Single cell transcriptomics reveals UAR codon reassignment in Palmarella salina (Metopida, Armophorea) and confirms Armophorida belongs to APM clade.

Anaerobes have emerged in several major lineages of ciliates, but the number of independent transitions to anaerobiosis among ciliates is unknown. The APM clade (Armophorea, Muranotrichea, Parablepharismea) represents the largest clade of obligate anaerobes among ciliates and contains free-living marine and freshwater representatives as well as gut endobionts of animals. The evolution of APM group has only recently started getting attention, and our knowledge on its phylogeny and genetics is still limited to a fraction of taxa. While ciliates portray a wide array of alternatives to the standard genetic code across numerous classes, the APM ciliates were considered to be the largest group using exclusively standard nuclear genetic code. In this study, we present a pan-ciliate phylogenomic analysis with emphasis on the APM clade, bringing the first phylogenomic analysis of the family Tropidoatractidae (Armophorea) and confirming the position of Armophorida within Armophorea. We include five newly sequenced single cell transcriptomes from marine, freshwater, and endobiotic APM ciliates - Palmarella salina, Anteclevelandella constricta, Nyctotherus sp., Caenomorpha medusula, and Thigmothrix strigosa. We report the first discovery of an alternative nuclear genetic code among APM ciliates, used by Palmarella salina (Tropidoatractidae, Armophorea), but not by its close relative, Tropidoatractus sp., and provide a comparative analysis of stop codon identity and frequency indicating the precedency to the UAG codon loss/reassignment over the UAA codon reassignment in the specific ancestor of Palmarella. Comparative genomic and proteomic studies of this group may help explain the constraints that underlie UAR stop-to-sense reassignment, the most frequent type of alternative nuclear genetic code, not only in ciliates, but eukaryotes in general.

Study of the interaction of the palmatine alkaloid with hybrid G-quadruplex/duplex and i-motif/duplex DNA structures.

There is an increasing interest in the study of guanine or cytosine-rich sequences that may fold into G-quadruplex (G4) or i-motif (iM) structures showing a short hairpin (or stem-loop) stabilized by Watson-Crick base pairs. These hybrid spatial arrangements may be target of ligands that have been shown to interact strongly with B-DNA. In this work, the interaction of the palmatine alkaloid with several sequences forming different G4s, iMs, and hybrid structures has been studied by means of spectroscopic and separation techniques, as well as multivariate data analysis methods. At the experimental conditions used in this work, the results have shown that this ligand strongly stabilizes parallel G4 structures, whereas a weaker interaction was observed with the antiparallel G4 adopted by the thrombin-binding aptamer or iMs. The presence of hairpins within the loops scarcely affects the affinity of this ligand for the hybrid G4/duplex or iM/duplex structures. Fluorescence measurements have provided evidence of a certain interaction with iMs at pH 5.1, despite the absence of thermal stabilization effects.

Alkaloid Escholidine and Its Interaction with DNA Structures.

Berberine, the most known quaternary protoberberine alkaloid (QPA), has been reported to inhibit the SIK3 protein connected with breast cancer. Berberine also appears to reduce the bcl-2 and XIAP expression-proteins responsible for the inhibition of apoptosis. As some problems in the therapy with berberine arose, we studied the DNA binding properties of escholidine, another QPA alkaloid. CD, fluorescence, and NMR examined models of i-motif and G-quadruplex sequences present in the n-myc gene and the c-kit gene. We provide evidence that escholidine does not induce stabilization of the i-motif sequences, while the interaction with G-quadruplex structures appears to be more significant.

Anaeramoebae are a divergent lineage of eukaryotes that shed light on the transition from anaerobic mitochondria to hydrogenosomes.

Discoveries of diverse microbial eukaryotes and their inclusion in comprehensive phylogenomic analyses have crucially re-shaped the eukaryotic tree of life in the 21st century.1 At the deepest level, eukaryotic diversity comprises 9-10 "supergroups." One of these supergroups, the Metamonada, is particularly important to our understanding of the evolutionary dynamics of eukaryotic cells, including the remodeling of mitochondrial function. All metamonads thrive in low-oxygen environments and lack classical aerobic mitochondria, instead possessing mitochondrion-related organelles (MROs) with metabolisms that are adapted to low-oxygen conditions. These MROs lack an organellar genome, do not participate in the Krebs cycle and oxidative phosphorylation,2 and often synthesize ATP by substrate-level phosphorylation coupled to hydrogen production.3,4 The events that occurred during the transition from an oxygen-respiring mitochondrion to a functionally streamlined MRO early in metamonad evolution remain largely unknown. Here, we report transcriptomes of two recently described, enigmatic, anaerobic protists from the genus Anaeramoeba.5 Using phylogenomic analysis, we show that these species represent a divergent, phylum-level lineage in the tree of metamonads, emerging as a sister group of the Parabasalia and reordering the deep branching order of the metamonad tree. Metabolic reconstructions of the Anaeramoeba MROs reveal many "classical" mitochondrial features previously not seen in metamonads, including a disulfide relay import system, propionate production, and amino acid metabolism. Our findings suggest that the cenancestor of Metamonada likely had MROs with more classical mitochondrial features than previously anticipated and demonstrate how discoveries of novel lineages of high taxonomic rank continue to transform our understanding of early eukaryote evolution.

