Morphological and molecular description of Dictyocaulus xanthopygus sp. nov. (Nematoda: Trichostrongyloidea) from the Manchurian wapiti Cervus elaphus xanthopygus.

Dictyocaulus xanthopygus sp. nov. (Nematoda: Trichostrongyloidea) was isolated from the lungs of the Manchurian wapiti in Primorsky kray, Russia. The newly described species exhibits morphological characteristics of Dictyocaulus but is distinct from congeneric species based on morphological (lengths of body and esophagus, distances from the anterior end to nerve ring and to excretory pore, the thickness of the buccal capsule, etc.) and molecular features. High levels of genetic divergence as well as Bayesian phylogenetic analyses based on 18S rRNA nuclear and cox1 mitochondrial genes supported the independence of Dictyocaulus xanthopygus sp. nov. Secondary structures of helix 39 of 18S rRNA were identical, while ES9 adjacent to the helix has a unique conformation for newly described worms. Energy-efficient conformational rearrangements of rRNA secondary structures can be applicable in studies on the pathogenesis, epidemiology, taxonomy and evolutionary biology of parasites. Additionally, bracketed dichotomous keys to six valid species of Dictyocaulus were prepared.

Prospects for the use of olfactory mucosa cells in bioprinting for the treatment of spinal cord injuries.

The review focuses on the most important areas of cell therapy for spinal cord injuries. Olfactory mucosa cells are promising for transplantation. Obtaining these cells is safe for patients. The use of olfactory mucosa cells is effective in restoring motor function due to the remyelination and regeneration of axons after spinal cord injuries. These cells express neurotrophic factors that play an important role in the functional recovery of nerve tissue after spinal cord injuries. In addition, it is possible to increase the content of neurotrophic factors, at the site of injury, exogenously by the direct injection of neurotrophic factors or their delivery using gene therapy. The advantages of olfactory mucosa cells, in combination with neurotrophic factors, open up wide possibilities for their application in three-dimensional and four-dimensional bioprinting technology treating spinal cord injuries.

Integrative approach for discovering of the new species within the genus Allocreadium Looss, 1900 (Trematoda: Allocreadiidae) and framing of biogeographical hypotheses for the genus.

In July 2012 new Allocreadium species was isolated from Carassius gibelio caught in the Arsenyevka River, Primorsky region, Russia. Analyses on the morphometrics and internal organs' topology revealed that these worms are morphologically closest with A. isoporum but both species are independent according to high genetic distances based on the 28S gene fragment (5.434±0.0073%). Unlike A. isoporum found earlier in Europe, the new species named A. pseudoisoporum sp. nov. has a shorter body length and the vitellarium not reaching the posterior end of the body at some distance and its anterior border is on the level of the ventral sucker. Allocreadium pseudoisoporum sp. nov. differs from seven species previously found in the Russian Far East with the following features: smaller size of the body, suckers' ratio, range values, and topology of internal structures. Newly localities in the Pavlovka River and the Artyomovka River were discovered for A. khankaiensis. Morphological variability of the worms from the Pavlovka River was observed in comparison with A. khankaiensis from the Komissarovka River. Using scanning electron microscope, we examined external surfaces of three species (A. pseudoisoporum sp. nov., A. khankaiensis, A. hemibarbi) and observed structures reminiscent sensory receptors. This study was aimed to describe species diversity of allocreadiids inhabiting the south of Primorsky region, and to clarify phylogenetic relationships between the species from the genus Allocreadium Looss, 1900 using molecular genetic methods. The phylogenetic Bayesian tree based on the 28S gene showed a clear separation of ten Allocreadium species and confirmed the validity of A. pseudoisoporum sp. nov. Allocreadium pseudoisoporum sp. nov. is most similar to A. gotoi (genetic distances - 3.578±0.0051% in 28S, and 18.777±0.0149% in cox1), and represented the earliest divergent lineage in Allocreadium clade on the phylogenetic tree based on the 28S gene, thereby indicating its proximity to the ancestral node. Also, dichotomous keys for 25 Palearctic species of Allocreadium were prepared based on the morphology of the adult worms.

Molecular identification of the trematode P. ichunensis stat. n. from lungs of siberian tigers justified reappraisal of Paragonimus westermani species complex.

