Correction: Pinging the brain with visual impulses reveals electrically active, not activity-silent, working memories.

[This corrects the article DOI: 10.1371/journal.pbio.3001436.].

Polo regulates Spindly to prevent premature stabilization of kinetochore-microtubule attachments.

Accurate chromosome segregation in mitosis requires sister kinetochores to bind to microtubules from opposite spindle poles. The stability of kinetochore-microtubule attachments is fine-tuned to prevent or correct erroneous attachments while preserving amphitelic interactions. Polo kinase has been implicated in both stabilizing and destabilizing kinetochore-microtubule attachments. However, the mechanism underlying Polo-destabilizing activity remains elusive. Here, resorting to an RNAi screen in Drosophila for suppressors of a constitutively active Polo mutant, we identified a strong genetic interaction between Polo and the Rod-ZW10-Zwilch (RZZ) complex, whose kinetochore accumulation has been shown to antagonize microtubule stability. We find that Polo phosphorylates Spindly and impairs its ability to bind to Zwilch. This precludes dynein-mediated removal of the RZZ from kinetochores and consequently delays the formation of stable end-on attachments. We propose that high Polo-kinase activity following mitotic entry directs the RZZ complex to minimize premature stabilization of erroneous attachments, whereas a decrease in active Polo in later mitotic stages allows the formation of stable amphitelic spindle attachments. Our findings demonstrate that Polo tightly regulates the RZZ-Spindly-dynein module during mitosis to ensure the fidelity of chromosome segregation.

Pinging the brain with visual impulses reveals electrically active, not activity-silent, working memories.

Persistently active neurons during mnemonic periods have been regarded as the mechanism underlying working memory maintenance. Alternatively, neuronal networks could instead store memories in fast synaptic changes, thus avoiding the biological cost of maintaining an active code through persistent neuronal firing. Such "activity-silent" codes have been proposed for specific conditions in which memories are maintained in a nonprioritized state, as for unattended but still relevant short-term memories. A hallmark of this "activity-silent" code is that these memories can be reactivated from silent, synaptic traces. Evidence for "activity-silent" working memory storage has come from human electroencephalography (EEG), in particular from the emergence of decodability (EEG reactivations) induced by visual impulses (termed pinging) during otherwise "silent" periods. Here, we reanalyze EEG data from such pinging studies. We find that the originally reported absence of memory decoding reflects weak statistical power, as decoding is possible based on more powered analyses or reanalysis using alpha power instead of raw voltage. This reveals that visual pinging EEG "reactivations" occur in the presence of an electrically active, not silent, code for unattended memories in these data. This crucial change in the evidence provided by this dataset prompts a reinterpretation of the mechanisms of EEG reactivations. We provide 2 possible explanations backed by computational models, and we discuss the relationship with TMS-induced EEG reactivations.

Walking the talk for dementia: A unique immersive, embodied, and multi-experiential initiative.

Coping with dementia requires an integrated approach encompassing personal, health, research, and community domains. Here we describe "Walking the Talk for Dementia," an immersive initiative aimed at empowering people with dementia, enhancing dementia understanding, and inspiring collaborations. This initiative involved 300 participants from 25 nationalities, including people with dementia, care partners, clinicians, policymakers, researchers, and advocates for a 4-day, 40 km walk through the Camino de Santiago de Compostela, Spain. A 2-day symposium after the journey provided novel transdisciplinary and horizontal structures, deconstructing traditional hierarchies. The innovation of this initiative lies in its ability to merge a physical experience with knowledge exchange for diversifying individuals' understanding of dementia. It showcases the transformative potential of an immersive, embodied, and multi-experiential approach to address the complexities of dementia collaboratively. The initiative offers a scalable model to enhance understanding, decrease stigma, and promote more comprehensive and empathetic dementia care and research.

A practical guide for studying human behavior in the lab.

In the last few decades, the field of neuroscience has witnessed major technological advances that have allowed researchers to measure and control neural activity with great detail. Yet, behavioral experiments in humans remain an essential approach to investigate the mysteries of the mind. Their relatively modest technological and economic requisites make behavioral research an attractive and accessible experimental avenue for neuroscientists with very diverse backgrounds. However, like any experimental enterprise, it has its own inherent challenges that may pose practical hurdles, especially to less experienced behavioral researchers. Here, we aim at providing a practical guide for a steady walk through the workflow of a typical behavioral experiment with human subjects. This primer concerns the design of an experimental protocol, research ethics, and subject care, as well as best practices for data collection, analysis, and sharing. The goal is to provide clear instructions for both beginners and experienced researchers from diverse backgrounds in planning behavioral experiments.

