Cystidicola farionis, a Swim Bladder Parasite of European Smelt: Characterization of the Nematode Trehalose Strategy.

The molecular identification of Cystidicola farionis (a swim bladder nematode of European smelt from the Vistula Lagoon in Poland) was performed. Their prevalence level was determined, and changes in the trehalose synthesis pathway in larvae and adult nematodes were demonstrated. The trehalose level was almost four times higher in adult nematodes than in larvae. In contrast, the activity of both enzymes (trehalose 6-phosphate synthase, TPS and trehalose 6-phosphate phosphatase, TPP) involved in the synthesis of trehalose was higher in larvae than in adults under optimal conditions. The optimum pH for TPS isolated from larvae and adults was pH 7.0. The optimum pH for TPP from larvae and adults was pH 7.0 and pH 8.0, respectively. The optimal temperature was 20 °C, and Mg2+ ions were an activator for trehalose-synthetizing enzymes from both sources. Enzymes isolated from adult nematodes were less susceptible to divalent ion chelator and inorganic phosphate than larval enzymes. The dynamic transformation of trehalose in the nematode developing inside the swim bladder of the smelt appears to be an important metabolic pathway in the nematode survival strategy. These studies are aimed at a better understanding of the issue of the metabolic adaptation of parasites, which, in the future, may indirectly contribute to the elimination of the parasite from aquacultures, which will impact public health.

A new microsporidian parasite, Microsporidium theragrae n. sp., infecting Alaska pollock Gadus chalcogrammus (Teleostei: Gadidae).

A new microsporidian infecting Gadus chalcogrammus Pallas, 1814 (Gadidae), is described based on morphological, ultrastructural, and molecular studies. This microsporidian parasite develops inside intramuscular spindle-shaped lesions measuring approximately 1-2 mm in width and 4-8 mm in length. Infected cells encapsulated by a host-produced wall containing a sponge-like acellular zone. Sporogony presumably proceeds via segmentation of sporogonial plasmodium, resulting in a variable number of spores. Sporogonial stages develop in sporophorous vesicles (SVs), abutting a moderately electron-dense thick walled coat of a homogeneous amorphous material. SVs space contains rare granular and tubular inclusions. Neighboring SVs often interconnected by bridges of the host cell cytoplasm that were limited by membrane comparable with SV coat. The elongate-ovoid spores, measuring 4.29 ± 0.38 × 2.51 ± 0.26 µm (N 104), possess a bipartite polaroplast and polar tube with 15-16 coils arranged in 2-3 layers. The angle of tilt of the polar tube coils is less than 30°. The sequence analysis of SSU rDNA coding region showed that the studied microsporidians differs from other fish muscle-infecting species at least in 17 bp (2.58%) and is closely related to Microsporidium cypselurus Yokoyama et al. (2002) infecting the flying fish from East China Sea. The parasite is provisionally positioned as Microsporidium theragrae sp. n.

Molecular dynamics simulations suggest why the A2058G mutation in 23S RNA results in bacterial resistance against clindamycin.

Clindamycin, a lincosamide antibiotic, binds to 23S ribosomal RNA and inhibits protein synthesis. The A2058G mutation in 23S RNA results in bacterial resistance to clindamycin. To understand the influence of this mutation on short-range interactions of clindamycin with 23S RNA, we carried out full-atom molecular dynamics simulations of a ribosome fragment containing clindamycin binding site. We compared the dynamical behavior of this fragment simulated with and without the A2058G mutation. Molecular dynamics simulations suggest that clindamycin in the native ribosomal binding site is more internally flexible than in the A2058G mutant. Only in the native ribosome fragment did we observe intramolecular conformational change of clindamycin around its C7-N1-C10-C11 dihedral. In the mutant, G2058 makes more stable hydrogen bonds with clindamycin hindering its conformational freedom in the ribosome-bound state. Clindamycin binding site is located in the entrance to the tunnel through which the newly synthesized polypeptide leaves the ribosome. We observed that in the native ribosome fragment, clindamycin blocks the passage in the tunnel entrance, whereas in the mutated fragment the aperture is undisturbed due to a different mode of binding of clindamycin in the mutant. Restricted conformational freedom of clindamycin in a position not blocking the tunnel entrance in the A2058G mutant could explain the molecular mechanism of bacterial resistance against clindamycin occurring in this mutant.

