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Our knowledge of ciliate endosymbiont diversity greatly expanded over the past decades due to the development of characterization methods for uncultivable bacteria. Chlamydia-like bacteria have been described as symbionts of free-living amoebae and other phylogenetically diverse eukaryotic hosts. In the present work, a systematic survey of the bacterial diversity associated with the ciliate Euplotes octocarinatus strain Zam5b-1 was performed, using metagenomic screening as well as classical full-cycle rRNA approach, and a novel chlamydial symbiont was characterized. The metagenomic screening revealed 16S rRNA gene sequences from Polynucleobacter necessarius, three previously reported accessory symbionts, and a novel chlamydia-like bacterium. Following the full-cycle rRNA approach, we obtained the full-length 16S rRNA gene sequence of this chlamydia-like bacterium and developed probes for diagnostic fluorescence in situ hybridizations. The phylogenetic analysis of the 16S rRNA gene sequences unambiguously places the new bacterium in the family Rhabdochlamydiaceae. This is the first report of chlamydia-like bacterium being found in Euplotes. Based on the obtained data, the bacterium is proposed as a new candidate genus and species: "Candidatus Euplotechlamydia quinta.", (© 2022 International Society of Protistologists.)

Ciliates are a rich source of molecules synthesized to socialize, compete ecologically, and interact with prey and predators. Their isolation from laboratory cultures is often straightforward, permitting the study of their mechanisms of action and their assessment for applied research. This review focuses on three classes of these bioactive molecules: (i) water-borne, cysteine-rich proteins that are used as signaling pheromones in self/nonself recognition phenomena; (ii) cell membrane-associated lipophilic terpenoids that are used in interspecies competitions for habitat colonization; (iii) cortical granule-associated molecules of various chemical nature that primarily serve offence/defense functions., (© 2022 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists.)

Background: Pediatric hepatic cancer is a rare malignancy, comprising only approximately 2% of all cancers diagnosed in children aged <15 years. The authors sought to describe trends in pediatric hepatic cancer incidence and survival in Ontario, Canada; the United States; and Australia., Methods: Children aged <15 years who were diagnosed with hepatic cancer from 1985 through 2013 were ascertained through population-based registries and followed from the time of diagnosis until December 31, 2015. Age-standardized incidence and 5-year relative survival were calculated for each jurisdiction. Multivariable flexible parametric survival models were used to explore predictors of hepatic cancer mortality., Results: A total of 794 children were identified in Ontario (148 children), the United States (400 children), and Australia (246 children). The average annual incidence increased by 2.2% (95% CI, 0.5%-4.0%) in Australia, 2.1% (95% CI, 0.9%-3.3%) in the United States, and 1.3% (95% CI, -0.4% to 3.0%) in Ontario. The 5-year relative survival rate improved from 60% to 82% (P = .08) in Ontario and 62% to 78% (P = .02) in the United States between the diagnostic periods 1985 through 1994 and 2005 through 2013, whereas in Australia the rate remained constant (between 74% and 77%) during the study period. On multivariable analysis, there was no significant difference noted with regard to the hazard of death between jurisdictions (P = .06). Older age, the presence of metastatic disease, and being diagnosed with hepatocellular carcinoma were found to be associated with mortality., Conclusions: The incidence of hepatic cancer in children appears to have increased over the last 30 years in Australia and North America. Survival differences between Australia; Ontario, Canada; and the United States observed in the 1980s and 1990s were no longer apparent and only marginal geographical differences in the hazard of mortality were observed., (© 2020 American Cancer Society.)

The ciliate Euplotes deviates from the universal genetic code by translating UGA as cysteine and using UAA and UAG as the termination codon. Here, we cloned and sequenced the Cathepsin B gene of Euplotes octocarinatus (Eo-CTSB) which containing several in-frame stop codons throughout the coding sequence. We provide evidences, based on 3'-RACE method and Western blot, that the Eo-CTSB gene is actively expressed. Comparison of the derived amino acid sequence with the homologs in other eukaryotes revealed that UAA and UAG may code for glutamine in Eo-CTSB. These findings imply an evolutionary complexity of stop codon reassignment in eukaryotes., (© 2019 International Society of Protistologists.)

