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BACKGROUND: Sleep duration and quality have been associated with increased cardiovascular risk. However, large studies linking objectively measured sleep and subclinical atherosclerosis assessed in multiple vascular sites are lacking. OBJECTIVES: The purpose of this study was to evaluate the association of actigraphy-measured sleep parameters with subclinical atherosclerosis in an asymptomatic middle-aged population, and investigate interactions among sleep, conventional risk factors, psychosocial factors, dietary habits, and inflammation. METHODS: Seven-day actigraphic recording was performed in 3,974 participants (age 45.8 ± 4.3 years; 62.6% men) from the PESA (Progression of Early Subclinical Atherosclerosis) study. Four groups were defined: very short sleep duration 8 h. Sleep fragmentation index was defined as the sum of the movement index and fragmentation index. Carotid and femoral 3-dimensional vascular ultrasound and cardiac computed tomography were performed to quantify noncoronary atherosclerosis and coronary calcification. RESULTS: When adjusted for conventional risk factors, very short sleep duration was independently associated with a higher atherosclerotic burden with 3-dimensional vascular ultrasound compared to the reference group (odds ratio: 1.27; 95% confidence interval: 1.06 to 1.52; p = 0.008). Participants within the highest quintile of sleep fragmentation presented a higher prevalence of multiple affected noncoronary territories (odds ratio: 1.34; 95% confidence interval: 1.09 to 1.64; p = 0.006). No differences were observed regarding coronary artery calcification score in the different sleep groups. CONCLUSIONS: Lower sleeping times and fragmented sleep are independently associated with an increased risk of subclinical multiterritory atherosclerosis. These results highlight the importance of healthy sleep habits for the prevention of cardiovascular disease. The PESA study is cofunded equally by the CNIC and Banco Santander. The study also receives funding from the Institute of Health Carlos III (PI15/02019) and the European Regional Development Fund. The CNIC is supported by the Ministry of Economy, Industry and Competitiveness (MEIC) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505). Dr. Bueno has received consulting fees from AstraZeneca, Bayer, Bristol-Myers Squibb-Pfizer, and Novartis; has received speaking fees or support for attending scientific meetings from AstraZeneca, Bayer, Bristol-Myers Squibb-Pfizer, Ferrer, Novartis, and MEDSCAPE-the heart.org; and has received research funding from the Instituto de Salud Carlos III, Spain (PIE16/00021 and PI17/01799), AstraZeneca, Bristol-Myers Squibb, Janssen, and Novartis. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Sí

This study was supported by competitive grants from the Spanish Ministry of Science, Innovation and Universities (BIO2015-67580-P, PGC2018-097019-B-I00, PID2019-106814RB-I00 and SAF2016-80843-R), through the Carlos III Institute of Health-Fondo de Investigacion Sanitaria grant PRB3 (IPT17/0019 - ISCIII-SGEFI / ERDF, ProteoRed), CIBERCV and CIBERDEM, the Fundacio MaratoTV3 (grant 122/C/2015) and “la Caixa” Banking Foundation (project HR17-00247). The PESA study is co-funded equally by the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain, and Banco Santander, Madrid, Spain. The ILERVAS study was funded by the Diputacio de Lleida. The study also receives funding from the Instituto de Salud Carlos III (PI15/02019; PI18/00610; RD16/0009) and the FEDER funds. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovacion y Universidades (MCNU) and the Pro CNIC Foundation. Imaging of subclinical atherosclerosis improves cardiovascular risk prediction on top of traditional risk factors. However, cardiovascular imaging is not universally available. This work aims to identify circulating proteins that could predict subclinical atherosclerosis. Hypothesis-free proteomics was used to analyze plasma from 444 subjects from PESA cohort study (222 with extensive atherosclerosis on imaging, and 222 matched controls) at two timepoints (three years apart) for discovery, and from 350 subjects from AWHS cohort study (175 subjects with extensive atherosclerosis on imaging and 175 matched controls) for external validation. A selected three-protein panel was further validated by immunoturbidimetry in the AWHS population and in 2999 subjects from ILERVAS cohort study. PIGR, IGHA2, APOA, HPT and HEP2 were associated with subclinical atherosclerosis independently from traditional risk factors at both timepoints in the discovery and validation cohorts. Multivariate analysis rendered a potential three-protein biomarker panel, including IGHA2, APOA and HPT. Immunoturbidimetry confirmed the independent associations of these three proteins with subclinical atherosclerosis in AWHS and ILERVAS. A machine-learning model with these three proteins was able to predict subclinical atherosclerosis in ILERVAS (AUC [95%CI]:0.73 [0.70-0.74], p

Molecular data have considerably contributed to building the taxonomy of protists. Recently, the systematics of Hyalospheniidae (Amoebozoa; Tubulinea; Arcellinida) has been widely revised, with implications extending to ecological, biogeographical and evolutionary investigations. Certain taxa, however, still have an uncertain phylogenetic position, including the common and conspicuous species Nebela militaris. A phylogenetic reconstruction of the Hyalospheniidae using partial sequences of the mitochondrial Cytochrome Oxidase Subunit 1 (COI) gene shows that N. militaris does not belong to genus Nebela, but should be placed in its own genus. The morphological singularities (strongly curved pseudostome and a marked notch in lateral view) and phylogenetic placement of our isolates motivated the creation of a new genus: Alabasta gen. nov. Based on their morphology, we include in this genus Nebela kivuense and Nebela longicollis. We discuss the position of genus Alabasta within Hyalospheniidae, and the species that could integrate this new genus based on their morphological characteristics.

