Surviving in a multistressor world: Gene expression changes in earthworms exposed to heat, desiccation, and chemicals.

An investigation of the effects of anthropogenic stress on terrestrial ecosystems is urgently needed. In this work, we explored how exposure to heat, desiccation, and chemical stress alters the expression of genes that encode heat shock proteins (HSPs), an enzyme that responds to oxidative stress (CAT), hypoxia-related proteins (HIF1 and HYOU), and a DNA repair-related protein (PARP1) in the earthworm Eisenia fetida. Exposure to heat (31°C) for 24 h upregulated HSPs and hypoxia-related genes, suggesting possible acquired thermotolerance. Desiccation showed a similar expression profile; however, the HSP response was activated to a lesser extent. Heat and desiccation activated the small HSP at 24 h, suggesting that they may play a role in adaptation. Simultaneous exposure to endosulfan and temperature for 7 h upregulated all of the evaluated genes, implicating a coordinated response involving multiple biological processes to ensure survival and acclimation. These results highlight the relevance of multistress analysis in terrestrial invertebrates.

mRNA COVID-19 Vaccination Does Not Exacerbate Symptoms or Trigger Neural Antibody Responses in Multiple Sclerosis.

BACKGROUND AND OBJECTIVE: In people with multiple sclerosis (pwMS), concern for potential disease exacerbation or triggering of other autoimmune disorders contributes to vaccine hesitancy. We assessed the humoral and T-cell responses to SARS-CoV-2 after mRNA vaccination, changes in disease activity, and development of antibodies against central or peripheral nervous system antigens. METHODS: This was a prospective 1-year longitudinal observational study of pwMS and a control group of patients with other inflammatory neurologic disorders (OIND) who received an mRNA vaccine. Blood samples were obtained before the first dose (T1), 1 month after the first dose (T2), 1 month after the second dose (T3), and 6 (T4), 9 (T5), and 12 (T6) months after the first dose. Patients were assessed for the immune-specific response, annualized relapse rate (ARR), and antibodies to onconeuronal, neural surface, glial, ganglioside, and nodo-paranodal antigens. RESULTS: Among 454 patients studied, 390 had MS (22 adolescents) and 64 OIND; the mean (SD) age was 44 (14) years; 315 (69%) were female; and 392 (87%) were on disease-modifying therapies. Antibodies to the receptor-binding domain were detected in 367 (86%) patients at T3 and 276 (83%) at T4. After a third dose, only 13 (22%) of 60 seronegative patients seroconverted, and 255 (92%) remained seropositive at T6. Cellular responses were present in 381 (93%) patients at T3 and in 235 (91%) patients at T6 including all those receiving anti-CD20 therapies and in 79% of patients receiving fingolimod. At T3 (429 patients) or T6 (395 patients), none of the patients had developed CNS autoantibodies. Seven patients had neural antibodies that were already present before immunization (3 adult patients with MS had MOG-IgG, 2 with MG and 1 with MS had neuronal cell surface antibodies [unknown antigen], and 1 with MS had myelin antibody reactivity [unknown antigen]. Similarly, no antibodies against PNS antigens were identified at T3 (427 patients). ARR was lower in MS and not significantly different in patients with OIND. Although 182 (40%) patients developed SARS-CoV-2 infection, no cases of severe COVID-19 or serious adverse events occurred. DISCUSSION: In this study, mRNA COVID-19 vaccination was safe and did not exacerbate the autoimmune disease nor triggered neural autoantibodies or immune-mediated neurologic disorders. The outcome of patients who developed SARS-CoV-2 infection was favorable.

Contribution to the knowledge of the genus Boucheona (Oligochaeta, Hormogastridae) in France with a newly described species and a redescribed species.