Helminths of European smelt Osmerus eperlanus (Linnaeus, 1758) in Lake Hancza and the Vistula Lagoon, with special regard to their zoonotic threats.

Reports published in recent years on the increased risk in the Baltic Sea of fish infection with larvae of nematodes potentially pathogenic to humans have prompted a study of European smelt Osmerus eperlanus with respect to the presence of these parasites in the Vistula Lagoon. Additionally, samples of this fish from Lake Hancza were comparatively considered. The body cavity, the surface of internal organs, the alimentary tract and the swimbladder of fish from two environmentally different water bodies were taken into account in the analysis. Only the tapeworm Proteocephalus longicollis was found in the alimentary tract of the fish from Lake Hancza. Depending on the period of study, the prevalence ranged from 60% to 100%, and the mean intensity from 11.5 to 42.0. The helminth fauna of smelt from the Vistula Lagoon was more diverse: Cystidicola farionis occurred with a prevalence from 25.9 to 75.0% and a mean intensity of infection from 2.7 to 66.0%, Proteocephalus longicollis and cystacanths of Corynosoma with a prevalence ranging in different years from 12.0 to 44.0% and from 14.8 to 50.0%, respectively. Zoonotic parasites, such as nematode larvae of the Anisakidae family (Contracaecum sp., Anisakis simplex), occurred in the intestine with a prevalence of 31%, 72% and 22% in consecutive years of the study.

Conformational space of clindamycin studied by ab initio and full-atom molecular dynamics.

Molecular dynamics (MD) simulations allow determining internal flexibility of molecules at atomic level. Using ab initio Born-Oppenheimer molecular dynamics (BOMD), one can simulate in a reasonable time frame small systems with hundreds of atoms, usually in vacuum. With quantum mechanics/molecular mechanics (QM/MM) or full-atom molecular dynamics (FAMD), the influence of the environment can also be simulated. Here, we compare three types of MD calculations: ab initio BOMD, hybrid QM/MM, and classical FAMD. As a model system, we use a small antibiotic molecule, clindamycin, which is one of the lincosamide antibiotics. Clindamycin acquires two energetically stable forms and we investigated the transition between these two experimentally known conformers. We performed 60-ps BOMD simulations in vacuum, 50-ps QM/MM, and 100-ns FAMD in explicit water. The transition between two antibiotic conformers was observed using both BOMD and FAMD methods but was not noted in the QM/MM simulations.

Endurance Exercise Mobilizes Developmentally Early Stem Cells into Peripheral Blood and Increases Their Number in Bone Marrow: Implications for Tissue Regeneration.

Endurance exercise has been reported to increase the number of circulating hematopoietic stem/progenitor cells (HSPCs) in peripheral blood (PB) as well as in bone marrow (BM). We therefore became interested in whether endurance exercise has the same effect on very small embryonic-like stem cells (VSELs), which have been described as a population of developmentally early stem cells residing in BM. Mice were run daily for 1 hour on a treadmill for periods of 5 days or 5 weeks. Human volunteers had trained in long-distance running for one year, six times per week. FACS-based analyses and RT-PCR of murine and human VSELs and HSPCs from collected bone marrow and peripheral blood were performed. We observed that endurance exercise increased the number of VSELs circulating in PB and residing in BM. In parallel, we observed an increase in the number of HSPCs. These observations were subsequently confirmed in young athletes, who showed an increase in circulating VSELs and HSPCs after intensive running exercise. We provide for the first time evidence that endurance exercise may have beneficial effects on the expansion of developmentally early stem cells. We hypothesize that these circulating stem cells are involved in repairing minor exercise-related tissue and organ injuries.

The Effects of Cadmium at Low Environmental Concentrations on THP-1 Macrophage Apoptosis.

