Production and Characterization of Graphene Oxide Surfaces against Uropathogens

Graphene and its functionalized derivatives have been increasingly applied in the biomedi-cal field, particularly in the production of antimicrobial and anti-adhesive surfaces. This study aimed to evaluate the performance of graphene oxide (GO)/polydimethylsiloxane (PDMS) composites against Staphylococcus aureus and Pseudomonas aeruginosa biofilms. GO/PDMS composites containing different GO loadings (1, 3, and 5 wt.%) were synthesized and characterized regarding their morphol-ogy, roughness, and hydrophobicity, and tested for their ability to inhibit biofilm formation under conditions that mimic urinary tract environments. Biofilm formation was assessed by determining the number of total and culturable cells. Additionally, the antibacterial mechanisms of action of GO were investigated for the tested uropathogens. Results indicated that the surfaces containing GO had greater roughness and increased hydrophobicity than PDMS. Biofilm analysis showed that the 1 wt.% GO/PDMS composite was the most effective in reducing S. aureus biofilm formation. In oppo-sition, P. aeruginosa biofilms were not inhibited by any of the synthesized composites. Furthermore, 1% (w/v) GO increased the membrane permeability, metabolic activity, and endogenous reactive oxygen species (ROS) synthesis in S. aureus. Altogether, these results suggest that GO/PDMS com-posites are promising materials for application in urinary catheters, although further investigation is required

Microglial Sirtuin 2 shapes long-term potentiation in hippocampal slices

Copyright © 2020 Sa de Almeida, Vargas, Fonseca-Gomes, Tanqueiro, Belo, Miranda-Lourenço, Sebastião, Diógenes and Pais. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Microglial cells have emerged as crucial players in synaptic plasticity during development and adulthood, and also in neurodegenerative and neuroinflammatory conditions. Here we found that decreased levels of Sirtuin 2 (Sirt2) deacetylase in microglia affects hippocampal synaptic plasticity under inflammatory conditions. The results show that long-term potentiation (LTP) magnitude recorded from hippocampal slices of wild type mice does not differ between those exposed to lipopolysaccharide (LPS), a pro-inflammatory stimulus, or BSA. However, LTP recorded from hippocampal slices of microglial-specific Sirt2 deficient (Sirt2-) mice was significantly impaired by LPS. Importantly, LTP values were restored by memantine, an antagonist of N-methyl-D-aspartate (NMDA) receptors. These results indicate that microglial Sirt2 prevents NMDA-mediated excitotoxicity in hippocampal slices in response to an inflammatory signal such as LPS. Overall, our data suggest a key-protective role for microglial Sirt2 in mnesic deficits associated with neuroinflammation.This study was supported by Santa Casa da Misericórdia de Lisboa (MB37-2017), GAPIC Research Program of the University of Lisbon Medical School (n° 2014002 and n° 2015028) and the following doctoral grants: PD/BD/128091/2016, SFRH/BD/118238/2016, PD/BD/114337/2016, and PD/BD/1144- 41/2016.info:eu-repo/semantics/publishedVersio

The Neuroprotective Action of Amidated-Kyotorphin on Amyloid β Peptide-Induced Alzheimer’s Disease Pathophysiology

