Metrology supporting the European regulation for radiation protection.

The European Association of National Metrology Institutes (EURAMET) within its research programme European Metrology Programme for Innovation and Research (EMPIR) funded project EMPIR 19NET03 supportBSS that contributes to the establishment of a European Metrology Network (EMN) for Radiation Protection (RP). The EMN-RP was established in September 2021 with the intent to work as a meeting point for the metrology community and all stakeholders in the field of ionising radiation regulation, thus providing quality assurance for measurements in each of the exposure situations contemplated in the European Legislation. Within project EMPIR 19NET03, work package 3 aims at the preparation of a Strategic Research Agenda (SRA) by identifying the metrology needs to support the European legislation and regulation in Radiation Protection and of two Roadmaps for metrology services, one under the European Council Directive 2013/59/EURATOM and the other under the EURATOM Treaty. Following a Gaps Workshop held in September 2020 and a second internal workshop that took place in April 2022, a questionnaire was prepared for distribution to the stakeholders, e.g. RP platforms and authorities, academia, industry, among other, together with an accompanying paper. In this paper, the authors present the state of the art of European legislation in RP, address the importance of metrology, the practices and activities that need metrology to meet the requirements set in the regulations, emphasise the need for quality assured measurements in all fields, highlight the stakeholders contributions in their specific area and show their vision of the EMN-RP.

Demographic and Clinical Characteristics of Mpox Patients Attending an STD Clinic in Lisbon.

Mpox is a viral disease caused by the monkeypox virus, which marked the year of 2022 with a global outbreak. While previously considered to be a zoonosis of almost exclusive animal-to-human transmission, the current outbreak has been attributed to human-to-human transmission, particularly sexual transmission. As a new sexually transmissible disease, we studied the epidemiological and clinical features, as well as the concomitant occurrence of other sexually transmissible diseases, treatment approach, and outcome of our 291 patients, in the current outbreak. We found a total of 169 concomitant sexually transmissible infections of bacterial and viral origins, corresponding to 107 patients. Neisseria gonorrhoeae was the most common agent, particularly in the anal location. With this work, we emphasize the need for a thorough epidemiological and medical history, as well as a concomitant complete laboratorial screening for other STIs in patients with confirmed or suspected mpox.

Viral genetic clustering and transmission dynamics of the 2022 mpox outbreak in Portugal.

Pathogen genome sequencing during epidemics enhances our ability to identify and understand suspected clusters and investigate their relationships. Here, we combine genomic and epidemiological data of the 2022 mpox outbreak to better understand early viral spread, diversification and transmission dynamics. By sequencing 52% of the confirmed cases in Portugal, we identified the mpox virus sublineages with the highest impact on case numbers and fitted them into a global context, finding evidence that several international sublineages probably emerged or spread early in Portugal. We estimated a 62% infection reporting rate and that 1.3% of the population of men who have sex with men in Portugal were infected. We infer the critical role played by sexual networks and superspreader gatherings, such as sauna attendance, in the dissemination of mpox virus. Overall, our findings highlight genomic epidemiology as a tool for the real-time monitoring and control of mpox epidemics, and can guide future vaccine policy in a highly susceptible population.

Monkeypox Infection With Primarily Mucosal Involvement.

ABSTRACT: Since May 2022, several nonendemic countries face a monkeypox outbreak, with clinic and epidemiological characteristics distinct from the classic ones. Primarily affecting the sexually active population, these cases present with mucocutaneous lesions mainly localized in perioral, genital, and anal areas. A retrospective study was conducted in a tertiary center in Lisbon, to characterize the population diagnosed with monkeypox infection with primarily mucosal involvement.

Targeting of Cellular Organelles by Fluorescent Plasmid DNA Nanoparticles.