The anaerobic digestion of municipal sewage was studied at psychrophilic temperatures (19 °C and 17 °C) in three up-flow anaerobic sludge bed reactors (UASB). A pulsed feeding system was operated for 430 d, during which different hydraulic retention time (HRT) (6, 8, 10, 12, 14, 18, 24 and 36 h) were tested. Granulation of the sludge was observed under the pulsed feeding strategy and a 6 h HRT. Low hydrolysis rates were obtained at HRTs lower than 10 h, as a result of an increased solids accumulation inside the reactor. An HRT of 14 h was identified as the most suitable HRT under the studied temperatures (19 °C and 17 °C), with methane yields of 136 and 117 mL CH4/g of removed chemical oxygen demand (CODremoved), respectively.

This study aims to compare a conventional wastewater treatment plant and a hybrid alternative treatment plant for the treatment of real urban wastewater from the city of Almeria, Spain. The hybrid treatment plant combines a semi-extensive wastewater treatment systems such as a high rate algae pond (HRAP), a dissolved air flotation unit to separate algae biomass from treated wastewater and an extensive wastewater treatment systems such as vertical flow constructed wetlands. The conventional wastewater treatment plant has a treatment capacity of 4000 m3 d−1. The experimental demo plant with a 3000 m2 HRAP and 200 m2 of wetlands surface has been operated throughout one year in parallel with a conventional activated sludge full-scale plant, obtaining a complete comparison of both systems. Regarding removal efficiencies, the new hybrid system achieved values of 96.6%, 76.6% 89.8% and 99.9% for COD, TN, TP and TSS, respectively. The hybrid systems reach disinfection rates of 4 and 5 log units for total coliforms and E.Coli, respectively. Energy demand required in the hybrid system was five times lower than a conventional activated sludge (0.1 kWh m−3). The results here obtained demonstrated the capacity of using hybrid alternative treatment plant as an efficient way of treating wastewater in small populations.

High-throughput sequencing (HTS) of soil environmental DNA (eDNA) allows assessing the full diversity of soil micro-eukaryotes. The resulting operational taxonomic units (OTUs) can be assigned to potential taxonomic and functional identities using increasingly complete reference databases. HTS of soil eDNA is revealing a high diversity and abundance of potential eukaryovorous protists, thus challenging the paradigm of the predominantly bacterivorous function of soil phagotrophic protists (i.e. microbial loop). Using Illumina sequencing of soil eDNA and targeting the V9 region of the SSU rRNA gene, we investigated the taxonomic and functional diversities, distribution and co-occurrence patterns of soil micro-eukaryotes in three land-use categories: forests, meadows and croplands located in Switzerland. Each OTU was assigned to a broad functional category (phototrophs, phagotrophs, osmotrophs, or parasites). Total OTU richness was similar in the three land-use categories, but community composition differed significantly between forests and other land-uses. The proportion of fungal sequences (especially Basidiomycota) was highest, and phototroph (i.e. soil microalgae) sequences least abundant in forests. Seven OTUs representing phagotrophic protists, together accounting for >25% of all phagotroph sequences, were significantly correlated to the total number of phototroph sequences, thus suggesting algivory. At least three of these OTUs corresponded to known algal predators. These results suggest that beyond plants, soil microalgae represent a functionally significant but rarely considered input of carbon in soils that should be taken into account when modelling soil nutrient cycling.

Arcellinid testate amoebae include a wide variety of amoeboid organisms whose test (shell) varies in shape, composition and size. A decade ago, we initiated molecular phylogenetic analyses based on SSU rRNA gene sequences and a taxonomic revision of Arcellinida. However, many lineages within Arcellinida still lack molecular data, and the phylogeny of this group is largely incomplete. In this study, we obtained SSU rRNA gene sequences from seven taxa, of which six have agglutinated shell (Difflugia oblonga, D. labiosa, D. gramen, Mediolus corona, Netzelia wailesi, and N. tuberculata), and one has an entirely proteinaceous shell (Arcella intermedia). All species but Difflugia oblonga branched within the recently erected suborder Sphaerothecina, confirming the synapomorphic value of an oviform or discoid shell. Thus, we propose that species with an oviform or discoid shell currently classified within genus Difflugia must be transferred to other genera, thus continuing the process of taxonomic revision of genus Difflugia, the largest Arcellinida genus. We therefore transferred the current and the previously sequenced oviform Difflugia spp. to Netzelia spp., based on the shared globular/oviform shell shape and their monophyly. Another species, D. labiosa, formed an independent lineage that branched as a sister clade to Arcella spp.; based on the shell morphology and their phylogenetic position, we considered D. labiosa as incertae sedis.

Molecular phylogeny is an indispensable tool for assessing evolutionary relationships among protists. The most commonly used marker is the small subunit ribosomal RNA gene, a conserved gene present in many copies in the nuclear genomes. However, this marker is not variable enough at a fine-level taxonomic scale, and intra-genomic polymorphism has already been reported. Finding a marker that could be useful at both deep and fine taxonomic resolution levels seemed like a utopic dream. We designed Amoebozoa-specific primers to amplify a region including partial sequences of two subunits of the mitochondrial nicotinamide adenine dinucleotide dehydrogenase gene (NAD9/NAD7). We applied them to arcellinids belonging to distantly related genera (Arcella, Difflugia, Netzelia and Hyalosphenia) and to Arcellinid-rich environmental samples to obtain additional Amoebozoa sequences. Tree topology was congruent with previous phylogenies, all nodes being highly supported, suggesting that this marker is well-suited for deep phylogenies in Arcellinida and perhaps Amoebozoa. Furthermore, it enabled discrimination of close-related taxa. This short genetic marker (ca. 250 bp) can therefore be used at different taxonomic levels, due to a fast-varying intergenic region presenting either a small intergenic sequence or an overlap, depending on the species.