Kyotorphin (KTP, l-tyrosyl-l-arginine) is an endogenous dipeptide initially described to have analgesic properties. Recently, KTP was suggested to be an endogenous neuroprotective agent, namely for Alzheimer’s disease (AD). In fact, KTP levels were shown to be decreased in the cerebrospinal fluid of patients with AD, and recent data showed that intracerebroventricular (i.c.v.) injection of KTP ameliorates memory impairments in a sporadic rat model of AD. However, this administration route is far from being a suitable therapeutic strategy. Here, we evaluated if the blood-brain permeant KTP-derivative, KTP-NH2, when systemically administered, would be effective in preventing memory deficits in a sporadic AD animal model and if so, which would be the synaptic correlates of that action. The sporadic AD model was induced in male Wistar rats through i.c.v. injection of amyloid β peptide (Aβ). Animals were treated for 20 days with KTP-NH2 (32.3 mg/kg, intraperitoneally (i.p.), starting at day 3 after Aβ administration) before memory testing (Novel object recognition (NOR) and Y-maze (YM) tests). Animals were then sacrificed, and markers for gliosis were assessed by immunohistochemistry and Western blot analysis. Synaptic correlates were assessed by evaluating theta-burst induced long term potentiation (LTP) of field excitatory synaptic potentials (fEPSPs) recorded from hippocampal slices and cortical spine density analysis. In the absence of KTP-NH2 treatment, Aβ-injected rats had clear memory deficits, as assessed through NOR or YM tests. Importantly, these memory deficits were absent in Aβ-injected rats that had been treated with KTP-NH2, which scored in memory tests as control (sham i.c.v. injected) rats. No signs of gliosis could be detected at the end of the treatment in any group of animals. LTP magnitude was significantly impaired in hippocampal slices that had been incubated with Aβ oligomers (200 nM) in the absence of KTP-NH2. Co-incubation with KTP-NH2 (50 nM) rescued LTP toward control values. Similarly, Aβ caused a significant decrease in spine density in cortical neuronal cultures, and this was prevented by co-incubation with KTP-NH2 (50 nM). In conclusion, the present data demonstrate that i.p. KTP-NH2 treatment counteracts Aβ-induced memory impairments in an AD sporadic model, possibly through the rescuing of synaptic plasticity mechanisms.publishersversionpublishe

A small TAT-TrkB peptide prevents BDNF receptor cleavage and restores synaptic physiology in Alzheimer's disease

Publisher Copyright: Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.In Alzheimer's disease (AD), amyloid β (Aβ)-triggered cleavage of TrkB-FL impairs brain-derived neurotrophic factor (BDNF) signaling, thereby compromising neuronal survival, differentiation, and synaptic transmission and plasticity. Using cerebrospinal fluid and postmortem human brain samples, we show that TrkB-FL cleavage occurs from the early stages of the disease and increases as a function of pathology severity. To explore the therapeutic potential of this disease mechanism, we designed small TAT-fused peptides and screened their ability to prevent TrkB-FL receptor cleavage. Among these, a TAT-TrkB peptide with a lysine-lysine linker prevented TrkB-FL cleavage both in vitro and in vivo and rescued synaptic deficits induced by oligomeric Aβ in hippocampal slices. Furthermore, this TAT-TrkB peptide improved the cognitive performance, ameliorated synaptic plasticity deficits and prevented Tau pathology progression in vivo in the 5XFAD mouse model of AD. No evidence of liver or kidney toxicity was found. We provide proof-of-concept evidence for the efficacy and safety of this therapeutic strategy and anticipate that this TAT-TrkB peptide has the potential to be a disease-modifying drug that can prevent and/or reverse cognitive deficits in patients with AD.publishersversionpublishe

Oocyte and ovarian tissue cryopreservation in European countries : statutory background, practice, storage and use