The development of a suitable delivery system and the targeting of intracellular organelles are both essential for the success of drug and gene therapies. The conception of fluorescent ligands, displaying targeting specificity together with low toxicity, is an emerging and reliable tool to develop innovative delivery systems. Biocompatible BSA or pDNA/ligand nanoparticles were synthesized by a coprecipitation method and were shown to display adequate sizes and morphology for delivery purposes, and positive surface charges. Additionally, these fluorescent vectors can target specific intracellular organelles. In vitro transfection mediated by BSA or pDNA based carriers can result in the accumulation of BSA in the cytosol, lysosomes, and mitochondria or the expression of the plasmid-encoded protein, respectively. Moreover, the therapeutic effect of pDNA/ligand vectors in cancer gene therapy instigates further research aiming clinical translation.

Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons.

This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of cells, while offering minimal cell death that often occurs during dissociation of differentiated neurons. Cells plated under the appropriate conditions, either in Petri-dishes or in multi-well plates, will develop and establish synaptic contacts over time since the neuronal culture medium provides the nutrients and trophic factors required for differentiation. In this protocol we describe the methodology for the preparation of both cortical and hippocampal neuronal cultures.

The Role of Proteases in Hippocampal Synaptic Plasticity: Putting Together Small Pieces of a Complex Puzzle.

Long-term synaptic plasticity in the hippocampus is thought to underlie the formation of certain forms of memory, including spatial memory. The early phase of long-term synaptic potentiation and synaptic depression depends on post-translational modifications of synaptic proteins, while protein synthesis is also required for the late-phase of both forms of synaptic plasticity (L-LTP and L-LTD). Numerous pieces of evidence show a role for different types of proteases in synaptic plasticity, further increasing the diversity of mechanisms involved in the regulation of the intracellular and extracellular protein content. The cleavage of extracellular proteins is coupled to changes in postsynaptic intracellular mechanisms, and additional alterations in this compartment result from the protease-mediated targeting of intracellular proteins. Both mechanisms contribute to initiate signaling cascades that drive downstream pathways coupled to synaptic plasticity. In this review we summarize the evidence pointing to a role for extracellular and intracellular proteases, with distinct specificities, in synaptic plasticity. Where in the cells the proteases are located, and how they are regulated is also discussed. The combined actions of proteases and translation mechanisms contribute to a tight control of the synaptic proteome relevant for long-term synaptic potentiation and synaptic depression in the hippocampus. Additional studies are required to elucidate the mechanisms whereby these changes in the synaptic proteome are related with plasticity phenomena.

Role of the ubiquitin-proteasome system in brain ischemia: friend or foe?

The ubiquitin-proteasome system (UPS) is a catalytic machinery that targets numerous cellular proteins for degradation, thus being essential to control a wide range of basic cellular processes and cell survival. Degradation of intracellular proteins via the UPS is a tightly regulated process initiated by tagging a target protein with a specific ubiquitin chain. Neurons are particularly vulnerable to any change in protein composition, and therefore the UPS is a key regulator of neuronal physiology. Alterations in UPS activity may induce pathological responses, ultimately leading to neuronal cell death. Brain ischemia triggers a complex series of biochemical and molecular mechanisms, such as an inflammatory response, an exacerbated production of misfolded and oxidized proteins, due to oxidative stress, and the breakdown of cellular integrity mainly mediated by excitotoxic glutamatergic signaling. Brain ischemia also damages protein degradation pathways which, together with the overproduction of damaged proteins and consequent upregulation of ubiquitin-conjugated proteins, contribute to the accumulation of ubiquitin-containing proteinaceous deposits. Despite recent advances, the factors leading to deposition of such aggregates after cerebral ischemic injury remain poorly understood. This review discusses the current knowledge on the role of the UPS in brain function and the molecular mechanisms contributing to UPS dysfunction in brain ischemia with consequent accumulation of ubiquitin-containing proteins. Chemical inhibitors of the proteasome and small molecule inhibitors of deubiquitinating enzymes, which promote the degradation of proteins by the proteasome, were both shown to provide neuroprotection in brain ischemia, and this apparent contradiction is also discussed in this review.