Cadmium at environmental concentrations is a risk factor for many diseases, including cardiovascular and neurodegenerative diseases, in which macrophages play an important role. The aim of this study was to evaluate the effects of cadmium at low environmental (nanomolar) concentrations on apoptotic processes in THP-1(acute monocytic leukemia cells line)-derived macrophages, with special focus on mitochondrial events involved. Macrophages were incubated with various cadmium chloride (CdCl2) solutions for 48 h at final concentrations of 5 nM, 20 nM, 200 nM and 2 µM CdCl2. Cell viability was measured using flow cytometry. Flow cytometric measurement (annexin V/FITC (annexin V/fluorescein isothiocyanate) and PI (propidium iodide) double staining) was used to quantify the extent of apoptosis. Fluorescence and confocal microscopy were used for imaging of apoptosis process. Changes in mitochondrial membrane potential were monitored using cytofluorimetry after cell staining with JC-1(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyane iodide) probe. Mitochondrial ROS (reactive oxygen species) levels were measured cytofluorimetrically after incubation of cells with mitochondrial superoxide indicator (MitoSOX) red fluorescent marker. The mRNA expression of Bcl-2 and Bax was analysed with qRT-PCR. Our study demonstrates that cadmium, even at low environmental concentrations, exerts mitochondrial toxicity in THP-1 macrophages. Forty-eight-hour exposure to very low concentrations reduces cell viability and results in cell death by apoptosis and necrosis. The decrease in mitochondrial membrane potential, increased ROS production, increased Bax and decreased Bcl-2 mRNA expression are mitochondrial events involved in cadmium-induced apoptosis.

Hematopoietic stem/progenitor cells express several functional sex hormone receptors-novel evidence for a potential developmental link between hematopoiesis and primordial germ cells.

Evidence has accumulated that hematopoietic stem progenitor cells (HSPCs) share several markers with the germline, a connection supported by reports that prolactin, androgens, and estrogens stimulate hematopoiesis. To address this issue more directly, we tested the expression of receptors for pituitary-derived hormones, such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH), on purified murine bone marrow (BM) cells enriched for HSPCs and tested the functionality of these receptors in ex vivo signal transduction studies and in vitro clonogenic assays. We also tested whether administration of pituitary- and gonad-derived sex hormones (SexHs) increases incorporation of bromodeoxyuridine (BrdU) into HSPCs and expansion of hematopoietic clonogenic progenitors in mice and promotes recovery of blood counts in sublethally irradiated animals. We report for the first time that HSPCs express functional FSH and LH receptors and that both proliferate in vivo and in vitro in response to stimulation by pituitary SexHs. Furthermore, based on our observations that at least some of CD45(-) very small embryonic-like stem cells (VSELs) may become specified into CD45(+) HSPCs, we also evaluated the expression of pituitary and gonadal SexHs receptors on these cells and tested whether these quiescent cells may expand in vivo in response to SexHs administration. We found that VSELs express SexHs receptors and respond in vivo to SexHs stimulation, as evidenced by BrdU accumulation. Since at least some VSELs share several markers characteristic of migrating primordial germ cells and can be specified into HSPCs, this observation sheds new light on the BM stem cell hierarchy.

Positive effects of prolonged caloric restriction on the population of very small embryonic-like stem cells - hematopoietic and ovarian implications.

BACKGROUND: Low calorie intake, or calorie restriction (CR) without malnutrition, has been demonstrated in several animal species, including mice, to increase both median and maximum lifespan as well as delay reproductive senescence. Our previous work demonstrated a positive correlation between life span and the number of very small embryonic-like stem cells (VSELs) in long living Laron dwarf mice. These animals have very low levels of circulating insulin-like growth factor 1 (IGF-1) in peripheral blood (PB), maintain higher numbers of hematopoietic stem cells (HSPCs) in bone marrow (BM), and display prolonged fecundity compared with wild type littermates. Since CR lowers the level of IGF-1 in PB, we become interested in the effect of CR on the number of VSELs and HSPCs in BM as well as on the morphology of ovaries and testes. METHODS: In our studies four-week-old female and male mice were subjected to CR by employing an alternate-day ad libitum feeding diet for a period of 9 months. RESULTS: We observed that mice on CR had a higher number of BM-residing VSELs than control mice fed ad libitum. These changes correlated with higher numbers of HSPCs in BM, spleen, and peripheral blood (PB) as well as with an increase in the number of primordial and primary follicles in ovaries. At the same time, however, no changes were observed in the testes of mice under CR. CONCLUSION: We conclude that CR positively affects the pool of VSELs in adult tissues and explains the positive effect of CR on longevity.