A cerumenolomic approach to bovine trypanosomosis diagnosis.

INTRODUCTION: Trypanosomiasis caused by Trypanosoma vivax (T. vivax, subgenus Duttonella) is a burden disease in bovines that induces losses of billions of dollars in livestock activity worldwide. To control the disease, the first step is identifying the infected animals at early stages. However, convention tools for animal infection detection by T. vivax present some challenges, facilitating the spread of the pathogenesis. OBJECTIVES: This work aims to develop a new procedure to identify infected bovines by T. vivax using cerumen (earwax) in a volatilomic approach, here named cerumenolomic, which is performed in an easy, quick, accurate, and non-invasive manner. METHODS: Seventy-eight earwax samples from Brazilian Curraleiro Pé-Duro calves were collected in a longitudinal study protocol during health and inoculated stages. The samples were analyzed using Headspace/Gas Chromatography-Mass Spectrometry followed by multivariate analysis approaches. RESULTS: The cerumen analyses lead to the identification of a broad spectrum of volatile organic metabolites (VOMs), of which 20 VOMs can discriminate between healthy and infected calves (AUC = 0.991, sensitivity = 0.967, specificity = 1.000). Furthermore, 13 VOMs can indicate a pattern of discrimination between the acute and chronic phases of the T. vivax infection in the animals (AUC = 0.989, sensitivity = 0.944, specificity = 1.000). CONCLUSION: The cerumen volatile metabolites present alterations in their occurrence during the T.vivax infection, which may lead to identifying the infection in the first weeks of inoculation and discriminating between the acute and chronic phases of the illness. These results may be a breakthrough to avoid the T. vivax outbreak and provide a faster clinical approach to the animal.

Crystallographic approach to fragment-based hit discovery against Schistosoma mansoni purine nucleoside phosphorylase.

Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.

A systemic approach to the quantification of the phenotypic plasticity of plant physiological traits: the multivariate plasticity index.

The phenotype of an individual emerges from the interaction of its genotype with the environment in which it is located. Phenotypic plasticity (PP) is the ability of a specific genotype to present multiple phenotypes in response to the environment. Past and current methods for quantification of PP present limitations, mainly in what constitutes a systemic analysis of multiple traits. This research proposes an integrative index for quantifying and evaluating PP. The multivariate plasticity index (MVPi) was calculated based on the Euclidian distance between scores of a canonical variate analysis. It was evaluated for leaf physiological traits in two cases using Brazilian Cerrado species and sugarcane varieties, grown under diverse environmental conditions. The MVPi was sensitive to plant behaviour from simple to complex genotype-environment interactions and was able to inform coarse and fine changes in PP. It was correlated to biomass allocation, showing agreement between plant organizational levels. The new method proved to be elucidative of plant metabolic changes, mainly by explaining PP as an integrated process and emergent property. We recommend the MVPi method as a tool for analysis of phenotypic plasticity in the context of a systemic evaluation of plant phenotypic traits.

Dissecting protein domain variability in the core RNA interference machinery of five insect orders.

RNA interference (RNAi)-mediated gene silencing can be used to control specific insect pest populations. Unfortunately, the variable efficiency in the knockdown levels of target genes has narrowed the applicability of this technology to a few species. Here, we examine the current state of knowledge regarding the miRNA (micro RNA) and siRNA (small interfering RNA) pathways in insects and investigate the structural variability at key protein domains of the RNAi machinery. Our goal was to correlate domain variability with mechanisms affecting the gene silencing efficiency. To this end, the protein domains of 168 insect species, encompassing the orders Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera, were analysed using our pipeline, which takes advantage of meticulous structure-based sequence alignments. We used phylogenetic inference and the evolutionary rate coefficient (K) to outline the variability across domain regions and surfaces. Our results show that four domains, namely dsrm, Helicase, PAZ and Ribonuclease III, are the main contributors of protein variability in the RNAi machinery across different insect orders. We discuss the potential roles of these domains in regulating RNAi-mediated gene silencing and the role of loop regions in fine-tuning RNAi efficiency. Additionally, we identified several order-specific singularities which indicate that lepidopterans have evolved differently from other insect orders, possibly due to constant coevolution with plants and viruses. In conclusion, our results highlight several variability hotspots that deserve further investigation in order to improve the application of RNAi technology in the control of insect pests.

Mix of destinations and sedentary behavior among Brazilian adults: a cross-sectional study.

