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STUDY OBJECTIVE:s: To describe overall and income related trends in infant mortality inequalities in the Region of the Americas from 1955 to 1995. DESIGN: Infant mortality rates (IMRs) were computed and their trends assessed by ordinary least squares. Overall trends in IMR inequalities among countries were analysed by comparing 10 year period IMRs, Gini coefficients, and Lorenz curves. Income related trends in IMR inequalities were assessed using 10 year period IMR ratios between the highest and the lowest quintiles of the per capita gross national product (GNP) distributions (adjusted for purchasing power). SETTING: Aggregated country data were used for all countries with over 200 thousand inhabitants (33 geopolitical units). The 10 year period midpoint IMR estimates used for the 1955-1995 time series were those published by the United Nations in 1997. MAIN RESULTS: IMRs decreased from 90.34 to 31.31 per 1000 live births between 1955 and 1995 at an average of 15.3 every 10 years. In contrast, Lorenz curves and Gini coefficients were similar for the five 10 year periods. After grouping by adjusted GNP distribution, a similar decreasing trend of IMR was observed in all groups. The rate ratio between the group at the lowest quintile and that at the highest quintile ranged from 4 to 5. The analysis of variance for repeated observations showed that there is a significant reduction in the IMR (F=130.18; p<0.01), that trends did not differ significantly among groups (F=1.16; p=0.32), and that they were approximately linear (F=155.83; p<0.01). CONCLUSIONS: Despite a sizable reduction in the infant mortality, whether or not income related, levels of IMR inequality among countries have remained almost constant between 1955 and 1995 in the Region of the Americas. Further analysis and focused interventions are needed to tackle the challenges of reducing these persistent mortality inequalities. [ABSTRACT FROM AUTHOR]

Competing Interests: We declare no competing interests. We are responsible for the views expressed in this publication, and they do not necessarily represent the decisions or policies of our institutions.

Objective: Perfume (Parfum) or fragrance is a natural or synthetic cosmetic ingredient added to emit a pleasant aroma or to improve the odor of a cosmetic formula. It is a mixture of substances, not revealed by the manufacturer, which may contain ingredients with allergenic potential, endocrine disruptors, and other possible harmful effects on human health. This study aims to analyze children's cosmetics labels to assess the presence of Perfume., Methods: The researchers randomly visited points of sale in Curitiba, the capital of a southern Brazilian state; in order to catalog the largest possible number of children's cosmetics items., Results: 398 children's cosmetics were analyzed and found Parfum on 295 (74.1 %) of the labels, including 90.4 and 79,1 % of the shampoos and wet wipes, respectively., Conclusion: Exposure of children's skin to fragrances can lead to local side effects such as allergies, but also to systemic effects, and the lack of knowledge of the general population and health professionals about its possible deleterious effects emphasizes the importance of changes in the regulation of cosmetics aiming to reduce the use of this ingredient., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier Editora Ltda.)

In 1973, the National Rabies Program was created in Brazil through an agreement between the Ministry of Health and Agriculture. Since its beginning, it developed integrated action through access to free post-exposure prophylaxis (PEP) for people at risk, dog vaccination campaigns, a joint surveillance system, and awareness. This study aims to describe human rabies in Brazil under the One Health perspective in recent decades, including achievements in the control of dog-mediated cases and challenges in human cases transmitted by wild animals. This paper also explores possible drivers of human rabies in the Northeast Region with half of the cases. The first part of this study was descriptive, presenting data and examples by periods. Statistical analysis was performed in the last period (2010-2022) to explore possible drivers. Dog-mediated human cases decreased from 147 to 0, and dog cases decreased from 4500 to 7. A major challenge is now human cases transmitted by wild animals (bats, non-human primates, and wild canids). Most current human cases occur in municipalities with a tropical and subtropical moist broadleaf forest biome and a Gini index higher than 0.5. In the multivariable analysis, an association with temperature was estimated (OR = 1.739; CI 95% = 1.181-2.744), and primary healthcare coverage (OR = 0.947; CI 95% = 0.915-0.987) was identified as a protector. It is possible to significantly reduce the number of dog-mediated human rabies cases through the efforts presented. However, Brazil has wildlife variants of the rabies virus circulating. The association of human cases with higher temperatures in the Northeast is a concern with climate change. To reduce human cases transmitted by wild animals, it is important to continue distributing free PEP, especially in remote at-risk areas in the Amazon Region, and to increase awareness.