Flukes from the genus Paragonimus Braun, 1899 are medically important foodborne trematodes predominantly occurring throughout Asian countries. Providing molecular genetic characteristics based on ITS2 and partial 28 S rDNA of the paragonimids from the Russian Far East, Northeast, South, and Southeast Asian countries, we performed a partial reappraisal of Paragonimus westermani species complex. Members of this complex are genetically distinct worms with different divergence times and explosive expansion during Miocene-Pliocene epochs. We confirm the taxonomic status as valid species for P. ichunensis stat. n. (from the Russian Far East and Northern China), and P. filipinus (from the Philippines), which were previously considered subspecies of P. westermani, and reinstated the species name P. pulmonalis (from Japan). We suggest considering the worms from South Korea the Korean variety of P. ichunensis, because Korean specimens are sister and genetically closest to P. ichunensis from Northeast China and Primorsky region of Russia. Worms from South (India (type 2), Sri Lanka), Southeast (Malaysia, Vietnam, Thailand (types 1 and 2)) and East Asia (Taiwan) were left in the paragonimid systematics as Paragonimus sp. We propose to consider Indian worms of type 1 as true P. westermani, but in further revisions, due to the lack of holotype and unknown exact type locality, new type specimens (neotype) should be established.

Origins, phylogenetic relationships and host-parasite interactions of Troglotrematoidea since the cretaceous.

In the current study, we raise the issue concerning origins and historical relationships of the trematodes from the families Troglotrematidae and Paragonimidae using phylogenetic analysis and molecular-clock method for estimating evolutionary rates. For the first time we provided 28S rRNA gene fragment (1764 bp) for the type species Troglotrema acutum - zoonotic trematodes that cause cranial lesions (troglotremiasis) in mustelid and canid mammals of the Central Europe, Iberian Peninsula, and North-West Caucasus. Molecular genetic analysis revealed that T. acutum belongs to the monophyletic family Troglotrematidae sister with the family Paragonimidae. The family Troglotrematidae includes five genera: Nanophyetus, Troglotrema, Skrjabinophyetus, Nephrotrema, and Macroorchis; and the family Paragonimidae is monotypic including the only genus Paragonimus. We recover the superfamily Troglotrematoidea for these two families. Divergence of the common ancestor of the superfamily Troglotrematoidea (common troglotrematoid ancestor) likely occurred during the Cretaceous period of the Mesozoic Era and potentially originated in the Asiatic region. The lineage of the family Troglotrematidae is much closer to the common troglotrematoid ancestor than the species of the family Paragonimidae. The radiation time of the common troglotrematoid ancestor (126 Ma, the Early Cretaceous), and formation of the families Troglotrematidae and Paragonimidae (96 Ma and 73 Ma respectively, the Late Cretaceous) corresponds to the time of settling in East Asia by many species of mammaliaforms (about 130-70 Ma).

Study of the Therapeutic Efficiency of Transduced Olfactory Ensheathing Cells in Spinal Cord Cysts.

Posttraumatic spinal cord cysts are difficult to treat with medication and surgery. Gene-cell therapy is a promising area of treatment for such patients. However, optimal gene-cell construct for this therapy has not been developed. We investigated the therapeutic efficiency of human olfactory ensheathing cells (OECs) transduced by adenoviral vector encoding the mature form of brain-derived neurotrophic factor (mBDNF) in spinal cord cysts. The adenoviral vectors Ad5/35-CAG-mBDNF and Ad5/35-CAG-Fluc were constructed. Spinal cysts were modeled in female Wistar rats. We selected animals at the early and intermediate stages of recovery with scores to 13 according to the Basso, Beattie and Bresnahan (BBB) scale. The efficiency of therapy was evaluated by BBB tests. No cytotoxicity was detected using the Resazurin/AlamarBlue assay for both vectors at multiplicity of infection (MOIs) of 1, 5, and 25. There was an increase in the proliferation of cells treated with Ad5/35-CAG-mBDNF at MOIs of 5 and 25. The hind limb mobility after the transplantation of Ad5/35-CAG-mBDNF- and Ad5/35-CAG-Fluc-transduced human OECs and nontransduced OECs had approximately the same tendency to improve. Cyst reduction was observed with the transplantation of all the samples. Although Ad5/35-CAG-mBDNF-transduced OECs had high BDNF expression levels in vitro, these cells lacked positive effect in vivo because they did not exhibit significant effect concerning functional test when comparing the groups that received the same numbers of OECs. The therapeutic efficiency of transduced OECs appears to be due to the cell component. The autological and tissue-specific human OECs are promising for the personalized cell therapy. It is extremely important to test new gene-cell constructs based on these cells for further clinical use.

Antiparasitic Effects of Sulfated Polysaccharides from Marine Hydrobionts.

