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João Batista Santos Garcia, José Osvaldo Barbosa Neto, Thiago Alves RodriguesExperimental Laboratory to the Study of Pain, Federal University of Maranhão, São Luis, Maranhão, BrazilPurpose: Chronic pain remains undertreated in both developed and developing countries. There are various factors involved in this acknowledged health problem, including lack of pain education. In Brazil, a particular approach was used to mitigate the deficit in pain education. The academic leagues of pain (ALPs) were formed as associations of health undergraduate students with the goal of encouraging students in educational activities, patient care, and pain research. The aim of this study was to evaluate how ALP works and its contribution to pain education and to the inclusion of students in practical and scientific research activities, as well as its legacy in the field of pain.Patients and methods: An electronic survey was directed to the leagues representatives to collect data referent to how the leagues operate, their individual approach towards pain education, patient care, research activities, and its impact on students after they graduate.Results: A total of 17 leagues were identified and responded to the survey. Only three of the involved universities offered study of pain as a discipline in their mandatory curriculum. Patient care activities were carried out by 59% of the leagues, 94% provided educational activities. Twelve leagues reported that students were involved in one to four research projects in pain, and 59% of those chose pain as their subject for post-graduation programs. And, 47% of the leagues had students that sought specialization or residency in pain after graduation.Conclusion: The Brazilian experience with academic leagues of pain has shown that it is possible to address curricular deficiencies in pain education through a strategy not well known in other countries.Keywords: medical education, pain education, pain management, chronic pain

PEX5, the peroxisomal protein shuttling receptor, binds newly synthesized proteins in the cytosol and transports them to the organelle. During its stay at the peroxisomal protein translocon, PEX5 is monoubiquitinated at its cysteine 11 residue, a mandatory modification for its subsequent ATP-dependent extraction back into the cytosol. The reason why a cysteine and not a lysine residue is the ubiquitin acceptor is unknown. Using an established rat liver-based cell-free in vitro system, we found that, in contrast to wild-type PEX5, a PEX5 protein possessing a lysine at position 11 is polyubiquitinated at the peroxisomal membrane, a modification that negatively interferes with the extraction process. Wild-type PEX5 cannot retain a polyubiquitin chain because ubiquitination at cysteine 11 is a reversible reaction, with the E2-mediated deubiquitination step presenting faster kinetics than PEX5 polyubiquitination. We propose that the reversible nonconventional ubiquitination of PEX5 ensures that neither the peroxisomal protein translocon becomes obstructed with polyubiquitinated PEX5 nor is PEX5 targeted for proteasomal degradation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Francisco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Despite intensive research on peroxisome biochemistry, the role of glutathione in peroxisomal redox homeostasis has remained a matter of speculation for many years, and only recently has this issue started to be experimentally addressed. Here, we summarize and compare data from several organisms on the peroxisome-glutathione topic. It is clear from this comparison that the repertoire of glutathione-utilizing enzymes in peroxisomes of different organisms varies widely. In addition, the available data suggest that the kinetic connectivity between the cytosolic and peroxisomal pools of glutathione may also be different in different organisms, with some possessing a peroxisomal membrane that is promptly permeable to glutathione whereas in others this may not be the case. However, regardless of the differences, the picture that emerges from all these data is that glutathione is a crucial component of the antioxidative system that operates inside peroxisomes in all organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Despite the large amounts of H 2 O 2 generated in mammalian peroxisomes, cysteine residues of intraperoxisomal proteins are maintained in a reduced state. The biochemistry behind this phenomenon remains unexplored, and simple questions such as "is the peroxisomal membrane permeable to glutathione?" or "is there a thiol-disulfide oxidoreductase in the organelle matrix?" still have no answer. We used a cell-free in vitro system to equip rat liver peroxisomes with a glutathione redox sensor. The organelles were then incubated with glutathione solutions of different redox potentials and the oxidation/reduction kinetics of the redox sensor was monitored. The data suggest that the mammalian peroxisomal membrane is promptly permeable to both reduced and oxidized glutathione. No evidence for the presence of a robust thiol-disulfide oxidoreductase in the peroxisomal matrix could be found. Also, prolonged incubation of organelle suspensions with glutaredoxin 1 did not result in the internalization of the enzyme. To explore a potential role of glutathione in intraperoxisomal redox homeostasis we performed kinetic simulations. The results suggest that even in the absence of a glutaredoxin, glutathione is more important in protecting cysteine residues of matrix proteins from oxidation by H 2 O 2 than peroxisomal catalase itself., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: The perception of prejudice against, and stigmatization of, sickle cell disease (SCD) leads the patient to perceive a different treatment, due to the disease stigma and may be related to a worse quality of life (QoL)., Objectives: Describe and evaluate the perception of the prejudice against the disease and its impact on the quality of life of patients with sickle cell disease., Methods: This is a cross-sectional study conducted between March 2019 and February 2020, with patients diagnosed with SCD. Patients were questioned about the perception of prejudice in any kind of situation, choosing between "Yes" or "No", not differentiating situations related to prejudice. To assess the QoL and impact of the disease, the volunteers answered a version of the SF-36 questionnaire translated and validated into Brazilian Portuguese., Results: In this study, 113 patients with SCD were followed up, 92% were classified as HbSS and the rest, divided between HbSC and HbS-β-0. Regarding the SF-36, the worst scores were in the summary of the physical components (mean 48.19 ± 21.51) and the physical aspect had the lowest mean (30.75 ±€42.65). When questioned if they had already perceived any kind of prejudice, including the SCD, 32.74% answered "Yes". For this comparison, there was a significant difference in the summary of the physical and mental components, with worse QoL for those who had already suffered prejudice., Conclusion: Patients diagnosed with SCD who reported perception of prejudice had statistically significant worse QoL, revealing the negative impact, that might lead to sadness and social isolation., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2021 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier España, S.L.U. All rights reserved.)

