Per- and Poly-Fluoroalkyl Substances in Portuguese Rivers: Spatial-Temporal Monitoring.

Eighteen per-and polyfluoroalkyl substances (PFASs) were investigated in surface waters of four river basins in Portugal (Ave, Leça, Antuã, and Cértima) during the dry and wet seasons. All sampling sites showed contamination in at least one of the seasons. In the dry season, perfluorooctanoate acid (PFOA) and perfluoro-octane sulfonate (PFOS), were the most frequent PFASs, while during the wet season these were PFOA and perfluobutane-sulfonic acid (PFBS). Compounds detected at higher concentrations were PFOS (22.6 ng L-1) and perfluoro-butanoic acid (PFBA) (22.6 ng L-1) in the dry and wet seasons, respectively. Moreover, the prospective environmental risks of PFASs, detected at higher concentrations, were evaluated based on the Risk Quotient (RQ) classification, which comprises acute and chronic toxicity. The results show that the RQ values of eight out of the nine PFASs were below 0.01, indicating low risk to organisms at different trophic levels in the four rivers in both seasons, wet and dry. Nevertheless, in the specific case of perfluoro-tetradecanoic acid (PFTeA), the RQ values calculated exceeded 1 for fish (96 h) and daphnids (48 h), indicating a high risk for these organisms. Furthermore, the RQ values were higher than 0.1, indicating a medium risk for fish, daphnids and green algae (96 h).

Astrocyte regional diversity in ALS includes distinct aberrant phenotypes with common and causal pathological processes.

Astrocytes are major contributors of motor neuron (MN) degeneration in amyotrophic lateral sclerosis (ALS). We investigated whether regional and cell maturation differences influence ALS astrocyte malfunction. Spinal and cortical astrocytes from SOD1G93A (mSOD1) 7-day-old mice were cultured for 5 and 13 days in vitro (DIV). Astrocyte aberrancies predominated in 13DIV cells with region specificity. 13DIV cortical mSOD1 astrocytes showed early morphological changes and a predominant reactive and inflammatory phenotype, while repressed proteins and genes were found in spinal cells. Inflammatory-associated miRNAs, e.g. miR-155/miR-21/miR-146a, were downregulated in the first and upregulated in the later ones. Interestingly, depleted miR-155/miR-21/miR-146a in small extracellular vesicles (sEVs/exosomes) was a common pathological feature. Cortical mSOD1 astrocytes induced late apoptosis and kinesin-1 downregulation in mSOD1 NSC-34 MNs, whereas spinal cells upregulated dynein, while decreased nNOS and synaptic-related genes. Both regional-distinct mSOD1 astrocytes enhanced iNOS gene expression in mSOD1 MNs. We provide information on the potential contribution of astrocytes to ALS bulbar-vs. spinal-onset pathology, local influence on neuronal dysfunction and their shared miRNA-depleted exosome trafficking. These causal and common features may have potential therapeutic implications in ALS. Future studies should clarify if astrocyte-derived sEVs are active players in ALS-related neuroinflammation and glial activation.

Effects of early intervention on parenting stress after preterm birth: A meta-analysis.

BACKGROUND: Preterm infants have a higher risk of development disorders. Prematurity can be considered a source of stress, in both children and their parents, due to the high number of interventions that they require. Early intervention (EI) programmes have shown to have a positive influence on the neurodevelopment of children with neurological risk. On the other hand, parenting stress has a negative influence on the development of any child. This systematic review aimed to identify the effect of EI programmes on decreasing parenting stress suffered by parents of preterm babies. METHODS: Systematic review and meta-analysis of experimental studies in accordance with the PRISMA declaration guidelines were applied in this work. RESULTS: Fifteen randomized clinical trials were included whose methodological quality was assessed using the PEDro scale. Stress data extraction was meta-analysed using the inverse variance method in a random effects model. Statistical heterogeneity was assessed with the I2 heterogeneity statistic. The domains most commonly reported in the trials were the childcare-related stress (Child Domain), personal discomfort (Parent Domain) and computation of both (Total Stress). The results showed significant (P < 0,05) and clinically relevant differences in favour of the EI programme group at 18 months and 5 years. CONCLUSIONS: This review found moderate to strong evidence of the impact of EI programmes on the reduction of parenting stress in parents of preterm babies. These findings offer useful insights regarding the delivery of current support and the development of future family interventions. Finally, recommendations are provided for future intervention evaluation studies in this area.