High Rate Algae Ponds (HRAP) are the simplest way to grow microalgae biomass and an interesting alternative for wastewater treatment. In this work the performance of these systems was evaluated using anaerobically digested wastewater as culture medium. Two variables were studied in long-term mode: the carbon dioxide supply and the modification of the dilution rates. The results showed that CO2 supply increases the productivity but less than expected considering the potential biomass generation calculated based on the ratios of carbon to nitrogen of microalgae and wastewater. The assimilation into biomass only accounted for 57% of the inlet nitrogen under the best conditions because nitrification and volatilization reduced the availability of this element. The operation under short hydraulic retention times presented a more interesting performance with higher biomass productivities. The biomass produced was efficiently harvested with in a Dissolved Air Flotation (DAF) unit.

Introduction Improved outcome and longer life expectancy in patients with cystinosis and the intrinsic complexity of the disease, underline the need for a guided transition of patients from pediatric to adult care. The process aims to guarantee the continuum of care and enable the empowerment of patients from guardian to self-care. Methods Bibliography review, expert opinion and anonymous surveys of patients, relatives and patient advocacy groups. Results A new plan to support and coordinate the transition of cystinotic patients providing specific proposals for a variety of medical fields and improved treatment adherence. Nephrologists play a key role in the transition since most cystinotic patients have severe chronic kidney disease and require kidney transplantation before adulthood. Conclusion We present a proposal providing recommendations and a chronogram to aid the transition of adolescents and young adults with cystinosis in our area.

Oomycete diversity has been generally underestimated, despite their ecological and economic importance. Surveying unexplored natural ecosystems with up-to-date molecular diversity tools can reveal the existence of unsuspected organisms. Here, we have explored the molecular diversity of five microhabitats located in five different oligotrophic peat bogs in the Jura Mountains using a high-throughput sequencing approach (Illumina HiSeq 2500). We found a total of 34 different phylotypes distributed in all major oomycete clades, and comprising sequences affiliated to both well-known phylotypes and members of undescribed, basal clades. Parasitic species, including obligate forms were well-represented, and phylotypes related to highly damaging invasive pathogens (Aphanomyces astaci: X1100 and Saprolegnia parasitica: X1602) were retrieved. Microhabitats differed significantly in their community composition, and many phylotypes were strongly affiliated to free water habitats (pools). Our approach proved effective in screening oomycete diversity in the studied habitat, and could be applied systematically to other environments and other fungal and fungal-like groups., Instituto de Botánica "Dr. Carlos Spegazzini"

Background Dilated cardiomyopathy (DCM) is the most frequent cause of heart transplantation (HTx). The genetic basis of DCM among patients undergoing HTx has been poorly characterized. We sought to determine the genetic basis of familial DCM HTx and to establish the yield of modern next generation sequencing (NGS) technologies in this setting. Methods Fifty-two heart-transplanted patients due to familial DCM underwent NGS genetic evaluation with a panel of 126 genes related to cardiac conditions (59 associated with DCM). Genetic variants were initially classified as pathogenic mutations or as variants of uncertain significance (VUS). Final pathogenicity status was determined by familial cosegregation studies. Results Initially, 24 pathogenic mutations were found in 21 patients (40%); 25 patients (48%) carried 19 VUS and 6 (12%) did not show any genetic variant. Familial evaluation of 220 relatives from 36 of the 46 families with genetic variants confirmed pathogenicity in 14 patients and allowed reclassification of VUS as pathogenic in 17 patients, and as non-pathogenic in 3 cases. At the end of the study, the DCM-causing mutation was identified in 38 patients (73%) and 5 patients (10%) harbored only VUS. No genetic variants were identified in 9 cases (17%). Conclusions The genetic spectrum of familial DCM patients undergoing HTx is heterogeneous and involves multiple genes. NGS technology plus detailed familial studies allow identification of causative mutations in the vast majority of familial DCM cases. Detailed familial studies remain critical to determine the pathogenicity of underlying genetic defects in a substantial number of cases.

A high diversity of Apicomplexa was recently found in tropical soils presumably reflecting the diversity of their invertebrate hosts, but such patterns have not been explored in colder regions. We analysed the diversity of Apicomplexa and their potential metazoan hosts in litter and mosses collected in 11 different alpine habitats using an eDNA metabarcoding approach. The abundance and diversity of Apicomplexa phylotypes and of their potential invertebrate hosts were positively correlated. This confirms that eDNA metabarcoding is a useful tool to explore the unknown biodiversity of free-living eukaryotes, as well as potential host-parasite interactions. Future studies should aim at describing this diversity using a combination of morphological and molecular approaches.

High Rate Algae Ponds (HRAPs) are a promising technology for the treatment of municipal wastewater in locations with sufficient space and solar radiation. Algae-based processes do not require aeration, and thus have the potential to be less energy-intensive than activated sludge processes. We used a combination of LCA and LCCA analysis to evaluate the sustainability of HRAP systems, using data from the construction and operation of two demonstration-scale systems in Almería and Cádiz, Spain. As a reference for comparison, we used data from an activated sludge-based Sequencing Batch Reactor (SBR) treatment system in operation in Leppersdorf, Germany, which has comparable removal rates for a similar inflow. We focused solely on the actual wastewater treatment aspect of these technologies, excluding sludge treatment from this analysis. Based on our analysis, the current HRAP technology is more energy-efficient than activated sludge-based SBRs and requires only 22% of its electricity consumption. In addition, HRAP is more advantageous both economically (0.18 €/m3 versus 0.26 €/m3) and environmentally, with both lower global warming and eutrophication potentials (146.27 vs. 458.27 × 10−3 kg CO2 equiv./m3; 126.14 vs. 158.01 × 10−6 kg PO4 equiv./m3). However, the Net Environmental Benefit of SBR was slightly more favorable than of HRAP because of the higher removal rate for nutrients of SBR.

