Comparative study of blood pressure and echocardiographic values of male cynomolgus macaques (Macaca fascicularis).

BACKGROUND: Primates represent a unique object for biomedical research, in particular in the field of physiology and pathology of the cardiovascular system. Echocardiography is the most important non-invasive method for the intravital study of the heart structure and function, intracardiac and systemic haemodynamics. The available data on reference values of echocardiographic parameters in primates are limited. METHODS: We determined and described 29 structural and functional parameters in echocardiographic examination using B-mode (two-dimensional scanning), M-mode (one-dimensional scanning) and in various Doppler modes together with blood pressure in 17 male cynomolgus macaques with an average age of 5.7 ± 0.6 years. We compared available literature data on reference values of echocardiography in this species. RESULTS AND CONCLUSIONS: Echocardiographic values in cynomolgus macaques depend on age, sex composition and the anaesthesia method. There is lack of presentation in the published studies of complete list of parameters that can be obtained by echocardiographic examination.

Molecular phylogeny and new light microscopic data of Metchnikovella spiralis (Microsporidia: Metchnikovellidae), a hyperparasite of eugregarine Polyrhabdina sp. from the polychaete Pygospio elegans.

Metchnikovellids are a deep-branching group of microsporidia, parasites of gregarines inhabiting the alimentary tract of polychaetes and some other invertebrates. The diversity and phylogeny of these hyperparasites remain poorly studied. Modern descriptions and molecular data are still lacking for many species. The results of a light microscopy study and molecular data for Metchnikovella spiralis Sokolova et al., 2014, a hyperparasite of the eugregarine Polyrhabdina sp., isolated from the polychaete Pygospio elegans, were obtained. The original description of M. spiralis was based primarily on the analysis of stained preparations and transmission electron microscopy images. Here, the species description was complemented with the results of in vivo observations and phylogenetic analysis based on the SSU rRNA gene. It was shown that in this species, free sporogony precedes sac-bound sporogony, as it occurs in the life cycle of most other metchnikovellids. Spore sacs are entwined with spirally wound cords, and possess only one polar plug. Phylogenetic analyses did not group M. spiralis with M. incurvata, another metchnikovellid from the same gregarine species, but placed it as a sister branch to Amphiacantha. The paraphyletic nature of the genus Metchnikovella was discussed. The taxonomic summary for M. spiralis was emended.

Evolutionary relationships of Metchnikovella dogieli Paskerova et al., 2016 (Microsporidia: Metchnikovellidae) revealed by multigene phylogenetic analysis.

The species Metchnikovella dogieli (Paskerova et al. Protistology 10:148-157, 2016) belongs to one of the early diverging microsporidian groups, the metchnikovellids (Microsporidia: Metchnikovellidae). In relation to typical ('core') microsporidia, this group is considered primitive. The spores of metchnikovellids have no classical polar sac-anchoring disk complex, no coiled polar tube, no posterior vacuole, and no polaroplast. Instead, they possess a short thick manubrium that expands into a manubrial cistern. These organisms are hyperparasites; they infect gregarines that parasitise marine invertebrates. M. dogieli is a parasite of the archigregarine Selenidium pygospionis (Paskerova et al. Protist 169:826-852, 2018), which parasitises the polychaete Pygospio elegans. This species was discovered in samples collected in the silt littoral zone at the coast of the White Sea, North-West Russia, and was described based on light microscopy. No molecular data are available for this species, and the publicly accessible genomic data for metchnikovellids are limited to two species: M. incurvata Caullery & Mesnil, 1914 and Amphiamblys sp. WSBS2006. In the present study, we applied single-cell genomics methods with whole-genome amplification to perform next-generation sequencing of M. dogieli genomic DNA. We performed a phylogenetic analysis based on the SSU rRNA gene and reconstructed a multigene phylogeny using a concatenated alignment that included 46 conserved single-copy protein domains. The analyses recovered a fully supported clade of metchnikovellids as a basal group to the core microsporidia. Two members of the genus Metchnikovella did not form a clade in our tree. This may indicate that this genus is paraphyletic and requires revision.

Molecular Phylogeny of Polychaos annulatum (Amoebozoa, Tubulinea, Euamoebida) Shows that Genus Polychaos Belongs to the Family Hartmannellidae.