Matrix metalloproteinase (MMP)-sensitive hydrogels are promising for cartilage tissue engineering due to cell-mediated control over hydrogel degradation. However, any variability in MMP, tissue inhibitors of matrix metalloproteinase (TIMP), and/or extracellular matrix (ECM) production among donors will impact neotissue formation in the hydrogels. The goal for this study was to investigate the impact of inter- and intra-donor variability on the hydrogel-to-tissue transition. Transforming growth factor β3 was tethered into the hydrogel to maintain the chondrogenic phenotype and support neocartilage production, allowing the use of chemically defined medium. Bovine chondrocytes were isolated from two donor groups, skeletally immature juvenile and skeletally mature adult donors (inter-donor variability) and three donors within each group (intra-donor group variability). While the hydrogel supported neocartilaginous growth by all donors, donor age impacted MMP, TIMP, and ECM synthesis rates. Of the MMPs and TIMPs studied, MMP-1 and TIMP-1 were the most abundantly produced by all donors. Adult chondrocytes secreted higher levels of MMPs, which was accompanied by higher production of TIMPs. Juvenile chondrocytes exhibited more rapid ECM growth. By day 29, juvenile chondrocytes had surpassed the gel-to-tissue transition. On the contrary, the adult donors had a percolated polymer network indicating that despite higher levels of MMPs the gel-to-transition had not yet been achieved. The intra-donor group variability of MMP, TIMP, and ECM production was higher in adult chondrocytes but did not impact the extent of the gel-to-tissue transition. In summary, age-dependent inter-donor variations in MMPs and TIMPs significantly impact the timing of the gel-to-tissue transition in MMP-sensitive hydrogels.

Leptospirosis is a zoonotic disease that is primarily transmitted through close contact with contaminated environments or infected animals. Brazil has the highest number of reported cases of leptospirosis in the Americas (approximately 4,000 annual cases). The purpose of this study is to identify the occupational groups with a higher risk of leptospirosis in Brazil from 2010 through 2015 among suspected cases reported to the national surveillance system. Confirmed and unconfirmed cases of leptospirosis with laboratory diagnosis, 20,193 and 59,034 respectively, were classified into 12 occupational groups. Confirmed cases were predominantly male (79.4%), between 25 and 59 years of age (68.3%), white (53.4%), illiterate or with incomplete primary education (51.1%), and participating in agricultural work (19.9%). After controlling for age, sex, race, and area of residency, the multivariate analysis identified that between confirmed and unconfirmed cases of leptospirosis reported to the Brazilian national surveillance system, five occupational groups are at higher risk for leptospirosis: garbage and recycling collectors (odds ratio [OR] = 4.10; 95% CI = 3.36-4.99); agricultural, forestry, and fishery workers (OR = 1.65; 95% CI = 1.49-1.84); prisoners (OR = 1.56; 95% CI = 1.04-2.35); building workers (OR = 1.36; 95% CI = 1.22-1.51); cleaners and mining workers (OR = 1.25; 95% CI = 1.07-1.45). This is the first nationwide study to examine leptospirosis risk by occupational group in Brazil using national surveillance data. Our results suggest that among suspected cases there was an increased risk among occupational groups with low income and low educational levels.

In this work, we analyzed cytogenetically eight Chactidae and Buthidae, including the localization of repetitive DNA sequences. The chactids possess monocentric chromosomes and the highest diploid numbers (2n=50 in Brotheas amazonicus, 2n=36 in Chactopsis amazonica, 2n=30 in Neochactas sp.) when compared with buthids (2n=10 in Tityus bahiensis, 2n=14 in Tityus apiacas and Tityus metuendus, 2n=18 in Tityus aba, 2n=26 in Ischnotelson peruassu). The localization of rDNA genes and (TTAGG)n sequences exhibited a conserved pattern of two terminal/subterminal ribosomal cistrons and terminal telomere signals. However, the comparison between the data of C-banding, DAPI after FISH and Cot-DNA fraction indicated a variable quantity and distribution of these regions, as follow: (i) positive heterochromatin and Cot-DNA signals (B. amazonicus and I. peruassu), (ii) small blocks of heterochromatin with large Cot-DNA signals (T. metuendus), (iii) positive heterochromatic regions and absence of Cot-DNA signals (T. aba and T. apiacas), and (iv) negative heterochromatin and Cot-DNA signals (T. bahiensis). Therefore, our results revealed that there still is not a clear relation between quantity of heterochromatin and presence of monocentric or holocentric chromosomes and occurrence of chromosomal rearrangements, indicating that repetitive regions in scorpions must be analyzed using different cytogenetic approaches.

The COVID-19 pandemic offers an opportunity to examine public opinion regarding the allocation of scarce medical resources. In this conjoint experiment on a nationally representative sample of US adults, we examined how a range of patient characteristics affect respondents' willingness to allocate a ventilator between two patients with equal likelihood of short-term survival and how this differs by respondents' attributes. Respondents were 5.5 percentage points less likely to allocate a ventilator to a patient with a disability than to a nondisabled patient. Disability bias was correlated with older age cohorts and higher education levels of respondents. Liberal and moderate respondents were more likely to give a ventilator to Black and Asian patients than to White patients. Conservatives were much less likely to allocate a ventilator to transgender patients than to cisgender patients. These findings demonstrate the importance of bias mitigation and civil rights enforcement in health policy making, especially under conditions of scarcity.