Arcellinida (lobose testate amoebae) are abundant and diverse in many ecosystems, especially in moist to aquatic environments. Molecular phylogeny has shown that overall test morphology (e.g., spherical or elongate) is generally conserved in Arcellinida lineages, but the taxonomic value of other traits (e.g., size, ornamentation, mixotrophy/heterotrophy metabolism type) has not been systematically evaluated. Morphological and physiological traits that correspond to genetic differences likely represent adaptive traits of ecological significance. We combined high-resolution phylogenetics (NAD9-NAD7 genes) and advanced morphometrics to assess the phylogenetic signal of morphological traits of a group of elongate Difflugia species (Arcellinida). The phylogenetic analyses revealed two clades which could be reliably separated by test size and the presence/absence of mixotrophy. Differences in test size may reflect trophic level, with smaller organisms occupying lower trophic levels. In addition to having larger tests, elongate mixotrophic Difflugia are characterised by wide, flat bases and an inflation of the lower two thirds of their test. These morphological traits may provide additional volume for endosymbionts and/or increased surface area to aid light transmission. Our results showcase greater diversity within the elongate Difflugia and highlight morphological traits of ecological and evolutionary significance.

Bringing together more than 170 years of data, this study represents the first attempt to construct a species checklist and analyze the diversity and distribution of testate amoebae in Chile, a country that encompasses the southwestern region of South America, countless islands and part of the Antarctic. In Chile, known diversity includes 416 testate amoeba taxa (64 genera, 352 infrageneric taxa), 24 of which are here reported for the first time. Species-accumulation plots show that in Chile, the number of testate amoeba species reported has been continually increasing since the mid-19th century without leveling off. Testate amoebae have been recorded in 37 different habitats, though they are more diverse in peatlands and rainforest soils. Only 11% of species are widespread in continental Chile, while the remaining 89% of the species exhibit medium or short latitudinal distribution ranges. Also, species composition of insular Chile and the Chilean Antarctic territory is a depauperated subset of that found in continental Chile. Nearly, the 10% of the species reported here are endemic to Chile and many of them are distributed only within the so-called Chilean biodiversity hotspot (ca. 25° S-47° S). These findings are here thoroughly discussed in a biogeographical and evolutionary context.

Introduction Cystinosis is a rare systemic lysosomal storage disease that mainly affects the kidney and the eye. Renal replacement therapy is started in patients with cystinosis during the first decade of life in the absence of treatment. The prognosis of cystinosis depends on early diagnosis and the prompt start of and good compliance with cysteamine treatment. Kidney disease progression, extra-renal complications and shorter life expectancy are more pronounced in patients who do not adhere to treatment. Objective The aim of this work was to establish recommendations for the comprehensive care of cystinosis and facilitate patient transition from paediatric to adult medicine, based on clinical experience. The goal is to reduce the impact of the disease and improve prognosis and patient quality of life. Methods Bibliographic research and consensus meetings with a multidisciplinary professional team of clinical experts in cystinosis (T-CiS.bcn group) from 5 hospitals in Barcelona. Results This consensus document gathers specific recommendations for the diagnosis, treatment and multidisciplinary care of cystinotic patients in the following areas: nephrology, dialysis, kidney transplantation, ophthalmology, endocrinology, neurology, laboratory, genetic counselling, nursing and pharmacy. Conclusions Guidelines for the comprehensive care of cystinosis provide a support tool for health professionals who look after these patients. They are based on the following main pillars: (a) a multidisciplinary approach; (b) appropriate disease monitoring and control of white blood cell (WBC) cystine levels; (c) the importance of adherence to cysteamine treatment; and (d) the promotion of patient self-care by means of disease education programmes. All these recommendations will lead us, in a second phase, to create a coordinated model of transition from paediatric to adult care services which will cover the specific needs of cystinosis.

ResumenIntroducciónLa cistinosis es una enfermedad lisosomal minoritaria de expresión sistémica con especial afectación renal y oftalmológica, en la que los pacientes inician terapia renal sustitutiva en la primera década de la vida en ausencia de tratamiento. El pronóstico de la cistinosis depende del diagnóstico precoz, la pronta instauración del tratamiento con cisteamina y el buen cumplimiento terapéutico. La progresión de la enfermedad renal y de las complicaciones extrarrenales y una menor supervivencia, son más acentuadas en pacientes no adherentes.ObjetivoEl objetivo de este trabajo fue la elaboración de unas recomendaciones para la atención integral de la cistinosis y la transición del adolescente a la medicina del adulto, basadas en la experiencia clínica, con el fin de reducir el impacto de la enfermedad y mejorar la calidad de vida y el pronóstico del paciente.MétodoBúsqueda bibliográfica y reuniones de consenso de un equipo multidisciplinar de expertos en la práctica clínica con pacientes afectos de cistinosis (Grupo T-CiS.bcn), procedentes de 5 hospitales localizados en Barcelona.ResultadosEl documento recoge recomendaciones específicas y necesarias para el diagnóstico, tratamiento y seguimiento multidisciplinar de la cistinosis en las siguientes áreas: nefrología, diálisis, trasplante renal, oftalmología, endocrinología, neurología, laboratorio, consejo genético, enfermería y farmacia.ConclusionesDisponer de un documento de referencia para la atención integral de la cistinosis constituye una herramienta de soporte para los profesionales de la salud que asisten a estos pacientes. Los principales pilares en los que se sustenta son: a) el enfoque multidisciplinar, b) la adecuada monitorización de la enfermedad y control de los niveles de cistina intraleucocitarios, c) la importancia de la adherencia al tratamiento con cisteamina y d) la promoción del autocuidado del paciente mediante programas de educación en la enfermedad. Todo ello conducirá, en una segunda fase, a la elaboración de un modelo de transición coordinado entre los servicios de pediatría y de adultos que contemple las necesidades específicas de la cistinosis.AbstractIntroductionCystinosis is a rare lysosomal systemic disease that mainly affects the kidney and the eye. Patients with cystinosis begin renal replacement therapy during the first decade of life in absence of treatment. Prognosis of cystinosis depends on early diagnosis, and prompt starting and good compliance with cysteamine treatment. Kidney disease progression, extra-renal complications and shorter life expectancy are more pronounced in those patients that do not follow treatment.The objective of this work was to elaborate recommendations for the comprehensive care of cystinosis and the facilitation of patient transition from paediatric to adult treatment, based on clinical experience. The goal is to reduce the impact of the disease, and to improve patient quality of life and prognosis.MethodsBibliographic research and consensus meetings among a multidisciplinary professional team of experts in the clinical practice, with cystinotic patients (T-CiS.bcn group) from 5 hospitals located in Barcelona.ResultsThis document gathers specific recommendations for diagnosis, treatment and multidisciplinary follow-up of cystinotic patients in the following areas: nephrology, dialysis, renal transplant, ophthalmology, endocrinology, neurology, laboratory, genetic counselling, nursing and pharmacy.ConclusionsA reference document for the comprehensive care of cystinosis represents a support tool for health professionals who take care of these patients. It is based on the following main pillars: a) a multi-disciplinary approach, b) appropriate disease monitoring and control of intracellular cystine levels in leukocytes, c) the importance of adherence to treatment with cysteamine, and d) the promotion of patient self-care by means of disease education programmes. All these recommendations will lead us, in a second phase, to create a coordinated transition model between paediatric and adult care services which will cover the specific needs of cystinosis.