We have obtained a sequence of the 18S rRNA gene of the species Polychaos annulatum (Penard 1902) Smirnov et Goodkov 1998 using the isolation of a single nucleus from an amoeba cell. Attempts to amplify the 18S rRNA gene from the DNA of this species by conventional PCR were not successful, so we applied the whole genome amplification of the nuclear DNA followed by NGS sequencing. The 18S rRNA gene was found among the resulting contigs. The analysis unexpectedly shows that P. annulatum robustly groups within the family Hartmannellidae, but not Amoebidae. This finding warrants revision of the basic morphological criteria used to classify Euamoebida families and show that "proteus-type" amoebae may belong to other families rather than Amoebidae. This makes taxonomic assignments of such species more complex and the borders between Euamoebida families more nuanced. It is getting evident that molecular data are necessary to clarify the position of species even in this most "classical" order of naked lobose amoebae.

Mitochondrial Genome of Vannella croatica (Amoebozoa, Discosea, Vannellida).

Mitochondrial genome sequence of Vannella croatica (Amoebozoa, Discosea, Vannellida) was obtained using pulse-field gel electrophoretic isolation of the circular mitochondrial DNA, followed by the next-generation sequencing. The mitochondrial DNA of this species has the length of 28,933 bp and contains 12 protein-coding genes, two ribosomal RNAs, and 16 transfer RNAs. Vannella croatica mitochondrial genome is relatively short compared to other known amoebozoan mitochondrial genomes but is rather gene-rich and contains significant number of open reading frames.

Lineage-Specific and Highly Derived Gene Sequences Among Amoebozoa, Revealed by the Comparative Analysis of Transcriptomes from Twelve Amoebozoan Species.

Amoebozoa represent a difficult group for traditional morphology-based taxonomy. Molecular approaches, such as gene sequencing and DNA barcoding have greatly enhanced our knowledge of the diversity of these organisms. However, metagenomic studies of Amoebozoa still did not provide as impressive results as they did among some other groups of protists. In environmental DNA surveys done on fragments of SSU rDNA gene and other traditional DNA barcodes, Amoebozoa genes normally constitute a minor part of the total gene diversity and represent only the most abundant lineages. A potential way to resolve this problem is the usage of DNA barcodes based on genes, which are unique or highly derived in this group of organisms. In the present study, we attempted to find such genes and gene families with a low level of paralogy, potentially appropriate as Amoebozoa-specific DNA barcodes. For this we re-assembled transcriptomes of 12 amoebozoan species available from the public databases and performed gene annotation and identification of orthologous genes. In our analysis Amoebozoa-specific and highly derived sequences formed 53,182 clusters of orthologs, containing from 2 to 299 proteins each. Some of these genes may be a potential target for DNA barcoding of Amoebozoa.

A New Freshwater Naked Lobose Amoeba Korotnevella venosa n. sp. (Amoebozoa, Discosea).

A new freshwater species of naked lobose amoebae Korotnevella venosa n. sp. isolated from freshwater pond in St. Petersburg, Russia was studied with light and transmission electron microscopy. Basket scales of this species have six vertical columns supporting perforated rim. The latter has tongue-like broadening with membranous region. Vertical columns bifurcate at both ends so that neighboring columns are connected by their bifurcations forming combined structure. Basket scales of K. venosa are similar to those of Korotnevella hemistylolepis in having six full-length vertical columns and perforated rim. At the same time, they are different in having tongue-like broadening of perforated rim with membranous region and absence of six half-length columns and an intermediate crosspiece. Phylogenetic trees based on 18S rDNA gene placed K. venosa either at the base of the whole Korotnevella clade, next to K. hemistylolepis, or as a sister to the clade comprising Korotnevella species with latticework basket in large scales.

Taxonomic position of holothurian Eupentactafraudatrix (Echinodermata, Holothuroidea).

Samples of the holothurian Eupentactafraudatrix (Djakonov & Baranova in Djakonov, Baranova & Saveljeva, 1958) from the Sea of Japan were studied and the relationships of the genera Eupentacta and Sclerodactyla, as well as related taxa, were evaluated on the basis of phylogenetic analysis of the mitochondrial DNA COI and 16S rRNA genes. Using three methods, phylogenetic trees were constructed, and the degree of reliability of topological reconstructions was estimated by means of a nonparametric bootstrap test for the neighbor joining (NJ) and maximum likelihood (ML) techniques, as well as by a posteriori probability for Bayesian inference (BI) analysis. Genetic data confirm the validity of the assignment of Cucumariafraudatrix to the genus Eupentacta Deichmann, 1938. The study of sequences obtained from the holothurian specimens collected in Russian waters, near the city of Vladivostok, and determined by morphological characters clearly indicate that these specimens belong to the genus Eupentacta and are assigned as E.fraudatrix . The specimens from China in GenBank named as Sclerodactylamultipes and used in the present study, were likely misidentified, and after re-examination they may be assigned to the genus Eupentacta, either as E.fraudatrix or another taxon. Analyses of morphological characters of S.multipes unequivocally affirm that this species must be excluded from Sclerodactyla Ayres, 1851 and is provisionally assigned to the genus Sclerothyone Thandar, 1989 based on the external morphological characters and the body wall ossicles.