BACKGROUND: Sedentary behavior is influenced by contextual, social, and individual factors, including the built environment. However, associations between the built environment and sitting time have not been extensively investigated in countries with economies in transition such as Brazil. The objective of this study is to examine the relationship between sitting-time and access to a mix of destinations for adults from Sao Paulo city, Brazil. METHODS: This study uses data from the Health Survey of Sao Paulo. Sedentary behavior was assessed by a questionnaire using two questions: total sitting time in minutes on a usual weekday; and on a usual weekend day. The mix of destinations was measured by summing the number of facilities (comprising bus stops, train/subway stations, parks, squares, public recreation centres, bike paths, primary health care units, supermarkets, food stores, bakeries, and coffee-shops) within 500 m of each participant's residence. Minutes of sitting time in a typical weekday and weekend day were the outcomes and the mix of destinations score in 500 m buffers was the exposure variable. Associations between the mix of destinations and sitting time were examined using multilevel linear regression: these models accounted for clustering within census tracts and households and adjusted for environmental, sociodemographic, and health-related factors. RESULTS: After adjustment for covariates, the mix of destinations was inversely associated with minutes of sitting time on a weekday (ß=- 8.8, p=0.001) and weekend day (ß=- 6.1, p=0.022). People who lived in areas with a greater mix of destinations had shorter average sitting times. CONCLUSION: Greater mix of destinations within 500 m of peoples' residences was inversely associated with sitting time on a typical weekday and weekend day. In Latin American cities like Sao Paulo built environments more favorable for walking may contribute to reducing sedentary behavior and prevent associated chronic disease.

Identification of cattle poisoning by Bifenthrin via earwax analysis by HS/GC-MS.

The use of pyrethroids has increased over recent years, and corresponds to a higher exposure of animals to pesticide residues in the environment and diet. Here, an outbreak of pyrethroid poisoning in beef cattle was reported occurring in Midwestern Brazil. After veterinary evaluation, it was observed that the bovines presented common pyrethroid intoxication symptoms. Aiming to identify the cattle poising by pyrethroid, earwax samples were collected from two groups: exposed and nonexposed animals from the same farm. Blind earwax analyses of the bovines were carried out using headspace/gas chromatography-mass spectrometry (HS/GC-MS). The HS/GC-MS analysis detected the presence of bifenthrin in the earwax analysis of the exposed animals, confirmed by the comparison of its MS fragments with a bifenthrin standard, and also by its retention time relative to the internal standard. In summary, HS/GC-MS analysis of earwax emerges as a tool that can be used in the detection and monitoring of bifenthrin poisoning in cattle, as a useful veterinary diagnosis that ensures animal health and the safety of their products.

The structure of the extended E2 DNA-binding domain of the bovine papillomavirus-1.

Bovine papillomavirus proteins were extensively studied as a prototype for the human papillomavirus. Here, the crystal structure of the extended E2 DNA-binding domain of the dominant transcription regulator from the bovine papillomavirus strain 1 is described in the space group P31 21. We found two protein functional dimers packed in the asymmetric unit. This new protein arrangement inside the crystal led to the reduction of the mobility of a previously unobserved loop directly involved in the protein-DNA interaction, which was then modeled for the first time.

A volatolomic approach using cerumen as biofluid to diagnose bovine intoxication by Stryphnodendron rotundifolium.

An innovative volatolomic approach employs the detection of biomarkers present in cerumen (earwax) to identify cattle intoxication by Stryphnodendron rotundifolium Mart., Fabaceae (popularly known as barbatimão). S. rotundifolium is a poisonous plant with the toxic compound undefined and widely distributed throughout the Brazilian territory. Cerumen samples from cattle of two local Brazilian breeds ('Curraleiro Pé-Duro' and 'Pantaneiro') were collected during an experimental intoxication protocol and analyzed using headspace (HS)/GC-MS followed by multivariate analysis (genetic algorithm for a partial least squares, cluster analysis, and classification and regression trees). A total of 106 volatile organic metabolites were identified in the cerumen samples of bovines. The intoxication by S. rotundifolium influenced the cerumen volatolomic profile of the bovines throughout the intoxication protocol. In this way, it was possible to detect biomarkers for cattle intoxication. Among the biomarkers, 2-octyldecanol and 9-tetradecen-1-ol were able to discriminate all samples between intoxicated and nonintoxicated bovines. The cattle intoxication diagnosis by S. rotundifolium was accomplished by applying the cerumen analysis using HS/GC-MS, in an easy, accurate, and noninvasive way. Thus, the proposed bioanalytical chromatography protocol is a useful tool in veterinary applications to determine this kind of intoxication.