Oropouche virus (OROV) is an emerging vector-borne arbovirus with high epidemic potential, causing illness in more than 500,000 people. Primarily contracted through its midge and mosquito vectors, OROV remains prevalent in its wild, non-human primate and sloth reservoir hosts as well. This virus is spreading across Latin America; however, the majority of cases occur in Brazil. The aim of this research is to document OROV's presence in Brazil using the One Health approach and geospatial techniques. A scoping review of the literature (2000 to 2021) was conducted to collect reports of this disease in humans and animal species. Data were then geocoded by first and second subnational levels and species to map OROV's spread. In total, 14 of 27 states reported OROV presence across 67 municipalities (second subnational level). However, most of the cases were in the northern region, within the tropical and subtropical moist broadleaf forests biome. OROV was identified in humans, four vector species, four genera of non-human primates, one sloth species, and others. Utilizing One Health was important to understand the distribution of OROV across several species and to suggest possible environmental, socioeconomic, and demographic drivers of the virus's presence. As deforestation, climate change, and migration rates increase, further study into the spillover potential of this disease is needed.

The human mind shows extraordinary capability at recognizing patterns, while at the same time tending to underestimate the natural scope of random processes. Taken together, this easily misleads researchers in judging whether the observed characteristics of their data are of significance or just the outcome of random effects. One of the best tools to assess whether observed features fall into the scope of pure randomness is statistical significance testing, which quantifies the probability to falsely reject a chosen null hypothesis. The central parameter in this context is the p -value, which can be calculated from the recorded data sets. In case of p -values smaller than the level of significance, the null hypothesis is rejected, otherwise not. While significance testing has found widespread application in many sciences including the life sciences, it is hardly used in (bio-)physics. We propose here that significance testing provides an important and valid addendum to the toolbox of quantitative (single molecule) biology. It allows to support a quantitative judgement (the hypothesis) about the data set with a probabilistic assessment. In this manuscript we describe ways for obtaining valid p -values in two selected applications of single molecule microscopy: (i) Nanoclustering in single molecule localization microscopy. Previously, we developed a method termed 2-CLASTA, which allows to calculate a valid p -value for the null hypothesis of an underlying random distribution of molecules of interest while circumventing overcounting issues. Here, we present an extension to this approach, yielding a single overall p -value for data pooled from multiple cells or experiments. (ii) Single molecule trajectories. Data from a single molecule trajectory are inherently correlated, thus prohibiting a direct analysis via conventional statistical tools. Here, we introduce a block permutation test, which yields a valid p -value for the analysis and comparison of single molecule trajectory data. We exemplify the approach based on FRET trajectories., 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 Schneider  and Schütz .)

Single molecule localization microscopy (SMLM) has the potential to resolve structural details of biological samples at the nanometer length scale. Compared to room temperature experiments, SMLM performed under cryogenic temperature achieves higher photon yields and, hence, higher localization precision. However, to fully exploit the resolution it is crucial to account for the anisotropic emission characteristics of fluorescence dipole emitters with fixed orientation. In case of slight residual defocus, localization estimates may well be biased by tens of nanometers. We show here that astigmatic imaging in combination with information about the dipole orientation allows to extract the position of the dipole emitters without localization bias and down to a precision of 1 nm, thereby reaching the corresponding Cramér Rao bound. The approach is showcased with simulated data for various dipole orientations, and parameter settings realistic for real life experiments., Competing Interests: The authors have declared that no competing interests exist.

DNA origami structures provide flexible scaffolds for the organization of single biomolecules with nanometer precision. While they find increasing use for a variety of biological applications, the functionalization with proteins at defined stoichiometry, high yield, and under preservation of protein function remains challenging. In this study, we applied single molecule fluorescence microscopy in combination with a cell biological functional assay to systematically evaluate different strategies for the site-specific decoration of DNA origami structures, focusing on efficiency, stoichiometry, and protein functionality. Using an activating ligand of the T-cell receptor (TCR) as the protein of interest, we found that two commonly used methodologies underperformed with regard to stoichiometry and protein functionality. While strategies employing tetravalent wildtype streptavidin for coupling of a biotinylated TCR-ligand yielded mixed populations of DNA origami structures featuring up to three proteins, the use of divalent (dSAv) or DNA-conjugated monovalent streptavidin (mSAv) allowed for site-specific attachment of a single biotinylated TCR-ligand. The most straightforward decoration strategy, via covalent DNA conjugation, resulted in a 3-fold decrease in ligand potency, likely due to charge-mediated impairment of protein function. Replacing DNA with charge-neutral peptide nucleic acid (PNA) in a ligand conjugate emerged as the coupling strategy with the best overall performance in our study, as it produced the highest yield with no multivalent DNA origami structures and fully retained protein functionality. With our study we aim to provide guidelines for the stoichiometrically defined, site-specific functionalization of DNA origami structures with proteins of choice serving a wide range of biological applications.

