariance and uncertainty realism in space surveillance and tracking. Technical report, Numerica Corporation Fort Collins United States, 2016. [6] T. Kelecy and K. Hamada. Satellite maneuver detection using tow-line element (tle) data. In AMOS, 2007.

We study diagonally implicit Runge-Kutta (DIRK) schemes when applied to abstract evolution problems that fit into the Gelfand-triple framework. We introduce novel stability notions that are well-suited to this setting and provide simple, necessary and sufficient, conditions to verify that a DIRK scheme is stable in our sense and in Bochner-type norms. We use several popular DIRK schemes in order to illustrate cases that satisfy the required structural stability properties and cases that do not. In addition, under some mild structural conditions on the problem we can guarantee compactness of families of discrete solutions with respect to time discretization.

Gender affirmation surgeries are performed to decrease the patient’s dysphoria and improve quality of life. Preservation of the vaginal canal with reproductive organs is uncommon though becoming increasingly discussed in trans men. This series examines surgical complexities of vaginal and/or reproductive organ preservation in patients undergoing phalloplasty, highlighting considerations for safety and well-being. Two patients who underwent phalloplasty met inclusion and exclusion criteria for the study and were treated in accordance with World Professional Association for Transgender Health standards. We retrospectively reviewed patients’ medical records and extracted demographic data. Phallus and clitoral sensation, ability for penetrative intercourse and treatment of gender incongruence were assessed postoperatively. At an average of 22 months postoperatively, both flaps survived with tactile sensation two-thirds down the shaft, and the clitoris of both maintained erogenous sensation from stimulation. Gender incongruence was described to be reduced, and both patients were able to receive penetrative vaginal intercourse.

The low regeneration potential of the central nervous system (CNS) represents a challenge for the development of new therapeutic strategies for neurodegenerative diseases, including spinocerebellar ataxias. Spinocerebellar ataxia type 3 (SCA3)—or Machado–Joseph disease (MJD)—is the most common dominant ataxia, being mainly characterized by motor deficits; however, SCA3/MJD has a complex and heterogeneous pathophysiology, involving many CNS brain regions, contributing to the lack of effective therapies. Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for CNS disorders. Beyond their differentiation potential, MSCs secrete a broad range of neuroregulatory factors that can promote relevant neuroprotective and immunomodulatory actions in different pathophysiological contexts. The objective of this work was to study the effects of (1) human MSC transplantation and (2) human MSC secretome (CM) administration on disease progression in vivo, using the CMVMJD135 mouse model of SCA3/MJD. Our results showed that a single CM administration was more beneficial than MSC transplantation—particularly in the cerebellum and basal ganglia—while no motor improvement was observed when these cell-based therapeutic approaches were applied in the spinal cord. However, the effects observed were mild and transient, suggesting that continuous or repeated administration would be needed, which should be further tested.

Parkinson’s disease (PD) is a prevalent movement disorder characterized by the progressive loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). The 6-hydroxydopamine (6-OHDA) lesion is still one of the most widely used techniques for modeling Parkinson’s disease (PD) in rodents. Despite commonly used in rats, it can be challenging to reproduce a similar lesion in mice. Moreover, there is a lack of characterization of the extent of behavioral deficits and of the neuronal loss/neurotransmitter system in unilateral lesion mouse models. In this study, we present an extensive behavioral and histological characterization of a unilateral intrastriatal 6-OHDA mouse model. Our results indicate significant alterations in balance and fine motor coordination, voluntary locomotion, and in the asymmetry’s degree of forelimb use in 6-OHDA lesioned animals, accompanied by a decrease in self-care and motivational behavior, common features of depressive-like symptomatology. These results were accompanied by a decrease in tyrosine hydroxylase (TH)-labelling and dopamine levels within the nigrostriatal pathway. Additionally, we also identify a marked astrocytic reaction, as well as proliferative and reactive microglia in lesioned areas. These results confirm the use of unilateral intrastriatal 6-OHDA mice for the generation of a mild model of nigrostriatal degeneration and further evidences the recapitulation of key aspects of PD, thereby being suitable for future studies beholding new therapeutical interventions for this disease., This work was supported by the Foundation for Science and Technology (FCT) through PhD Fellowships attributed to B.M.P. (SFRH/BD/120124/2016), E.L.C. (SFRH/BD/131278/2017), J.C. (SFRH/BD/5813/2020), S.B.A. (PD/BDE/135568/2018), D.M.F. (SFRH/BD/147947/2019), and CEEC attributed to C.S.C. (CEECIND/03887/2017), S.D.S. (CEECIND/00685/2020), and L.P. (CEECIND/ 02855/2020). ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI— Portuguese Platform of Bioimaging PPBI-POCI-01-0145-FEDER-022122; National funds, through the FCT—project UIDB/50026/2020 and UIDP/50026/2020; the projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); Competitiveness Internationalization Operational Programme (POCI), and by National funds, through the FCT, under the scope of the project POCI-01-0145-FEDER-032619.

Human mesenchymal stem cells (hMSCs) secretome has been have been at the forefront of a new wave of possible therapeutic strategies for central nervous system neurodegenerative disorders, as Parkinson's disease (PD). While within its protein fraction, several promising proteins were already identified with therapeutic properties on PD, the potential of hMSCs-secretome vesicular fraction remains to be elucidated. Such highlighting is important, since hMSCs secretome-derived vesicles can act as biological nanoparticles with beneficial effects in different pathological contexts. Therefore, in this work, we have isolated hMSCs secretome vesicular fraction, and assessed their impact on neuronal survival, and differentiation on human neural progenitors' cells (hNPCs), and in a 6-hydroxydopamine (6-OHDA) rat model of PD when compared to hMSCs secretome (as a whole) and its protein derived fraction. From the results, we have found hMSCs vesicular fraction as polydispersity source of vesicles, which when applied in vitro was able to induce hNPCs differentiation at the same levels as the whole secretome, while the protein separated fraction was not able to induce such effect. In the context of PD, although distinct effects were observed, hMSCs secretome and its derived fractions displayed a positive impact on animals' motor and histological performance, thereby indicating that hMSCs secretome and its different fractions may impact different mechanisms and pathways. Overall, we concluded that the use of the secretome collected from hMSCs and its different fractions might be active modulators of different neuroregeneration mechanisms, which could open new therapeutical opportunities for their future use as a treatment for PD., Prémios Santa Casa Neurociências Prize Mantero Belard for Neurodegenerative Diseases Research (MB-28-2019). This work was supported by the European Regional Development Fund (FEDER), through the Competitiveness Internationalization Operational Programme (POCI), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of projects UIDB/50026/2020; UIDP/50026/2020 and POCI-01-0145-FEDER-029751. This article has also been developed under the scope of the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This work has been funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI–POCI-01-0145-FEDER-022122), info:eu-repo/semantics/publishedVersion