The Bayesian Cut.

An important task in the analysis of graphs is separating nodes into densely connected groups with little interaction between each other. Prominent methods here include flow based graph cutting procedures as well as statistical network modeling approaches. However, adequately accounting for this, the so-called community structure, in complex networks remains a major challenge. We present a novel generic Bayesian probabilistic model for graph cutting in which we derive an analytical solution to the marginalization of nuisance parameters under constraints enforcing community structure. As a part of the solution a large scale approximation for integrals involving multiple incomplete gamma functions is derived. Our multiple cluster solution presents a generic tool for Bayesian inference on Poisson weighted graphs across different domains. Applied on three real world social networks as well as three image segmentation problems our approach shows on par or better performance to existing spectral graph cutting and community detection methods, while learning the underlying parameter space. The developed procedure provides a principled statistical framework for graph cutting and the Bayesian Cut source code provided enables easy adoption of the procedure as an alternative to existing graph cutting methods.

EukRef-excavates: seven curated SSU ribosomal RNA gene databases.

The small subunit ribosomal RNA (SSU rRNA) gene is a widely used molecular marker to study the diversity of life. Sequencing of SSU rRNA gene amplicons has become a standard approach for the investigation of the ecology and diversity of microbes. However, a well-curated database is necessary for correct classification of these data. While available for many groups of Bacteria and Archaea, such reference databases are absent for most eukaryotes. The primary goal of the EukRef project (eukref.org) is to close this gap and generate well-curated reference databases for major groups of eukaryotes, especially protists. Here we present a set of EukRef-curated databases for the excavate protists-a large assemblage that includes numerous taxa with divergent SSU rRNA gene sequences, which are prone to misclassification. We identified 6121 sequences, 625 of which were obtained from cultures, 3053 from cell isolations or enrichments and 2419 from environmental samples. We have corrected the classification for the majority of these curated sequences. The resulting publicly available databases will provide phylogenetically based standards for the improved identification of excavates in ecological and microbiome studies, as well as resources to classify new discoveries in excavate diversity.

Genomics of New Ciliate Lineages Provides Insight into the Evolution of Obligate Anaerobiosis.

Oxygen plays a crucial role in energetic metabolism of most eukaryotes. Yet adaptations to low-oxygen concentrations leading to anaerobiosis have independently arisen in many eukaryotic lineages, resulting in a broad spectrum of reduced and modified mitochondrion-related organelles (MROs). In this study, we present the discovery of two new class-level lineages of free-living marine anaerobic ciliates, Muranotrichea, cl. nov. and Parablepharismea, cl. nov., that, together with the class Armophorea, form a major clade of obligate anaerobes (APM ciliates) within the Spirotrichea, Armophorea, and Litostomatea (SAL) group. To deepen our understanding of the evolution of anaerobiosis in ciliates, we predicted the mitochondrial metabolism of cultured representatives from all three classes in the APM clade by using transcriptomic and metagenomic data and performed phylogenomic analyses to assess their evolutionary relationships. The predicted mitochondrial metabolism of representatives from the APM ciliates reveals functional adaptations of metabolic pathways that were present in their last common ancestor and likely led to the successful colonization and diversification of the group in various anoxic environments. Furthermore, we discuss the possible relationship of Parablepharismea to the uncultured deep-sea class Cariacotrichea on the basis of single-gene analyses. Like most anaerobic ciliates, all studied species of the APM clade host symbionts, which we propose to be a significant accelerating factor in the transitions to an obligately anaerobic lifestyle. Our results provide an insight into the evolutionary mechanisms of early transitions to anaerobiosis and shed light on fine-scale adaptations in MROs over a relatively short evolutionary time frame.

Dialysis patient in internal medicine inpatient ward.