Objectives: to assess the factors associated with the safety culture of patients under dialysis in the context of the COVID-19 pandemic., Methods: a cross-sectional and analytical study, carried out in Minas Gerais, with 134 professionals from three dialysis services. The Hospital Survey on Patient Safety Culture, adapted for Brazil, was used., Results: only variable type of management was associated with the highest percentage of positive response in public and private services. Patient safety was rated as good by 55.7% of respondents. In dimension assessment, the public service presented one strength and five weaknesses, the private service did not present weak areas, and the philanthropic service presented a weakness. The priority areas for improvement actions are represented by dimensions "Nonpunitive response to error" and "Staffing"., Conclusions: interventions should consider the type of service management, as it is a factor associated with safety culture.

The AAA ATPases PEX1•PEX6 extract PEX5, the peroxisomal protein shuttling receptor, from the peroxisomal membrane so that a new protein transport cycle can start. Extraction requires ubiquitination of PEX5 at residue 11 and involves a threading mechanism, but how exactly this occurs is unclear. We used a cell-free in vitro system and a variety of engineered PEX5 and ubiquitin molecules to challenge the extraction machinery. We show that PEX5 modified with a single ubiquitin is a substrate for extraction and extend previous findings proposing that neither the N- nor the C-terminus of PEX5 are required for extraction. Chimeric PEX5 molecules possessing a branched polypeptide structure at their C-terminal domains can still be extracted from the peroxisomal membrane thus suggesting that the extraction machinery can thread more than one polypeptide chain simultaneously. Importantly, we found that the PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and, accordingly, an intra-molecularly cross-linked ubiquitin blocked extraction when conjugated to residue 11 of PEX5. Collectively, our data suggest that the PEX5-linked monoubiquitin is the extraction initiator and that the complete ubiquitin-PEX5 conjugate is threaded by PEX1•PEX6., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cell-free in vitro systems are invaluable tools to study the molecular mechanisms of protein translocation across biological membranes. We have been using such a strategy to dissect the mechanism of the mammalian peroxisomal matrix protein import machinery. Here, we provide a detailed protocol to import proteins containing a peroxisomal targeting signal type 2 (PTS2) into the organelle. The in vitro system consists of incubating a 35 S-labeled reporter protein with a post-nuclear supernatant from rat/mouse liver. At the end of the incubation, the organelle suspensions are generally treated with an aggressive protease to degrade reporter proteins that did not enter peroxisomes, and the organelles are isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. This in vitro system is particularly suited to characterize the functional consequences of PEX5 and PEX7 mutations found in patients affected with a peroxisomal biogenesis disorder., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The recent development and mass administration of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines allowed for disease control, reducing hospitalizations and mortality. Most of these vaccines target the SARS-CoV-2 Spike (S) protein antigens, culminating with the production of neutralizing antibodies (NAbs) that disrupt the attachment of the virus to ACE2 receptors on the host cells. However, several studies demonstrated that the NAbs typically rise within a few weeks after vaccination but quickly reduce months later. Thus, multiple booster administration is recommended, leading to vaccination hesitancy in many populations. Detecting serum anti-SARS-CoV-2 NAbs can instruct patients and healthcare providers on correct booster strategies. Several in vitro diagnostics kits are available; however, their high cost impairs the mass NAbs diagnostic testing. Recently, we engineered an ACE2 mimetic that interacts with the Receptor Binding Domain (RBD) of the SARS-2 S protein. Here we present the use of this engineered mini-protein (p-deface2 mut) to develop a detection assay to measure NAbs in patient sera using a competitive ELISA assay. Serum samples from twenty-one patients were tested. Nine samples (42.8%) tested positive, and twelve (57.1%) tested negative for neutralizing sera. The data correlated with the result from the standard commercial assay that uses human ACE2 protein. This confirmed that p-deface2 mut could replace human ACE2 in ELISA assays. Using bacterially expressed p-deface2 mut protein is cost-effective and may allow mass SARS-CoV-2 NAbs detection, especially in low-income countries where economical diagnostic testing is crucial. Such information will help providers decide when a booster is required, reducing risks of reinfection and preventing the administration before it is medically necessary.