STUDY QUESTION: What is known in Europe about the practice of oocyte cryopreservation (OoC), in terms of current statutory background, funding conditions, indications (medical and ‘non-medical’) and specific number of cycles? SUMMARY ANSWER: Laws and conditions for OoC vary in Europe, with just over half the responding countries providing this for medical reasons with state funding, and none providing funding for ‘non-medical’ OoC. WHAT IS ALREADY KNOWN: The practice of OoC is a well-established and increasing practice in some European countries, but data gathering on storage is not homogeneous, and still sparse for use. Ovarian tissue cryopreservation (OtC) is only practiced and registered in a few countries. STUDY DESIGN, SIZE, AND DURATION: A transversal collaborative survey on OoC and OtC, was designed, based on a country questionnaire containing information on statutory or professional background and practice, as well as available data on ovarian cell and tissue collection, storage and use. It was performed between January and September 2015. PARTICIPANTS/MATERIALS, SETTING AND METHODS: All ESHRE European IVF Monitoring (EIM) consortium national coordinators were contacted, as well as members of the ESHRE committee of national representatives, and sent a questionnaire. The form included national policy and practice details, whether through current existing law or code of practice, criteria for freezing (age, health status), availability of funding and the presence of a specific register. The questionnaire also included data on both the number of OoC cycles and cryopreserved oocytes per year between 2010 and 2014, specifically for egg donation, fertility preservation for medical disease, ‘other medical’ reasons as part of an ART cycle, as well as for ‘non-medical reasons’ or age-related fertility decline. Another question concerning data on freezing and use of ovarian tissue over 5 years was added and sent after receiving the initial questionnaire. MAIN RESULTS AND THE ROLE OF CHANCE: Out of 34 EIM members, we received answers regarding OoC regulations and funding conditions from 27, whilst 17 countries had recorded data for OoC, and 12 for OtC. The specific statutory framework for OoC and OtC varies from absent to a strict frame. A total of 34 705 OoC cycles were reported during the 5-year-period, with a continuous increase. However, the accurate description of numbers was concentrated on the year 2013 because it was the most complete. In 2013, a total of 9126 aspirations involving OoC were reported from 16 countries. Among the 8885 oocyte aspirations with fully available data, the majority or 5323 cycles (59.9%) was performed for egg donation, resulting in the highest yield per cycle, with an average of 10.4 oocytes frozen per cycle. OoC indication was ‘serious disease’ such as cancer in 10.9% of cycles, other medical indications as ‘part of an ART cycle’ in 16.1%, and a non-medical reason in 13.1%. With regard to the use of OoC, the number of specifically recorded frozen oocyte replacement (FOR) cycles performed in 2013 for all medical reasons was 14 times higher than the FOR for non-medical reasons, using, respectively, 8.0 and 8.4 oocytes per cycle. Finally, 12 countries recorded storage following OtC and only 7 recorded the number of grafted frozen/thawed tissues. LIMITATIONS, REASONS FOR CAUTION: Not all countries have data regarding OoC collection, and some data came from voluntary collaborating centres, rather than a national authority or register. Furthermore, the data related to use of OoC were not included for two major players in the field, Italy and Spain, where numbers were conflated for medical and non-medical reasons. Finally, the number of cycles started with no retrieval is not available. Data are even sparser for OtC. WIDER IMPLICATIONS OF THE FINDINGS: There is a need for ART authorities and professional bodies to record precise data for practice and use of OoC (and OtC), according to indications and usage, in order to reliably inform all stakeholders including women about the efficiency of both methods. Furthermore, professional societies should establish professional standards for access to and use of OoC and OtC, and give appropriate guidance to all involved. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by ESHRE. There are no conflicts of interest.peer-reviewe