Overexpression of miR-124 in Motor Neurons Plays a Key Role in ALS Pathological Processes.

miRNA(miR)-124 is an important regulator of neurogenesis, but its upregulation in SOD1G93A motor neurons (mSOD1 MNs) was shown to associate with neurodegeneration and microglia activation. We used pre-miR-124 in wild-type (WT) MNs and anti-miR-124 in mSOD1 MNs to characterize the miR-124 pathological role. miR-124 overexpression in WT MNs produced a miRNA profile like that of mSOD1 MNs (high miR-125b; low miR-146a and miR-21), and similarly led to early apoptosis. Alterations in mSOD1 MNs were abrogated with anti-miR-124 and changes in their miRNAs mostly recapitulated by their secretome. Normalization of miR-124 levels in mSOD1 MNs prevented the dysregulation of neurite network, mitochondria dynamics, axonal transport, and synaptic signaling. Same alterations were observed in WT MNs after pre-miR-124 transfection. Secretome from mSOD1 MNs triggered spinal microglia activation, which was unno-ticed with that from anti-miR-124-modulated cells. Secretome from such modulated MNs, when added to SC organotypic cultures from mSOD1 mice in the early symptomatic stage, also coun-teracted the pathology associated to GFAP decrease, PSD-95 and CX3CL1-CX3CR1 signaling im-pairment, neuro-immune homeostatic imbalance, and enhanced miR-124 expression levels. Data suggest that miR-124 is implicated in MN degeneration and paracrine-mediated pathogenicity. We propose miR-124 as a new therapeutic target and a promising ALS biomarker in patient sub-populations.

Eco-friendly LC-MS/MS method for analysis of multi-class micropollutants in tap, fountain, and well water from northern Portugal.

Organic micropollutants present in drinking water (DW) may cause adverse effects for public health, and so reliable analytical methods are required to detect these pollutants at trace levels in DW. This work describes the first green analytical methodology for multi-class determination of 21 pollutants in DW: seven pesticides, an industrial compound, 12 pharmaceuticals, and a metabolite (some included in Directive 2013/39/EU or Decision 2015/495/EU). A solid-phase extraction procedure followed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (offline SPE-UHPLC-MS/MS) method was optimized using eco-friendly solvents, achieving detection limits below 0.20 ng L-1. The validated analytical method was successfully applied to DW samples from different sources (tap, fountain, and well waters) from different locations in the north of Portugal, as well as before and after bench-scale UV and ozonation experiments in spiked tap water samples. Thirteen compounds were detected, many of them not regulated yet, in the following order of frequency: diclofenac > norfluoxetine > atrazine > simazine > warfarin > metoprolol > alachlor > chlorfenvinphos > trimethoprim > clarithromycin ≈ carbamazepine ≈ PFOS > citalopram. Hazard quotients were also estimated for the quantified substances and suggested no adverse effects to humans. Graphical Abstract Occurrence and removal of multi-class micropollutants in drinking water, analyzed by an eco-friendly LC-MS/MS method.

Investigations on the Surface Integrity and Wear Mechanisms of TiAlYN-Coated Tools in Inconel 718 Milling Operations.