The need for discovering new compounds that can act selectively on pathogens is becoming increasingly evident, given the number of deaths worldwide due to bacterial infections or tumor cells. New multifunctional biotechnological tools are being sought, including compounds present in spider venoms, which have high biotechnological potential. The present work aims to perform the rational design and functional evaluation of synthetic peptides derived from Lachesana tarabaevi spider toxin, known as latarcin-3a. The antimicrobial activity was tested against Gram-positive and -negative bacteria, with minimum inhibitory concentrations (MIC) between 4 and 128 μg.ml -1 . Anti-biofilm tests were then performed to obtain MICs, where the peptides demonstrated activity from 4 to 128 μg.ml -1 . In vitro cell cytotoxicity assays were carried out from tumor cell lines, lineages C1498, Kasumi-1, K-562, Jurkat, MOLT4, and Raji. Erythrocyte integrity was evaluated in the presence of synthetic peptides analog, which did not promote hemolysis at 128 μg.ml -1 . The peptide that showed the best antibacterial activity was Lt-MAP3 and the best antitumor was Lt-MAP2. In conclusion, rational design of multifunctional antimicrobial peptides may be promising alternative tools in the treatment of emerging diseases such as bacterial infections and tumor cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 de Moraes, Silva, Pereira, Almeida Rodrigues, Farias Frihling, da Costa, Torquato, Lima, Paredes-Gamero and Migliolo.)

In Brief: Elevated temperatures disturbed sperm physiology. Bovine sperm cells exposed to heat shock led to diminished mitochondrial activity, fertilizing ability, increased oxidative stress and caspase activity concomitant with a delay in embryonic developmental kinetics and modulation of sperm-borne microRNAsmiRNAs., Abstract: Sperm function is susceptible to adverse environmental conditions. It has been demonstrated that in vivo and in vitro exposure of bovine sperm to elevated temperature reduces sperm motility and fertilizing potential. However, the cascade of functional, cellular, and molecular events triggered by elevated temperature in the mature sperm cell remains not fully understood. Therefore, the aim of this study was to determine the effect of heat shock on mature sperm cells. Frozen-thawed Holstein sperm were evaluated immediately after Percoll purification (0 h non-incubation control) or after incubation at 35, 38.5, and 41°C for 4 h. Heat shock reduced sperm motility after 3-4 h at 41°C while mitochondrial activity was reduced by 38.5 and 41°C when compared to the control. Heat shock also increased sperm reactive oxygen species production and caspase activity. Heat-shocked sperm had lower fertilizing ability, which led to diminished cleavage and blastocyst rates. Preimplantation embryo developmental kinetics was also slowed and reduced by sperm heat shock. The microRNA (miR) profiling identified >300 miRs in bovine sperm. Among these, three and seven miRs were exclusively identified in sperm cells exposed to 35 and 41°C, respectively. Moreover, miR-181d was enriched in sperm cells exposed to higher temperatures. Hence, elevated temperature altered the physiology of mature sperm cells by perturbing cellular processes and the miR profile, which collectively led to lower fertilizing ability and preimplantation development.

Uncrewed aerial vehicles (UAVs) for last-mile deliveries will affect the energy productivity of delivery and require new methods to understand energy consumption and greenhouse gas (GHG) emissions. We combine empirical testing of 188 quadcopter flights across a range of speeds with a first-principles analysis to develop a usable energy model and a machine-learning algorithm to assess energy across takeoff, cruise, and landing. Our model shows that an electric quadcopter drone with a very small package (0.5 kg) would consume approximately 0.08 MJ/km and result in 70 g of CO 2 e per package in the United States. We compare drone delivery with other vehicles and show that energy per package delivered by drones (0.33 MJ/package) can be up to 94% lower than conventional transportation modes, with only electric cargo bicycles providing lower GHGs/package. Our open model and coefficients can assist stakeholders in understanding and improving the sustainability of small package delivery., Competing Interests: The authors declare no competing interests. C.S. has a patent for a system, method, and computer program product for transporting an unmanned vehicle, US patent 11300978B1, 2022. The patent’s methods were not used this study., (© 2022 The Author(s).)

Objective: To describe, in the mothers' perception, the experiences lived by families in the care of children with autism spectrum disorder., Method: Qualitative study, carried out with 20 mothers of children diagnosed with autistic disorder accompanied by an institution in Teresina-Piauí, Brazil. Semi-structured interviews were conducted between February and March 2019 and subjected to content analysis., Results: Five central ideas related to the stages experienced by family members after the diagnosis were identified, ranging from denial to acceptance. Family members and caregivers experience feelings of sadness and mourning for the discovery of the impossibility of curing the syndrome, revealing the need for care for this family. The search for help and adaptations of the routine are constant experiences., Conclusion: Caring for children who live with autistic disorder involves learning ranging from structural to emotional aspects, such as dealing with limitations and impossibility of cure, pointing out to the need for family care.