Chronic kidney disease has been recognized as a worldwide epidemic. Its prevalence in developed countries is about 10 %, the number of patients treated by dialysis is on average 1.000 person per 1 million of population. Kidney failure is associated with high mortality due to high prevalence of cardiovascular disease. Cardiovascular risk factors differ in renal patients and general population. Most important of them are chronic overhydratation, anemia, disturbed calcium-phosphate metabolism, uremic toxins and protein-calorie malnutrition. Hospitalization of dialysis patient in internal medicine ward has some specific problems including dialysis tactics, care of vascular access and inpatient logistics. Dialysis tactics is expressed in dialysis prescription, where the ultrafiltration, anticoagulation and dialysate composition must be carefully planned. Peripheral veins should be saved for future vascular access, early diagnostics of thrombotic and infectious complications is necessary. Proper timing of blood sampling for laboratory investigation and drug administration must be related to dialysis schedule. Effective communication between inpatient ward and dialysis unit is essential.

Dactylomonas gen. nov., a Novel Lineage of Heterolobosean Flagellates with Unique Ultrastructure, Closely Related to the Amoeba Selenaion koniopes Park, De Jonckheere & Simpson, 2012.

We report the discovery of a new genus of heterolobosean flagellates, Dactylomonas gen. nov., with two species, D. venusta sp. nov. and D. crassa sp. nov. Phylogenetic analysis of the SSU rRNA gene showed that Dactylomonas is closely related to the amoeba Selenaion, the deepest-branching lineage of Tetramitia. Dactylomonads possess two flagella, and ultrastructural studies revealed an unexpected organization of the flagellar apparatus, which resembled Pharyngomonada (the second lineage of Heterolobosea) instead of Tetramitia: basal bodies were orthogonal to each other and a putative root R1 was present in the mastigont. On the other hand, Dactylomonas displayed several features uncommon in Heterolobosea: a microtubular corset, a distinctive rostrum supported by the main part of the right microtubular root, a finger-like projection on the proximal part of the recurrent flagellum, and absence of a ventral groove. In addition, Dactylomonas is anaerobic and seems to have lost mitochondrial cristae. Dactylomonas and Selenaion are accommodated in the family Selenaionidae fam. nov. and order Selenionida ord. nov. The taxonomy of Tetramitia is partially revised, and the family Neovahlkampfiidae fam. nov. is established.

Fe-S Cluster Assembly in Oxymonads and Related Protists.

The oxymonad Monocercomonoides exilis was recently reported to be the first eukaryote that has completely lost the mitochondrial compartment. It was proposed that an important prerequisite for such a radical evolutionary step was the acquisition of the SUF Fe-S cluster assembly pathway from prokaryotes, making the mitochondrial ISC pathway dispensable. We have investigated genomic and transcriptomic data from six oxymonad species and their relatives, composing the group Preaxostyla (Metamonada, Excavata), for the presence and absence of enzymes involved in Fe-S cluster biosynthesis. None possesses enzymes of mitochondrial ISC pathway and all apparently possess the SUF pathway, composed of SufB, C, D, S, and U proteins, altogether suggesting that the transition from ISC to SUF preceded their last common ancestor. Interestingly, we observed that SufDSU were fused in all three oxymonad genomes, and in the genome of Paratrimastix pyriformis. The donor of the SUF genes is not clear from phylogenetic analyses, but the enzyme composition of the pathway and the presence of SufDSU fusion suggests Firmicutes, Thermotogae, Spirochaetes, Proteobacteria, or Chloroflexi as donors. The inventory of the downstream CIA pathway enzymes is consistent with that of closely related species that retain ISC, indicating that the switch from ISC to SUF did not markedly affect the downstream process of maturation of cytosolic and nuclear Fe-S proteins.

Naturally occurring quaternary benzo[c]phenanthridine alkaloids selectively stabilize G-quadruplexes.

In this work, the interaction of six natural benzo[c]phenanthridine alkaloids (macarpine, sanguilutine, sanguirubine, chelerythrine, sanguinarine and chelirubine) with parallel and antiparallel G-quadruplex DNA structures was studied. HT22 corresponding to the end of human telomeres and the modified promoter oncogene c-kit21 and Pu22 sequences have been used. Spectroscopically-monitored melting experiments and fluorescence titrations, competitive dialysis and nuclear magnetic resonance spectroscopy were used for this purpose. The results showed that these alkaloids stabilized G-quadruplex structures in terms of increments of Tm values (from 15 to 25 °C) with high selectivity over duplexes and unfolded DNA. The mode of binding was mainly by stacking on the terminal G-tetrads with stoichiometries of 1 : 2 (DNA : ligand). The presence of non-specific electrostatic interactions was also observed. Overall, the results pointed to a strong stabilization of G-quadruplex structures by these alkaloids.

