Next step in Monachacantiana (Montagu, 1803) phylogeography: northern French and Dutch populations (Eupulmonata, Stylommatophora, Hygromiidae).

Features of shell and genitalia as well as nucleotide sequences of selected mitochondrial and nuclear genes of specimens of Monachacantiana from ten northern French and two Dutch populations were compared with the same features of British and Italian populations. They were found to be very similar to populations previously identified as belonging to the CAN-1 lineage of M.cantiana. This confirms previous suggestions that M.cantiana was introduced to western Europe (England, France and the Netherlands) in historical times.

The complete mitochondrial genome of the terrestrial snail Monachacartusiana (O.F. Müller, 1774) (Gastropoda, Eupulmonata, Hygromiidae).

The mitochondrial genome of Monachacartusiana is the first complete mitochondrial sequence described for the pulmonate snail genus Monacha and for the family Hygromiidae. The identified mitogenome has a length of 13,894 bp and encodes 13 proteins, 22 tRNAs, and two rRNAs. A phylogenetic analysis of available mitogenomes from representatives of helicoid families shows a sister group relationship of Hygromiidae and Geomitridae, which have been recently recognised as separate families.

Ligand-Sensitised LaF3 :Eu3+ and SrF2 :Eu3+ Nanoparticles and in Vitro Haemocompatiblity Studies.

Luminescent Ln3+ -doped nanoparticles (NPs) functionalised with the desired organic ligand molecules for haemocompatibility studies were obtained in a one-pot synthesis. Chelated aromatic organic ligands such as isophthalic acid, terephthalic acid, ibuprofen, aspirin, 1,2,4,5-benzenetetracarboxylic acid, 2,6-pyridine dicarboxylic acid and adenosine were applied for surface functionalisation. The modification of the nanoparticles is based on the donor-acceptor character of the ligand-nanoparticle system, which is an alternative to covalent functionalisation by peptide bonding as presented in our recent report. The aromatic groups of selected ligands absorb UV light and transfer their excited-state energy to the dopant Eu3+ ions in LaF3 and SrF2 NPs. Herein, we discuss the structural and spectroscopic characterisation of the NPs and the results of haemocompatibility studies. Flow cytometry analysis of the nanoparticles' membrane-binding is also presented.

Redescription of Monacha pantanellii (De Stefani, 1879), a species endemic to the central Apennines, Italy (Gastropoda, Eupulmonata, Hygromiidae) by an integrative molecular and morphological approach.

Specimens obtained from ten populations of a Monacha species from the central Apennines were compared with six molecular lineages of Monacha cantiana s. l. (CAN-1, CAN-2, CAN-3, CAN-4, CAN-5, CAN-6) and two other Monacha species (M. cartusiana and M. parumcincta), treated as outgroup, by molecular (nucleotide sequences of two mitochondrial COI and 16S rDNA as well as two nuclear ITS2 and H3 gene fragments) and morphological (shell and genital anatomy) analysis. The results strongly suggest that these populations represent a separate species for which two names are available: the older Helix pantanellii De Stefani, 1879 and the junior M. ruffoi Giusti, 1973. The nucleotide sequences created well separated clades on each phylogenetic tree. Genital anatomy included several distinctive features concerning vaginal appendix, penis, penial papilla and flagellum; instead, shell characters only enabled them to be distinguished from M. cartusiana and M. parumcincta. Remarkably, populations of M. pantanellii show high morphological variability. Shell variability mainly concerns size, some populations having very small dimensions. Genital variability shows a more intricate pattern of all anatomical parts, being higher as regards the vagina and vaginal appendix. Despite this morphological variability, the K2P distance range of COI sequences between populations is narrow (0.2-4.5%), if we consider all but three of the 53 sequences obtained. This research confirmed that the species of Monacha and their molecularly distinguished lineages can only occasionally be recognised morphologically and that they have significant inter- and intra-population variability. The possibility of using an overall approach, including shell, genital and molecular evidence, was taken in order to establish a reliable taxonomic setting.

Surface Modification of Luminescent LnIII Fluoride Core-Shell Nanoparticles with Acetylsalicylic acid (Aspirin): Synthesis, Spectroscopic and in Vitro Hemocompatibility Studies.