Background: Covid-19 has the respiratory tract as the main target of infection, and patients present mainly dyspnea, pneumonia, dry cough, and fever. Nevertheless, organs outside the respiratory tract had been reported in recent studies, including the gastrointestinal tract and liver. The host innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through their pattern recognition receptor (PRRs). Toll-like receptor 7 (TLR-7) is a pattern recognition receptor recognizing ssRNA (SARS-CoV-2 is an ssRNA). Polymorphisms are characterized by two or more alternative forms of a distinct phenotype in the same population. Polymorphisms in tlrs genes can negatively influence the immune response to infectious diseases. There are several references in the literature to non-synonymous single nucleotide (rs) polymorphisms related to several genes. Some of them are important for the innate immunity, as rs 179008 (tlr-7), rs3775291 (tlr3), rs8177374 (tir domain-containing adaptor protein, tirap), rs1024611 (monocyte chemoattractant protein-1, mcp-1) and rs61942233 (2'-5'-oligoadenylate synthase-3, oas-3)., Case Presentation: We identified a 5-year-old-male child with gastrointestinal symptoms and fever presenting acholic stool and jaundice, who was positive for SARS-CoV-2 IgM, IgA, and IgG and presenting the Gln11Leu rs 179008 in tlr-7. The child presented high levels of aspartate aminotransferase, alanine aminotransferase, bilirubin, C-reactive protein, D-dimer, gamma-glutamyl transferase, alkaline phosphatase, and was negative for serological tests for hepatitis A, B, C, E, HIV 1 and 2, herpes virus, cytomegalovirus, Epstein-Barr virus, and negative for RTqPCR for Influenza A and B, RSV and SARS-CoV-2. We also investigated other SNPs in the tlr-3 (rs3775291), tirap (rs8177374), mcp-1 (rs1024611), and oas-3 (rs61942233) genes, and no mutation was detected. After an interview with the child's caregivers, any possible accidental ingestion of drugs or hepatotoxic substances was ruled out., Conclusion: To our knowledge, this is the first report of a SARS-CoV-2 caused hepatitis in a male child that has the tlr-7 Gln11Leu rs 179008, which could impair an efficient initial immune response. The knowledge of the patient's immune deficiency could improve the treatment to correct this deficiency with specific medications., (© 2021. The Author(s).)

Defect detection in additive manufacturing (AM) is of paramount importance to improve the reliability of products. Nondestructive testing is not yet widely used for defect detection. The main challenges are a lack of standards and methods, the types and location of defects, and the complex geometry of many parts. During selective laser melting (SLM), several types of defects can occur such as porosity, cracking, and lack of fusion. In this study, several nondestructive tests were conducted in a highly complex shaped part in AISI 316L stainless steel with real defects manufactured by SLM. Two additional artificial defects (one horizontal and one flat bottom hole) were produced and the defect detectability was evaluated. The techniques used were as follows: dye penetrant, infrared thermography, immersion ultrasonic, eddy current, and X-ray microcomputed tomography to assess different types of defects in the as-built part. We conclude that no single technique can detect every type of defect, although multiple techniques provide complementary and redundant information to critically evaluate the integrity of the parts. This approach is fundamental for improving the reliability of defect detection, which will help expand the potential for using AM to produce parts for critical structural applications., Competing Interests: No competing financial interests exist., (Copyright 2021, Mary Ann Liebert, Inc., publishers.)

We autonomously directed a small quadcopter package delivery Uncrewed Aerial Vehicle (UAV) or "drone" to take off, fly a specified route, and land for a total of 209 flights while varying a set of operational parameters. The vehicle was equipped with onboard sensors, including GPS, IMU, voltage and current sensors, and an ultrasonic anemometer, to collect high-resolution data on the inertial states, wind speed, and power consumption. Operational parameters, such as commanded ground speed, payload, and cruise altitude, were varied for each flight. This large data set has a total flight time of 10 hours and 45 minutes and was collected from April to October of 2019 covering a total distance of approximately 65 kilometers. The data collected were validated by comparing flights with similar operational parameters. We believe these data will be of great interest to the research and industrial communities, who can use the data to improve UAV designs, safety, and energy efficiency, as well as advance the physical understanding of in-flight operations for package delivery drones.