Heavy water enhancement of fluorescence signal in reversed-phase liquid chromatography.

Liquid chromatography with fluorescence detection has been used in analyses demanding high sensitivity and selectivity. As majority systems rely on reversed-phase columns with water being the main component of the mobile phase, fluorescent compounds with emission maxima higher than 500 nm might be dynamically quenched. A simple replacement of H2O with D2O enhanced the sensitivity for selected compounds by 10-200%. Affected compounds included an anti-cancer drug doxorubicin, a luminescent probe fluorescein, and naturally occurring forms of vitamin B2. Similar levels of enhancement were obtained by fluorescence spectrometry. Such simple yet effective approach may greatly improve HPLC analyses coupled to fluorescence detection.

Alumazene Reactivity with Quinolinols - Addition and Substitution Reactions on the Al3N3 Ring.

Molecular structures of two compounds obtained in reactions of alumazene [DippNAlMe]3 (1, Dipp = 2,6-i-Pr2C6H3) with substituted quinolinols have been elucidated by the single-crystal X-ray diffraction analysis. Quinolin-8-ol (Hq) provides a dinuclear complex [(DippNH)2Al2Me2(q)2] (2) with a central Al2O2 ring and five-coordinate Al atoms. The compound 2×THF crystallizes in the orthorhombic Pbca space group. The molecular structure of a mononuclear complex [(DippNH)Al(Meq)2] (3) obtained in the reaction of 1 with 2-methylquinolin-8-ol (HMeq) possesses a five-coordinate Al center. The structure was solved in a triclinic cell P-1. The dinuclear complex 2 can be considered as a model product of alcohol addition on the formal Al-N double bond, while the mononuclear complex 3 represents subsequent reaction of remaining Al-Me moiety with proton. Both complexes 2 and 3 are highly luminescent showing emission around 500 nm in solid state.

Sanguinarine is reduced by NADH through a covalent adduct.

Sanguinarine is a benzo[c]phenanthridine alkaloid with interesting cytotoxic properties, such as induction of oxidative DNA damage and very rapid apoptosis, which is not mediated by p53-dependent signaling. It has been previously documented that sanguinarine is reduced with NADH even in absence of any enzymes while being converted to its dihydro form. We found that the dark blue fluorescent species, observed during sanguinarine reduction with NADH and misinterpreted by Matkar et al. (Arch. Biochem. Biophys. 2008, 477, 43-52) as an anionic form of the alkaloid, is a covalent adduct formed by the interaction of NADH and sanguinarine. The covalent adduct is then converted slowly to the products, dihydrosanguinarine and NAD+, in the second step of reduction. The product of the reduction, dihydrosanguinarine, was continually re-oxidized by the atmospheric oxygen back to sanguinarine, resulting in further reacting with NADH and eventually depleting all NADH molecules. The ability of sanguinarine to diminish the pool of NADH and NADPH is further considered when explaining the sanguinarine-induced apoptosis in living cells.

Anaeramoebidae fam. nov., a Novel Lineage of Anaerobic Amoebae and Amoeboflagellates of Uncertain Phylogenetic Position.

We report discovery of a new lineage of anaerobic marine amoebae and amoeboflagellates, Anaeramoeba gen. nov., represented by six newly described species. The trophic form of Anaeramoeba spp. is an amoeba corresponding to the uncommon flabellate or flamellian morphotype - it is fan-shaped and produces an anterior, flattened hyaline zone and posterior hyaline projections. In contrast to other representatives of these morphotypes, cells of Anaeramoeba spp. possess acristate mitochondrion-related organelles associated with prokaryotic symbionts, and a large acentriolar centrosome. Surprisingly, two Anaeramoeba species form morphologically unique flagellates with two or four isokont, thickened flagella. Phylogenetic analyses of the SSU rRNA gene showed that Anaeramoeba spp. form a clade, which is not robustly related to any other eukaryotic lineage. We accommodate Anaeramoeba in a new family Anaeramoebidae, which we classify as Eukaryota incertae sedis.

Combined Culture-Based and Culture-Independent Approaches Provide Insights into Diversity of Jakobids, an Extremely Plesiomorphic Eukaryotic Lineage.