Animal stings are environmental hazards that threaten millions annually and cause a significant socioeconomic impact. Snakebite envenoming affects 2.7 million people globally every year, mostly the poorest and rural communities, with approximately 27,000 annual cases in Brazil. This study's objective is to identify the most exposed racial group for snakebites in rural areas of Brazil and analyze possible differences in the outcome of an accident. A retrospective epidemiological study was conducted using a database of rural snakebite cases from Brazil's Ministry of Health (2017). Descriptive analysis and a regression model were performed to examine the association of bad outcomes after a snakebite with several covariables. While mixed-race individuals presented the highest number of cases (61.79%), indigenous and white populations were the racial groups with the highest and lowest exposure rates (194.3 and 34.1 per 100,000 population, respectively). The fatality rate was 3.5 times higher in the indigenous population compared to the white population. In the multivariable model, the number of hours between the accident and health care received and the case classification suggested an association with a bad outcome. Snakebite is prominent in Brazil, particularly among indigenous groups. Antivenom is available in the Brazilian Health System; however, efforts need to be made for decentralization.

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.

Background: Leptospirosis is one of the most widespread zoonosis in the world and Brazil has the highest number of cases in Latin America. Transmission occurs mainly through exposure to water and soil contaminated by the urine of infected animals. The goals of this study are to describe the geographic distribution, demographic characteristics and exposure factors of urban and rural cases of leptospirosis, and identify spatial clusters in urban and rural areas of Brazil., Methods/results: A retrospective epidemiological study was carried out using 16 years (2000-2015) of surveillance data from the Brazilian Ministry of Health. Cases were described by age, sex and race, and exposure factors were characterized in urban and rural areas. A spatial autocorrelation analysis was conducted using local Moran's I to identify urban and rural clusters of disease. On average 3,810 leptospirosis cases were reported annually with higher numbers in urban areas. National urban and rural incidence rates were the same (1.9 cases/100,000 population), however, regional differences were observed. Urban incidence rates were higher in the North and Northeast regions, while rural incidence rates were higher in the Southeast and South. The main exposure factor reported in urban and rural areas was exposure to places with signs of rodents, followed by flood in urban areas and agriculture and animal farming in rural areas. Clusters of leptospirosis were identified in densely populated urban areas of the North, Southeast and South regions, while rural clusters were concentrated in of the Southern region with large agriculture and animal farming practices., Conclusions: This study highlights that leptospirosis is an important public health problem in both urban and rural areas of Brazil. The results provide decision-makers with detailed information about where disease incidence is high and can be used in the development of prevention and control strategies for priority areas and risk groups., Competing Interests: The authors have declared that no competing interests exist.

Objective: To describe a founder mutation effect and the clinical phenotype of homozygous FRRS1L c.737&#95;739delGAG (p.Gly246del) variant in 15 children of Puerto Rican (Boricua) ancestry presenting with early infantile epileptic encephalopathy (EIEE-37) with prominent movement disorder., Background: EIEE-37 is caused by biallelic loss of function variants in the FRRS1L gene, which is critical for AMPA-receptor function, resulting in intractable epilepsy and dyskinesia., Methods: A retrospective, multicenter chart review of patients sharing the same homozygous FRRS1L (p.Gly246del) pathogenic variant identified by clinical genetic testing. Clinical information was collected regarding neurodevelopmental outcomes, neuroimaging, electrographic features and clinical response to antiseizure medications., Results: Fifteen patients from 12 different families of Puerto Rican ancestry were homozygous for the FRRS1L (p.Gly246del) pathogenic variant, with ages ranging from 1 to 25 years. The onset of seizures was from 6 to 24 months. All had hypotonia, severe global developmental delay, and most had hyperkinetic involuntary movements. Developmental regression during the first year of life was common (86%). Electroencephalogram showed hypsarrhythmia in 66% (10/15), with many older children evolving into Lennox-Gastaut syndrome. Six patients demonstrated progressive volume loss and/or cerebellar atrophy on brain magnetic resonance imaging (MRI)., Conclusions: We describe the largest cohort to date of patients with epileptic encephalopathy. We estimate that 0.76% of unaffected individuals of Puerto Rican ancestry carry this pathogenic variant due to a founder effect. Children homozygous for the FRRS1L (p.Gly246del) Boricua variant exhibit a very homogenous phenotype of early developmental regression and epilepsy, starting with infantile spasms and evolving into Lennox-Gastaut syndrome with hyperkinetic movement disorder.