Peroxisomes, single-membrane intracellular organelles, play an important role in various metabolic pathways. The translocation of proteins from the cytosol to peroxisomes depends on peroxisome import receptor proteins and defects in peroxisome transport result in a wide spectrum of peroxisomal disorders. Here, we report a large consanguineous family with autosomal recessive congenital cataracts and developmental defects. Genome-wide linkage analysis localized the critical interval to chromosome 12p with a maximum two-point LOD score of 4.2 (θ = 0). Next-generation exome sequencing identified a novel homozygous missense variant (c.653 T > C; p.F218S) in peroxisomal biogenesis factor 5 (PEX5), a peroxisome import receptor protein. This missense mutation was confirmed by bidirectional Sanger sequencing. It segregated with the disease phenotype in the family and was absent in ethnically matched control chromosomes. The lens-specific knockout mice of Pex5 recapitulated the cataractous phenotype. In vitro import assays revealed a normal capacity of the mutant PEX5 to enter the peroxisomal Docking/Translocation Module (DTM) in the presence of peroxisome targeting signal 1 (PTS1) cargo protein, be monoubiquitinated and exported back into the cytosol. Importantly, the mutant PEX5 protein was unable to form a stable trimeric complex with peroxisomal biogenesis factor 7 (PEX7) and a peroxisome targeting signal 2 (PTS2) cargo protein and, therefore, failed to promote the import of PTS2 cargo proteins into peroxisomes. In conclusion, we report a novel missense mutation in PEX5 responsible for the defective import of PTS2 cargo proteins into peroxisomes resulting in congenital cataracts and developmental defects.

Biotic resistance mediated by native plant diversity has long been hypothesized to reduce the success of invading plant species in terrestrial systems in temperate regions. However, still little is known about the mechanisms driving invasion patterns in other biomes or latitudes. We help to fill this gap by investigating how native plant community presence and diversity, and the presence of native phylogenetically closely related species to an invader, would affect invader Hydrilla verticillata establishment success in tropical freshwater submerged plant communities. The presence of a native community suppressed the growth of H. verticillata, but did not prevent its colonisation. Invader growth was negatively affected by native plant productivity, but independent of native species richness and phylogenetic relatedness to the invader. Native plant production was not related to native species richness in our study. We show that resistance in these tropical aquatic submerged plant communities is mainly driven by the presence and biomass of a native community independent of native species diversity. Our study illustrates that resistance provided by these tropical freshwater submerged plant communities to invasive species contrasts to resistance described for other ecosystems. This emphasizes the need to include understudied systems when predicting patterns of species invasiveness and ecosystem invasibility across biomes.

CLASPs are conserved microtubule plus-end-tracking proteins that suppress microtubule catastrophes and independently localize to kinetochores during mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore-microtubule dynamics required for chromosome segregation fidelity, but the underlying mechanism remains unknown. Here, we found that human CLASP2 exists predominantly as a monomer in solution, but it can self-associate through its C-terminal kinetochore-binding domain. Kinetochore localization was independent of self-association, and driving monomeric CLASP2 to kinetochores fully rescued normal kinetochore-microtubule dynamics, while partially sustaining mitosis. CLASP2 kinetochore localization, recognition of growing microtubule plus-ends through EB-protein interaction, and the ability to associate with curved microtubule protofilaments through TOG2 and TOG3 domains independently sustained normal spindle length, timely spindle assembly checkpoint satisfaction, chromosome congression, and faithful segregation. Measurements of kinetochore-microtubule half-life and poleward flux revealed that CLASP2 regulates kinetochore-microtubule dynamics by integrating distinctive microtubule-binding properties at the kinetochore-microtubule interface. We propose that kinetochore CLASP2 suppresses microtubule depolymerization and detachment by binding to curved protofilaments at microtubule plus-ends., (© 2019 Girão et al.)

In contrast to many protein translocases that use ATP or GTP hydrolysis as the driving force to transport proteins across biological membranes, the peroxisomal matrix protein import machinery relies on a regulated self-assembly mechanism for this purpose and uses ATP hydrolysis only to reset its components. The ATP-dependent protein complex in charge of resetting this machinery-the Receptor Export Module (REM)-comprises two members of the "ATPases Associated with diverse cellular Activities" (AAA+) family, PEX1 and PEX6, and a membrane protein that anchors the ATPases to the organelle membrane. In recent years, a large amount of data on the structure/function of the REM complex has become available. Here, we discuss the main findings and their mechanistic implications.

Additive manufacturing has revolutionized the manufacturing paradigm in recent years due to the possibility of creating complex shaped three-dimensional parts which can be difficult or impossible to obtain by conventional manufacturing processes. Among the different additive manufacturing techniques, wire and arc additive manufacturing (WAAM) is suitable to produce large metallic parts owing to the high deposition rates achieved, which are significantly larger than powder-bed techniques, for example. The interest in WAAM is steadily increasing, and consequently, significant research efforts are underway. This review paper aims to provide an overview of the most significant achievements in WAAM, highlighting process developments and variants to control the microstructure, mechanical properties, and defect generation in the as-built parts; the most relevant engineering materials used; the main deposition strategies adopted to minimize residual stresses and the effect of post-processing heat treatments to improve the mechanical properties of the parts. An important aspect that still hinders this technology is certification and nondestructive testing of the parts, and this is discussed. Finally, a general perspective of future advancements is presented.