Hormogastrid earthworms are found in the diversity hotspot of the Franco-Iberian domain, together with the better-known family Lumbricidae. Integrative systematics (the combination of morphological, molecular and ecological data) have increased our knowledge of the diversity and evolutionary history of these earthworms, highlighting unresolved taxonomic conflicts. One example of a species group in need of integrative taxonomic revision is the genus Boucheona in France. In this work, we analyzed their diversity using previously published data together with additional data obtained from recently sampled localities. Molecular data including DNA barcodes and additional markers enabled us to reconstruct Bayesian and time-calibrated phylogenies to discuss the evolutionary relationships among the different taxa, and to propose hypotheses regarding their biogeographical history. Based on our results, four species of Boucheona are present in Southern France, including two new taxa. Morphological distinctness and molecular phylogenetics results supported the status of four populations as the newly described Boucheona corbierensis sp. nov., as well as the status of "Hormogaster pretiosa var. nigra" as an independent species, redescribed as Boucheona tenebrae sp. nov. These results provide a new perspective of the importance of the genus Boucheona in southern France, as the possible evolutionary origin of a clade of giant anecic earthworms with unknown (but probably remarkable) impact on ecosystem functioning across their range.

Terrestrial Zen master: Transcriptomics in long-term aestivation and arousal of Mediterranean earthworms.

Earthworms have a crucial role in the maintenance of the biotic and abiotic soil properties, which is important for the biodiversity and productivity of terrestrial ecosystems, especially in the current scenario of climate change. Aestivation is a form of dormancy witnessed in organisms living in deserts or semiarid environments such as the ones found in the central part of the Iberian Peninsula. This work employs next-generation sequencing techniques to explore the changes in gene expression of different aestivation times (1 month and 1 year) as well as changes in gene expression upon arousal. Not surprisingly, the more the aestivation persisted the higher levels of gene downregulation were observed. Conversely, upon arousal, a quick recovery of the levels of gene expression were noted, comparable to the control. Transcriptional changes related to immune responses coming predominantly from abiotic stressors in aestivating earthworms and from biotic stressors in aroused earthworms triggered regulation of the cell fate via apoptosis. Long-term aestivation seemed to be enabled by remodeling of the extracellular matrix, activity of DNA repair mechanisms, and inhibitory neurotransmitters, which could also play a role in lifespan increase. Arousal from 1-month aestivation was on the other hand, characterized by regulation of the cell division cycle. Since aestivation is considered as an unfavorable metabolic state, aroused earthworms probably go through a damage removal process and a subsequent reparation process. This study provides the first transcriptomic investigation done on earthworms in such long aestivation times as well as arousal demonstrating the resilience and adaptability of Carpetania matritensis.

Ovary micromorphology in hormogastrid earthworms with a particular emphasis on the organization of the germline cysts.

There is a gap in our knowledge of microorganization and the functioning of ovaries in earthworms (Crassiclitellata) and allied taxa. Recent analyses of ovaries in microdriles and leech-like taxa revealed that they are composed of syncytial germline cysts accompanied by somatic cells. Although the pattern of cyst organization is conserved across Clitellata - each cell is connected via one intercellular bridge (ring canal) to the central and anuclear cytoplasmic mass termed the cytophore - this system shows high evolutionary plasticity. In Crassiclitellata, only the gross morphology of ovaries and their segmental localization is well known, whereas ultrastructural data are limited to lumbricids like Dendrobaena veneta. Here we present the first report about ovarian histology and ultrastructure in Hormogastridae, a small family of earthworms inhabiting the western parts of the Mediterranean sea basin. We analyzed three species from three different genera and showed that the pattern of ovary organization is the same within this taxon. Ovaries are cone-like, with a broad part connected to the septum and a narrow distal end forming an egg string. Ovaries are composed of numerous cysts uniting a small number of cells, eight in Carpetania matritensis. There is a gradient of cysts development along the long ovary axis, and three zones can be distinguished. In zone I, cysts develop in complete synchrony and unite oogonia and early meiotic cells (till diplotene). Then (zone II), the synchrony is lost, and one cell (prospective oocyte) grows faster than the rest (prospective nurse cells). In zone III, oocytes pass the growth phase and gather nutrients; at this time, their contact with the cytophore is lost. Nurse cells grow slightly, eventually die via apoptosis, and are removed by coelomocytes. The most characteristic feature of hormogastrid germ cysts is the inconspicuous cytophore in the form of thread-like thin cytoplasmic strands (reticular cytophore). We found that the ovary organization in studied hormogastrids is very similar to that described for D. veneta and propose the term "Dendrobaena" type of ovaries. We expect the same microorganization of ovaries will be found in other hormogastrids and lumbricids.