Diabetes is not a single and homogeneous disease, but a cluster of metabolic diseases characterized by the common feature of hyperglycemia. The pathogenesis of type 1 diabetes (T1D) and type 2 diabetes (T2D) (and all other intermediate forms of diabetes) involves the immune system, in terms of inflammation and autoimmunity. The past decades have seen an increase in all types of diabetes, accompanied by changes in eating habits and consequently a structural evolution of gut microbiota. It is likely that all these events could be related and that gut microbiota alterations might be involved in the immunomodulation of diabetes. Thus, gut microbiota seems to have a direct, even causative role in mediating connections between the environment, food intake, and chronic disease. As many conditions that increase the risk of diabetes modulate gut microbiota composition, it is likely that immune-mediated reactions, induced by alterations in the composition of the microbiota, can act as facilitators for the onset of diabetes in predisposed subjects. In this review, we summarize recent evidence in the field of gut microbiota and the role of the latter in modulating the immune reactions involved in the pathogenesis of diabetes., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2019 Simona Moffa et al.)

The risk perception for developing diabetes has not been well established. The aim of this study is to evaluate knowledge and perception of risk for developing diabetes. A cross-sectional study was conducted among a sample of 527 parents of children attending public schools in Naples (Italy). A self-administered anonymous questionnaire was used to collect the data. In total, 97.3% of participants have heard about diabetes, but only 16.7% knew the main risk and protective factors. This knowledge was statistically significantly higher in those who had close relatives with diabetes. Moreover, those who had middle school or lower and high school education, compared with those who had a college degree or higher, were less knowledgeable. The mean total value of the risk perception for developing diabetes was 1.9. Females those who had more than 40 years of age, those who needed of additional information, those who had a higher BMI, those who had close relatives with diabetes, those who had at least one chronic disease, and those who reported a lower value of self-rated health status were more likely to perceive a higher risk for developing diabetes. Moreover, this perception was statistically significantly lower among those who had a middle school or lower and high school education, compared with those who had a college degree or higher. The knowledge about diabetes needs to be improved, and the low risk perception for developing diabetes among the sample is worrying given the severity of the disease and the preventive measures available., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2019 Concetta P. Pelullo et al.)

The aims of this cross-sectional survey were to document the prevalence, the determinants, and the reasons of oral medication use without the prescription of a physician among a random sample of 672 parents of students attending randomly selected public schools in Italy. A total of 69.2% practiced self-medication at least once. The odds of having performed a self-medication were higher in females, in younger population, and in those who have had a health problem in the preceding year and were lower in respondents with a middle or lower school level of education. Among those reporting experience of self-medication, 53.4% have practiced at least once in the last year and this was more likely for those who have had a health problem. Nonsteroidal anti-inflammatory drugs were more frequently used without a prescription in the last year. Two-thirds inappropriately self-medicated in the last year at least once. Of those who did not report a self-medication, 13.1% were willing to practice it. Females were more willing and those with a secondary school level of education less willing to practice self-medication. The frequency of oral self-medication was quite high and in most cases inappropriate with a potential impact on the health status and educative programs are needed.

Background: The objectives of the study were to document knowledge, attitudes, and behaviors of women regarding cardiovascular diseases (CVDs) and the determinants associated., Materials and Methods: The cross-sectional survey was conducted among a random sample of 830 women older than 18 years from the general population in Italy., Results: Almost all participants reported having heard about CVDs, and among them 89.4% and 74.7% identified smoking and high cholesterol level as risk factors. Only 26.5% identified the main CVDs risk factors. Women more knowledgeable were married and better educated and self-perceived a worse health status. Only 23% knew the main CVDs preventive measures and this knowledge was significantly higher in women who are unemployed, who are more educated, who have received information about CVDs from physicians, and who know the main risk factors. Respondents with lower education, those with at least three children, those who self-perceived a worse health status, and those who need information were most likely to have a positive attitude toward the perceived risk of developing CVDs. Women with two or three children or more were at high risk profiles 49% and 56% lower than women with one child., Conclusions: Educational programs are needed among women as support to improve knowledge and appropriate behavior about CVDs.