Two new species of the genus Leptomyxa (Goodey, 1915) - Leptomyxa botanica n. sp. and Leptomyxa monrepos n. sp. from the soil habitats of Northwestern Russia.

Amoebae of the genus Leptomyxa have variable morphologies and can only be reliably identified using molecular data. However, species distinction based on the 18S rRNA gene sequence is difficult due to the very low level of sequence divergence among morphologically different species. The database for other genes is much smaller, and genomic data on Leptomyxa is almost absent. In this study, we describe two new terrestrial species of the genus Leptomyxa isolated from Northwestern Russia, Leptomyxa botanica n. sp. and Leptomyxa monrepos n. sp. Both species easily adopt an expanded fan-shaped form and have a complex structure of the nucleolar material. Phylogenetic analyses show a derived status of these two species. They form a clade with Leptomyxa valladaresi. Our tree confirms that the 18S rRNA gene sequences of Leptomyxa species are split into two large clades. The morphological synapomorphies of these clades are not obvious. This analysis is complicated by the lack of reliable morphological data on many sequenced strains and probable misidentification of some isolates.

Diversity, Distribution, and Development of Hyperparasitic Microsporidia in Gregarines within One Super-Host.

Metchnikovellids (Microsporidia: Metchnikovellida) are poorly studied hyperparasitic microsporidia that live in gregarines inhabiting the intestines of marine invertebrates, mostly polychaetes. Our recent studies showed that diversity of metchnikovellids might be significantly higher than previously thought, even within a single host. Four species of metchnikovellids were found in the gregarines inhabiting the gut of the polychaete Pygospio elegans from littoral populations of the White and Barents Seas: the eugregarine Polyrhabdina pygospionis is the host for Metchnikovella incurvata and M. spiralis, while the archigregarine Selenidium pygospionis is the host for M. dogieli and M. dobrovolskiji. The most common species in the White Sea is M. incurvata, while M. dobrovolskiji prevails in the Barents Sea. Gregarines within a single worm could be infected with different metchnikovellid species. However, co-infection of one and the same gregarine with several species of metchnikovellids has never been observed. The difference in prevalence and intensity of metchnikovellid invasion apparently depends on the features of the life cycle and on the development strategies of individual species.

Neuroprotective action of Cortexin, Cerebrolysin and Actovegin in acute or chronic brain ischemia in rats.

This study was the first to compare the neuroprotective activity of Cerebrolysin®, Actovegin® and Cortexin® in rodent models of acute and chronic brain ischemia. The neuroprotective action was evaluated in animals with acute (middle cerebral artery occlusion) or chronic (common carotid artery stenosis) brain ischemia models in male rats. Cortexin® (1 or 3 mg/kg/day), Cerebrolysin® (538 or 1614 mg/kg/day) and Actovegin® (200 mg/kg/day) were administered for 10 days. To assess the neurological and motor impairments, open field test, adhesive removal test, rotarod performance test and Morris water maze test were performed. Brain damage was assessed macro- and microscopically, and antioxidant system activity was measured in brain homogenates. In separate experiments in vitro binding of Cortexin® to a wide panel of receptors was assessed, and blood-brain barrier permeability of Cortexin® was assessed in mice in vivo. Cortexin® or Cerebrolysin® and, to a lesser extent, Actovegin® improved the recovery of neurological functions, reduced the severity of sensorimotor and cognitive impairments in rats. Cortexin® reduced the size of necrosis of brain tissue in acute ischemia, improved functioning of the antioxidant system and prevented the development of severe neurodegenerative changes in chronic ischemia model. Radioactively labeled Cortexin® crossed the blood-brain barrier in mice in vivo with concentrations equal to 6-8% of concentrations found in whole blood. During in vitro binding assay Cortexin® (10 µg/ml) demonstrated high or moderate binding to AMPA-receptors (80.1%), kainate receptors (73.5%), mGluR1 (49.0%), GABAA1 (44.0%) and mGluR5 (39.7%), which means that effects observed in vivo could be related on the glutamatergic and GABAergic actions of Cortexin®. Thus, Cortexin, 1 or 3 mg/kg, or Cerebrolysin®, 538 or 1614 mg/kg, were effective in models acute and chronic brain ischemia in rats. Cortexin® contains compounds acting on AMPA, kainate, mGluR1, GABAA1 and mGluR5 receptors in vitro, and readily crosses the blood-brain barrier in mice.