Transcriptome analysis of aphids exposed to glandular trichomes in tomato reveals stress and starvation related responses.

Understanding the responses of insect herbivores to plant chemical defences is pivotal for the management of crops and pests. However, the mechanisms of interaction are not entirely understood. In this study, we compared the whole transcriptome gene expression of the aphid Macrosiphum euphorbiae grown on two different varieties of tomato that differ in their inducible chemical defences. We used two isogenic lines of tomato with a shared genetic background that only differ in the presence of type IV glandular trichomes and their associated acylsucrose excretions. This works also reports a de novo transcriptome of the aphid M. euphorbiae. Subsequently, we identified a unique and distinct gene expression profile for the first time corresponding to aphid´s exposure to type IV glandular trichomes and acylsugars. The analysis of the aphid transcriptome shows that tomato glandular trichomes and their associated secretions are highly efficient in triggering stress-related responses in the aphid, and demonstrating that their role in plant defence goes beyond the physical impediment of herbivore activity. Some of the differentially expressed genes were associated with carbohydrate, lipid and xenobiotic metabolisms, immune system, oxidative stress response and hormone biosynthesis pathways. Also, the observed responses are compatible with a starvation syndrome. The transcriptome analysis puts forward a wide range of genes involved in the synthesis and regulation of detoxification enzymes that reveal important underlying mechanisms in the interaction of the aphid with its host plant and provides a valuable genomic resource for future study of biological processes at the molecular level using this aphid.

Understanding Diversity, Evolution, and Structure of Small Heat Shock Proteins in Annelida Through in Silico Analyses.

Small heat shock proteins (sHsps) are oligomeric stress proteins characterized by an α-crystallin domain (ACD). These proteins are localized in different subcellular compartments and play critical roles in the stress physiology of tissues, organs, and whole multicellular eukaryotes. They are ubiquitous proteins found in all living organisms, from bacteria to mammals, but they have never been studied in annelids. Here, a data set of 23 species spanning the annelid tree of life, including mostly transcriptomes but also two genomes, was interrogated and 228 novel putative sHsps were identified and manually curated. The analysis revealed very high protein diversity and showed that a significant number of sHsps have a particular dimeric architecture consisting of two tandemly repeated ACDs. The phylogenetic analysis distinguished three main clusters, two of them containing both monomeric sHsps, and ACDs located downstream in the dimeric sHsps, and the other one comprising the upstream ACDs from those dimeric forms. Our results support an evolutionary history of these proteins based on duplication events prior to the Spiralia split. Monomeric sHsps 76) were further divided into five subclusters. Physicochemical properties, subcellular location predictions, and sequence conservation analyses provided insights into the differentiating elements of these putative functional groups. Strikingly, three of those subclusters included sHsps with features typical of metazoans, while the other two presented characteristics resembling non-metazoan proteins. This study provides a solid background for further research on the diversity, evolution, and function in the family of the sHsps. The characterized annelid sHsps are disclosed as essential for improving our understanding of this important family of proteins and their pleotropic functions. The features and the great diversity of annelid sHsps position them as potential powerful molecular biomarkers of environmental stress for acting as prognostic tool in a diverse range of environments.

How to thrive in unstable environments: Gene expression profile of a riparian earthworm under abiotic stress.