Addition of follicular fluid to oocyte maturation medium can affect cumulus cell function, increase competence of the oocytes to be fertilised and develop to the blastocyst stage and protect the oocyte from heat shock. Here, it was tested whether exosomes in follicular fluid are responsible for the effects of follicular fluid on the function of the cumulus-oocyte complex (COC). This was accomplished by culturing COCs during oocyte maturation at 38.5°C (body temperature of the cow) or 41°C (heat shock) with follicular fluid or exosomes derived from follicular fluid and evaluating various aspects of function of the oocyte and the embryo derived from it. Negative effects of heat shock on cleavage and blastocyst development, but not cumulus expansion, were reduced by follicular fluid and exosomes. The results support the idea that exosomes in follicular fluid play important roles during oocyte maturation to enhance oocyte function and protect it from stress.

Despite having a membrane that is impermeable to all but the smallest of metabolites, peroxisomes acquire their newly synthesized (cytosolic) matrix proteins in an already folded conformation. In some cases, even oligomeric proteins have been reported to translocate the organelle membrane. The protein sorting machinery that accomplishes this feat must be rather flexible and, unsurprisingly, several of its key components have large intrinsically disordered domains. Here, we provide an overview on these domains and their interactions trying to infer their functional roles in this protein sorting pathway., (© 2018 Federation of European Biochemical Societies.)

PEX13 and PEX14 are two core components of the so-called peroxisomal docking/translocation module, the transmembrane hydrophilic channel through which newly synthesized peroxisomal proteins are translocated into the organelle matrix. The two proteins interact with each other and with PEX5, the peroxisomal matrix protein shuttling receptor, through relatively well characterized domains. However, the topologies of these membrane proteins are still poorly defined. Here, we subjected proteoliposomes containing PEX13 or PEX14 and purified rat liver peroxisomes to protease-protection assays and analyzed the protected protein fragments by mass spectrometry, Edman degradation and western blotting using antibodies directed to specific domains of the proteins. Our results indicate that PEX14 is a bona fide intrinsic membrane protein with a N in -C out topology, and that PEX13 adopts a N out -C in topology, thus exposing its carboxy-terminal Src homology 3 [SH3] domain into the organelle matrix. These results reconcile several enigmatic findings previously reported on PEX13 and PEX14 and provide new insights into the organization of the peroxisomal protein import machinery. ENZYMES: Trypsin, EC3.4.21.4; Proteinase K, EC3.4.21.64; Tobacco etch virus protease, EC3.4.22.44., (© 2018 Federation of European Biochemical Societies.)

Background: The impact of rapid on-site evaluation (ROSE) on thyroid aspirates has been a matter of extensive debate. In the current study, the authors reviewed all thyroid fine-needle aspiration biopsies (FNABs) performed in their service in recent years to evaluate the impact of ROSE on final adequacy and diagnostic rates., Methods: All ultrasound-guided FNABs of the thyroid performed between July 2015 and July 2017 were included retrospectively. ROSE was performed by experienced cytopathologists, with production of Romanowsky-stained slides for immediate evaluation. When ROSE was not performed, a total of 3 needle passes were performed as the default. Final specimen adequacy and the risk of malignancy (ROM) of each The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) category were calculated in the 2 groups (ROSE and non-ROSE) and compared using the chi-square test., Results: An initial search obtained 4649 cytology specimens, 3469 of which (74.6%) underwent ROSE and 1180 of which (25.4%) did not. Patients were predominantly female (85.4%), with a mean age of 53 years. Specimen adequacy was found to be significantly higher in the ROSE group (93.4% vs 69.4%; P<.0001), with a mean number of needle passes necessary for an adequate diagnosis of 1.48 ± 0.71 (median, 1.0 needle passes; range, 1-5 needle passes). No statistical difference was observed with regard to the ROM for each TBSRTC category when the 2 groups (ROSE and non-ROSE) were compared., Conclusions: The current study data support ROSE as a valuable technique in thyroid FNAB. It was proven to significantly improve specimen adequacy with a decreased mean number of needle passes necessary to achieve an adequate cytological diagnosis and no impact on the ROM for any TBSRTC category., (© 2018 American Cancer Society.)