Background: Lassa fever (LF) is one of the most devastating rodent-borne diseases in West Africa, causing thousands of deaths annually. The geographical expansion of LF is also a concern; cases were recently identified in Ghana and Benin. Previous ecological studies have suggested that high natural-host biodiversity reduces the likelihood of spillover transmission of rodent-borne diseases, by suppressing the activities of reservoir species. However, the association of biodiversity with the geographical expansion of LF has not been the subject of epidemiological studies., Methodology/principal Findings: We conducted a spatial analysis based on sociodemographic, geographical, and ecological data, and found that higher rodent species richness was significantly associated with a lower risk of LF emergence in West Africa from 2008 to 2017 (Odds Ratio = 0.852, 95% Credible Interval = 0.745-0.971)., Conclusions/significance: The results reinforce the importance of the 'One Health' approach by demonstrating that a high level of biodiversity could benefit human health., Competing Interests: The authors have declared that no competing interests exist.

Snakebite envenoming affects close to 2.7 million people globally every year. In Brazil, snakebites are reported to the Ministry of Health surveillance system and cases receive antivenom free of charge. There is an urgent need to identify higher risk areas for antivenom distribution, and to develop prevention activities. The objective of this study is to provide an overview of the epidemiological situation of snakebite envenoming in Brazil and explore possible drivers; as well as to create a flowchart tool to support decision-makers identify higher risk areas. An ecological-type study was carried out using data by municipality (2013-2017). Study parts: 1) Create a geocoded database and perform a descriptive and cluster analysis; 2) Statistical analysis to measure the association of snakebite and possible environmental and socioeconomic drivers; 3) Develop a flowchart to support decision-makers and the application of this tool in one state (Rio Grande do Sul) as an example. An average of 27,120 snakebite cases per year were reported at the country level. Clusters of municipalities with high numbers of snakebites are mostly found in the Amazon Legal Region. The negative binomial regression model showed association with the snakebite case count: the type of major habitat, tropical or non-tropical; temperature; percentage of urbanization; precipitation; elevation; GDP per capita; a weaker relation with forest loss; and with venomous snake richness. The state where the instrument was applied reported 4,227 snakebites in the period. Most municipalities were considered as medium risk and 56/496 as high risk according to the tool created. Snakebite cases are distributed across the entire country with the highest concentration in the Legal Amazon Region. This creates a complex situation both for better understanding of the association of environmental and socioeconomic factors with snakebites and for the distribution and maintenance of antivenom to remote areas. Research into types of antivenom with a longer shelf life without the need for refrigeration is needed., Competing Interests: The authors have declared that no competing interests exist.

T cells detect with their T cell antigen receptors (TCRs) the presence of rare agonist peptide/MHC complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested to be a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. When targeting TCRs via stably binding monovalent antibody fragments, we found the minimum signaling unit promoting efficient T cell activation to consist of two antibody-ligated TCRs within a distance of 20 nm. In contrast, transiently engaging antigenic pMHCs stimulated T cells robustly as well-isolated entities. These results identify pairs of antibody-bound TCRs as minimal receptor entities for effective TCR triggering yet validate the exceptional stimulatory potency of single isolated pMHC molecules., Competing Interests: The authors declare no competing interest.

Single molecule localization microscopy (SMLM) has enormous potential for resolving subcellular structures below the diffraction limit of light microscopy: Localization precision in the low digit nanometer regime has been shown to be achievable. In order to record localization microscopy data, however, sample fixation is inevitable to prevent molecular motion during the rather long recording times of minutes up to hours. Eventually, it turns out that preservation of the sample's ultrastructure during fixation becomes the limiting factor. We propose here a workflow for data analysis, which is based on SMLM performed at cryogenic temperatures. Since molecular dipoles of the fluorophores are fixed at low temperatures, such an approach offers the possibility to use the orientation of the dipole as an additional information for image analysis. In particular, assignment of localizations to individual dye molecules becomes possible with high reliability. We quantitatively characterized the new approach based on the analysis of simulated oligomeric structures. Side lengths can be determined with a relative error of less than 1% for tetramers with a nominal side length of 5 nm, even if the assumed localization precision for single molecules is more than 2 nm., Competing Interests: The authors have declared that no competing interests exist.

Are predators of rodents beneficial for public health? This question focuses on whether predators regulate the spillover transmission of rodent-borne diseases. No clear answer has emerged because of the complex linkages across multiple trophic levels and the lack of accessible data. Although previous empirical findings have suggested ecological mechanisms, such as resource partitioning, which implies protective effects from predator species richness, epidemiological evidence is needed to bolster these arguments. Thus, we investigated the association between predator species richness and incidence of rodent-borne haemorrhagic fever with renal syndrome in the human population using district-level longitudinal data of 13 years for South Korea. With the exception of districts with low species richness, we found a significant negative association between the incidence of haemorrhagic fever with renal syndrome and the species richness of both avian and mammalian predators; the trends for both predator types were similar. Thus, biodiversity conservation may benefit public health.