Evidence that the population of quiescent bone marrow-residing very small embryonic/epiblast-like stem cells (VSELs) expands in response to neurotoxic treatment.

The concept that bone marrow (BM)-derived cells may participate in neural regeneration remains controversial, and the identity of the specific cell type(s) involved remains unknown. We recently reported that the adult murine BM contains a highly mobile population of Sca-1(+) Lin(-) CD45(-) cells known as very small embryonic/epiblast-like stem cells (VSELs) that express several markers of pluripotency such as Oct-4. In the BM microenvironment, these cells are kept quiescent because of epigenetic modification of certain paternally imprinted genes. However, as reported, these cells can be mobilized in mice in an experimental model of stroke and express several genes involved in neurogenesis while circulating in peripheral blood (PB). In the current work, we employed a model of toxic brain damage, which is induced by administration of kainic acid, to see not only whether VSELs can be mobilized into PB in response to this neurotoxin, but, more importantly, whether they proliferate and expand in BM tissue. We report here for the first time that brain damage leads to activation and expansion of the BM pool of quiescent VSELs, which precedes their subsequent egress into PB. Harnessing these cells in neural tissue regeneration is currently one of the challenges in regenerative medicine.

Parasitization of invasive gobiids in the eastern part of the Central trans-European corridor of invasion of Ponto-Caspian hydrobionts.

Four gobiid species, Babka gymnotrachelus, Neogobius melanostomus, Neogobius fluviatilis, and Proterorhinus semilunaris, were parasitologically studied in different localities of the Dnieper and Vistula river basins. The highest number of parasitic species was found in N. fluviatilis (35 taxa). The parasite fauna of N. melanostomus, B. gymnotrachelus, and P. semilunaris consists of 23, 22, and 15 taxa, respectively. The species accumulation curves show stable accumulation of parasite species by all four fish hosts along the studied part of the corridor, from the Dnieper Estuary to the Vistula River delta. The plot reveals also that the studied gobies lose the parasites common in the host native range and accept new parasites from the colonized area. In the case of N. melanostomus, it complies with the enemy release hypothesis, as the parasite load was low in the invaded area if compared to the native range. The three other alien gobies are vector for Gyrodactylus proterorhini in the Baltic basin. Moreover, populations of this alien monogenean tend to be more abundant in their new range in comparison with the Black Sea basin. In general, the number of parasite species in the colonized area was of the same rank as in the native one for N. fluviatilis, and even higher for B. gymnotrachelus. This results from accumulating new parasite species along the gobiid invasion route. In particular, the N. fluviatilis, B. gymnotrachelus, and P. semilunaris lost some of their native parasites and gained the local ones after entering the post-dam part of the Vistula River; it can be interpreted as a partial escape from parasites.

Genome-wide analysis of murine bone marrow­derived very small embryonic-like stem cells reveals that mitogenic growth factor signaling pathways play a crucial role in the quiescence and ageing of these cells.

It has been postulated that the most primitive population of stem cells, Oct4(+)Sca-1(+)Lin(-)CD45(-) very small embryonic-like stem cells (VSELs), differentiate into tissue-committed stem cells in adult mice. However, Oct4(+) VSELs remain quiescent in adult tissues and do not form teratomas. In thi study, we report the characteristics of the VSEL transcriptome by gene set enrichment analysis employing a microarray database established from 20 murine bone marrow-derived, FACS-sorted VSELs in comparison with hematopoietic stem cells and embryonic stem cells. In the Oct4(+) VSELs, we observed the upregulation of tissue-specific gene sets and a gene set encoding the complement-coagulation cascade. By contrast, in the VSELs, we observed the downregulation of genes involved in the UV radiation response, mRNA processing and mitogenic growth factor signaling [e.g., insulin-like growth factor­1 (IGF-1) and neurotrophic tyrosine kinase receptor A (TRKA), as well as the ERK and PI3K pathways]. Employing leading-edge subset analysis and real-time PCR assays, we observed that several genes, such as growth factor receptor-bound protein 2 (Grb2), son of sevenless homolog 1 (Sos1), SHC (Src homology 2 domain containing) transforming protein 1 (Shc1), mitogen-activated protein kinase kinase 1 (Map2k1), v-akt murine thymoma viral oncogene homolog 3 (Akt3), Elk1, ribosomal protein S6 kinase, 90 kDa, polypeptide 3 (Rps6kA3), glycogen synthase kinase 3ß (Gsk3ß) and casein kinase 2, alpha 1 polypeptide (Csnk2A1), which are involved in mitogenic growth factor signaling pathways, were commonly downregulated in the VSELs. Notably, this repression was reversed in the VSELs co-cultured over a C2C12 supportive cell-line, whereby they are induced to form VSEL-derived spheres (VSEL-DSs); thus, they are enriched, forming more differentiated stem cells. Therefore, we suggest that the repression of mitogenic growth factor signaling (e.g., through the IGF-1 receptor) may prevent uncontrolled Oct4(+) VSEL proliferation and teratoma formation. Thus, restoring the responsiveness to mitogenic growth factors may be a crucial step in employing these cells in regenerative medicine.