Introduction: Strontium ranelate (SrRan) has the potential to interfere in the progression of osteoarthritis (OA), multifactorial disease associated with mechanical problems and articular inflammatory changes. Objectives: This study aimed to test the effects of prophylactic and therapeutic use of SrRan on clinical parameters of pain, the inflammatory process, and degradation of the articular cartilage. Methods: This was an experimental study, using a model of knee OA induced by intra-articular injection of monoiodoacetate. Thirty Wistar rats were divided into five groups and treated as indicated: control, without intervention; prophylactic, received SrRan at a daily oral dose of 250 mg/kg for 28 days before OA induction; SrRan treatments, administered 250 or 500 mg/kg/day for 28 days after the induction; and model control, received saline solution after the induction. Behavioral tests (joint incapacity, mechanical hyperalgesia, tactile sensitivity, and forced ambulation), histological evaluation of articular cartilage, and determination of inflammatory cytokines in the synovial fluid (interleukin [IL]-6, IL-10, tumor necrosis factor [TNF]-α, and interferon [INF]-γ) were performed. Results: Both prophylactic and therapeutic treatments improved the articular discomfort. A prophylactic dose of 500 mg/kg/day also improved mechanical hyperalgesia and the same dose was beneficial on tactile sensitivity. SrRan did not improve ambulation. Levels of IL-6, IL-10, TNF-α, and IFN-γ in SrRan-treated groups with OA were not significantly different compared with those in the normal control animals. The histopathological evaluation showed less articular damage in the SrRan-treated and control groups compared to the saline-treated group. Conclusion: The prophylactic and therapeutic administration of SrRan was associated with improved behavioral patterns of pain, especially joint discomfort. SrRan administration mitigated histological changes in the articular cartilage and reduced the inflammatory process, which beneficially reduced the progression of OA in the experimental model studied.

The cellular mechanisms induced by elevated temperature on oocytes are not fully understood. However, there is evidence that some of the deleterious effects of heat shock are mediated by a heat-induced increase in reactive oxygen species (ROS). In this context, carotenoid antioxidants might have a thermoprotective effect. Therefore, the objective of this study was to determine the role of astaxanthin (AST) on oocyte ROS production and on the redox profile and developmental competency of cumulus-oocyte complexes (COCs) after 14h heat shock (41°C) during in vitro maturation (IVM). Exposure of oocytes to heat shock during IVM increased ROS and reduced the ability of the oocyte to cleave and develop to the blastocyst stage. However, 12.5 and 25nM astaxanthin rescued these negative effects of heat shock; astaxanthin counteracted the heat shock-induced increase in ROS and restored oocyte developmental competency. There was no effect of astaxanthin on maturation medium lipid peroxidation or on glutathione peroxidase and catalase activity in oocytes and cumulus cells. However, astaxanthin stimulated superoxide dismutase (SOD) activity in heat-shocked cumulus cells. In conclusion, direct heat shock reduced oocyte competence, which was restored by astaxanthin, possibly through regulation of ROS and SOD activity in oocytes and COCs.

PEX1 and PEX6 are two members of the A TPases a ssociated with diverse cellular a ctivities (AAA) family and the core components of the receptor export module of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein-shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation can start. Recent data have shown that PEX1 and PEX6 form a heterohexameric complex that unfolds substrates by processive threading. However, whether the natural substrate of the PEX1-PEX6 complex is monoubiquitinated PEX5 (Ub-PEX5) itself or some Ub-PEX5-interacting component(s) of the DTM remains unknown. In this work, we used an established cell-free in vitro system coupled with photoaffinity cross-linking and protein PEGylation assays to address this problem. We provide evidence suggesting that DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through its ubiquitin moiety and that the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event. These findings strongly suggest that DTM-embedded Ub-PEX5 is a bona fide substrate of the PEX1-PEX6 complex., (© 2018 Pedrosa et al.)

Considering that osteoarthritis (OA) is the most prevalent joint disease worldwide, multiple pharmacological treatments have been proposed to alter the articular structure with potential benefit in the progression of the disease. The so-called disease-modifying OA drugs have been frequently investigated but conclusive findings are rare. Strontium ranelate (SrRan) is a drug usually prescribed to treat osteoporosis, with proven effects in decreasing the risk of fractures and possible effect in reducing the progression of OA. The objective of this review was to demonstrate the current panorama of knowledge on the use of SrRan in clinical and experimental models, clarifying its mechanisms of action and describing possible anti-nociceptive and anti-inflammatory effects. The systematic review was based on the PRISMA statement and included articles that are indexed in scientific databases. Fifteen studies were included: seven pre-clinical and eight clinical studies. Despite the limited number of studies, the results suggest a positive effect of SrRan in patients with OA, through changes in functional capacity and reduction of progression of morphological parameters and joint degradation, with moderate quality of evidence for those clinical outcomes. Novel studies are necessary to elucidate the molecular targets of SrRan, focusing on anti-inflammatory effects and histological changes promoted by SrRan, which seemed to reduce the progression of OA in the experimental and clinical studies.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and rapidly transported into the organelle by a complex machinery. The data gathered in recent years suggest that this machinery operates through a syringe-like mechanism, in which the shuttling receptor PEX5 - the "plunger" - pushes a newly synthesized protein all the way through a peroxisomal transmembrane protein complex - the "barrel" - into the matrix of the organelle. Notably, insertion of cargo-loaded receptor into the "barrel" is an ATP-independent process, whereas extraction of the receptor back into the cytosol requires its monoubiquitination and the action of ATP-dependent mechanoenzymes. Here, we review the main data behind this model., (© 2017 WILEY Periodicals, Inc.)