The high-multiple mating system of Euplotes crassus is known to be controlled by multiple alleles segregating at a single locus and manifesting relationships of hierarchical dominance, so that heterozygous cells would produce a single mating-type substance (pheromone). In strain L-2D, now known to be homozygous at the mating-type locus, we previously identified two pheromones (Ec-α and Ec-1) characterized by significant variations in their amino acid sequences and structure of their macronuclear coding genes. In this study, pheromones and macronuclear coding genes have been analyzed in strain POR-73 characterized by a heterozygous genotype and strong mating compatibility with L-2D strain. It was found that POR-73 cells contain three distinct pheromone coding genes and, accordingly, secrete three distinct pheromones. One pheromone revealed structural identity in amino acid sequence and macronuclear coding gene to the Ec-α pheromone of L-2D cells. The other two pheromones were shown to be new and were designated Ec-2 and Ec-3 to denote their structural homology with the Ec-1 pheromone of L-2D cells. We interpreted these results as evidence of a phenomenon of gene duplication at the E. crassus mating-type locus, and lack of hierarchical dominance in the expression of the macronuclear pheromone genes in cells with heterozygous genotypes., (© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.)

Ciliates comprise species synthesizing water-diffusible mating type factors or pheromones and species synthesizing insoluble, cell membrane-bound pheromones. Euplotes crassus has traditionally been placed in the latter group. In contrast with this notion, we found that E. crassus is a constitutive pheromone-secreting ciliate, like other Euplotes species. From cell-free filtrate preparations of the E. crassus strain L-2D, we isolated two distinct pheromones, designated as Ec-α and Ec-1, and determined their complete amino acid sequences by combined chemical and genetic approaches. The Ec-α pheromone sequence extends for 56 amino acid residues with six cysteines and shows a molecular mass of 6,183 Da, while the Ec-1 pheromone sequence extends for 45 amino acid residues with 10 cysteines and shows a molecular mass of 4,840 Da. Marked structural differences distinguish the full-length Ec-α and Ec-1 coding sequences, which have been cloned and characterized from the transcriptionally active macronuclear genome. They were taken as clear indication that the Ec-α and Ec-1 pheromones are specified by genes that are not allelic, but likely derived from a duplicated genetic locus of the transcriptionally silent micronuclear genome., (© 2011 The Author(s). Journal of Eukaryotic Microbiology© 2011 International Society of Protistologists.)

The sesquiterpenoid euplotin C is a secondary metabolite produced by the ciliated protist Euplotes crassus and provides a mechanism for damping populations of potential competitors. Indeed, E. crassus is virtually resistant to its own product while different non-producer species representing an unbiased sample of the marine, interstitial, ciliate diversity are sensitive. For instance, euplotin C exerts a marked disruption of different homeostatic mechanisms in Euplotes vannus. We demonstrate by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay that euplotin C quickly decreases viability and mitochondrial function of E. vannus with a very high efficacy and at micromolar potency. In addition, euplotin C induces apoptosis in E. vannus as 4,6-diamino-2-phenylindole and terminal transferase dUTP nick end labeling staining show the rapid condensation and fragmentation of nuclear material in cells treated with euplotin C. These effects occur without detectable permeabilisation or rupture of cell membranes and with no major changes in the overall morphology, although some traits, such as vacuolisation and disorganized microtubules, can be observed by transmission electron microscopy. In particular, E. vannus show profound changes of the mitochondrial ultrastructure. Finally, we also show that caspase activity in E. vannus is increased by euplotin C. These data elucidate the pro-apoptotic role of euplotin C and suggest a mechanism for its impact on natural selection.

Among secondary metabolites, the acetylated hemiacetal sesquiterpene euplotin C has been isolated from the marine, ciliated protist Euplotes crassus, and provides an effective mechanism for reducing populations of potential competitors through its cytotoxic properties. However, intracellular signaling mechanisms and their functional correlates mediating the ecological role of euplotin C are largely unknown. We report here that, in E. vannus (an Euplotes morphospecies that does not produce euplotin C and shares with E. crasssus the same interstitial habitat), euplotin C rapidly increases the intracellular concentration of both Ca(2+) and Na(+), suggesting a generalized effect of this metabolite on cation transport systems. In addition, euplotin C does not induce oxidative stress, but modulates the electrical properties of E. vannus through an increase of the amplitude of graded action potentials. These events parallel the disassembling of the ciliary structures, the inhibition of cell motility, the occurrence of aberrant cytoplasmic vacuoles, and the rapid inhibition of phagocytic activity. Euplotin C also increases lysosomal pH and decreases lysosomal membrane stability of E. vannus. These results suggest that euplotin C exerts a marked disruption of those homeostatic mechanisms whose efficiency represents the essential prerequisite to face the challenges of the interstitial environment.