Triterpene glycosides from Antarctic sea cucumbers. 1. Structure of liouvillosides A1, A2, A3, B1, and B2 from the sea cucumber Staurocucumis liouvillei: new procedure for separation of highly polar glycoside fractions and taxonomic revision.

Five new triterpene glycosides, liouvillosides A1 (1), A2 (2), A3 (3), B1 (4), and B2 (5), have been isolated from the Antarctic sea cucumber Staurocucumis liouviellei along with the known liouvilloside A(6), isolated earlier from the same species, and hemoiedemosides A (7) and B (8), isolated earlier from the Patagonian sea cucumber Hemioedema spectabilis. The isolation was carried out using a new chromatographic procedure including application of ion-pair reversed-phase chromatography followed by chiral chromatography on a cyclodextrin ChiraDex column. The structures of the new glycosides were elucidated using extensive NMR spectroscopy (1H and 13C NMR spectrometry, DEPT, 1H-(1)H COSY, HMBC, HMQC, and NOESY), ESI-FTMS, and CID MS/MS, and chemical transformations. Glycosides 1-3 are disulfated tetraosides and glycosides 4 and 5 are trisulfated tetraosides. Glycosides 2 and 3 contain 3-O-methylquinovose, found for the first time as a natural monosaccharide in sea cucumber glycosides. On the basis of analyses of glycoside structures a taxonomic revision is proposed.

Correct identification of species makes the amoebozoan rRNA tree congruent with morphology for the order Leptomyxida Page 1987; with description of Acramoeba dendroida n. g., n. sp., originally misidentified as 'Gephyramoeba sp.'.

Morphological identification of protists remains an expert task, especially for little known and poorly described species. Culture collections normally accept organisms under the name provided by depositors and are not responsible for identification. Uncritical acceptance of these names by molecular phylogeneticists may result in serious errors of interpretation of phylogenetic trees based on DNA sequences, making them appear more incongruent with morphology than they really are. Several cases of misidentification in a major culture collection have recently been reported. Here we provide evidence for misidentifications of two more gymnamoebae. The first concerns "Gephyramoeba sp." ATCC 50654; it is not Gephyramoeba, a leptomyxid with lobose pseudopods, but a hitherto undescribed branching amoeba with fine, filamentous subpseudopods named here Acramoeba dendroida gen. et sp. nov. We also sequenced 18S rRNA of Page's strain of Rhizamoeba saxonica (CCAP 1570/2) and show that it is the most deeply branching leptomyxid and is not phylogenetically close to 'Rhizamoeba saxonica' ATCC 50742, which was misidentified. Correcting these misidentifications improves the congruence between morphological diversity of Amoebozoa and their rRNA-based phylogenies, both for Leptomyxida and for the Acramoeba part of the tree. On morphological grounds we transfer Gephyramoebidae from Varipodida back to Leptomyxida and remove Flamella from Leptomyxida; sequences are needed to confirm these two revisions.

Morphology, biology and phylogeny of Phalansterium arcticum sp. n. (Amoebozoa, Variosea), isolated from ancient Arctic permafrost.

A new species, Phalansterium arcticum sp. n., was isolated from an 8580-year-old Arctic permafrost layer. This organism typically lives as a sedentary uniflagellated cell enclosed in a thin flexible mucilaginous sheath, but can form naked swimming cells and amoeboid cells with eruptive pseudopodia accompanied with the formation of short, filopodia-like projections. In an SSU rDNA phylogenetic tree, it robustly groups with other species of this genus. Along with a description of the species, we also add new details to the description of the cell division of Phalansterium and the feeding process in this organism.

Phylogeny, evolution, and taxonomy of vannellid amoebae.

We sequenced 18S rRNA genes from 21 vannellid amoebae (Amoebozoa; Vannellidae), including nearly all available type cultures, and performed a comprehensive phylogenetic analysis for 57 Vannellidae sequences. The results show that species of Vannella and Platyamoeba are completely mixed and do not form distinct clades. Several very closely related species pairs exist, each with a Vannella and a Platyamoeba species differing in only a few nucleotides. Therefore, presence (Vannella) or absence (Platyamoeba) of glycostyles in the cell surface coat is an invalid generic distinction; the genera must be merged. As Vannella has priority, we formally transferred Platyamoeba species into Vannella, except for the non-vannellid P. stenopodia, here renamed Stenamoeba stenopodia gen. n. comb. n. and transferred to the family Thecamoebidae. Our trees show that Vannella glycostyles were probably easily and repeatedly evolutionarily lost. We have established a new genus Ripella, with distinct morphology and sequence signatures for Vannella platypodia and morphologically similar species that form a clearly separate clade, very distant from other Vannellidae. Vannellids form four well-separated single-genus clades: Vannella sensu stricto, Ripella, Clydonella, and Lingulamoeba. Species of the revised genus Vannella comprise four closely related, well-supported subclades: one marine and three freshwater. Here, we provide an illustrated checklist for all 40 known Vannellidae species.