Crystallographic structure of a complex between trypsin and a nonapeptide derived from a Bowman-Birk inhibitor found in Vigna unguiculata seeds.

Natural inhibitors of proteases have been classified into different families, among them is the Bowman-Birk Inhibitor (BBI) family. Members of BBI have two structurally reactive loops that simultaneously inhibit trypsin and chymotrypsin. Here, we have investigated the binding of bovine trypsin by a cyclic nonapeptide, named PTRY9 (CTKSIPPQC), derived of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) from Vigna unguiculata seeds. This peptide was synthetically produced with the disulfide bond restraining its conformation to mimic the reactive loop that inhibits trypsin. PTRY9 complexed to pancreatic bovine trypsin was crystallized in orthorhombic and trigonal space groups, P212121 and P3221, with maximum resolutions of 1.15 and 1.61 Å, respectively. The structures presented refinement parameters of Rwork = 14.52 % and Rfree = 15.59 %; Rwork = 15.60 % and Rfree = 18.78 %, and different surface area between the peptide and the enzyme of 1024 Å2 and 1070 Å2, respectively. The binding site of the PTRY9 is similar to that found for BTCI as shown by a r.m.s.d. of 0.358 Šbetween the superimposed structures and the electrostatic complementary pattern at the enzyme-peptide interface. Additionally, enzyme inhibition assays show that the affinity of trypsin for PTRY9 is smaller than that for BTCI. In vitro assays revealed that, like BTCI, this synthetic peptide is not cytotoxic for normal mammary epithelial MCF-10A cells, but exerts cytotoxic effects on MDA.MB.231 invasive human breast cancer cells.

Full-arch implant-supported rehabilitations: A prospective study comparing porcelain-veneered zirconia frameworks to monolithic zirconia.

OBJECTIVES: To evaluate the performance of two types of zirconia frameworks. MATERIAL AND METHODS: From 2014 to 2016, in a prospective clinical trial, 150 patients were rehabilitated with 83 and 110 implant-supported, screw-retained, full-arch ceramic-veneered zirconia (PVZ) rehabilitations and monolithic zirconia with porcelain veneering limited to buccal (MZ) rehabilitations, respectively. Patients were consecutively enlisted according to pre-defined inclusion criteria and evaluated on 4 months intervals. A Kaplan-Meier estimator was adopted, and the log-rank test and Wilcoxon test used to test differences in survival and successful function in the two different groups. RESULTS: The average follow-up time (±SD) and implant success rate was 608.80 ± 172.52 days with 99.53% implant success for the PVZ group and 552.63 ± 197.57 days with 99.83% success for the MZ group. According to the Kaplan-Meier estimator, the mean cumulative survival rate at the 2-year follow-up for framework fracture, major chipping, minor chipping, or any of the former combined to occur was 0.99, 0.95, 0.93 and 0.85 for the PVZ group (n = 18) and 0.99, 0.95, 0.95 and 0.89 for the MZ group (n = 15). No significant differences were found between the two groups. CONCLUSIONS: Results suggest zirconia as a suitable material for frameworks in full-arch implant-supported rehabilitations. Both groups presented a low incidence of technical complications. When comparing the two different designs, the MZ group presented a lower technical complication rate, thus presenting itself as a viable alternative for full-arch implant-supported rehabilitations. Further clinical studies with longer follow-ups (5 years) should be performed to evaluate the long-term stability of such rehabilitations.

Schoolbag weight carriage in Portuguese children and adolescents: a cross-sectional study comparing possible influencing factors.

BACKGROUND: Schoolbags and the consequences of carrying them, particularly those associated with overload, are often studied as a health concern. Modifications in gait and posture were reported when children carried loads that corresponded to more than 10% of their body weight (BW). The aims of this study were to verify the load that is carried by Portuguese students and how it is influenced by factors such as school grade, school schedule, lunch site, physical education, sex and body mass index (BMI). Acquiring a more specific knowledge of the Portuguese context and understanding the influence of these factors may allow us to generate proposals to control them in ways that benefit students. METHODS: The load carried by students in the 5th grade (10.6 ± 0.4 years) and 9th grade (14.7 ± 0.6 years) were weighed with a luggage scale on all days of the week, resulting in 680 evaluations. Data related to the school day were also collected, such as the student's lunch site, how he or she got to school and his or her school schedule for that day. Individual height and weight were also assessed. RESULTS: The 5th grade students carried greater loads than the 9th grade students, resulting in a substantial difference relative to their BW. The school loads of the 5th grade students were mostly greater than 10% of their BWs. Girls tended to carry heavier loads than boys, and overweight students also tended to carry heavier loads. Students who could eat lunch at home carried less weight, and on physical education days, the total load carried increased, but the backpacks of the 5th grade students were lighter. CONCLUSIONS: The results of the current study describe excessive schoolbag weight among Portuguese students and expound on some of the factors that influence it, which can help researchers and professionals design a solution to decrease children's schoolbag loads.