We describe here a new species of sphagnicolous testate amoeba found abundantly in the forested part of the Le Cachot peatland (Jura Mountains, Neuchâtel, Switzerland) based on microscopical observations (LM, SEM). The new species, called Nebela gimlii was placed in a phylogenetic tree based on mitochondrial cytochrome oxidase sequences (COI), and branched robustly within the N. collaris complex next to the morphologically similar N. guttata and N. tincta. It is however genetically clearly distinct from these two species, and differs morphologically from them by its smaller size and stouter shape of the shell. This new species completes the phylogeny of the Nebela collaris species complex, with now eight species described, mostly from peatlands and acidic forest litter, and further demonstrates the existence of an unknown diversity within testate amoebae. Improving the taxonomy of testate amoebae in peatlands and clarifying the ecology of newly discovered species should make these organisms even more valuable as bioindicator and for palaeoecological reconstruction.

We characterized molecularly the first freshwater member ever reported for the family Bathycoccaceae in Lake Musters (Argentinean Patagonia). Members of this family are extremely numerous and play a key ecological role in marine systems as primary producers. We cloned a fragment comprising the SSU rRNA gene + ITS region from environmental DNA using specific mamiellophyte primers. The unique SSU rRNA gene sequence obtained clustered robustly with Bathycoccus prasinos. Analysis of the two-dimensional structure of the ITS region showed the presence of a typical supplementary helix in the ITS-2 region, a synapomorphy of Bathycoccaceae, which confirmed further its phylogenetic placement. We finally discuss the possible causes for the presence of this organism in Lake Musters. Fil: Lara, Enrique. Universite de Neuchatel. Faculte de Sciences; Suiza. Consejo Superior de Investigaciones Científicas. Real Jardín Botánico; España Fil: Fernández, Leonardo D.. Universidad Bernardo O’Higgin; Chile Fil: Schiaffino, María Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires; Argentina Fil: Izaguirre, Irina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentina

Life was microbial for the majority of Earth's history, but as very few microbial lineages leave a fossil record, the Precambrian evolution of life remains shrouded in mystery. Shelled (testate) amoebae stand out as an exception with rich documented diversity in the Neoproterozoic as vase-shaped microfossils (VSMs). While there is general consensus that most of these can be attributed to the Arcellinida lineage in Amoebozoa, it is still unclear whether they can be used as key fossils for interpretation of early eukaryotic evolution. Here, we present a well-resolved phylogenomic reconstruction based on 250 genes, obtained using single-cell transcriptomic techniques from a representative selection of 19 Arcellinid testate amoeba taxa. The robust phylogenetic framework enables deeper interpretations of evolution in this lineage and demanded an updated classification of the group. Additionally, we performed reconstruction of ancestral morphologies, yielding hypothetical ancestors remarkably similar to existing Neoproterozoic VSMs. We demonstrate that major lineages of testate amoebae were already diversified before the Sturtian glaciation (720 mya), supporting the hypothesis that massive eukaryotic diversification took place in the early Neoproterozoic and congruent with the interpretation that VSM are arcellinid testate amoebae.Arcellinid testate amoebae are some of the earliest eukaryotic microbes. Lahr et al. show that these amoebae have seven major deep phylogenetic lineages, probably diverged as early as 730 mya. Diversification of these microbes in the mid-Neoproterozoic was probably happening alongside the gradual oxygenation of oceans.

Ciliates (or Ciliophora) are probably the most popular group of protists, with some classroom lab favorites such as Paramecium spp. These highly conspicuous micro-organisms are easier to identify than many other eukaryotic microbes, at least for trained specialists. As a result, a large amount of data has been produced on their taxonomy, ecology and biogeography. Their relatively short generation times and their ubiquity in virtually all ecosystems on Earth make them excellent bioindicator organisms, particularly in soil systems. However, their practical use is hampered by long and tedious procedures of handling and preparing of specimens for identification, a task which is rendered even more difficult by the existence of cryptic species. A comfortable way to overcome these pitfalls is through the application of environmental molecular diversity screening methods that have been developed in the last two decades to the ciliate communities, and also more modern approaches such as next generation sequencing (454 pyrosequencing). As a complement, several approaches already developed for the prokaryotic communities (functional gene screening, environmental RNA clone libraries, fluorescent in situ hybridization) have not been considered today but could be easily applied to ciliate communities in soils. Still, before these methods can be applied to monitor natural ciliate communities, a considerable effort in species description and culturing (i.e. “classical protistology”) must be achieved in order to interpret optimally the data obtained by molecular-based techniques. Here, we provide an overview of these methods as potential monitor strategies of environmental ciliate communities and its contribution to bioindication in soils, and also their limitations.