Kyotorphin as a new pharmacological therapeutic strategy for Alzheimer’s Disease

Kyotorphin (KTP, L-tyrosyl-L-arginine) is an endogenous dipeptide, described for the first time in 1979, as a potent analgesic molecule. Its naloxone-reversible opioid-like analgesic effect is indirectly mediated by the inducing the release of Met-enkephalin (Met-enk). It is currently accepted that KTP acts through a specific Gi-coupled receptor (KTPr), inducing Ca2+ influx in a phospholipase C-mediated process. Moreover, this KTPr-mediated action can be antagonized by the dipeptide L-Leucine-L-Arginine (KTPant). Over the last decades, several studies have been revealing KTP role in the modulation of several mechanisms in the central and peripheric nervous systems. KTP has been described as having an antiepileptic, thermoregulatory, anti-hibernation, behavioral and stress modulatory actions, being these non-opioid-mediated effects. However, the vast majority of KTP research has been explored its potential application in pain treatment. More recently, the link between pain and Alzheimer’s Disease (AD), two conditions with high epidemiologic relevance, supported the use of KTP-related drugs as new therapeutic strategies for AD. Evidence shows that chronic pain aggravates AD, and the limited capacity of AD patients to verbally express and perceive pain can worsen disease progression. AD has proven to be a highly complex neurodegenerative disease, of which brain amyloid plaques, mainly constituted by amyloid beta (Aβ) peptide, and neurofibrillary tangles formed by hyperphosphorylated tau (p-Tau) protein, are the two histopathological hallmarks of this disease. Additionally, excitotoxicity, as well as dysregulation of brain-derived neurotrophic factor (BDNF) signaling, are known to be involved in neurodegenerative disorders such as AD. Similarly to what was observed in patients with persistent pain, AD patients have decreased cerebrospinal fluid (CSF) KTP levels, which are inversely correlated to the increase of p-Tau levels in CSF of those patients. Recently, KTP was suggested to be an endogenous neuroprotective agent. In particular, when intracerebroventricular (i.c.v.) injected, KTP ameliorated memory impairments in a rat model of sporadic AD. However, KTP has a limited capacity to cross the blood-brain barrier (BBB). The potential therapeutic value of KTP as a central nervous system (CNS) drug led to the development of synthetic KTP derivatives, which might cross the BBB. Accordingly, the KTP amidated-derivative, the Amidated-Kyotorphin (KTP-NH2), was designed and produced to overcome the BBB. In this work, the main goal was to explore the therapeutic use of KTP-NH2 as a new drug for AD treatment. It started with the characterization and comparison of the impact of KTP and KTP-NH2 in synaptic function under physiological mimetic conditions. Then, given the particular interest of this work in AD treatment, the neuroprotective potential of both peptides was evaluated upon Aβ-induced AD (Chapter 3). Finally, this work correlated the synaptic mechanisms protected by KTP-NH2 action against Aβ-induced toxicity, and the ameliorated memory impairments observed after the systemic administration of KTP-NH2 in a model of sporadic AD in rat (Chapter 4). Electrophysiological recordings obtained in the CA1 area of hippocampal slices prepared from adult male C57BL/6J mice, pre-exposed or superfused with KTP and KTP-NH2, allowed the characterization of the effects of both peptides on synaptic function under non pathological conditions. Results revealed that for concentrations ranging from 5 nM to 50 µM, the peptides affected the basal synaptic transmission in a concentration-dependent manner. While KTP slightly increased synaptic transmission, with a maximal effect measured at 50 nM, KTP-NH2 had a gradual inhibitory effect. At concentrations of 5 mM, largely in excess of what is likely to occur endogenously, peptides’ action rapidly inhibited synaptic transmission, being these effects reversible. In fact, this inhibitory effect was totally or partially eliminated with the respective removal of KTP or KTP-NH2. Thus, under nonpathological conditions, these findings suggested a different effect on synaptic mechanisms. However, neither KTP (50 nM), nor KTP-NH2 (50 nM), significantly affected: 1) synaptic transmission efficiency, evaluated by input/output curves; 2) short-term plasticity, evaluated by post-tetanic potentiation and paired-pulse facilitation; or 3) glutamate release. Together, these findings suggested that the effects of both peptides on synaptic transmission were likely not directly mediated by pre- or post-synaptic mechanisms. Synaptic plasticity is a key mechanism in memory and learning processes. Long-term potentiation (LTP) was evaluated in hippocampal slices, which were either pre-exposed for 3h or acutely superfused with KTP (50 nM) or KTP-NH2 (50 nM). For testing mimetic AD pathophysiological conditions, hippocampal slices were pre-treated with oligomeric Aβ peptide species (200 nM), one of the most soluble toxic Aβ species. Additionally, dendritic spines were evaluated in cultured cortical neurons, after the treatment with KTP (50 nM) or KTP-NH2 (50 nM), and in the presence or absence of Aβ peptide (25 µM) for 24h. Results demonstrated that KTP-NH2, but not KTP, had a neuroprotective effect against Aβ-induced impairments on LTP magnitude. However, these differences contrasted with the similar molecular neuroprotective effect. The action of both peptides restored the density of dendritic spines affected by the action of the Aβ peptide, without inducing toxic effects on neurons. To evaluate whether the neuroprotective effect of KTP-NH2 (50 nM) over LTP could be antagonized by KTPant, slices were pre-treated for 30 min with KTPant (250 nM). The results revealed that KTPant antagonized the neuroprotective effect of KTP-NH2 over Aβ peptide-induced impairments in LTP. However, it is still unclear if KTPant, alone, had a preventive neuroprotective action against Aβ peptide, or which affected mechanism might prompt toxicity when KTPant was added prior to KTP-NH2. Elevation of intracellular Ca2+ levels are a hallmark of KTP-mediated processes. Thus, calcium imaging technique was used to understand whether the increase in Ca2+ levels could be directly caused by KTP activity and/or whether such increases would be necessary for the emergence of KTP-mediated actions. The results revealed that neither the presence of KTP (50 nM) nor KTP-NH2 (50 nM) affected Ca2+ intracellular levels in neurons, so there were no significant changes in neuronal calcium homeostasis. Calpain overactivation happens due to increased intracellular Ca2+ levels, usually as a consequence of Aβ peptide-induced excitotoxicity. As such, the action of both peptides (50 nM) over calpain in vitro activity was assessed using mice cortical tissue homogenates. Results revealed that neither KTP nor KTP-NH2 impacted calpain activation and, consequently, did not prevent calpain-induced cleavage of the BDNF receptor, the tropomyosin receptor kinase B-full length (TrkB-FL), which has a well-known neuroprotective role, and it is a substrate for calpains under Aβ-induced toxicity. Finally, in a rat model of sporadic AD, systemic administration of KTP-NH2 protected against spatial working-memory and episodic memory deficits, without affecting motor activity or inducing an anxiety-like behavior in the animals. Moreover, this KTP-NH2-induced neuroprotective effect was correlated with the prevention of Aβ-induced deficits, in both LTP magnitude of pre-treated hippocampal slices and spine density of cortical neuronal cultures. In conclusion, the present work collected novel evidence of KTP and KTP-NH2 neuromodulatory effects over synaptic function, highlighting the neuroprotective actions of KTP-NH2. Under physiological mimetic conditions, the absence of effects over synaptic function bolsters the therapeutic potential of both peptides, suggesting the absence of sideeffects upon synaptic transmission. At molecular and functional levels, these findings supported the KTP-NH2 neuroprotective effect (Chapter 3), confirming the results obtained through its systemic administration in a rat model of sporadic AD (Chapter 4), which provides important evidence for the use of KTP-NH2 as a drug for treatment of AD and, eventually, other diseases. In light of the complex pathophysiology of AD, in the future, it will be important to determine whether KTP-NH2 neuroprotective effect is potentiated as a standalone treatment, or if its actions should be included as a broader multidrug treatment regimen. Among other aspects, in the treatment of AD, identifying the time-window for therapeutic intervention with KTP-NH2 will be essential to ascertain whether its action relies on the prevention or if this drug is able to recover the molecular and cognitive deficits present in AD pathophysiology.Marie Sklodowska-Curie Mobility Grant (INPACT-H2020-RISE, 2014)POCI-01-0145-FEDER-022184, supported by COMPETE 202