Cryptic freshwater amoeba species in the bottom sediments of Nivå Bay (Øresund, Baltic Sea).

An aquarium containing a portion of freshly collected brackish-water sediment from Nivå Bay (Øresund, Baltic Sea) was sampled to determine the species of amoebae present and refilled with inorganic salt medium to achieve freshwater salinity. After 2 months incubation, the diversity of amoebae was again studied, and the salinity was restored to the original level. The aquarium was incubated for a further 2 months and the amoeba fauna was studied for the last time. A number of freshwater species appeared in the sample after the first salinity shift, while some marine species disappeared. Most marine species did not re-appear after the salinity in the aquarium was restored, but one marine species not noted previously was recorded. The experiment illustrates the presence of 'cryptic diversity' of amoebae in natural habitats and demonstrates that laboratory manipulation of the salinity of a sample prior to inoculation may achieve an increased recovery of species from a brackish-water habitat.

Scoliorhapis stepanovi-new species of sea cucumber from the North-West Pacific (Holothuroidea: Synaptida: Chiridotidae: Taeniogyrinae) and some remarks on the genus Scoliorhapis.

New species Scoliorhapis stepanovi has been collected from depths of 10-23 m on the sandy bottom of Avacha Bay (east coast of Kamchatka), Paramushir Island (North Kuril Islands), and Matua Island (Middle Kuril Islands). It is unique in having sigmoid ossicles in the body wall with points at both ends (without an open-eye, and with the two points lying in a perpendicular plane). Such sigmoids do not occur in any other taeniogyrinid species. The two-pointed sigmoids are 80-115 µm in length, and are scattered in the body wall and not clustered. Probably the two-pointed sigmoids are underdeveloped typical sigmoid with open-eye. In the tentacles there are straight and C-shaped rods, sometimes branched at the ends, 70-90 µm in length. It is assumed that atypical two-pointed sigmoid ossicles may have been originated due to deviating from the usual course of sigmoid development in the middle stages. Scoliorhapis stepanovi resembles S. lindbergi. A key for species of Scoliorhapis is provided.

ZB-16, a Novel GPR119 Agonist, Relieves the Severity of Streptozotocin-Nicotinamide-Induced Diabetes in Rats.

GPR119 is involved in the regulation of incretin and insulin secretion, so the GPR119 agonists have been suggested as novel antidiabetic medications. The purpose of this work was to assess the influence of novel GPR119 agonist ZB-16 on the glucose utilization, insulin, and glucagon-like peptide-1 (GLP-1) secretion and the morphology of pancreas in rats with streptozotocin-nicotinamide-induced diabetes. 45 male Wistar rats were used in the study. The criteria of streptozotocin-nicotinamide-induced diabetes were blood glucose levels of 9-14 mmol/l measured in fasting conditions on the third day since administration of streptozotocin (65 mg/kg) and nicotinamide (230 mg/kg). Animals failed to reach the criteria were excluded from the experiment. The substances were administered per os once per day for 28 days. Measurements included blood glucose monitoring (every 7 days), glucose tolerance test (every 14 days), the assessment of insulin and GLP-1 levels in blood plasma (28 days after beginning), and the results of immunohistochemical staining of pancreas. It was found that ZB-16 (1 mg/kg per os, once a day) decreases the blood glucose levels under fasting conditions and improves the glucose utilization. These changes were associated with the increase in stimulated secretion of GLP-1 and insulin, accompanied by the growth of insulin-positive cells in pancreas. Thus, ZB-16 could be a promising antidiabetic drug for oral administration.

Morphology and phylogeny of Vannella croatica n. sp. (Amoebozoa, Discosea, Vannellida).

We isolated and described a new species of freshwater vannellid amoeba from Krka natural reserve in Croatia--Vannella croatica n. sp. This species has certain morphological differences from all known vannellids and differs at the level of SSU sequence. It resembles in size and morphology Vannella lata; to facilitate direct comparison we publish images of V. lata CCAP 1589/12 strain (type strain, which is now lost) taken in 1999. Vannela croatica feeds on bacteria and can be easily grown in large amount in relatively pure culture and thus is suitable for molecular and biochemical studies requiring large amounts of material.