Nowadays, extreme weather events caused by climate change are becoming more frequent. This leads to the occurrence of extreme habitats to which species must adapt. This challenge becomes crucial for species living in unstable environments, such as the riparian earthworm Eiseniella tetraedra. Its cosmopolitan distribution exposes it to various environmental changes, such as freezing in subarctic regions or droughts in Mediterranean areas. Transcriptional changes under cold and desiccation conditions could therefore shed light on the adaptive mechanisms of this species. An experiment was performed for each condition. In the cold experiment, the temperature was lowered to -14 °C ± 2 °C (compared to 8 °C for control samples), and in the desiccation treatment, humidity was lowered from 60% to 15%. Comparisons of gene expression levels between earthworms under freezing conditions and control earthworms revealed a total of 84 differentially expressed genes and comparisons between the desiccation experiment and the control yielded 163 differentially expressed genes. However, no common responses were found between the two treatments. The results suggest that E. tetraedra can acclimate to low temperatures due to the upregulation of genes involved in glucose accumulation. However, downregulation of the respiratory chain suggests that this earthworm does not tolerate freezing conditions. Under desiccation conditions, genes involved in cell protection from apoptosis and DNA repair were upregulated. In contrast, lipid metabolism was downregulated, presumably to conserve resources by reducing the rate at which they are consumed.

How to resist soil desiccation: Transcriptional changes in a Mediterranean earthworm during aestivation.

Earthworms have a central role in ministering the terrestrial ecosystems and are proving to have an important role in modulating the effects climate change has on soil. Aestivation is a form of dormancy employed by the organisms living in deserts and arid environments, when confronted with prolonged periods of drought. Understanding global metabolic adjustments required for withstanding the harsh conditions of the ever more severe Iberian drought, we performed a global transcriptomic exploration of the endogeic earthworm Carpetania matritensis during aestivation. There were a total of 6352 differentially expressed transcripts in the aestivating group, with 65% being downregulated. Based on GO and KEGG enrichment analyses, downregulated genes seem to be indicative of an overall metabolic depression during aestivation. Indeed we noted a reduction of protein turnover and macromolecule metabolism coupled with suppression of genes involved in digestion. Upregulated genes, namely antioxidant genes and DNA repair genes showed clear signs of abiotic stress caused by ROS generation. Abiotic stress led to transcriptomic changes of genes involved in immune response, mostly affecting the NF-kb signaling pathway as well as changes in apoptotic genes indicating the necessity of investigating these processes in a tissue specific manner. Lastly we uncovered a possible mechanism for water retention by nitrogenous waste accumulation. This study provides the first ever transcriptomic investigation done on aestivating earthworms and as such serves as a general framework for investigation on other earthworm species and other soil invertebrates, which is becoming increasingly important with the current scenario of climate change.

Persistent pesticides: effects of endosulfan at the molecular level on the aquatic invertebrate Chironomus riparius.

Although banned in multiple areas, due to its persistence in the environment, endosulfan constitutes a significant environmental concern. In this work, fourth instar Chironomus riparius larvae were exposed at environmentally relevant endosulfan concentrations of 0.1, 1, and 10 µg/L for 24 h to analyze the possible effects of this acaricide on gene expression and enzymatic activity. Transcriptional changes were studied through the implementation of a real-time polymerase chain reaction array with 42 genes related to several metabolic pathways (endocrine system, detoxification response, stress response, DNA reparation, and immune system). Moreover, glutathione-S-transferase (GST), phenoloxidase (PO), and acetylcholinesterase (AChE) activities were assessed. The five pathways were differentially altered by endosulfan exposure with significant changes in the E93, Dis, MAPR, Met, InR, GSTd3, GSTt3, MRP1, hsp70, hsp40, hsp24, ATM, PARP, Proph, and Def genes. Besides, all of the measured enzymatic activities were modified, with increased activity of GST, followed by PO and AChE. In summary, the results reflected the effects provoked in C. riparius at molecular level despite the absence of lethality. These data raise concerns about the strong alteration on different metabolic routes despite the low concentrations used. Therefore, new risk assessment strategies should consider include the effects at the sub-organismal level as endpoints in addition to the classical ecologically relevant parameters (such as survival). This endeavor will facilitate a comprehensive evaluation of toxicants in the environment.