Inconel 718 is a Ni superalloy with superior mechanical properties, even at high temperatures. However, due to its high hardness and low thermal conductivity, it is considered a difficult-to-machine material. This material is widely used in applications that require good dimensional stability, making the milling process the most used in machining this alloy. The wear resulting from this process and the quality of the machined surface are still challenging factors when it comes to Inconel 718. TiAlN-based coating has been used on cutting tools with Yttrium as a doping element to improve the process performance. Based on this, this work evaluated the machined surface integrity and wear resistance of cutting tools coated using Physical Vapor Deposition (PVD) HiPIMS with TiAlYN in the end milling of Inconel 718, varying the process parameters such as cutting speed (vc), feed per tooth (fz), and cutting length (Lcut). It was verified that the Lcut is the parameter that exerts the most significant influence since, even at small distances, Inconel 718 already generates high tool wear (TW). Furthermore, the main wear mechanisms were abrasive and adhesive wear, with the development of a built-up edge (BUE) under a125 m/min feed rate (f) and a Lcut = 15 m. Chipping, cracking, and delamination of the coating were also observed, indicating a lack of adhesion between the coating and the substrate, suggesting the need for a good interlayer or the adjustment of the PVD parameters.

Mechanisms of ventilatory limitation to maximum exercise in children and adolescents with chronic airway diseases.

INTRODUCTION: Exercise intolerance is common in chronic airway diseases (CAD), but its mechanisms are still poorly understood. The aim of this study was to evaluate exercise capacity and its association with lung function, ventilatory limitation, and ventilatory efficiency in children and adolescents with cystic fibrosis (CF) and asthma when compared to healthy controls. METHODS: Cross-sectional study including patients with mild-to-moderate asthma, CF and healthy children and adolescents. Anthropometric data, lung function (spirometry) and exercise capacity (cardiopulmonary exercise testing) were evaluated. Primary outcomes were peak oxygen consumption (VO2 peak), forced expiratory volume in 1 s (FEV1 ), breathing reserve (BR), ventilatory equivalent for oxygen consumption (VE /VO2 ) and for carbon dioxide production (VE /VCO2 ), both at the ventilatory threshold (VT1 ) and peak exercise. RESULTS: Mean age of 147 patients included was 11.8 ± 3.0 years. There were differences between asthmatics and CF children when compared to their healthy peers for anthropometric and lung function measurements. Asthmatics showed lower VO2 peak when compared to both healthy and CF subjects, although no differences were found between healthy and CF patients. A lower BR was found when CF patients were compared to both healthy and asthmatic. Both CF and asthmatic patients presented higher values for VE /VO2 and VE /VCO2 at VT1 when compared to healthy individuals. For both VE /VO2 and VE /VCO2 at peak exercise CF patients presented higher values when compared to their healthy peers. CONCLUSION: Patients with CF achieved good exercise capacity despite low ventilatory efficiency, low BR, and reduced lung function. However, asthmatics reported reduced cardiorespiratory capacity and normal ventilatory efficiency at peak exercise. These results demonstrate differences in the mechanisms of ventilatory limitation to maximum exercise testing in children and adolescents with CAD.

Studying the Degradation of Three Polymers under Different Chlorine Concentrations and Exposure Times.

Due to chlorine's ability to kill bacteria and fungi through a chemical reaction, chlorine solutions are commonly used to clean and disinfect numerous public facilities, although these actions are also dependent to the equipment present in those facilities. Accordingly, the interest in studying its effect when in contact with different materials is obvious. This study was carried out through accelerated degradation tests and various analysis methods (optical microscope, scanning electron microscope, and tensile tests). The objective was to observe the wear presented by three polymeric materials, polyvinyl chloride (PVC), high-density polyethylene (HDPE), and polypropylene (PP), when exposed to chlorine's action in swimming pools and drinking water treatment plants. The resulting effect depends on the chlorine content and the type of contact between the chemical agent and the material. The aim was to select the material less likely to be affected by chlorine through tests and analyses, allowing a longer component life. The use of certain more resistant polymeric materials can drastically reduce maintenance, reducing fundamental factors such as costs, the downtime of municipal facilities, and also the risk to public health. It was concluded that PVC has the most stable behaviour overall when in contact with chlorine solutions.

Study of the Chlorine Influence on the Corrosion of Three Steels to Be Used in Water Treatment Municipal Facilities.