USP9X is an X-chromosome gene that escapes X-inactivation. Loss or compromised function of USP9X leads to neurodevelopmental disorders in males and females. While males are impacted primarily by hemizygous partial loss-of-function missense variants, in females de novo heterozygous complete loss-of-function mutations predominate, and give rise to the clinically recognisable USP9X-female syndrome. Here we provide evidence of the contribution of USP9X missense and small in-frame deletion variants in USP9X-female syndrome also. We scrutinise the pathogenicity of eleven such variants, ten of which were novel. Combined application of variant prediction algorithms, protein structure modelling, and assessment under clinically relevant guidelines universally support their pathogenicity. The core phenotype of this cohort overlapped with previous descriptions of USP9X-female syndrome, but exposed heightened variability. Aggregate phenotypic information of 35 currently known females with predicted pathogenic variation in USP9X reaffirms the clinically recognisable USP9X-female syndrome, and highlights major differences when compared to USP9X-male associated neurodevelopmental disorders.

Hantavirus infection is a rodent-borne disease (RBD) transmitted by urine or feces (as the natural reservoirs) with an annual estimated worldwide incidence of ~150,000 cases. Previous ecological studies suggested that higher species richness of rodents could decrease the risk of RBDs in humans, because the regulatory pressure of predators could reduce contact among rodents, and between humans and rodents. Using surveillance data, we investigated the association of predator species richness with hantavirus infection emergence in 5562 Brazilian inland municipalities between 2007 and 2017. Multivariable logistic regression models were used for the analyses. Diurnal and non-diurnal predator species were independent explanatory variables in the models. Rodent species richness and demographic, socioeconomic, and environmental factors were used as covariates. During the study period, 1084 cases were reported in 429 municipalities. The results showed a reverse-U-shaped association between diurnal predator species richness and hantavirus infection emergence (odds ratio [OR] 0.463, 0.688, and 0.553 for the first [lowest], third, and fourth [highest] quartiles, respectively, using the second quartile as a reference), while higher non-diurnal predator species richness tended to be associated with higher emergence risk (OR 0.134, 1.065, and 2.708 for the first, third, and fourth quartiles, respectively). The difference in these associations illustrates the complexity of the effects of predator species richness on human RBDs, which require further investigation in follow-up studies. The results showed a close link between environmental factors and public health, emphasizing that the One Health concept should be employed to understand the dynamics of RBDs., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore's blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.

Objective: This review describes the geographic and temporal distribution of, detection methods for, and other epidemiological features of published leptospirosis outbreaks, with the aim of informing efforts to standardize outbreak-reporting practices., Methods: We conducted a systematic review of leptospirosis outbreaks reported in the scientific literature and ProMED during 1970-2012. Predefined criteria were used to identify and classify outbreaks and a standard form was used to extract information., Results: During 1970-2012, we identified 318 outbreaks (average: 7 outbreaks/year; range: 1-19). Most outbreaks were reported in the Latin America and the Caribbean region (36%), followed by Southern Asia (13%), and North America (11%). Most outbreaks were located in tropical and subtropical ecoregions (55%). Quality classification showed that there was clear description of laboratory-confirmed cases in 40% of outbreaks. Among those, the average outbreak size was 82 cases overall (range: 2-2 259) but reached 253 cases in tropical/subtropical ecoregions. Common risk factors included outdoor work activities (25%), exposure to floodwaters (23%), and recreational exposure to water (22%). Epidemiologic investigation was conducted in 80% of outbreaks, mainly as case interviews. Case fatality was 5% overall (range: 0%-60%)., Conclusions: Outbreak reporting increased over the study period with outbreaks covering tropical and non-tropical regions. Outbreaks varied by size, setting, and risk factors; however, data reviewed often had limited information regarding diagnosis and epidemiology. Guidelines are recommended to develop standardized procedures for diagnostic and epidemiological investigations during an outbreak and for reporting., Competing Interests: Conflicts of interest. The authors declare no conflict of interest.