This review presents materials characterizing sulfated polysaccharides (SPS) of marine hydrobionts (algae and invertebrates) as potential means for the prevention and treatment of protozoa and helminthiasis. The authors have summarized the literature on the pathogenetic targets of protozoa on the host cells and on the antiparasitic potential of polysaccharides from red, brown and green algae as well as certain marine invertebrates. Information about the mechanisms of action of these unique compounds in diseases caused by protozoa has also been summarized. SPS is distinguished by high antiparasitic activity, good solubility and an almost complete absence of toxicity. In the long term, this allows for the consideration of these compounds as effective and attractive candidates on which to base drugs, biologically active food additives and functional food products with antiparasitic activity.

Infrainguinal bypass under triple nerve block in patients with severely compromised left ventricular ejection fraction and chronic limb-threatening ischemia.

A severely compromised left ventricular ejection fraction (LVEF) is a major limitation for lower extremity bypass reconstruction both under general anesthesia or neuraxial anesthesia (NA). A series of eight infrainguinal bypass procedures were performed under peripheral nerve block in five patients (three males and two females; median age, 67 years) with chronic limb-threatening ischemia and a preoperative LVEF of 35% or less (median, 27%; range, 20%-35%). There were no conversions to neuraxial anesthesia/general anesthesia or early postoperative complications. This study showed that open infrainguinal reconstructions can be performed safely under peripheral nerve blockade in this vulnerable category of patients.

Systematics of Crepidostomum species from the Russian Far East and northern Japan, with description of a new species and validation of the genus Stephanophiala.

Current article touched upon the issue of the complicated taxonomic status of some species from the genus Crepidostomum collected from the freshwater fish in the rivers of Primorsky region, Sakhalin, and Hokkaido Islands. Primary morphological analyses showed affiliation of the worms to the species C. farionis (Müller, 1784) Lühe, 1909; C. metoecus Braun, 1900b; C. chaenogobii Yamaguti and Matsumura, 1942; C. nemachilus Krotov, 1959. We described the new species Crepidostomum achmerovi sp. nov. that is a sibling species of C. nemachilus. Molecular-genetic investigation have shown that C. nemachilus and C. achmerovi sp. nov. are closely related to C. metoecus in both 28S rDNA and cox1 mtDNA markers. Crepidostomum nemachilus forms a separate branch within the C. metoecus clade on the 28S BI tree with strong statistical support and separate clade in relation to C. metoecus clade on the cox1 BI tree. Values of p-distances between Crepidostomum species were at intergeneric level. Crepidostomum metoecus species complex including five species (C. metoecus, C. nemachilus, C. oschmarini, C. brinkmanni, and C. achmerovi sp. nov.) was reconsidered as independent genus Crepidostomum sensu stricto. Minimum Spanning Network showed that C. nemachilus, C. metoecus and C. achmerovi sp. nov. were separated by large number of mutational events and represent independent phyletic lines. An amended diagnosis is provided for the subfamily Crepidostomatinae, the genera Crepidostomum s. str. and Stephanophiala Nicoll, 1909, along with keys to species of both genera.

First report on molecular identification of Anisakis simplex in Oncorhynchus nerka from the fish market, with taxonomical issues within Anisakidae.

Alive anisakids cause acute gastrointestinal diseases, and dead worms contained in food can provoke sensibilization and allergic reactions in humans. Detected in the purchased minced salmon Oncorhynchus nerka nematodes were identified as Anisakis simplex sensu stricto (Anisakidae). We found that recently published phylogenetic trees (reconstructed using different ribosomal and mitochondrial genetic markers) showed independent clusterization of species recognized in the A. simplex sensu lato species complex. This prompted us to undertake this full-fledged molecular genetics study of anisakids from Kamchatka with phylogenetic reconstructions (NJ/ML) and calculated ranges of interspecific and intergeneric p-distances using ITS1-5.8S-ITS2 sequences. We confirmed that molecular markers based on the ITS region of rDNA were able to recognize 'pure' specimens belonging to the cryptic species. We offer new insights into the systematics of anisakids. The genus Anisakis sensu stricto should include Anisakis simplex sensu stricto, Anisakis pegreffii, Anisakis berlandi, Anisakis ziphidarum, and Anisakis nascettii. Presumably, two genera should be restored in the structure of the subfamily Anisakinae: Skrjabinisakis for the species Anisakis paggiae, Anisakis brevispiculata, and Anisakis physeteris; and Peritrachelius for the species Anisakis typica. In addition, we provide the short annotated list of some genera of the family Anisakidae, including their diagnoses.

Recombinant Adenoviruses for Delivery of Therapeutics Following Spinal Cord Injury.