We used culture-based and culture-independent approaches to discover diversity and ecology of anaerobic jakobids (Excavata: Jakobida), an overlooked, deep-branching lineage of free-living nanoflagellates related to Euglenozoa. Jakobids are among a few lineages of nanoflagellates frequently detected in anoxic habitats by PCR-based studies, however only two strains of a single jakobid species have been isolated from those habitats. We recovered 712 environmental sequences and cultured 21 new isolates of anaerobic jakobids that collectively represent at least ten different species in total, from which four are uncultured. Two cultured species have never been detected by environmental, PCR-based methods. Surprisingly, culture-based and culture-independent approaches were able to reveal a relatively high proportion of overall species diversity of anaerobic jakobids-60 or 80%, respectively. Our phylogenetic analyses based on SSU rDNA and six protein-coding genes showed that anaerobic jakobids constitute a clade of morphologically similar, but genetically and ecologically diverse protists-Stygiellidae fam. nov. Our investigation combines culture-based and environmental molecular-based approaches to capture a wider extent of species diversity and shows Stygiellidae as a group that ordinarily inhabits anoxic, sulfide- and ammonium-rich marine habitats worldwide.

Marine Isolates of Trimastix marina Form a Plesiomorphic Deep-branching Lineage within Preaxostyla, Separate from Other Known Trimastigids (Paratrimastix n. gen.).

Trimastigids are free-living, anaerobic protists that are closely related to the symbiotic oxymonads, forming together the taxon Preaxostyla (Excavata: Metamonada). We isolated fourteen new strains morphologically corresponding to two species assigned to Trimastix (until now the only genus of trimastigids), Trimastix marina and Trimastix pyriformis. Unexpectedly, marine strains of Trimastix marina branch separately from freshwater strains of this morphospecies in SSU rRNA gene trees, and instead form the sister group of all other Preaxostyla. This position is confirmed by three-gene phylogenies. Ultrastructural examination of a marine isolate of Trimastix marina demonstrates a combination of trimastigid-like features (e.g. preaxostyle-like I fibre) and ancestral characters (e.g. absence of thickened flagellar vane margins), consistent with inclusion of marine T. marina within Preaxostyla, but also supporting its distinctiveness from 'freshwater T. marina' and its deep-branching position within Preaxostyla. Since these results indicate paraphyly of Trimastix as currently understood, we transfer the other better-studied trimastigids to Paratrimastix n. gen. and Paratrimastigidae n. fam. The freshwater form previously identified as T. marina is described as Paratrimastix eleionoma n. sp., and Trimastix pyriformis becomes Paratrimastix pyriformis n. comb. Because of its phylogenetic position, 'true' Trimastix is potentially important for understanding the evolution of mitochondrion-related organelles in metamonads.

Influence of Solvent Polarity and DNA-Binding on Spectral Properties of Quaternary Benzo[c]phenanthridine Alkaloids.

Quaternary benzo[c]phenanthridine alkaloids are secondary metabolites of the plant families Papaveraceae, Rutaceae, and Ranunculaceae with anti-inflammatory, antifungal, antimicrobial and anticancer activities. Their spectral changes induced by the environment could be used to understand their interaction with biomolecules as well as for analytical purposes. Spectral shifts, quantum yield and changes in lifetime are presented for the free form of alkaloids in solvents of different polarity and for alkaloids bound to DNA. Quantum yields range from 0.098 to 0.345 for the alkanolamine form and are below 0.033 for the iminium form. Rise of fluorescence lifetimes (from 2-5 ns to 3-10 ns) and fluorescence intensity are observed after binding of the iminium form to the DNA for most studied alkaloids. The alkanolamine form does not bind to DNA. Acid-base equilibrium constant of macarpine is determined to be 8.2-8.3. Macarpine is found to have the highest increase of fluorescence upon DNA binding, even under unfavourable pH conditions. This is probably a result of its unique methoxy substitution at C12 a characteristic not shared with other studied alkaloids. Association constant for macarpine-DNA interaction is 700000 M(-1).

Alkaloid chelirubine and DNA: blue and red luminescence.

Extracts from Himalayan herb Dicranostigma lactucoides containing alkaloid chelirubine have been used for centuries in Chinese herbal medicine. We have found a new utilization for the alkaloid: it can be used as a DNA fluorescent probe showing blue (free form) and red (intercalated to DNA) luminescence emission after irradiation by near-UV light. Besides quantification of DNA (LOD = 6 ng ml(-1)) it can also be used as a supravital cell probe because chelirubine molecules can effectively enter into the living cell through the cell membrane.