Enzyme-sensitive hydrogels containing encapsulated chondrocytes are a promising platform for cartilage tissue engineering. However, the growth of neotissue is closely coupled to the degradation of the hydrogel and is further complicated due to the encapsulated cells serving as the enzyme source for hydrogel degradation. To better understand these coupled processes, this study combined experimental and computational methods to analyze the transition from hydrogel to neotissue in a biomimetic MMP-sensitive poly(ethylene glycol) (PEG) hydrogel with encapsulated chondrocytes. A physics-based computational model that describes spatial heterogeneities in cell distribution was used. Experimentally, cell-laden hydrogels were cultured for six weeks under free swelling or subjected daily to one-hour of dynamic compressive loading. Extracellular matrix (ECM) synthesis rates were used as model inputs, and the model was fit to the experimentally determined construct modulus over time for the free swelling condition. Experimentally, ECM accumulation comprising collagen II and aggrecan increased over time concomitant with hydrogel degradation observed by a loss in PEG. Simulations demonstrated rapid degradation in regions of high cell density (i.e., cell clusters) reaching complete degradation by day 13, which facilitated localized ECM growth. Regions of low cell density degraded more slowly, had limited ECM, and led to the decrease in construct modulus during the first two weeks. The primary difference between the two culture environments was greater ECM accumulation in the clusters under free swelling, which facilitated a faster recovery in construct modulus. By 6 weeks the compressive modulus increased 2.5-fold to 107 kPa under free swelling, but dropped 1.6-fold to 26 kPa under loading. In summary, this biomimetic MMP-sensitive hydrogel supports neocartilage growth by facilitating rapid ECM growth within cell clusters, which was followed by slower growth in the rest of the hydrogel. Subtle temporal differences in hydrogel degradation and ECM accumulation, however, had a significant impact on the evolving mechanical properties.

The family Sicariidae comprises the genera Hexophthalma , Sicarius and Loxosceles . This latter is subdivided in eight monophyletic groups based on genitalia morphology and molecular analyses: amazonica , gaucho , laeta , and spadicea (South America); reclusa (North America); rufescens (Mediterranean); spinulosa and vonwredei (Africa). In Brazil, the genus Loxosceles is represented by 50 species. The mitotic and meiotic characteristics of eight Loxosceles species were analyzed in order to discuss the chromosome evolution, as well as the correspondence between cytogenetic data and morphological/molecular data for the delimitation of the South American groups of species belonging to this genus. All species studied in this work showed 2n♂ = 23, including a X1X2Y sex chromosome system (SCS). Despite the similarity of diploid number and SCS, the species studied here differed regarding the chromosome morphology of some autosomal pairs, presence of secondary constrictions, size of X chromosomes and localization of Ag-NOR/rDNA sites. Based on all these chromosomal data, we verified a close relationship between Loxosceles species belonging to the amazonica and gaucho groups. Transmission electron microscopy (TEM) analysis of spread pachytene cells of L. gaucho showed regular synapsis between homologous autosomal chromosomes, but asynaptic behavior of the sex chromosomes. The axial elements of the sex chromosomes undergo conspicuous morphological modifications resulting in shortening of their length.

While single-molecule localization microscopy (SMLM) offers the invaluable prospect to visualize cellular structures below the diffraction limit of light microscopy, its potential has not yet been fully capitalized due to its inherent susceptibility to blinking artifacts. Particularly, overcounting of single molecule localizations has impeded a reliable and sensitive detection of biomolecular nanoclusters. Here we introduce a 2-Color Localization microscopy And Significance Testing Approach (2-CLASTA), providing a parameter-free statistical framework for the qualitative analysis of two-dimensional SMLM data via significance testing methods. 2-CLASTA yields p-values for the null hypothesis of random biomolecular distributions, independent of the blinking behavior of the chosen fluorescent labels. The method is parameter-free and does not require any additional measurements nor grouping of localizations. We validated the method both by computer simulations as well as experimentally, using protein concatemers as a mimicry of biomolecular clustering. As the new approach is not affected by overcounting artifacts, it is able to detect biomolecular clustering of various shapes at high sensitivity down to a level of dimers.

We describe a case of 5,10-methenyltetrahydrofolate synthetase (MTHFS) deficiency characterized by microcephaly, global developmental delay, epilepsy, and cerebral hypomyelination. Whole exome sequencing (WES) demonstrated homozygosity for the R74X mutation in the MTHFS gene. The patient had the unexpected finding of elevated cerebrospinal fluid (CSF) neopterin. The novel finding of macrocytic anemia in this patient may provide a clue to the diagnosis of this rare neurometabolic disorder., Competing Interests: We, J. Romero, I. Abdelmoumen, D. Hasbani, D. Khurana, and M. Schneider, declare that we have no conflicts of interest., (© 2019 Published by Elsevier Inc.)