Excitotoxic stimulation downregulates the ubiquitin-proteasome system through activation of NMDA receptors in cultured hippocampal neurons.

Overactivation of glutamate receptors contributes to neuronal damage (excitotoxicity) in ischemic stroke but the detailed mechanisms are not fully elucidated. Brain ischemia is also characterized by an impairment of the activity of the proteasome, one of the major proteolytic systems in neurons. We found that excitotoxic stimulation with glutamate rapidly decreases ATP levels and the proteasome activity, and induces the disassembly of the 26S proteasome in cultured rat hippocampal neurons. Downregulation of the proteasome activity, leading to an accumulation of ubiquitinated proteins, was mediated by calcium entry through NMDA receptors and was only observed in the nuclear fraction. Furthermore, excitotoxicity-induced proteasome inhibition was partially sensitive to cathepsin-L inhibition and was specifically induced by activation of extrasynaptic NMDA receptors. Oxygen and glucose deprivation induced neuronal death and downregulated the activity of the proteasome by a mechanism dependent on the activation of NMDA receptors. Since deubiquitinating enzymes may regulate proteins half-life by counteracting ubiquitination, we also analyzed how their activity is regulated under excitotoxic conditions. Glutamate stimulation decreased the total deubiquitinase activity in hippocampal neurons, but was without effect on the activity of Uch-L1, showing that not all deubiquitinases are affected. These results indicate that excitotoxic stimulation with glutamate has multiple effects on the ubiquitin-proteasome system which may contribute to the demise process in brain ischemia and in other neurological disorders.

Role of the proteasome in excitotoxicity-induced cleavage of glutamic acid decarboxylase in cultured hippocampal neurons.

Glutamic acid decarboxylase is responsible for synthesizing GABA, the major inhibitory neurotransmitter, and exists in two isoforms--GAD65 and GAD67. The enzyme is cleaved under excitotoxic conditions, but the mechanisms involved and the functional consequences are not fully elucidated. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent cleavage of GAD65 and GAD67 in the N-terminal region of the proteins, and decrease the corresponding mRNAs. The cleavage of GAD67 was sensitive to the proteasome inhibitors MG132, YU102 and lactacystin, and was also abrogated by the E1 ubiquitin ligase inhibitor UBEI-41. In contrast, MG132 and UBEI-41 were the only inhibitors tested that showed an effect on GAD65 cleavage. Excitotoxic stimulation with glutamate also increased the amount of GAD captured in experiments where ubiquitinated proteins and their binding partners were isolated. However, no evidences were found for direct GADs ubiquitination in cultured hippocampal neurons, and recombinant GAD65 was not cleaved by purified 20S or 26S proteasome preparations. Since calpains, a group of calcium activated proteases, play a key role in GAD65/67 cleavage under excitotoxic conditions the results suggest that GADs are cleaved after ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons was significantly reduced after excitotoxic injury, and the total GAD activity measured in extracts from the cerebellum or cerebral cortex at 24h postmortem (when there is a partial cleavage of GADs) was also decreased. The results show a role of the UPS in the cleavage of GAD65/67 and point out the deregulation of GADs under excitotoxic conditions, which is likely to affect GABAergic neurotransmission. This is the first time that the UPS has been implicated in the events triggered during excitotoxicity and the first molecular target of the UPS affected in this cell death process.

Comparative study of Curiementor ionization chambers using Monte Carlo simulations.