Many municipal facilities, such as pools and drinking water treatment facilities, are subject to ongoing maintenance due to the corrosion of their metallic materials caused by chlorine, leading to high costs and a possible risk to public health. A proper study of the employed product's effect could lead to the use of better materials, which significantly increase the lifetime of metallic equipment more attacked by corrosion, through studies evaluating their cost-effectiveness. This paper was carried out with the objective of studying the degradation of some metallic materials (AISI 316L, AISI 321 and Duplex 14462) used in the referred facilities in order to select the one that possessed a better behavior. It was observed that the introduction of some more adequate materials can drastically reduce maintenance operations, with Duplex 14462 showing the best results, ideal for greater chlorine concentrations, followed by AISI 321, which may be employed for components in less contact with chlorine, since it is more easily affordable.

The Impact of the Deterioration on Wood by Chlorine: An Experimental Study.

The use of disinfection and cleaning chemicals in several municipal facilities, such as swimming pools and drinking water treatment plants, causes the degradation of various types of wood, which leads to failures in equipment and the corresponding need for maintenance. This degradation creates added costs for municipalities, as well as the closure of certain facilities due to curative or preventive maintenance and, in many cases, public health issues, due to the water being contaminated with deteriorating products. Through a thorough study of the degradation effect on the products, more resistant materials can be found which are able to withstand these adversities and increase the lifespan of wood in regular contact with chemical agents. This is achievable by the determination of the cost-effectiveness of the substitute material to replace these components with alternative ones, with properties that better resist the deterioration effects promoted by aggressive environments. No studies have been found so far strictly focused on this matter. The objective of this study is to evaluate the degradation presented by two types of wood, beech and oak, which are exposed to the action of chlorine in municipal facilities. This degradation varies according to the chlorine content and the materials' time of contact with the chemical agent, allowing the selection of new materials which will provide an extended lifetime of the components, reducing maintenance drastically, as well as costs for the facilities and the risk to public health. The performed experimental tests have shown that the oak wood has the best results regarding chlorine degradation resistance.

Improvement of In-School Physical Activity with Active School-Based Interventions to Interrupt Prolonged Sitting: A Systematic Review and Meta-Analysis.

BACKGROUND: Sedentary behaviors have increased in recent years and their consequences have led the World Health Organization to make recommendations for promoting a more active lifestyle. The school environment has been defined as a key place for achieving this objective for children and adolescents. This systematic review and meta-analysis aims to analyze the effect of active-break interventions for interrupting prolonged sitting times during school-time on physical activity (PA) and sedentary behavior (SB), at school, in childhood and youth. METHODS: A systematic review and meta-analysis were carried out, including clinical trials aimed at assessing the effects of interrupting prolonged sitting at school with active breaks on in-school PA and/or SB. Multimodal and static interventions were excluded. Six databases were analyzed: Medline, WOS, Cochrane Library, SPORT Discus, CINAHL and EMBASE. PA, SB; moderate-to-vigorous physical activity (MVPA) were the variables considered. RESULTS: Nine studies were included, with a total of 2145 children between 6 and 12 years old. The heterogeneity in the duration (five-sixty min), the frequency (one-three times per-day up to three times per week), and duration (five days to three years) of the interventions was detected. The meta-analyses for in-school PA, MVPA, and SB were performed, showing a significant improvement in both PA and MVPA. CONCLUSIONS: Interrupting prolonged sitting with active-based school interventions could improve PA and MVPA levels during school time. (PROSPERO: CRD42022358933).

Intrathecal Injection of the Secretome from ALS Motor Neurons Regulated for miR-124 Expression Prevents Disease Outcomes in SOD1-G93A Mice.

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.

Active School-Based Interventions to Interrupt Prolonged Sitting Improve Daily Physical Activity: A Systematic Review and Meta-Analysis.