Derrame pleural y pericárdico como única manifestación de enfermedad relacionada con IgG4 / Pleural and Pericardial Effusion as the Only Manifestation of IgG4-Related Disease

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Local adaptation fuels cryptic speciation in terrestrial annelids.

Uncovering the genetic and evolutionary basis of cryptic speciation is a major focus of evolutionary biology. Next Generation Sequencing (NGS) allows the identification of genome-wide local adaptation signatures, but has rarely been applied to cryptic complexes - particularly in the soil milieu - as it is the case with integrative taxonomy. The earthworm genus Carpetania, comprising six previously suggested putative cryptic lineages, is a promising model to study the evolutionary phenomena shaping cryptic speciation in soil-dwelling lineages. Genotyping-By-Sequencing (GBS) was used to provide genome-wide information about genetic variability between 17 populations, and geometric morphometrics analyses of genital chaetae were performed to investigate unexplored cryptic morphological evolution. Genomic analyses revealed the existence of three cryptic species, with half of the previously-identified potential cryptic lineages clustering within them. Local adaptation was detected in more than 800 genes putatively involved in a plethora of biological functions (most notably reproduction, metabolism, immunological response and morphogenesis). Several genes with selection signatures showed shared mutations for each of the cryptic species, and genes under selection were enriched in functions related to regulation of transcription, including SNPs located in UTR regions. Finally, geometric morphometrics approaches partially confirmed the phylogenetic signal of relevant morphological characters such as genital chaetae. Our study therefore unveils that local adaptation and regulatory divergence are key evolutionary forces orchestrating genome evolution in soil fauna.

Toxicidad grave por metotrexato en el anciano: nuestra experiencia en los últimos 7 años / Severe methotrexate toxicity in elderly: 7 years experience

El metotrexato es uno de los fármacos más empleados en pacientes con procesos reumatológicos, debido a su eficacia y perfil de seguridad. Sin embargo, los pacientes tratados con este fármaco son en ocasiones de edad avanzada, por lo que el riesgo de toxicidad aumenta, así como el de intoxicación por error en la toma de la medicación. Presentamos el caso de una paciente de 87 años, polimedicada, con antecedentes de deterioro cognitivo y escaso apoyo social que sufrió una intoxicación aguda grave por metotrexato. Además, describimos las características de los casos de toxicidad por este fármaco ingresados en nuestro Hospital en los últimos 7 años

Methotrexate is one of the most widely used drugs in rheumatology due to its high efficacy-to-toxicity. However, patients treated with this drug are sometimes elderly, which increases toxicity risks, as well as mistakes in taking the medication. The case is presented of an 87 year-old patient, on multiple medications, with a history of cognitive impairment and low social support, who suffered acute methotrexate toxicity. A description is also presented on the characteristics of the toxicity cases due this drug admitted to this hospital in the last 7 years

Unravelling the molecular mechanisms of nickel in woodlice.

During the last few years, there has been an alarming increase in the amount of nickel (Ni) being released into the environment, primarily due to its use in the production of stainless steel but also from other sources such as batteries manufacturing and consequent disposal. The established biotic ligand models provide precise estimates for Ni bioavailability, in contrast, studies describing the mechanisms underpinning toxicological effect of Ni are scarce. This study exploits RNA-seq to determine the transcriptomic responses of isopods using Porcellionides pruinosus as an example of a terrestrial metal-resistant woodlouse. Furthermore, the recently proposed model for Ni adverse outcome pathways (Ni-AOP) presents an unprecedented opportunity to fit isopod responses to Ni toxicity and define Porcellionides pruinosus as a metalomic model. Prior to this study, P. pruinosus represented an important environmental sentinel, though lacking genetic/omic data. The reference transcriptome generated here thus represents a major advance and a novel resource. A detailed annotation of the transcripts obtained is presented together with the homology to genes/gene products from Metazoan and Arthropoda phylum, Gene Ontology (GO) classification, clusters of orthologous groups (COG) and assignment to KEGG metabolic pathways. The differential gene expression comparison was determined in response to nickel (Ni) exposure and used to derive the enriched pathways and processes. It revealed a significant impact on ion trafficking and storage, oxidative stress, neurotoxicity, reproduction impairment, genetics and epigenetics. Many of the processes observed support the current Ni-AOP although the data highlights that the current model can be improved by including epigenetic endpoints, which represents key chronic risks under a scenario of Ni toxicity.