Transforming growth factor-β family cytokines have diverse actions in the maintenance of cardiac homeostasis. Follistatin-like 3 (Fstl3) is an extracellular regulator of certain TGF-β family members, including activin A. The aim of this study was to examine the role of Fstl3 in cardiac hypertrophy. Cardiac myocyte-specific Fstl3 knock-out (KO) mice and control mice were subjected to pressure overload induced by transverse aortic constriction (TAC). Cardiac hypertrophy was assessed by echocardiography and histological and biochemical methods. KO mice showed reduced cardiac hypertrophy, pulmonary congestion, concentric LV wall thickness, LV dilatation, and LV systolic dysfunction after TAC compared with control mice. KO mice displayed attenuated increases in cardiomyocyte cell surface area and interstitial fibrosis following pressure overload. Although activin A was similarly up-regulated in KO and control mice after TAC, a significant increase in Smad2 phosphorylation only occurred in KO mice. Knockdown of Fstl3 in cultured cardiomyocytes inhibited PE-induced cardiac hypertrophy. Conversely, adenovirus-mediated Fstl3 overexpression blocked the inhibitory action of activin A on hypertrophy and Smad2 activation. Transduction with Smad7, a negative regulator of Smad2 signaling, blocked the antihypertrophic actions of activin A stimulation or Fstl3 ablation. These findings identify Fstl3 as a stress-induced regulator of hypertrophy that controls myocyte size via regulation of Smad signaling.

The mitochondrial Cytochrome Oxidase Subunit 1 gene (COI) has been promoted as an ideal "DNA barcode" for animal species and other groups of eukaryotes. However, the utility of the COI marker for species level discrimination and for phylogenetic analyses has yet to be tested within the Rhizaria. Accordingly, we analysed mitochondrial COI gene sequences and nuclear small subunit rDNA (SSU) sequences from several morphospecies of euglyphid testate amoebae (Cercozoa, Rhizaria) in order to evaluate the utility of these DNA markers for species discrimination and phylogenetic reconstructions. Sequences were obtained from eleven populations belonging to sixCyphoderiamorphospecies that were isolated from field samples in North America and Europe. Mean inter-population COI sequence dissimilarities were on average 2.9 times greater than in the SSU, while the intra-population sequence dissimilarities were higher in the SSU (0-0.95%) than in the COI (0%); this suggests that the COI fragment is valuable for discriminating Cyphoderiidae isolates. Our study also demonstrated that COI sequences are useful for inferring phylogenetic relationships among Cyphoderiidae isolates. COI and SSU tree topologies were very similar even though the COI fragment used in these analyses (500bp) was much shorter than the SSU sequences (1600bp). Altogether, these results demonstrate the utility of the COI as a potential taxonomic DNA barcode for assessing cyphoderiid species diversity and for inferring phylogenetic relationships within the group.

Marine and freshwater ecosystems are fundamentally different regarding many biotic and abiotic parameters. The physiological adaptations required for an organism to pass the salinity barrier are considerable. Many eukaryotic lineages are restricted to either freshwater or marine environments. Molecular phylogenetic analyses generally demonstrate that freshwater species and marine species segregate into different subclades, indicating that transitions between these two environments occur only rarely in the course of evolution. It is, however, unclear if the transitions between freshwater and environments characterized by highly variable salinities, such as the marine supralittoral zone, are also infrequent. Here, we use testate amoebae within the Euglyphida to assess the phylogenetic interrelationships between marine supralittoral and freshwater taxa. Euglyphid testate amoebae are mainly present in freshwater habitats but also occur in marine supralittoral environments. Accordingly, we generated and analyzed partial SSU rRNA gene sequences from 49 new marine/supralittoral and freshwater Cyphoderiidae sequences, 20 sequences of the Paulinellidae, Trinematidae, Assulinidae, and Euglyphidae families as well as 21 GenBank sequences of unidentified taxa derived from environmental PCR surveys. Both the molecular and morphological data suggest that the diversity of Cyphoderiidae is strongly underestimated. The results of our phylogenetic analyses demonstrated that marine supralittoral and freshwater euglyphid testate amoeba species are segregated into distinct sub- clades, suggesting that transitions between these two habitats occurred only infrequently.

Ciliates (or Ciliophora) are ubiquitous organisms which can be widely used as bioindicators in ecosystems exposed to anthropogenic and industrial influences. The evaluation of the environmental impact on soil ciliate communities with methods relying on morphology-based identification may be hampered by the large number of samples usually required for a statistically supported, reliable conclusion. Cultivation-independent molecular-biological diagnostic tools are a promising alternative to greatly simplify and accelerate such studies. In this present work a ciliate-specific fingerprint method based on the amplification of a phylogenetic marker gene (i.e. the 18S ribosomal RNA gene) with subsequent analysis by denaturing gradient gel electrophoresis (DGGE) was developed and used to monitor community shifts in a polycyclic aromatic hydrocarbon (PAH) polluted soil. The semi-nested approach generated ciliate-specific amplification products from all soil samples and allowed to distinguish community profiles from a PAH-polluted and a non-polluted control soil. Subsequent sequence analysis of excised bands provided evidence that polluted soil samples are dominated by organisms belonging to the class Colpodea. The general DGGE approach presented in this study might thus in principle serve as a fast and reproducible diagnostic tool, complementing and facilitating future ecological and ecotoxicological monitoring of ciliates in polluted habitats.