BACKGROUND: Sedentary behavior (SB) is a negative routine for health, especially during advancing age. Promoting an active lifestyle and reducing SB is a global endeavor. The aim of this study was to analyze the effects of active school-based programs to interrupt prolonged sitting for daily physical activity (PA) and daily SB in children and adolescents. METHODS: A systematic review was conducted. Clinical trials analyzing the effect of interrupting prolonged sitting with active breaks and classroom-based PA were included. Studies that implemented PA in class without interrupting prolonged sitting or those that implemented multimodal interventions were excluded. A systematic search was conducted in 6 databases: Medline, WOS, Cochrane Library, SPORTDiscus, CINAHL and EMBASE. Primary outcomes were daily PA and daily SB, while moderate-to vigorous physical activity (MVPA) was considered a secondary outcome. RESULTS: Six studies were included, with a total of 976 participants between 6-14 years. The interventions were heterogeneous in duration of the sitting time interruption (5-30 min), frequency (1-3 times per-day up to three times per-week) and total duration (five days to three years). 50% of the studies scored "high risk" of bias. Three meta-analyses were performed for daily PA, MVPA and SB, showing a significant improvement in the daily PA and MVPA. CONCLUSIONS: School-based programs aimed to interrupt prolonged sitting could be a good strategy to improve daily PA and MVPA levels. (Registration number: CRD42022358933).

One Year of COVID-19 in Pregnancy: A National Wide Collaborative Study.

INTRODUCTION: Even though the risk of COVID-19 in pregnancy may be increased, large-scale studies are needed to better understand the impact of the infection in this population. The aim of this study is to describe obstetric complications and the rate of vertical transmission in pregnant women with SARS-CoV-2 infection. MATERIAL AND METHODS: Detected cases of SARS-CoV-2 infection in pregnancy were registered in Portuguese hospitals by obstetricians. Epidemiological, pregnancy and childbirth data were collected. RESULTS: There were 630 positive cases in 23 Portuguese maternity hospitals, most at term (87.9%) and asymptomatic (62.9%). The most frequent maternal comorbidity was obesity. The rates of preterm birth and small-to-gestational-age were 12.1% and 9.9%, respectively. In the third trimester, 2.9% of pregnant women required respiratory support. There were eight cases (1.5%) of fetal death, including two cases of vertical transmission. There were five cases of postpartum respiratory degradation, but no maternal deaths were recorded. The caesarean section rate was higher in the first than in the second wave (68.5% vs 31.5%). RT-PCR SARS-CoV-2 positivity among newborns was 1.3%. CONCLUSION: SARS-Cov-2 infection in pregnancy may carry increased risks for both pregnant women and the fetuses. Individualized surveillance and the prophylaxis of this population with vaccination. is recommended in these cases.

Introdução: Apesar do risco da COVID-19 na gravidez poder ser acrescido, são necessários estudos em larga escala para o melhor conhecimento do impacto desta infeção nesta população. O objetivo deste estudo é descrever as complicações obstétricas e a taxa de transmissão vertical em grávidas com infeção a SARS-CoV-2. Material e Métodos: Os casos conhecidos de infeção por SARS-CoV-2 na gravidez foram registados nos hospitais portugueses por obstetras. Foram recolhidos dados epidemiológicos, da gravidez e do parto. Resultados: Registaram-se 630 casos positivos em 23 maternidades portuguesas, a maioria no termo (87,9%) e assintomática (62,9%). A comorbilidade materna mais frequente foi a obesidade. A taxa de parto pré-termo e de leves para a idade gestacional foi de 12,1% e 9,9%, respectivamente. No terceiro trimestre, 2,9% das grávidas necessitaram de suporte respiratório. Verificou-se uma taxa de 1,5% de morte fetal, incluindo dois casos de transmissão vertical. Houve cinco casos de degradação respiratória no pós-parto, mas sem mortes maternas registadas. A taxa de cesarianas foi mais elevada na primeira do que na segunda vaga (68,5% vs 31,5%). A positividade do RT-PCR SARS-CoV-2 entre os recém-nascidos foi de 1,3%. Conclusão: A infeção pelo SARS-Cov-2 na gravidez pode acarretar riscos aumentados para as grávidas e fetos. Recomenda-se uma vigilância individualizada nestes casos e a profilaxia desta população com a vacinação.

Multicenter Evaluation of the Idylla GeneFusion in Non-Small-Cell Lung Cancer.

Targeted therapy in lung cancer requires the assessment of multiple oncogenic driver alterations, including fusion genes. This retrospective study evaluated the Idylla GeneFusion prototype, an automated and ease-of-use (<2 minutes) test, with a short turnaround time (3 hours) to detect fusions involving ALK, ROS1, RET, and NTRK1/2/3 genes and MET exon 14 skipping. This multicenter study (18 centers) included 313 tissue samples from lung cancer patients with 97 ALK, 44 ROS1, 20 RET, and 5 NTRKs fusions, 32 MET exon 14 skipping, and 115 wild-type samples, previously identified with reference methods (RNA-based next-generation sequencing/fluorescence in situ hybridization/quantitative PCR). Valid results were obtained for 306 cases (98%), overall concordance between Idylla and the reference methods was 89% (273/306); overall sensitivity and specificity were 85% (165/193) and 96% (108/113), respectively. Discordances were observed in 28 samples, where Idylla did not detect the alteration identified by the reference methods; and 5 samples where Idylla identified an alteration not detected by the reference methods. All of the ALK-, ROS1-, and RET-specific fusions and MET exon 14 skipping identified by Idylla GeneFusion were confirmed by reference method. To conclude, Idylla GeneFusion is a clinically valuable test that does not require a specific infrastructure, allowing a rapid result. The absence of alteration or the detection of expression imbalance only requires additional testing by orthogonal methods.

Carbon xerogels combined with nanotubes as solid-phase extraction sorbent to determine metaflumizone and seven other surface and drinking water micropollutants.

Carbon xerogels (CXs) were synthesized by polycondensation of resorcinol and formaldehyde, followed by thermal annealing, and subjected to hydrothermal oxidation. Solid-phase extraction (SPE) cartridges were filled with CXs and tested for extraction of metaflumizone and other seven environmental micropollutants (acetamiprid, atrazine, isoproturon, methiocarb, carbamazepine, diclofenac, and perfluorooctanesulfonic acid) before chromatographic analysis. The recoveries obtained with the pristine CX were low for most analytes, except for metaflumizone (69 ± 5%). Moreover, it was concluded that the adsorption/desorption process of the micropollutants performed better on CXs with a less acidic surface (i.e., pristine CX). Thus, cartridges were prepared with pristine CX and multi-walled carbon nanotubes (MWCNTs) in a multi-layer configuration. This reusable cartridge was able to simultaneously extract the eight micropollutants and was used to validate an analytical methodology based on SPE followed by ultra-high performance liquid chromatography-tandem mass spectrometry. A widespread occurrence of 6/8 target compounds was observed in surface water collected in rivers supplying three drinking water treatment plants and in the resulting drinking water at the endpoint of each distribution system. Therefore, the first study employing CXs and MWCNTs as sorbent in multi-layer SPE cartridges is herein reported as a proof of concept for determination of multi-class water micropollutants.

Recovery of Depleted miR-146a in ALS Cortical Astrocytes Reverts Cell Aberrancies and Prevents Paracrine Pathogenicity on Microglia and Motor Neurons.