Background and Purpose: Robot-assisted locomotor training on a bodyweight-supported treadmill is a rehabilitation intervention that compels repetitive practice of gait movements. Standard treadmill speed may elicit rhythmic movements generated primarily by spinal circuits. Slower-than-standard treadmill speed may elicit discrete movements, which are more complex than rhythmic movements and involve cortical areas., Objective: Compare effects of fast (i.e., rhythmic) versus slow (i.e., discrete) robot-assisted locomotor training on a bodyweight-supported treadmill in subjects with chronic, severe gait deficit after stroke., Methods: Subjects (N = 18) were randomized to receive 30 sessions (5 d/wk) of either fast or slow robot-assisted locomotor training on a bodyweight-supported treadmill in an inpatient setting. Functional ambulation category, time up and go, 6-min walk test, 10-m walk test, Berg Balance Scale, and Fugl-Meyer Assessment were administered at baseline and postintervention., Results: The slow group had statistically significant improvement on functional ambulation category (first quartile-third quartile, P = 0.004), 6-min walk test (95% confidence interval [CI] = 1.8 to 49.0, P = 0.040), Berg Balance Scale (95% CI = 7.4 to 14.8, P < 0.0001), time up and go (95% CI = -79.1 to 5.0, P < 0.0030), and Fugl-Meyer Assessment (95% CI = 24.1 to 45.1, P < 0.0001). The fast group had statistically significant improvement on Berg Balance Scale (95% CI = 1.5 to 10.5, P = 0.02)., Conclusions: In initial stages of robot-assisted locomotor training on a bodyweight-supported treadmill after severe stroke, slow training targeting discrete movement may yield greater benefit than fast training.

A remarkable property of the machinery for import of peroxisomal matrix proteins is that it can accept already folded proteins as substrates. This import involves binding of newly synthesized proteins by cytosolic peroxisomal biogenesis factor 5 (PEX5) followed by insertion of the PEX5-cargo complex into the peroxisomal membrane at the docking/translocation module (DTM). However, how these processes occur remains largely unknown. Here, we used truncated PEX5 molecules to probe the DTM architecture. We found that the DTM can accommodate a larger number of truncated PEX5 molecules comprising amino acid residues 1-197 than full-length PEX5 molecules. A shorter PEX5 version (PEX5(1-125)) still interacted correctly with the DTM; however, this species was largely accessible to exogenously added proteinase K, suggesting that this protease can access the DTM occupied by a small PEX5 protein. Interestingly, the PEX5(1-125)-DTM interaction was inhibited by a polypeptide comprising PEX5 residues 138-639. Apparently, the DTM can recruit soluble PEX5 through interactions with different PEX5 domains, suggesting that the PEX5-DTM interactions are to some degree fuzzy. Finally, we found that the interaction between PEX5 and PEX14, a major DTM component, is stable at pH 11.5. Thus, there is no reason to assume that the hitherto intriguing resistance of DTM-bound PEX5 to alkaline extraction reflects its direct contact with the peroxisomal lipid bilayer. Collectively, these results suggest that the DTM is best described as a large cavity-forming protein assembly into which cytosolic PEX5 can enter to release its cargo., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Strontium ranelate (SrRan) is a drug usually prescribed to treat osteoporosis, with proven effects of decreasing the risk of fractures and an indication of reducing the progression of osteoarthritis (OA). This study aimed to investigate the effects of SrRan as either a prophylactic or a treatment drug, using an OA rat model to assess pain behavior. A monoiodoacetate (MIA)-induced knee joint OA model in Wistar rats was used. Thirty Wistar rats (both sexes, 60 days old) were distributed in five groups of 6 rats each: the control group, that received no intervention; a prophylactic group, that received oral administration of 25 mg·kg-1·day-1 of SrRan for 28 days before induction of OA; a group treated with 25 mg·kg-1·day-1 of SrRan for 28 days after OA induction; a group treated with 50 mg·kg-1·day-1 during 28 days after OA induction; and a group that received oral saline for 28 days after induction. The assessment of pain behavior was performed considering articular incapacitation (weight-bearing test), mechanical hyperalgesia (Randall Selitto test) and motor activity (rotarod test), on days 0, 7, 14, 21, and 28. This experiment did not yield a significant difference when comparing the group that received SrRan prophylactically with the groups treated with 25 or 50 mg·kg-1·day-1 and the group that received oral saline. Thus, SrRan did not provide analgesia in either treated rats or as a prophylactic drug with the tested doses. Higher doses should be tested further to achieve possible significant results.

Protease protection assays are powerful tools to determine the topology of organelle proteins. Their simplicity, together with the fact that they are particularly suited to characterize endogenous proteins, are their major advantages and the reason why these assays have been in use for so many years. Here, we provide a detailed protocol to use with mammalian peroxisomes. Suggestions on how these assays can be controlled, and how to identify some technical pitfalls, are also presented.