Background Abnormalities in the extracellular matrix (ECM) can occur in heart failure. In this study we analyzed ECM gene expression in patients with advanced dilated cardiomyopathy who did and did not develop sustained myocardial recovery after left ventricular asst device (LVAD) unloading combined with pharmacologic therapy. Methods Myocardial gene expression of collagens (COL1A1 and COL3A1), fibronectin (FN), matrix metalloproteinases (MMPs 1 to 14), tissue inhibitors of metalloproteinases (TIMPs 1 to 4), connective tissue growth factor (CTGF), transforming growth factor-beta1 (TGF-β1) and THY1 was measured by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) at LVAD implantation and again at explantation (recovery, n = 11) or transplantation (non-recovery, n = 5). Results The non-recovery group had higher levels of pro-fibrotic markers (COL1A1, TGF-β1 and THY1) at implantation compared with the recovery group (1.82 ± 0.74-, 1.81 ± 0.69- and 3.01 ± 1.70-fold, respectively; p ≤ 0.05). Both recovery and non-recovery groups showed no significant difference in gene expression after treatment, but levels of pro-fibrotic genes (COL1A1, COL3A1, FN and THY1) correlated negatively with post-explant ejection fraction in the recovery group. Of all genes analyzed only TIMP4 showed a significant change, with expression reduced during recovery (0.55 ± 0.25-fold at explant vs implant, p = 0.001). All other genes showed complex patterns between individuals with both increased and decreased expression of pro-fibrotic markers, MMPs and TIMPs in recovery patients. Conclusions Patients who did not recover had higher myocardial expression of pro-fibrotic genes at LVAD implantation, and in recovered patients higher levels at explant were negatively associated with subsequent ejection fraction. However, individual patients showed complex expression patterns and a decrease in pro-fibrotic markers was not required for recovery.

alpha-Tubulin is one of the most widely used markers for estimating deep-level phylogenetic relationships amongst eukaryotes. We sequenced 6-7 nuclear protein-coding genes, including alpha-tubulin, from the two described species of the enigmatic jakobid(-like) excavate protist Andalucia. Concatenated protein phylogenies place Andalucia in a clade with other jakobids, Euglenozoa and Heterolobosea. Individual gene trees, except that of alpha-tubulin, do not conflict strongly with this position. In alpha-tubulin trees, Andalucia instead falls in a strongly supported clade with diplomonads, parabasalids and opisthokonts (including animals and fungi), and branches with diplomonads. This clade is robust to changes in taxon sampling, and is unlikely to represent long-branch attraction, compositional heterogeneity artefact, or segmental gene conversion. Phylogenies estimated without alpha-tubulin strongly support the original position for Andalucia, and also reinforce recent studies in placing diplomonads and parabasalids with Preaxostyla, not opisthokonts. alpha-Tubulin seems to have experienced two or more eukaryote-to-eukaryote lateral gene transfer (LGT) events, one perhaps from an ancestral opisthokont to an ancestor of diplomonads and parabasalids, or vice versa, and one probably from the diplomonad lineage to Andalucia. Like EF-1alpha/EFL, alpha-tubulin has a complex history that needs to be taken into account when using this marker for deep-level phylogenetic inference.

The existing data on the molecular phylogeny of filose testate amoebae from order Euglyphida has revealed contradictions between traditional morphological classification and SSU rRNA phylogeny and, moreover, the position of several important genera remained unknown. We therefore carried out a study aiming to fill several important gaps and better understand the relationships among the main euglyphid testate amoebae and the evolutionary steps that led to the present diversity at a higher level. We obtained new SSU rRNA sequences from five genera and seven species. This new phylogeny obtained shows that (1) the clade formed by species of genera Assulina and Placocista branches unambiguously at the base of the subclade of Euglyphida comprising all members of the family Trinematidae and genus Euglypha, (2) family Trinematidae (Trachelocorythion, Trinema, and Corythion) branches as a sister group to genus Euglypha, (3) three newly sequenced Euglypha species (E. cf. ciliata, E. penardi, and E. compressa) form a new clade within the genus. Since our results show that Assulina and Placocista do not belong to the Euglyphidae (unless the Trinematidae are also included in this family), we propose the creation of a new family named Assulinidae. Consequently, we give a family status to the genera Euglypha and (tentatively) Scutiglypha, which become the new family Euglyphidae. The evolutionary pattern suggested by SSU rRNA phylogeny shows a clear tendency towards increasing morphological complexity of the shell characterised by changes in the symmetry (migration of the aperture to a ventral position and/or compression of the shell) and the appearance of specialised scales at the aperture (in families Trinematidae and Euglyphidae).

We compared the abundance and diversity of cultivable protozoa (flagellates and amoebae) in a polycyclic aromatic hydrocarbon (PAH) polluted soil and an unpolluted control, by isolating and cultivating clonal strains. The number of cultivable protozoa was higher in the polluted soil; however, the polluted soil displayed an impoverished community, dominated by certain taxa, such as Acanthamoeba sp. We isolated a total of 31 protozoan strains to characterize them morphologically and by 18S rRNA gene sequence analysis. This approach, i.e. combining morphological and molecular information had the advantage of providing quantitative data, information on morphology but also an accurate positioning of the isolates in 18S rRNA trees.