Reactive astrocytes in Amyotrophic Lateral Sclerosis (ALS) change their molecular expression pattern and release toxic factors that contribute to neurodegeneration and microglial activation. We and others identified a dysregulated inflammatory miRNA profile in ALS patients and in mice models suggesting that they represent potential targets for therapeutic intervention. Such cellular miRNAs are known to be released into the secretome and to be carried by small extracellular vesicles (sEVs), which may be harmful to recipient cells. Thus, ALS astrocyte secretome may disrupt cell homeostasis and impact on ALS pathogenesis. Previously, we identified a specific aberrant signature in the cortical brain of symptomatic SOD1-G93A (mSOD1) mice, as well as in astrocytes isolated from the same region of 7-day-old mSOD1 mice, with upregulated S100B/HMGB1/Cx43/vimentin and downregulated GFAP. The presence of downregulated miR-146a on both cases suggests that it can be a promising target for modulation in ALS. Here, we upregulated miR-146a with pre-miR-146a, and tested glycoursodeoxycholic acid (GUDCA) and dipeptidyl vinyl sulfone (VS) for their immunoregulatory properties. VS was more effective in restoring astrocytic miR-146a, GFAP, S100B, HMGB1, Cx43, and vimentin levels than GUDCA, which only recovered Cx43 and vimentin mRNA. The miR-146a inhibitor generated typical ALS aberrancies in wild type astrocytes that were abolished by VS. Similarly, pre-miR-146a transfection into the mSOD1 astrocytes abrogated aberrant markers and intracellular Ca2+ overload. Such treatment counteracted miR-146a depletion in sEVs and led to secretome-mediated miR-146a enhancement in NSC-34-motor neurons (MNs) and N9-microglia. Secretome from mSOD1 astrocytes increased early/late apoptosis and FGFR3 mRNA in MNs and microglia, but not when derived from pre-miR-146a or VS-treated cells. These last strategies prevented the impairment of axonal transport and synaptic dynamics by the pathological secretome, while also averted microglia activation through either secretome, or their isolated sEVs. Proteomic analysis of the target cells indicated that pre-miR-146a regulates mitochondria and inflammation via paracrine signaling. We demonstrate that replenishment of miR-146a in mSOD1 cortical astrocytes with pre-miR-146a or by VS abrogates their phenotypic aberrancies and paracrine deleterious consequences to MNs and microglia. These results propose miR-146a as a new causal and emerging therapeutic target for astrocyte pathogenic processes in ALS.

Development and Content Validity of the Physical Activity Questionnaire-Young Children (PAQ-YC) to Assess Physical Activity in Children between 5 and 7 Years.

Childhood is a critical period in the development and consolidation of healthy habits, such as the practice of physical activity (PA). It is essential to have valid instruments to measure PA from an early age. The aim of this study was to design and evaluate the content validity of the Physical Activity Questionnaire-Young Children (PAQ-YC) to measure the PA level in children aged 5-7 years. The first version of the questionnaire was tested by a 2-round Delphi study. It was established as a consensus criterion that the relative interquartile range (RIR) and/or the coefficient of variation (CV) were ≤20%. The most significant discrepancies in the Delphi survey (n = 11-13) were observed for items about hours of Physical Education or similar activities at school (item 7: RIR = 20, CV = 38.73) and for items about participation in Physical Education (item 8: RIR = 25, CV = 15.45). The cognitive interviews (n = 5) confirmed the version agreed by the experts. The results show that the PAQ-YC presents adequate content validity in terms of relevance, comprehensiveness and comprehensibility.

A new methodology combining PCR, cloning, and sequencing of clones discriminated by RFLP for the study of microbial populations: application to an UASB reactor sample.

This work describes a methodology combining DNA extraction, polymerase chain reaction amplification with primers targeting 16S ribosomal RNA genes, cloning, and sequencing of clones previously analyzed by restriction fragment length polymorphism (RFLP), which can be applied to study the microbial diversity in a given habitat. The methodology allows the minimization of the sequencing effort, which is particularly relevant when analyzing large numbers of clones. The methodology does not require particularly skilled personnel and can easily be adaptable to the molecular characterization of virtually any particular microbial population, provided that both adequate primers and suitable restriction enzymes for RFLP analysis of the clone library have been chosen. An example of application is presented, in which a sample taken from a continuously operating upflow anaerobic sludge blanket reactor was analyzed. RFLP analysis of the initial 162 clones with HaeIII allowed the identification of only 28 distinct profiles. As expected, identical RFLP profiles corresponded to identical nucleotide sequences.