[Severe methotrexate toxicity in elderly: 7 years experience]. / Toxicidad grave por metotrexato en el anciano: nuestra experiencia en los últimos 7 años.

Methotrexate is one of the most widely used drugs in rheumatology due to its high efficacy-to-toxicity. However, patients treated with this drug are sometimes elderly, which increases toxicity risks, as well as mistakes in taking the medication. The case is presented of an 87 year-old patient, on multiple medications, with a history of cognitive impairment and low social support, who suffered acute methotrexate toxicity. A description is also presented on the characteristics of the toxicity cases due this drug admitted to this hospital in the last 7 years.

Gut-derived lipopolysaccharides increase post-prandial oxidative stress via Nox2 activation in patients with impaired fasting glucose tolerance: effect of extra-virgin olive oil.

PURPOSE: Post-prandial phase is characterized by enhanced oxidative stress but the underlying mechanism is unclear. We investigated if gut-derived lipopolysaccharide (LPS) is implicated in this phenomenon and the effect of extra virgin olive oil (EVOO) in patients with impaired fasting glucose (IFG). METHODS: This is a randomized cross-over interventional study including 30 IFG patients, to receive a lunch with or without 10 g of EVOO. Serum LPS, Apo-B48, markers of oxidative stress such as oxidized LDL (oxLDL) and soluble Nox2-derived peptide (sNox2-dp), a marker of nicotinamide-adenine-dinucleotide-phosphate oxidase isoform Nox2 activation, and plasma polyphenols were determined before, 60 and 120 min after lunch. RESULTS: In patients not given EVOO oxidative stress as assessed by sNox2-dp and oxLDL significantly increased at 60 and 120 min concomitantly with an increase of LPS and Apo-B48. In these patients, changes of LPS were correlated with Apo-B48 (Rs = 0.542, p = 0.002) and oxLDL (Rs = 0.463, p = 0.010). At 120 min, LPS (ß - 15.73, p < 0.001), Apo-B48 (ß - 0.14, p = 0.004), sNox2-dp (ß - 5.47, p = 0.030), and oxLDL (ß - 42.80, p < 0.001) significantly differed between the two treatment groups. An inverse correlation was detected between polyphenols and oxLDL (R - 0.474, p < 0.005). In vitro study showed that LPS, at the same concentrations found in the human circulation, up-regulated Nox2-derived oxidative stress via interaction with Toll-like receptor 4. CONCLUSIONS: Post-prandial phase is characterized by an oxidative stress-related inflammation potentially triggered by LPS, a phenomenon mitigated by EVOO administration.

Effects on tadpole snail gene expression after exposure to vinclozolin.

The fungicide vinclozolin (Vz) is an endocrine disruptor with known anti-androgenic activity in vertebrates. However, there is a lack of information about the Vz mode of action in invertebrates, although some studies have shown that this compound can produce alterations in different species. Transcriptional activity was analyzed in the freshwater snail Physella acuta in order to elucidate putative cellular processes altered by this chemical during a response. In order to identify potential molecular biomarkers, a de novo transcriptome was generated for this species that constitutes a valuable source for future studies. This data, together with some already available data, permitted the identification of several genes related to detoxification mechanisms (Cyp2u1, Cyp3a7, Cyp4f22, GSTo1, GSTt2, and MRP1), stress response (Hsp20.4, Hsp17, Hsp16.6, and Cu,Zn-SOD), the hormonal system (Estrogen Receptor and Hsp90), apoptosis (Casp3), and copper homeostasis (ATOX1). Using quantitative Real-Time polymerase chain reaction, mRNA levels of these genes were examined in snails exposed to 20 or 200 µg/L Vz for 24 h. The results showed an overall weak response, with downregulation of Hsp20.4 and no statistically significant change for the other genes. These findings suggest that P. acuta can manage the concentrations of Vz found in the environment with no relevant activation of the pathways analyzed, although additional studies are needed for longer exposure times and including other metabolic pathways. The new genes described open the range of processes that can be studied at the molecular level in toxicity tests.