Predictors of complication after adrenalectomy.

PURPOSE: To investigate risk factors for complications in patients undergoing adrenalectomy. MATERIALS AND METHODS: A retrospective search of our institutional database was performed of patients who underwent adrenalectomy, between 2014 and 2018. Clinical parameters and adrenal disorder characteristics were assessed and correlated to intra and post-operative course. Complications were analyzed within 30-days after surgery. A logistic regression was performed in order to identify independent predictors of morbidity in patients after adrenalectomy. RESULTS: The files of 154 patients were reviewed. Median age and Body Mass Index (BMI) were 52-years and 27.8kg/m2, respectively. Mean tumor size was 4.9±4cm. Median surgery duration and estimated blood loss were 140min and 50mL, respectively. There were six conversions to open surgery. Minor and major post-operative complications occurred in 17.5% and 8.4% of the patients. Intra-operative complications occurred in 26.6% of the patients. Four patients died. Mean hospitalization duration was 4-days (Interquartile Range: 3-8). Patients age (p=0.004), comorbidities (p=0.003) and pathological diagnosis (p=0.003) were independent predictors of post-operative complications. Tumor size (p<0.001) and BMI (p=0.009) were independent predictors of intra-operative complications. Pathological diagnosis (p<0.001) and Charlson score (p=0.013) were independent predictors of death. CONCLUSION: Diligent care is needed with older patients, with multiple comorbidities and harboring unfavorable adrenal disorders (adrenocortical carcinoma and pheocromocytoma), who have greater risk of post-operative complications. Patients with elevated BMI and larger tumors have higher risk of intra, but not of post-operative complications.

Synthesizing the role of epigenetics in the response and adaptation of species to climate change in freshwater ecosystems.

Freshwater ecosystems are amongst the most threatened ecosystems on Earth. Currently, climate change is one of the most important drivers of freshwater transformation and its effects include changes in the composition, biodiversity and functioning of freshwater ecosystems. Understanding the capacity of freshwater species to tolerate the environmental fluctuations induced by climate change is critical to the development of effective conservation strategies. In the last few years, epigenetic mechanisms were increasingly put forward in this context because of their pivotal role in gene-environment interactions. In addition, the evolutionary role of epigenetically inherited phenotypes is a relatively recent but promising field. Here, we examine and synthesize the impacts of climate change on freshwater ecosystems, exploring the potential role of epigenetic mechanisms in both short- and long-term adaptation of species. Following this wrapping-up of current evidence, we particularly focused on bringing together the most promising future research avenues towards a better understanding of the effects of climate change on freshwater biodiversity, specifically highlighting potential molecular targets and the most suitable freshwater species for future epigenetic studies in this context.

Transgenerational Inheritance of DNA Hypomethylation in Daphnia magna in Response to Salinity Stress.

Epigenetic mechanisms have been found to play important roles in environmental stress response and regulation. These can, theoretically, be transmitted to future unexposed generations, yet few studies have shown persisting stress-induced transgenerational effects, particularly in invertebrates. Here, we focus on the aquatic microcrustacean Daphnia, a parthenogenetic model species, and its response to salinity stress. Salinity is a serious threat to freshwater ecosystems and a relevant form of environmental perturbation affecting freshwater ecosystems. We exposed one generation of D. magna to high levels of salinity (F0) and found that the exposure provoked specific methylation patterns that were transferred to the three consequent nonexposed generations (F1, F2, and F3). This was the case for the hypomethylation of six protein-coding genes with important roles in the organisms' response to environmental change: DNA damage repair, cytoskeleton organization, and protein synthesis. This suggests that epigenetic changes in Daphnia are particularly targeted to genes involved in coping with general cellular stress responses. Our results highlight that epigenetic marks are affected by environmental stressors and can be transferred to subsequent unexposed generations. Epigenetic marks could therefore prove to be useful indicators of past or historic pollution in this parthenogenetic model system. Furthermore, no life history costs seem to be associated with the maintenance of hypomethylation across unexposed generations in Daphnia following a single stress exposure.