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During the SARS-CoV-2 pandemic, governments and public health authorities collected massive amounts of data on daily confirmed positive cases and incidence rates. These data sets provide relevant information to develop a scientific understanding of the pandemic's spatiotemporal dynamics. At the same time, there is a lack of comprehensive approaches to describe and classify patterns underlying the dynamics of COVID-19 incidence across regions over time. This seriously constrains the potential benefits for public health authorities to understand spatiotemporal patterns of disease incidence that would allow for better risk communication strategies and improved assessment of mitigation policies efficacy. Within this context, we propose an exploratory statistical tool that combines functional data analysis with unsupervised learning algorithms to extract meaningful information about the main spatiotemporal patterns underlying COVID-19 incidence on mainland Portugal. We focus on the timeframe spanning from August 2020 to March 2022, considering data at the municipality level. First, we describe the temporal evolution of confirmed daily COVID-19 cases by municipality as a function of time, and outline the main temporal patterns of variability using a functional principal component analysis. Then, municipalities are classified according to their spatiotemporal similarities through hierarchical clustering adapted to spatially correlated functional data. Our findings reveal disparities in disease dynamics between northern and coastal municipalities versus those in the southern and hinterland. We also distinguish effects occurring during the 2020-2021 period from those in the 2021-2022 autumn-winter seasons. The results provide proof-of-concept that the proposed approach can be used to detect the main spatiotemporal patterns of disease incidence. The novel approach expands and enhances existing exploratory tools for spatiotemporal analysis of public health data., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ribeiro 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.)

Hematogenous dissemination is a critical step in the evolution of local infection to systemic disease. The Lyme disease (LD) spirochete, which efficiently disseminates to multiple tissues, has provided a model for this process, in particular for the key early event of pathogen adhesion to the host vasculature. This occurs under shear force mediated by interactions between bacterial adhesins and mammalian cell-surface proteins or extracellular matrix (ECM). Using real-time intravital imaging of the Lyme spirochete in living mice, we previously identified BBK32 as the first LD spirochetal adhesin demonstrated to mediate early vascular adhesion in a living mouse; however, deletion of bbk32 resulted in loss of only about half of the early interactions, suggesting the existence of at least one other adhesin (adhesin-X) that promotes early vascular interactions. VlsE, a surface lipoprotein, was identified long ago by its capacity to undergo rapid antigenic variation, is upregulated in the mammalian host and required for persistent infection in immunocompetent mice. In immunodeficient mice, VlsE shares functional overlap with OspC, a multi-functional protein that displays dermatan sulfate-binding activity and is required for joint invasion and colonization. In this research, using biochemical and genetic approaches as well as intravital imaging, we have identified VlsE as adhesin-X; it is a dermatan sulfate (DS) adhesin that efficiently promotes transient adhesion to the microvasculature under shear force via its DS binding pocket. Intravenous inoculation of mice with a low-passage infectious B. burgdorferi strain lacking both bbk32 and vlsE almost completely eliminated transient microvascular interactions. Comparative analysis of binding parameters of VlsE, BBK32 and OspC provides a possible explanation why these three DS adhesins display different functionality in terms of their ability to promote early microvascular interactions., Competing Interests: The authors have declared that no competing interests exist.

Much evidence suggests that Amazonia and the Atlantic Forest were connected through at least three dispersion routes in the past: the Eastern route, the central route, and the Western route. However, few studies have assessed the use of these routes based on multiple species. Here we present a compilation of mammal species that potentially have dispersed between the two forest regions and which may serve to investigate these connections. We evaluate the present-day geographic distributions of mammals occurring in both Amazonia and the Atlantic Forest and the likely connective routes between these forests. We classified the species per habitat occupancy (strict forest specialists, species that prefer forest habitat, or generalists) and compiled the genetic data available for each species. We found 127 mammalian species presently occurring in both Amazonia and the Atlantic Forest for which, substantial genetic data was available. Hence, highlighting their potential for phylogeographic studies investigating the past connections between the two forests. Differently from what was previously proposed, the present-day geographic distribution of mammal species found in both Amazonia and the Atlantic Forest points to more species in the eastern portion of the dry diagonal (and adjoining forested habitats). The Central route was associated with the second most species. Although it remains to be seen how this present-day geography reflects the paleo dispersal routes, our results show the potential of using mammal species to investigate and bring new insights about the past connections between Amazonia and the Atlantic Forest., Competing Interests: The authors have declared that no competing interests exist.

Anurans exhibit limited dispersion ability and have physiological and behavioural characteristics that narrow their relationships with both environmental and spatial predictors. So, the relative contributions of environmental and spatial predictors in the patterns of taxonomic and functional anuran beta diversity were examined in a metacommunity of 33 ponds along the coast of south Brazil. We expected that neutral processes and, in particular, niche-based processes could have similar influence on the taxonomic and functional beta diversity patterns. Distance-based methods (db-RDA) with variation partitioning were conducted with abundance data to examine taxonomic and functional facets and components (total, turnover and nestedness) in relation to environmental and spatial predictors. Processes determining metacommunity structure differed between the components of beta diversity and among taxonomic and functional diversity. While taxonomic beta diversity was further accounted by both environmental and spatial predictors, functional beta diversity responded more strongly to spatial predictors. These two contrasting patterns were different to what we had predicted, suggesting that while there is a taxonomic turnover mediated by environmental filters, the spatial distance promotes the trait dissimilarity between sites. In addition, our data confirm that neutral and niche-based processes operate on anuran metacommunities even at short geographic scales. Our results reinforce the idea that studies aiming to evaluate the patterns of structure in metacommunities should include different facets of diversity so that better interpretations can be achieved., Competing Interests: The authors have declared that no competing interests exist.

Beta cell dysfunction accompanies and drives the progression of type 2 diabetes mellitus (T2D), but there are few clinical biomarkers available to assess islet cell stress in humans. Secretagogin, a protein enriched in pancreatic islets, demonstrates protective effects on beta cell function in animals. However, its potential as a circulating biomarker released from human beta cells and islets has not been studied. In this study primary human islets, beta cells and plasma samples were used to explore secretion and expression of secretagogin in relation to the T2D pathology. Secretagogin was abundantly and specifically expressed and secreted from human islets. Furthermore, T2D patients had an elevated plasma level of secretagogin compared with matched healthy controls, which was confirmed in plasma of diabetic mice transplanted with human islets. Additionally, the plasma secretagogin level of the human cohort had an inverse correlation to clinical assessments of beta cell function. To explore the mechanism of secretagogin release in vitro, human beta cells (EndoC-βH1) were exposed to elevated glucose or cellular stress-inducing agents. Secretagogin was not released in parallel with glucose stimulated insulin release, but was markedly elevated in response to endoplasmic reticulum stressors and cytokines. These findings indicate that secretagogin is a potential novel biomarker, reflecting stress and islet cell dysfunction in T2D patients.

The conservation and sustainable management of Annona coriacea requires knowledge of its floral and reproductive biology, and of its main pollinators and their life cycles. In this work, we analyzed these aspects in detail. Floral biology was assessed by observing flowers from the beginning of anthesis to senescence. The visiting hours and behavior of floral visitors in the floral chamber were recorded, as were the sites of oviposition. Excavations were undertaken around specimens of A. coriacea to determine the location of immature pollinators. Anthesis was nocturnal, starting at sunset, and lasted for 52-56 h. The flowers were bisexual, protogynous and emitted a strong scent similar to the plant´s own ripe fruit. There was pronounced synchrony among all floral events (the period and duration of stigmatic receptivity, release of odor, pollen release and drooping flowers) in different individuals, but no synchrony in the same individuals. All of the flowers monitored were visited by beetle species of the genera Cyclocephala and Arriguttia. Beetles arrived at the flowers with their bodies covered in pollen and these pollen grains were transferred to the stigmata while foraging on nutritious tissues at the base of the petals. With dehiscence of the stamens and retention within the floral chamber, the bodies of the floral visitors were again covered with pollen which they carried to newly opened flowers, thus promoting the cycle of pollination. After leaving the flowers, female beetles often excavated holes in the soil to lay eggs. Larvae were found between the leaf litter and the first layer of soil under specimens of A. coriacea. Cyclocephala beetles were the main pollinators of A. coriacea, but Arriguttia brevissima was also considered a pollinator and is the first species of this genus to be observed in Annonaceae flowers. Annona coriacea was found to be self-compatible with a low reproductive efficiency in the area studied. The results of this investigation provide ecological data that should contribute to the conservation and economic exploitation of A. coriacea., Competing Interests: The authors have declared that no competing interests exist.

Species range-limits are influenced by a combination of several factors. In our study we aimed to unveil the drivers underlying the expansion of the Egyptian mongoose in Portugal, a carnivore that was confined to southern Portugal and largely increased its range during the last three decades. We evaluated the expansion of the species in three periods (1980-1990, 1990-2000 and 2000-2010), by projecting the presence/absence data of the species in each temporal range and proposed four hypotheses to explain this sudden expansion associated to changes in the barrier effects of human infrastructure and topographic features, and in the availability of suitable areas due to climate change or land use. An exploratory analysis was made using Spearman rank correlation, followed by a hierarchical partitioning analysis to select uncorrelated potential explanatory variables associated with the different hypotheses. We then ran Generalized Linear Models (GLM) for every period for each hypothesis and for every combination of hypotheses. Our main findings suggest that dynamic transitions of land-use coupled with temperature and rainfall variations over the decades are the main drivers promoting the mongoose expansion. The geographic barriers and the human infrastructures functioned as barriers for mongoose expansion and have shaped its distribution. The expansion of the Egyptian mongoose across the Portuguese territory was due to a variety of factors. Our results suggest a rapid shift in species range in response to land-use and climate changes, underlining the close link between species ranges and a changing environment.