Delegating Sex: Differential Gene Expression in Stolonizing Syllids Uncovers the Hormonal Control of Reproduction.

Stolonization in syllid annelids is a unique mode of reproduction among animals. During the breeding season, a structure resembling the adult but containing only gametes, called stolon, is formed generally at the posterior end of the animal. When stolons mature, they detach from the adult and gametes are released into the water column. The process is synchronized within each species, and it has been reported to be under environmental and endogenous control, probably via endocrine regulation. To further understand reproduction in syllids and to elucidate the molecular toolkit underlying stolonization, we generated Illumina RNA-seq data from different tissues of reproductive and nonreproductive individuals of Syllis magdalena and characterized gene expression during the stolonization process. Several genes involved in gametogenesis (ovochymase, vitellogenin, testis-specific serine/threonine-kinase), immune response (complement receptor 2), neuronal development (tyrosine-protein kinase Src42A), cell proliferation (alpha-1D adrenergic receptor), and steroid metabolism (hydroxysteroid dehydrogenase 2) were found differentially expressed in the different tissues and conditions analyzed. In addition, our findings suggest that several neurohormones, such as methyl farnesoate, dopamine, and serotonin, might trigger stolon formation, the correct maturation of gametes and the detachment of stolons when gametogenesis ends. The process seems to be under circadian control, as indicated by the expression patterns of r-opsins. Overall, our results shed light into the genes that orchestrate the onset of gamete formation and improve our understanding of how some hormones, previously reported to be involved in reproduction and metamorphosis processes in other invertebrates, seem to also regulate reproduction via stolonization.

Digoxin and Platelet Activation in Patients With Atrial Fibrillation: In Vivo and In Vitro Study.

Background Digoxin use was shown to be associated with an increased risk of cardiovascular events in atrial fibrillation ( AF ). We hypothesized that digoxin may affect cardiovascular risk by increasing platelet activation. Methods and Results Post hoc analysis of a prospective study of anticoagulated patients with AF . Patients were divided into 2 groups balanced for age, sex, and cardiovascular risk factors: digoxin users (n=132) and nonusers (n=388). Urinary excretion of 11-dehydro-thromboxane B2 (TxB2), a marker of platelet activation, and serum digoxin concentration ( SDC ) were measured. In vitro experiments were performed on platelets from healthy subjects and AF patients, which were incubated with scalar doses of digoxin (0.6-2.4 ng/mL) with or without prestimulation with a sub-threshold of collagen. Median 11-dehydro-TxB2 was 105.0 ( interquartile range, 60.0-190.0) ng/mg creatinine, and median SDC was 0.65 ( interquartile range, 0.40-1.00) ng/mL. Urinary 11-dehydro-TxB2 and SDC were correlated ( rs=0.350, P<0.001). Patients in the upper tertile of SDC showed higher 11-dehydro-TxB2 compared with non-digoxin users ( P=0.019). In vitro study showed an increased basal platelet activation in patients with AF compared with healthy subjects . Digoxin (2.4 ng/mL) induced calcium mobilization, PAC -1 (procaspase-activating compound 1) and platelet aggregation in AF patients but not in healthy subjects . After pretreatment with a sub-threshold of collagen, digoxin dose-dependent induced calcium mobilization, arachidonic acid release, TxB2 biosynthesis, PAC -1 and soluble platelet selectin expression, and platelet aggregation, which were inhibited by antibody against digoxin. Conclusions We found a significant in vivo correlation between SDC and platelet activation. Supratherapeutic SDC increased in vitro platelet aggregation via calcium-related phospholipase A2 phosphorylation. Our findings may have clinical implications for AF patients treated with digoxin.