While matrix-assisted autologous chondrocyte implantation has emerged as a promising therapy to treat focal chondral defects, matrices that support regeneration of hyaline cartilage remain challenging. The goal of this work was to investigate the potential of a matrix metalloproteinase (MMP)-sensitive poly(ethylene glycol) (PEG) hydrogel containing the tethered growth factor, transforming growth factor β3 (TGF-β3), and compare cartilage regeneration in vitro and in vivo. The in vitro environment comprised chemically-defined medium while the in vivo environment utilized the subcutaneous implant model in athymic mice. Porcine chondrocytes were isolated and expanded in 2D culture for 10 days prior to encapsulation. The presence of tethered TGF-β3 reduced cell spreading. Chondrocyte-laden hydrogels were analyzed for total sulfated glycosaminoglycan and collagen contents, MMP activity, and spatial deposition of aggrecan, decorin, biglycan, and collagens type II and I. The total amount of extracellular matrix (ECM) deposited in the hydrogel constructs was similar in vitro and in vivo. However, the in vitro environment was not able to support long-term culture up to 64 days of the engineered cartilage leading to the eventual breakdown of aggrecan. The in vivo environment, on the other hand, led to more elaborate ECM, which correlated with higher MMP activity, and an overall higher quality of engineered tissue that was rich in aggrecan, decorin, biglycan and collagen type II with minimal collagen type I. Overall, the MMP-sensitive PEG hydrogel containing tethered TGF-β3 is a promising matrix for hyaline cartilage regeneration in vivo. STATEMENT OF SIGNIFICANCE: Regenerating hyaline cartilage remains a significant clinical challenge. The resultant repair tissue is often fibrocartilage, which long-term cannot be sustained. The goal of this study was to investigate the potential of a synthetic hydrogel matrix containing peptide crosslinks that can be degraded by enzymes secreted by encapsulated cartilage cells (i.e., chondrocytes) and tethered growth factors, specifically TGF-β3, to provide localized chondrogenic cues to the cells. This hydrogel led to hyaline cartilage-like tissue growth in vitro and in vivo, with minimal formation of fibrocartilage. However, the tissue formed in vitro, could not be maintained long-term. In vivo this hydrogel shows great promise as a potential matrix for use in regenerating hyaline cartilage., (Copyright © 2019. Published by Elsevier Ltd.)

Leptospirosis is an endemic zoonotic disease in Brazil and is widespread throughout rural populations in the state of Rio Grande do Sul. This study aimed to identify presumptive infecting Leptospira serogroups in human and animal cases and describe their occurrences within the ecoregions of the state by animal species. Data for human and animal leptospirosis cases were gathered from the government's passive surveillance systems and presumptive infecting serogroups were identified based on a two-fold titer difference in serogroups in the microscopic agglutination test (MAT) panel. A total of 22 different serogroups were reported across both human and animal cases. Serogroup Icterohaemorrhagiae was the most common among humans, while serogroup Sejroe predominated among animal cases, particularly bovines. Each ecoregion had a large distribution of cases, with 51% of the human cases in the Parana⁻Paraiba ecoregion, and 81% of the animal cases in the Savannah ecoregion. Identifying and mapping the serogroups circulating using the One Health approach is the first step for further understanding the distribution of the disease in the state. This study has the potential to aid in guiding public health and agricultural practices, furthering the need for a human vaccine in high-risk populations to complement control and prevention efforts.

Ion channel mutations can cause distinct neuropsychiatric diseases. We first studied the biophysical and neurophysiological consequences of four mutations in the human Na+ channel gene SCN8A causing either mild (E1483K) or severe epilepsy (R1872W), or intellectual disability and autism without epilepsy (R1620L, A1622D). Only combined electrophysiological recordings of transfected wild-type or mutant channels in both neuroblastoma cells and primary cultured neurons revealed clear genotype-phenotype correlations. The E1483K mutation causing mild epilepsy showed no significant biophysical changes, whereas the R1872W mutation causing severe epilepsy induced clear gain-of-function biophysical changes in neuroblastoma cells. However, both mutations increased neuronal firing in primary neuronal cultures. In contrast, the R1620L mutation associated with intellectual disability and autism-but not epilepsy-reduced Na+ current density in neuroblastoma cells and expectedly decreased neuronal firing. Interestingly, for the fourth mutation, A1622D, causing severe intellectual disability and autism without epilepsy, we observed a dramatic slowing of fast inactivation in neuroblastoma cells, which induced a depolarization block in neurons with a reduction of neuronal firing. This latter finding was corroborated by computational modelling. In a second series of experiments, we recorded three more mutations (G1475R, M1760I, G964R, causing intermediate or severe epilepsy, or intellectual disability without epilepsy, respectively) that revealed similar results confirming clear genotype-phenotype relationships. We found intermediate or severe gain-of-function biophysical changes and increases in neuronal firing for the two epilepsy-causing mutations and decreased firing for the loss-of-function mutation causing intellectual disability. We conclude that studies in neurons are crucial to understand disease mechanisms, which here indicate that increased or decreased neuronal firing is responsible for distinct clinical phenotypes.