High-caloric diet Induces memory impairment and disrupts synaptic plasticity in aged rats

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).The increasing consumption of sugar and fat seen over the last decades and the consequent overweight and obesity, were recently linked with a deleterious effect on cognition and synaptic function. A major question, which remains to be clarified, is whether obesity in the elderly is an additional risk factor for cognitive impairment. We aimed at unravelling the impact of a chronic high caloric diet (HCD) on memory performance and synaptic plasticity in aged rats. Male rats were kept on an HCD or a standard diet (control) from 1 to 24 months of age. The results showed that under an HCD, aged rats were obese and displayed significant long-term recognition memory impairment when compared to age-matched controls. Ex vivo synaptic plasticity recorded from hippocampal slices from HCD-fed aged rats revealed a reduction in the magnitude of long-term potentiation, accompanied by a decrease in the levels of the brain-derived neurotrophic factor receptors TrkB full-length (TrkB-FL). No alterations in neurogenesis were observed, as quantified by the density of immature doublecortin-positive neurons in the hippocampal dentate gyrus. This study highlights that obesity induced by a chronic HCD exacerbates age-associated cognitive decline, likely due to impaired synaptic plasticity, which might be associated with deficits in TrkB-FL signaling.This research was funded by Santa Casa da Misericórdia de Lisboa—MB37-2017; Fundação para a Ciência e a Tecnologia (FCT)—IF/01227/2015; and it received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952455. Researchers were supported by the following: S.L.P.—FCT (PD/BD/150341/2019); C.M.-L.—FCT (SFRH/BD/118238/2016) and Universidade de Lisboa (BD2015); R.F.B.—FCT (PD/BD/114337/2016); R.S.R.—FCT (SFRH/BD/129710/2017); S.R.T.—FCT (SFRH/BD/128091/2016).info:eu-repo/semantics/publishedVersio