The negative effect of prolonged somatotrophic/insulin signaling on an adult bone marrow-residing population of pluripotent very small embryonic-like stem cells (VSELs).

It is well known that attenuated insulin/insulin-like growth factor signaling (IIS) has a positive effect on longevity in several animal species, including mice. Here, we demonstrate that a population of murine pluripotent very small embryonic-like stem cells (VSELs) that reside in bone marrow (BM) is protected from premature depletion during aging by intrinsic parental gene imprinting mechanisms and the level of circulating insulin-like growth factor-I (IGF-I). Accordingly, an increase in the circulating level of IGF-I, as seen in short-lived bovine growth hormone (bGH)-expressing transgenic mice, which age prematurely, as well as in wild-type animals injected for 2 months with bGH, leads to accelerated depletion of VSELs from bone marrow (BM). In contrast, long-living GHR-null or Ames dwarf mice, which have very low levels of circulating IGF-I, exhibit a significantly higher number of VSELs in BM than their littermates at the same age. However, the number of VSELs in these animals decreases after GH or IGF-I treatment. These changes in the level of plasma-circulating IGF-I corroborate with changes in the genomic imprinting status of crucial genes involved in IIS, such as Igf-2-H19, RasGRF1, and Ig2R. Thus, we propose that a chronic increase in IIS contributes to aging by premature depletion of pluripotent VSELs in adult tissues.

Quantum mechanical studies of lincosamides.

Lincosamides are a class of antibiotics used both in clinical and veterinary practice for a wide range of pathogens. This group of drugs inhibits the activity of the bacterial ribosome by binding to the 23S RNA of the large ribosomal subunit and blocking protein synthesis. Currently, three X-ray structures of the ribosome in complex with clindamycin are available in the Protein Data Bank, which reveal that there are two distinct conformations of the pyrrolidinyl propyl group of the bound clindamycin. In this work, we used quantum mechanical methods to investigate the probable conformations of clindamycin in order to explain the two binding modes in the ribosomal 23S RNA. We studied three lincosamide antibiotics: clindamycin, lincomycin, and pirlimycin at the B3LYP level with the 6-31G** basis set. The focus of our work was to connect the conformational landscape and electron densities of the two clindamycin conformers found experimentally with their physicochemical properties. For both functional conformers, we applied natural bond orbital (NBO) analysis and the atoms in molecules (AIM) theory, and calculated the NMR parameters. Based on the results obtained, we were able to show that the structure with the intramolecular hydrogen bond C=O…H-O is the most stable conformer of clindamycin. The charge transfer between the pyrrolidine-derivative ring and the six-atom sugar (methylthiolincosamide), which are linked via an amide bond, was found to be the dominant factor influencing the high stability of this conformer.

Gyrodactylus proterorhini Ergens, 1967 (Monogenoidea, Gyrodactylidae) in gobiids from the Vistula River--the first record of the parasite in Poland.