The regeneration of nerve tissue after spinal cord injury is a complex and poorly understood process. Medication and surgery are not very effective treatments for patients with spinal cord injuries. Gene therapy is a popular approach for the treatment of such patients. The delivery of therapeutic genes is carried out in a variety of ways, such as direct injection of therapeutic vectors at the site of injury, retrograde delivery of vectors, and ex vivo therapy using various cells. Recombinant adenoviruses are often used as vectors for gene transfer. This review discusses the advantages, limitations and prospects of adenovectors in spinal cord injury therapy.

Paragonimus heterotremus Chen et Hsia, 1964 (Digenea: Paragonimidae): species identification based on the biological and genetic criteria, and pathology of infection.

As a result of the experimental infection of rats with metacercariae of Paragonimus heterotremus Chen et Hsia, 1964 from crabs (Potamiscus tannanti) caught in Yen Bai province, Vietnam, it was found that worms migrated into the lungs, to the liver and less frequently to the tissue that lines body cavities of the hosts, where they reached the adult stage, but in the muscles, worms stayed at the larval stage. Studies have shown that for P. heterotremus, rats can simultaneously play the role of the final and paratenic host; herewith, an infection with the trematode of this species can lead to the development of three forms of paragonimiasis: pulmonary, hepatic and muscular. Eggs from the adult worms localised in the liver, unlike eggs from the adult worms localised in the lungs, were not excreted into the external environment, but accumulated inside the organ. Histology and description of changes, which take place on the external surface of organs affected with P. heterotremus, are given in this study. Based on the behavioural characteristics of worms during rat infection and molecular genetic data, we established that worms from Vietnam and India should be assigned to different species of Paragonimus. P. heterotremus distribution is limited to the territory of the Southeast China, Northern Vietnam, Laos and Thailand.

Efficiency of Human Olfactory Ensheathing Cell Transplantation into Spinal Cysts to Improve Mobility of the Hind Limbs.

The pathological processes developing after spinal cord injuries often lead to formation of cysts. Existing surgical and medical methods are insufficient for treatment of post-traumatic spinal cord cysts. One of the emerging tools is cell therapy. Olfactory ensheathing cells (OECs) are perspective cells for cell therapy. In this study, we demonstrated that human OEC transplantation is effective in experimental spinal cysts. For our experiments, we selected animals only at the intermediate stage of recovery with scores from 8 to 13 according to the Basso, Beattie, and Bresnahan (BBB) scale. Cells were transplanted in different quantities (0.75 and 1.5 million) into the fully formed cysts and in the areas of injury without cysts. Improvement of limb mobility after human OEC transplantation into post-traumatic cysts was shown. In the group of rats with cysts, time-dependent increase in the BBB score was observed in subgroups treated with 0.75 and 1.5 million OECs with no statistically significant time-dependent dynamics of BBB values in the control group. When all three subgroups (control and two OEC doses) were compared, the Kruskal-Wallis test showed the presence of differences between subgroups after 1, 3, and 4 weeks of treatment with evidence of divergence increase. There was no statistically significant difference between the two doses of OEC treatment. The human OECs in the experiments without cysts were not effective. It was also shown that PKH26-labeled human OECs survive throughout the experiment and migrate to nearby areas of the cyst. Therefore, it was found that it is effective to transplant human OECs into fully formed cysts. In the future, autologous OECs can be used to personalize the treatment of patients with spinal cysts.

Genetic diversity and phylogenetic relations of salmon trematode Nanophyetus japonensis.

Nanophyetiasis is the severe zoonotic disease caused by parasitic worms from the genus Nanophyetus. Humans and carnivorous animals become infected when they ingest raw fish containing metacercariae, especially Pacific salmonids. Nanophyetiasis is detected in limited geographical areas which include the coastal regions of the North Pacific: the United States of America, Russian Federation and Japan. Despite the epidemiological significance, Nanophyetus species have not been well studied genetically. In this research, we for the first time explored genetic diversity of Nanophyetus japonensis from Japan in comparison with those of related species, N. salmincola from North America and N. schikhobalowi from the Russian Far East, based on sequence variation in the nuclear ribosomal gene family (18S, ITS1-5.8S-ITS2 and 28S) and mitochondrial nad1 gene, encoding subunit I of the respiratory chain NADH dehydrogenase. The results confirmed the independent species status for the compared flukes, demonstrated a greater genetic similarity of Asian species between themselves than each of them with the North American one, suggesting that N. japonensis and N. schikhobalowi are close sister species, and also revealed discrepancy between the levels of morphological and genetic differentiation.

Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer).

BACKGROUND: Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. METHODS: The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. RESULTS: In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440-640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. CONCLUSION: This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine.