Spring Ecosystems of the Alps: Isolated biodiversity islands with distinctive species assemblages

Freshwater springs are considered distinctive stream sections. Environmental conditions typical of subterranean groundwater habitats such as high temperature stability and nutrient scarcity predominate the spring mouth. Due to the rapidly increasing influence of surface processes such as solar radiation or precipitation downstream, springs exhibit steep environmental gradients and high microhabitat diversity on a small spatial scale. Furthermore, diverse species assemblages composed of crenobiontic (strictly spring-dwelling), stygobiontic (groundwater-adapted), and rhithrobiontic (stream-inhabiting) organisms are characteristic of springs. Due to their topographic distinctiveness and individual species assemblage compositions, particularly alpine springs are considered isolated island-like habitats and freshwater biodiversity hotspots. Water mites (Hydrachnidia) show many crenobiontic species adapted to different microhabitats such as gravel, moss, lentic, or lotic flow regimes in springs and can be seen as a model system to study crenobiology. Their taxonomy is currently re-investigated intensively and is still mainly morphology-based, although molecular genetic methods can reveal novel insights. Therefore, the a priori morphological species delimitation of the most abundant crenobiontic water mites was assessed with molecular genetic tools. The phylogenetic inferences, including a mitochondrial (cox1) and a nuclear (28S) marker, corresponded to the traditional taxonomy and confirmed the monophyly of Hydrachnidia. Moreover, species putatively new to science were discovered, a genetic species identification reference database was generated, and basic methods for further genetic work were established. The genomic structure of Partnunia steinmanni Walter, 1906, a strictly crenobiontic water mite species, was then investigated to study degree of spring habitat isolation. Populations were sampled in protected areas across the Alps and restriction site-associated DNA sequencing (RADseq) was performed. The admixture and RAxML analysis revealed a pronounced population genomic structure and distinctiveness of P. steinmanni spring populations between and within the different areas. Combined with the strong isolation by distance that has been found, a high degree of insularity of alpine springs can be concluded. Furthermore, a genetic principal component analysis of individuals between the different protected areas revealed a western genotype extending into eastern populations, likely caused by post-glacial recolonization. Finally, an environmental DNA (eDNA) bioindication methodology was established to monitor and assess alpine spring ecosystem integrity and detect a potential loss of crenobionts. For that purpose, sequence libraries were generated and novel qPCR primer and probe sets were designed to detect indicator species in eDNA filtered water samples. The final assays targeting spring-dwelling Trichoptera, Plecoptera, and Hydrachnidia species showed to be highly specific and sensitive. Furthermore, equal detection rates were revealed by comparing the qPCR eDNA assays with the conventional approach, which relies on direct sampling and morphological identification of organisms. Due to its non- invasive and time-efficient character, the newly developed spring bioindication method circumvents drawbacks of the conventional techniques and is particularly applicable in protected areas

New insights into the distribution, ecology, and systematic position of the rare water mite Rutripalpus limicola Sokolow, 1934 (Acari: Rutripalpidae)

The water mite Rutripalpus limicola Sokolow, 1934, can be considered exceptionally rare. It shows a scattered distribution range and, until recently, was known from six sites across Europe only. This strictly spring-dwelling species has a highly localised distribution, presumably due to its specific ecological requirements. We present the first records of R. limicola in the Netherlands and review the current state of knowledge on its distribution and biology. In addition to the previously existing morphology-based investigations, we provide a phylogenetic placement based on 28S rRNA gene data and shed light on the controversial systematic position of R. limicola : In contrast to the previously hypothesised Lebertioidea-relatedness of the isolated, monotypic family Rutripalpidae, our results reveal a putative Hydryphantoidea association. Moreover, we discuss the host-parasite association of R. limicola larvae and the dipteran family Ptychopteridae. Combined with additional information derived from the new records from the Netherlands, we contribute to a better understanding of this elusive species' biology and phylogenetic position

Hidden biodiversity revealed by integrated morphology and genetic species delimitation of spring dwelling water mite species (Acari, Parasitengona: Hydrachnidia)

Background: Water mites are among the most diverse organisms inhabiting freshwater habitats and are considered as substantial part of the species communities in springs. As parasites, Hydrachnidia influence other invertebrates and play an important role in aquatic ecosystems. In Europe, 137 species are known to appear solely in or near spring- heads. New species are described frequently, especially with the help of molecular species identification and delimi- tation methods. The aim of this study was to verify the mainly morphology-based taxonomic knowledge of spring- inhabiting water mites of central Europe and to build a genetic species identification library. Methods: We sampled 65 crenobiontic species across the central Alps and tested the suitability of mitochondrial ( cox 1) and nuclear ( 28S ) markers for species delimitation and identification purposes. To investigate both markers, distance- and phylogeny-based approaches were applied. The presence of a barcoding gap was tested by using the automated barcoding gap discovery tool and intra- and interspecific genetic distances were investigated. Further- more, we analyzed phylogenetic relationships between different taxonomic levels. Results: A high degree of hidden diversity was observed. Seven taxa, morphologically identified as Bandakia con- creta Thor, 1913, Hygrobates norvegicus (Thor, 1897), Ljania bipapillata Thor, 1898, Partnunia steinmanni Walter, 1906, Wandesia racovitzai Gledhill, 1970, Wandesia thori Schechtel, 1912 and Zschokkea oblonga Koenike, 1892, showed high intraspecific cox 1 distances and each consisted of more than one phylogenetic clade. A clear intraspecific threshold between 5.6-6.0% K2P distance is suitable for species identification purposes. The monophyly of Hydrachnidia and the main superfamilies is evident with different species clearly separated into distinct clades. cox 1 separates water mite species but is unsuitable for resolving higher taxonomic levels. Conclusions: Water mite species richness in springs is higher than has been suggested based on morphological species identification alone and further research is needed to evaluate the true diversity. The standard molecular species identification marker cox 1 can be used to identify species but should be complemented by a nuclear marker, e.g. 28S , to resolve taxonomic relationships. Our results contribute to the taxonomical knowledge on spring inhabiting Hydrachnida, which is indispensable for the development and implementation of modern environment assessment methods, e.g. metabarcoding, in spring ecology

Small-scale, semi-automated purification of eukaryotic proteins for structure determination

A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 μg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 μg/ml, and of purified selenomethione-labeled AIA–GFP (His8 removed by treatment with TEV protease) was 172 μg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10–50 ml) cell growth and automated purification. 1H–15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA–GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 Å. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination

Fate of the H-NS–Repressed bgl Operon in Evolution of Escherichia coli

In the enterobacterial species Escherichia coli and Salmonella enterica, expression of horizontally acquired genes with a higher than average AT content is repressed by the nucleoid-associated protein H-NS. A classical example of an H-NS–repressed locus is the bgl (aryl-β,D-glucoside) operon of E. coli. This locus is “cryptic,” as no laboratory growth conditions are known to relieve repression of bgl by H-NS in E. coli K12. However, repression can be relieved by spontaneous mutations. Here, we investigated the phylogeny of the bgl operon. Typing of bgl in a representative collection of E. coli demonstrated that it evolved clonally and that it is present in strains of the phylogenetic groups A, B1, and B2, while it is presumably replaced by a cluster of ORFans in the phylogenetic group D. Interestingly, the bgl operon is mutated in 20% of the strains of phylogenetic groups A and B1, suggesting erosion of bgl in these groups. However, bgl is functional in almost all B2 isolates and, in approximately 50% of them, it is weakly expressed at laboratory growth conditions. Homologs of bgl genes exist in Klebsiella, Enterobacter, and Erwinia species and also in low GC-content Gram-positive bacteria, while absent in E. albertii and Salmonella sp. This suggests horizontal transfer of bgl genes to an ancestral Enterobacterium. Conservation and weak expression of bgl in isolates of phylogenetic group B2 may indicate a functional role of bgl in extraintestinal pathogenic E. coli

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

Contributions to grounding systems modelling by the finite element method in the harmonic regime.

Este trabalho tem como objetivo fornecer contribuições ao estudo de sistemas de aterramento submetidos a correntes elétricas no regime permanente senoidal pelo Método dos Elementos Finitos. Nele se discutem as motivações para a utilização deste procedimento numérico em problemas desta categoria, e mais especificamente para o uso da variante deste método com elementos finitos de aresta. O tratamento completo necessário à construção das funções de forma para a aproximação local de grandezas vetoriais com elementos hexaédricos é nele desenvolvido e detalhado. Com estes resultados, uma aplicação computacional capaz de modelar o comportamento de uma haste de aterramento verticalmente enterrada no solo foi desenvolvida, utilizando-se para a discretização do domínio elementos finitos hexaédricos tri-lineares e quadráticos incompletos. Esta aplicação computacional foi utilizada para o estudo da resposta em freqüência da impedância equivalente a aterramentos desta categoria em diferentes condições de solo e com as duas variedades de elemento hexaédrico anteriormente mencionados. Os resultados obtidos são então discutidos, bem como a validade da modelagem adotada para o problema físico aqui considerado.This work aims to provide contributions to the study of grounding systems subjected to electric currents at the sinusoidal steady state by the Finite Element Method. Motivations for the use of this numerical procedure in problems of this category, and more specifically for the usage of the Edge Finite Element Method variant, are discussed in it. The complete treatment required for building the shape functions for the local approximation of vector physical quantities with hexahedral finite elements is here developed and detailed. With these results a computer application capable of modeling the behavior of vertically buried grounding rods was developed, in which domain discretization is accomplished by the usage of both tri-linear and incomplete quadratic hexahedral finite elements. This computer application was employed in the study of the frequency response of the impedance equivalent to grounding systems of this class at different soil conditions and with both hexahedral element varieties previously mentioned. The results obtained are then discussed, as well as the validity of the modeling adopted for this physical problem

Modélisation numérique des phénomènes de couplage électromagnétique dans les alentours des lignes aériennes de transmission d\'énergie.

Electromagnetic coupling phenomena between overhead power transmission lines and other nearby structures are inevitable, especially in densely populated areas. The undesired effects resulting from this proximity are manifold and range from the establishment of hazardous potentials to the outbreak of alternate current corrosion phenomena. The study of this class of problems is necessary for ensuring security in the vicinities of the interaction zone and also to preserve the integrity of the equipment and of the devices there present. However, the complete modeling of this type of application requires the three- -dimensional representation of the region of interest and needs specific numerical methods for field computation. In this work, the modeling of problems arising from the flow of electrical currents in the ground (the so-called conductive coupling) will be addressed with the finite element method. Those resulting from the time variation of the electromagnetic fields (the so-called inductive coupling) will be considered as well, and they will be treated with the generalized PEEC (Partial Element Equivalent Circuit) method. More specifically, a special boundary condition on the electric potential is proposed for truncating the computational domain in the finite element analysis of conductive coupling problems, and a complete PEEC formulation for modeling inductive coupling problems is presented. Test configurations of increasing complexities are considered for validating the foregoing approaches. These works aim to provide a contribution to the modeling of this class of problems, which tend to become common with the expansion of power grids.Les phénomènes de couplage électromagnétique entre les lignes aé- riennes de transmission d\'énergie et des structures voisines sont inévitables, surtout dans les zones densément peuplées. Les effets indésirables découlants de cette proximité sont variés, allant de l\'établissement des tensions dangereuses à l\'apparition de phénomènes de corrosion dus au courant alternatif. L\'étude de cette classe de problèmes est nécessaire pour assurer la sécurité dans les alentours de la zone d\'interaction et aussi pour préserver l\'intégrité des équipements et des dispositifs présents. Cependant, la modélisation compl ète de ce type d\'application implique la représentation tridimensionnelle de la région d\'intérêt et nécessite des méthodes numériques de calcul de champs spécifiques. Dans ces travaux, des problèmes liés à la circulation de courants électriques dans le sol (ou de couplage dit conductif) seront abordés avec la méthode des éléments finis. D\'autres problèmes résultants de la variation temporelle des champs électromagnétiques (ou de couplage dit inductif) seront aussi considérés et traités avec la méthode PEEC (Partial Element Equivalent Circuit) généralisée. Plus précisément, une condition limite particulière sur le potentiel électrique est proposée pour tronquer le domaine de calcul dans l\'analyse par éléments finis des problèmes de couplage conductif et une formulation PEEC complète pour la modélisation de problèmes de couplage inductif est présentée. Des con gurations tests de complexités croissantes sont considérées pour valider les approches précédentes. Ces travaux visent ainsi à apporter une contribution à la modélisation de cette classe de problèmes, qui tendent à devenir communs avec l\'expansion des réseaux électriques

CO1 reference sequences dataset of the sedDNA Chironomidae study by Blattner et al. 2024

<p>This dataset contains the CO1 reference sequences used to identify the sedDNA metabarcoding reads in the study entitled "Sediment core DNA-Metabarcoding and chitinous remain identification: Integrating complementary methods to characterise Chironomidae biodiversity in lake sediment archives", which was submitted to Molecular Ecology Resources.</p&gt

Modelagem numérica de fenômenos de acoplamento eletromagn ético nas imediações de linhas aéreas de transmissão de energia

Electromagnetic coupling phenomena between overhead power transmission lines and other nearby structures are inevitable, especially in densely populated areas. The undesired effects resulting from this proximity are manifold and range from the establishment of hazardous potentials to the outbreak of alternate current corrosion phenomena. The study of this class of problems is necessary for ensuring security in the vicinities of the interaction zone and also to preserve the integrity of equipment and devices there present. However, the complete modeling of this type of application requires the three-dimensional representation of the region of interest and needs specific numerical methods for field computation. In this work, the modeling of problems arising from the flow of electrical currents in the ground (the so-called conductive coupling) will be addressed with the finite element method. Those resulting from the time variation of the electromagnetic fields (the so-called inductive coupling) will be considered as well, and they will be treated with the generalized PEEC (Partial Element Equivalent Circuit) method. More specifically, a special boundary condition on the electric potential is proposed for truncating the computational domain in the finite element analysis of conductive coupling problems, and a complete PEEC formulation for modeling inductive coupling problems is presented. Test configurations of increasing complexities are considered for validating the foregoing approaches. These works aim to provide a contribution to the modeling of this class of problems, which tend to become common with the expansion of power grids.Les phénomènes de couplage électromagnétique entre les lignes aériennes de transmission d'énergie et des structures voisines sont inévitables, surtout dans les zones densément peuplées. Les effets indésirables découlants de cette proximité sont variés, allant de l'établissement des tensions dangereuses à l'apparition de phénomènes de corrosion dus au courant alternatif. L'étude de cette classe de problèmes est nécessaire pour assurer la sécurité dans les alentours de la zone d'interaction et aussi pour préserver l'intégrité des équipements et des dispositifs présents. Cependant, la modélisation complète de ce type d'application implique la représentation tridimensionnelle de la région d'intérêt et nécessite des méthodes numériques de calcul de champs spécifiques. Dans ces travaux, des problèmes liés à la circulation de courants électriques dans le sol (ou de couplage dit conductif) seront abordés avec la méthode des éléments finis. D'autres problèmes résultants de la variation temporelle des champs électromagnétiques (ou de couplage dit inductif) seront aussi considérés et traités avec la méthode PEEC (Partial Element Equivalent Circuit) généralisée. Plus précisément, une condition limite particulière sur le potentiel électrique est proposée pour tronquer le domaine de calcul dans l'analyse par éléments finis des problèmes de couplage conductif et une formulation PEEC complète pour la modélisation de problèmes de couplage inductif est présentée. Des configurations tests de complexités croissantes sont considérées pour valider les approches précédentes. Ces travaux visent ainsi à apporter une contribution à la modélisation de cette classe de problèmes, qui tendent à devenir communs avec l'expansion des réseaux électriques.Fenômenos de acoplamento eletromagnético entre linhas aéreas de transmissão de energia e outras estruturas vizinhas são inevitáveis, sobretudo emáreas densamente povoadas. Os efeitos indesejados decorrentes desta proximidadesão variados, indo desde o estabelecimento de potenciais perigosos até o surgimento de processos de corrosão por corrente alternada. O estudo desta classe de problemas é necessária para a garantia da segurança nas imediações da zona de interação e também para se preservar a integridade de equipamentos e dispositivos ali presentes. Entretanto, a modelagem completa deste tipo de aplicação requer a representação tridimensional da região de interesse e necessita de métodos numéricos de cálculo de campos específicos. Neste trabalho, serão abordadas as modelagens de problemas decorrentes da circulação de correntes elétricas no solo (ditos de acoplamentocondutivo) com o método dos elementos finitos. Também serão considerados problemas produzidos pela variação temporal dos campos eletromagnéticos (ditos de acoplamento indutivo), que serão tratados com o método PEEC(Partial Element Equivalent Circuit) generalizado. Mais especificamente, uma condição de contorno particular sobre o potencial elétrico é proposta para o truncamento do domínio de cálculo na análise de problemas de acoplamento condutivo com o método dos elementos finitos, e uma formulação completa tipo PEEC para a modelagem de problemas de acoplamento indutivo é apresentada. Problemas teste de complexidades crescentes são considerados para a validação das abordagens precedentes. Estes trabalhos visam fornecer desta forma uma contribuição à modelagem desta classe de problemas, que tendem a se tornar comuns com a expansão das redes elétricas