During a parasitological survey of two non-native fish species-racer goby Neogobius gymnotrachelus and monkey goby Neogobius fluviatilis-in the Wloclawek Reservoir on the lower Vistula River, the monogenean Gyrodactylus proterorhini was recorded for the first time in Poland, and for the first time, the racer goby was listed as the parasite host. Specimens of G. proterorhini were detected on body surface, fins, and gills of the fish studied. In 2006, the monkey goby was infected with the prevalence 41%, racer goby with the prevalence of 47%, at maximal intensity of 6 and 9 parasites per fish, (respectively). The infection level in relation to the fish size and sampling season was also discussed. The presented study supports the hypothesis of progressive introduction of the parasite with gobiids to the colonized areas.

First record of Nippotaenia mogurndae Yamaguti and Miyata, 1940 (Cestoda, Nippotaeniidae), a parasite introduced with Chinese sleeper to Poland.

A parasitological survey of an Asian fish, Chinese sleeper Perccottus glenii, inhabiting the Wloclawek Reservoir on the lower Vistula River (central Poland) revealed the occurrence of an exotic cestode Nippotaenia mogurndae Yamaguti and Miyata, 1940, which spreads spontaneously with a host to European waters. This paper documented the first record of the parasite in Poland. Specimens were described, and the effect of fish size and season on the infection level was analyzed. Within the 3 years, 112 fish were examined. Samples were collected by electrofishing in the upper part of the Wloclawek Reservoir three times (seasonally) in 2006 and 2007 and once in autumn 2008. Parasitological indices (prevalence, intensity of infection, and abundance) in particular samples as well as in two size groups of fish were calculated. Statistical tests were applied to analyze the results (Spearman's correlation coefficient and Mann-Whitney's U test). The infection level of Chinese sleeper with N. mogurndae in the Wloclawek Reservoir (prevalence 54.7%, mean intensity of infection 7.2, maximum 29) was comparable to infection noted in the area of the natural range of the host. The infection of smaller fish (less than 80 mm, TL) increased gradually with increasing body size. The infection of bigger specimens was in general higher, but the parasite recruitment was of a more incidental character. The spreading of N. mogurndae with Chinese sleeper is still in progress. The Wloclawek Reservoir provides suitable conditions for parasite development.

Fish monogeneans from a shallow, eutrophic Oswin Lake in Poland.

BACKGROUND: A study was carried out in a shallow, eutrophic Oswin Lake, within Seven Islands Reserve (north-eastern Poland), in order to determine the infection of fish with monogeneans as sensitive indicators of water quality. This paper presents comparative analysis of the occurrence of Monogenea in fishes from two, distinctly separated pools of the lake, differing in environmental conditions. The impact of fish size and sampling season on the infection parameters was also analyzed. MATERIAL AND METHODS: In 1998 and 1999, a total of 1091 fish representing 8 dominant species were examined. Samples were collected four times a year (in May, July, August, and October) simultaneously from the eastern and western parts of the lake. RESULTS: 16 species of Monogenea (and some unidentified specimeus) occurred in the fish examined, most of them from the Dactylogyridae family: Dactylogyrus sphyrna, D. auriculatus, D. intermedius, D. anchoratus, D. falcatus, D. tincae, D. wunderi, D. zandti, D. difformis, D. nanus, D. distinguendus, D. crucifer, D. caballeroi. Furthermore, Tetraonchus monenteron (Tetraonchidae), Gyrodactylus elegans (Gyrodactylidae), and Paradiplozoon megan (Diplozoidae) were detected. Monogenea occurred abundantly on the gills of pike, roach, rudd, white bream, carp bream and crucian carp, but rather sporadically in tench and perch. The highest species variety and diversity of monogenean guilds were detected in carp bream and roach. It was proved that D. crucifer and D. difformis were significantly more abundant in the western pool compared to the eastern one. Infection of fish with Dactylogyridae was season-dependent, contrary to the infection of pike with T. monenteron. Intensity of infection of white bream with D. sphyrna and roach with D. crucifer increased with fish body length; such a relationship did not appear for the other monogeneans and their hosts. CONCLUSION: Presence of common Monogenea in the fish examined, lack of rare species, poor diversity of monogenean guilds and relatively low infection rate could be connected with the low stability of the lake environment. Location-dependent occurrence of D. crucifer and D. difformis indicated that the exchange between fish groups from the eastern and western parts of the lake is limited.