The progression of chronic hepatitis B (CHB) is related to fibrosis and to the emergence of intrahepatic anomalous vascular structures. Hepatitis B virus (HBV) X protein transactivator (HBx) may play a significant role in both processes. To analyze how HBV induces vascular growth and remodeling in vivo, we assessed the expression of angiopoietin-2 (Ang2) in liver biopsies from CHB patients by reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry because of the relevant role of Ang2 in vascular development, remodeling, and tumor promotion. In addition, we analyzed the influence of HBx in the expression of Ang2 in HBx-expressing hepatocyte cell lines and in hepatic stellate cells stimulated with conditional medium from HBx-hepatocytes. Ang2 expression was clearly up-regulated at both mRNA and protein levels in the liver of CHB patients, showing an intense staining of inflammatory infiltrates and vascular structures at inflamed portal areas. HBx-expressing hepatocytes and stimulated stellate cells showed a significant induction of Ang2 expression. PI3K inhibitor and antioxidants repressed the 64-kd Ang2 form but further enhanced the inflammation-related 50-kd molecular species. Therefore, HBx could account for the induction of Ang2 observed in CHB, especially the 50-kd form, contributing to pathological angiogenesis and hepatocellular carcinoma progression.

Cohesins hold sister chromatids together from DNA replication until they are segregated. Although cohesins Smc1, Smc3, and Scc1/Rad21 are involved in chromatid cohesion and other cellular processes, little is known about the other mitotic cohesin subunit, Scc3/STAG. Here we describe STAG/Scc3, which may act as a transcriptional co-activator. STAG2 is able to enhance the activity of the tumor necrosis factor alpha, the CD69, and the human immunodeficiency virus long terminal repeat promoters in a NF-kappaB-dependent manner. In addition, STAG2 interacts with the viral transactivator Tat and enhances the Tat-mediated activation of the human immunodeficiency virus long terminal repeat promoter. Moreover, STAG2 co-activates a multimeric NF-kappaB reporter construct and enhances the activity of the transactivation domain of p65/RelA in a Gal4 system. This function is dependent on one of the LXXLL co-activation motives present in this cohesin and is substantiated by the interaction of STAG2 with the p65 subunit of NF-kappaB. These results describe a novel activity for cohesins, suggesting a role for STAG/Scc3 in transcriptional regulation.

Co-infection with hepatitis B virus (HBV) and human immunodeficiency virus type-1 (HIV-1) is relatively common. However, the impact of this co-infection on the clinical outcome of HIV infection has not been elucidated. We herein demonstrate that the HBV X protein (HBx) superinduces ongoing HIV-1 replication and HIV-1 long terminal repeat (LTR) transcription by synergizing with Tat protein and with T-cell activation signals. Although HBx cooperated with mitogenic stimuli in the induction of reporter plasmids harboring the HIV-1 kappaB enhancer, in both a NF-kappaB-dependent manner and a NF-AT-dependent manner, deletion of this element from the LTR did not affect the HBx-mediated up-regulation in the presence of Tat and/or mitogens. In contrast, mutation of the proximal LTR Sp1-binding sites abolished the HBx-mediated synergistic activation, but only when it was accompanied by deletion of the kappaB enhancer. When HBx was targeted to the nucleus, its ability to synergize with cellular activation stimuli was maintained. Furthermore, mutations of HBx affecting its interaction with the basal transcription machinery abrogated the synergistic activation by HBx, suggesting that this protein exerts its function by acting as a nuclear co-activator. These results indicate that HBx could contribute to a faster progression to AIDS in HBV-HIV co-infected individuals.

Urinalysis is one of the most common studies performed on the diabetic patient at every visit. The presence of leukocyturia is relatively common but it is not clear what the attitude of the physician toward this particular finding should be. The main objective of the present study was to investigate the clinical significance of leukocyturia in diabetic women.Ninety-eight diabetic women (84.7% type 2) aged 57 +/- 13 years who were being seen at the diabetic out-patient clinic were randomly selected. All patients underwent a clinical and gynecologic examination and a urinalysis. A Papanicolaou smear and a urine culture were also obtained.The overall prevalence of leukocyturia (5 cells/high power field (hpf)) was 46.5%. Patients with urinary tract infections (UTI) were 7.5 times more likely to have leukocyturia, while a leukocyte count5cells/hpf predicted the absence of UTI in 96% of the women. In the comparison of patients with and without leukocyturia, we found that proteinuria (p = 0.06) and bacteriuria (p0.002) were more common in the women with leukocyturia. A significant association with leukorrhea was not demonstrated. The empirical use of antibiotics was 12 times more frequent in the patients with leukocyturia.A urinary culture should be requested in all diabetic patients with leukocyturia. The possibility of a UTI is remote when leukocyturia is absent.

Reduction of delayed graft function (DGF) is critical to the success of renal transplantation. We report graft outcomes with antithymocyte globulin (ATG) induction compared with using interleukin-2 receptor antagonist basiliximab (IL-2RA). Twelve pediatric patients received a pediatric deceased donor kidney. We treated them with ATG (group A, ATG; n = 6) or IL-2RA (group B, n = 6). DGF was observed in 0 cases (group A) and in 6 cases (group B), which showed a mean of 7 +/- 3 days (P.001). In group A, lymphopenia occurred routinely resolving after 3-6 months, none with serious infection. The incidence of opportunistic infections was 0% in both groups. One-year patient and graft survivals were 100% in both groups. ATG induction significantly reduced the incidence of delayed graft function. Both induction treatments led to a good patient and graft survival.

HAND1 Gene Expression Is Associated Mitochondrial Energy Pathways and Is Altered during Myocardial Recovery from Heart Failure Following LVAD Combination Therapy E. Lara-Pezzi, L.E. Felkin, R. George, M.H. Yacoub, E.J. Birks, P.J. Barton, Harefield Heart Science Centre, Imperial College London, Harefield, Middlesex, United Kingdom; Harefield Hospital, Royal Brompton and Harefield NHS Trust, Harefield, Middlesex, United Kingdom