Ionization chambers (ICs) are the most commonly used instrument for measuring activities of solutions containing radiopharmaceuticals used in nuclear medicine, due to their simplicity of operation and accurate measurement results. As the dose delivered to the patient is determined by these measurements, for optimal results, the activity of these radiopharmaceuticals must be determined as accurately as possible, so that the radioprotection requirements are accomplished and at the same time good therapy or imaging results are achieved. In this work, the response of the ionization chamber Curiementor 2, from PTW-Freiburg, was studied by simulation with Monte Carlo (MC) code MCNPX and compared with experimental results for several radionuclides. The good agreement between experimental and simulation results validates the used methodology. The energy dependent sensitivity function, S (E), was established. Several ionization chambers were studied for (18)F and (99m)Tc, through Monte Carlo simulation method. In particular, some alterations to the original design of the Curiementor 2 were analysed, as well as the geometry of more recent chambers developed by PTW, namely Curiementor 3 and PET Curiementor 4. Calibration coefficients were compared for sources at different positions. Importance volumes were defined and determined for the Curiementors 2 and 3 and its ratio related with the corresponding ratio of sensitivity functions.

BDNF regulates the expression and traffic of NMDA receptors in cultured hippocampal neurons.

The neurotrophin BDNF regulates the activity-dependent modifications of synaptic strength in the CNS. Physiological and biochemical evidences implicate the NMDA glutamate receptor as one of the targets for BDNF modulation. In the present study, we investigated the effect of BDNF on the expression and plasma membrane abundance of NMDA receptor subunits in cultured hippocampal neurons. Acute stimulation of hippocampal neurons with BDNF differentially upregulated the protein levels of the NR1, NR2A and NR2B NMDA receptor subunits, by a mechanism sensitive to transcription and translation inhibitors. Accordingly, BDNF also increased the mRNA levels for NR1, NR2A and NR2B subunits. The neurotrophin NT3 also upregulated the protein levels of NR2A and NR2B subunits, but was without effect on the NR1 subunit. The amount of NR1, NR2A and NR2B proteins associated with the plasma membrane of hippocampal neurons was differentially increased by BDNF stimulation for 30 min or 24 h. The rapid upregulation of plasma membrane-associated NMDA receptor subunits was correlated with an increase in NMDA receptor activity. The results indicate that BDNF increases the abundance of NMDA receptors and their delivery to the plasma membrane, thereby upregulating receptor activity in cultured hippocampal neurons.

Brain-derived neurotrophic factor regulates the expression and synaptic delivery of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits in hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity in the hippocampus, but the mechanisms involved are not fully understood. The neurotrophin couples synaptic activation to changes in gene expression underlying long term potentiation and short term plasticity. Here we show that BDNF acutely up-regulates GluR1, GluR2, and GluR3 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits in 7-day in vitro cultured hippocampal neurons. The increase in GluR1 and GluR2 protein levels in developing cultures was impaired by K252a, a tropomyosin-related [corrected] kinase (Trk) inhibitor, and by translation (emetine and anisomycin) and transcription (alpha-amanitine and actinomycin D) inhibitors [corrected] The increase in GluR1 and GluR2 protein levels in developing cultures was impaired by K252a, a Trk inhibitor, and by translation (emetine and anisomycin) and transcription (alpha-amanitine and actinomycin D) inhibitors. Accordingly, BDNF increased the mRNA levels for GluR1 and GluR2 subunits. Biotinylation studies showed that stimulation with BDNF for 30 min selectively increased the amount of GluR1 associated with the plasma membrane, and this effect was abrogated by emetine. Under the same conditions, BDNF induced GluR1 phosphorylation on Ser-831 through activation of protein kinase C and Ca(2+)-calmodulin-dependent protein kinase II. Chelation of endogenous extracellular BDNF with TrkB-IgG selectively decreased GluR1 protein levels in 14-day in vitro cultures of hippocampal neurons. Moreover, BDNF promoted synaptic delivery of homomeric GluR1 AMPA receptors in cultured organotypic slices, by a mechanism independent of NMDA receptor activation. Taken together, the results indicate that BDNF up-regulates the protein levels of AMPA receptor subunits in hippocampal neurons and induces the delivery of AMPA receptors to the synapse.