Previous studies have shown that assortative mating acts as a driver of speciation by countering hybridization between two populations of the same species (pre-zygotic isolation) or through mate choice among the hybrids (hybrid speciation). In both speciation types, assortative mating promotes speciation over a transient hybridization stage. We studied mate choice in a hybrid vertebrate complex, the allopolyploid fish Squalius alburnoides. This complex is composed by several genomotypes connected by an intricate reproductive dynamics. We developed a model that predicts the hybrid complex can persist when females exhibit particular mate choice patterns. Our model is able to reproduce the diversity of population dynamic outcomes found in nature, namely the dominance of the triploids and the dominance of the tetraploids, depending on female mate choice patterns and frequency of the parental species. Experimental mate choice trials showed that females exhibit the preferences predicted by the model. Thus, despite the known role of assortative mating in driving speciation, our findings suggest that certain mate choice patterns can instead hinder speciation and support the persistence of hybrids over time without speciation or extinction.

Mist netting is a widely used technique to sample bird and bat assemblages. However, captures often decline with time because animals learn and avoid the locations of nets. This avoidance or net shyness can substantially decrease sampling efficiency. We quantified the day-to-day decline in captures of Amazonian birds and bats with mist nets set at the same location for four consecutive days. We also evaluated how net avoidance influences the efficiency of surveys under different logistic scenarios using re-sampling techniques. Net avoidance caused substantial declines in bird and bat captures, although more accentuated in the latter. Most of the decline occurred between the first and second days of netting: 28% in birds and 47% in bats. Captures of commoner species were more affected. The numbers of species detected also declined. Moving nets daily to minimize the avoidance effect increased captures by 30% in birds and 70% in bats. However, moving the location of nets may cause a reduction in netting time and captures. When moving the nets caused the loss of one netting day it was no longer advantageous to move the nets frequently. In bird surveys that could even decrease the number of individuals captured and species detected. Net avoidance can greatly affect sampling efficiency but adjustments in survey design can minimize this. Whenever nets can be moved without losing netting time and the objective is to capture many individuals, they should be moved daily. If the main objective is to survey species present then nets should still be moved for bats, but not for birds. However, if relocating nets causes a significant loss of netting time, moving them to reduce effects of shyness will not improve sampling efficiency in either group. Overall, our findings can improve the design of mist netting sampling strategies in other tropical areas.

The increase in species diversity from the Poles to the Equator is a major biogeographic pattern, but the mechanisms underlying it remain obscure. Our aim is to contribute to their clarification by describing the latitudinal gradients in species richness and in evolutionary age of species of New World bats, and testing if those patterns may be explained by the niche conservatism hypothesis. Maps of species ranges were used to estimate species richness in a 100 x 100 km grid. Root distances in a molecular phylogeny were used as a proxy for the age of species, and the mean root distance of the species in each cell of the grid was estimated. Generalised additive models were used to relate latitude with both species richness and mean root distance. This was done for each of the three most specious bat families and for all Chiroptera combined. Species richness increases towards the Equator in the whole of the Chiroptera and in the Phyllostomidae and Molossidae, families that radiated in the tropics, but the opposite trend is observed in the Vespertilionidae, which has a presumed temperate origin. In the whole of the Chiroptera, and in the three main families, there were more basal species in the higher latitudes, and more derived species in tropical areas. In general, our results were not consistent with the predictions of niche conservatism. Tropical niche conservatism seems to keep bat clades of tropical origin from colonizing temperate zones, as they lack adaptations to survive cold winters, such as the capacity to hibernate. However, the lower diversity of Vespertilionidae in the Neotropics is better explained by competition with a diverse pre-existing community of bats than by niche conservatism.

Due to the energetic frustration of RNA folding, tertiary structured RNA is typically characterized by a rugged folding free energy landscape where deep kinetic barriers separate numerous misfolded states from one or more native states. While most in vitro studies of RNA rely on (re)folding chemically and/or enzymatically synthesized RNA in its entirety, which frequently leads into kinetic traps, nature reduces the complexity of the RNA folding problem by segmental, co-transcriptional folding starting from the 5' end. We here have developed a simplified, general, nondenaturing purification protocol for RNA to ask whether avoiding denaturation of a co-transcriptionally folded RNA can reduce commonly observed in vitro folding heterogeneity. Our protocol bypasses the need for large-scale auxiliary protein purification and expensive chromatographic equipment and involves rapid affinity capture with magnetic beads and removal of chemical heterogeneity by cleavage of the target RNA from the beads using the ligand-induced glmS ribozyme. For two disparate model systems, the Varkud satellite (VS) and hepatitis delta virus (HDV) ribozymes, we achieve >95% conformational purity within one hour of enzymatic transcription, without the need for any folding chaperones. We further demonstrate that in vitro refolding introduces severe conformational heterogeneity into the natively-purified VS ribozyme but not into the compact, double-nested pseudoknot fold of the HDV ribozyme. We conclude that conformational heterogeneity in complex RNAs can be avoided by co-transcriptional folding followed by nondenaturing purification, providing rapid access to chemically and conformationally pure RNA for biologically relevant biochemical and biophysical studies.