A new microsporidian species, Euplotespora binucleata n. gen., n. sp., from the brackish-water ciliate Euplotes woodruffi is described and defined on the basis of life history characteristics, light and electron microscopic features, and small subunit (SSU) ribosomal DNA (rDNA) sequencing. The life cycle of E. binucleata n. sp. probably has rather short merogonic and relatively long sporogonic phases. Some uninuclear meronts and sporonts, along with diplokaryotic sporoblasts and spores, were found in experimentally infected host cells. Such a peculiar life cycle has been induced experimentally in Euplotes eurystomus and constitutively microsporidian-free stocks of E. woodruffi. Spores of E. binucleata n. sp. are monomorphic, ovoid-cylindrical in shape, 3.44+/-0.17 x 1.65+/-0.22 microm in size, and characterized by a diplokaryotic condition and a large posterior vacuole. The polar tube is isofilar, 4.5-5.5 microm in length when ejected, and lacking a distinctive coiled region (half-coiled). The polaroplast is divided into two regions: the anterior part has a few lamellae close to the anchoring disc; and the posterior part is a rounded body (sack), about one-quarter of the spore length. Spores do not appear to cluster together as a group. Each spore is surrounded by a sporophorous membrane closely adjacent to the exospore layer. A phylogenetic analysis of SSU rDNA sequences by different methods placed E. binucleata n. sp. in a clade with representatives of the microsporidian genera Cystosporogenes and Vittaforma. Observations of microsporidia in several other ciliates are discussed in view of the microsporidian infection frequency in the phylum Ciliophora.

We have characterized a macronuclear gene of the ciliate protozoan Euplotes raikovi, which encodes an acidic ribosomal protein of the P protein family. This gene shows the typical organization of the hypotrich ciliate macronuclear "gene-sized" molecules with Euplotes telomeres at the ends. The longest open reading frame encodes a conceptual protein of 113 amino acid residues, with a molecular mass and pI value of 11.45 kDa and 3.97, respectively. By using sequence homology analysis, the protein was found to belong to the ribosomal P2 protein family and was named Er P2, where Er stands for Euplotes raikovi. These proteins, generally called A (acidic/alanine rich) proteins in prokaryotes and P (phosphorylated) proteins in eukaryotes, in which they are divided into P1 and P2 families, play a role in the elongation step of protein synthesis. Approximately 40% amino acid sequence identity was found between the cloned protein and other known protozoan ribosomal P2 proteins. Within its N-terminal half, this protein contains several potential kinase phosphorylation sites. Protein Er P2 differs markedly from the consensus P protein sequence in its C-terminal region, usually highly conserved among eukaryotic ribosomal P proteins, and shows similarities with the C-terminus of the archaebacterial ribosomal A proteins. To our knowledge, this E. raikovi protein represents the first demonstration of a ribosome-associated protein of the P2 family in a ciliate protozoan.

Four different tubulin genes were identified in the somatic nucleus (macronucleus) of Euplotes focardii, a strictly cold-adapted, Antarctic ciliate: one of 1,800 bp for alpha-tubulin and three of 2,150, 1,900, and 1,600 bp, respectively, for beta-tubulin. Preliminarily analysed for restriction fragment length polymorphisms, these genes showed remarkable differences in organisation from tubulin genes of other ciliates which live in temperate areas and were analysed in parallel with E. focardii. The complete coding sequence of the 1,600 bp beta-tubulin gene was then determined and shown to contain unique structural features of potential importance for E. focardii microtubule organization and activity. Of eight unique substitutions detected, seven were concentrated in the large amino terminal domain of the molecule that directly interacts with the carboxy terminal region of alpha-tubulin for heterodimer formation. Sequence analysis of the cloned gene revealed, in addition, a potential new exception in the use of the genetic code by ciliates. A TAG codon was aligned in correspondence with Trp-21 which is strictly conserved in every tubulin sequence so far determined.