Luminescent lanthanide fluoride core-shell (LaF3 :Tb3+ ,Ce3+ @SiO2 -NH2 ) nanoparticles, with acetylsalicylic acid (aspirin) coated on the surface have been obtained. The synthesized products, which combine the potential located in the silica shell with the luminescent activity of the core, were characterized in detail with the use of luminescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and transmission electron microscopy (TEM) methods. The in vitro effects of the modified luminescent nanoparticles on human red blood cell (RBC) membrane permeability, RBC shape, and sedimentation rate were investigated to assess the hemocompatibility of the obtained compounds. This study demonstrates that LaF3 : Tb3+ 5 %, Ce3+ 10 %@SiO2 -NH2 nanoparticles with acetylsalicylic acid (aspirin) coated on the surface are very good precursors for multifunctional drug-delivery systems or bio-imaging probes that can be used safely in potential biomedical applications.

Exploration of phylogeography of Monachacantiana s.l. continues: the populations of the Apuan Alps (NW Tuscany, Italy) (Eupulmonata, Stylommatophora, Hygromiidae).

Two new lineages CAN-5 and CAN-6 were recognised in four populations of Monachacantiana (Montagu, 1803) s.l. from the Italian Apuan Alps by joint molecular and morphological analysis. They are different from other M.cantiana lineages known from English, Italian, Austrian and French populations, i.e. CAN-1, CAN-2, CAN-3 and CAN-4, as well as from the other Italian Monacha species used for comparisons (M.parumcincta and M.cartusiana). Although a definite taxonomic and nomenclatural setting seems to be premature, we suggest that the name or names for these new lineages as one or two species should be found among 19th century names (Helixsobara Mabille, 1881, H.ardesa Mabille, 1881, H.apuanica Mabille, 1881, H.carfaniensis De Stefani, 1883 and H.spallanzanii De Stefani, 1884).

Behavior of Caenorhabditis elegans in a nicotine gradient modulated by food.

Nicotine decreases food intake, and smokers often report that they smoke to control their weight. To see whether similar phenomena could be observed in the model organism Caenorhabditis elegans, we challenged drug-naïve nematodes with a chronic low (0.01 mM) and high (1 mM) nicotine concentration for 55 h (from hatching to adulthood). After that, we recorded changes in their behavior in a nicotine gradient, where they could choose a desired nicotine concentration. By using a combination of behavioral and morphometric methods, we found that both nicotine and food modulate worm behavior. In the presence of food (E. coli OP50) the nematodes adapted to the low nicotine concentration, when placed in the gradient, chose a similar nicotine concentration like C. elegans adapted to the high nicotine concentration. However, in the absence of food, the nematodes adapted to the low nicotine concentration, when placed in the gradient of this alkaloid, chose a similar nicotine concentration like naïve worms. The nematodes growing up in the presence of high concentrations of nicotine had a statistically smaller body size, compared to the control condition, and the presence of food did not cause any enhanced slowing movement. These results provide a platform for more detailed molecular and cellular studies of nicotine addiction and food intake in this model organism.

Exploring Monacha cantiana (Montagu, 1803) phylogeography: cryptic lineages and new insights into the origin of the English populations (Eupulmonata, Stylommatophora, Hygromiidae).

Molecular analysis of nucleotide sequences of mitochondrial cytochrome oxidase subunit 1 (COI) and 16S ribosomal DNA (16SrDNA) as well as nuclear histone 3 (H3) and internal transcribed spacer 2 of rDNA (ITS2) gene fragments together with morphological analysis of shell and genitalia features showed that English, French and Italian populations usually assigned to Monacha cantiana consist of four distinct lineages (CAN-1, CAN-2, CAN-3, CAN-4). One of these lineages (CAN-1) included most of the UK (five sites) and Italian (five sites) populations examined. Three other lineages represented populations from two sites in northern Italy (CAN-2), three sites in northern Italy and Austria (CAN-3), and two sites in south-eastern France (CAN-4). The taxonomic and nomenclatural setting is only currently available for lineages CAN-1 and CAN-4; a definitive frame for the other two requires much more research. The lineage CAN-1 corresponds to the true M. cantiana (Montagu, 1803) because it is the only one that includes topotypical English populations. The relationships and genetic distances support the hypothesis of the Italian origin of this lineage which was probably introduced to England by the Romans. The lineage CAN-4 is attributed to M. cemenelea (Risso, 1826), for which a neotype has been designated and deposited. Its diagnostic sequences of COI, 16SrDNA, H3 and ITS2 genes have also been deposited in GenBank. Molecular and morphological (shell and genitalia) features showed that M. parumcincta (Rossmässler, 1834) is a distinct taxon from the M. cantiana lineages.

Shell colour, temperature, (micro)habitat structure and predator pressure affect the behaviour of Cepaea nemoralis.

Although shell colour polymorphism of the land snail Cepaea nemoralis is a well-known phenomenon, proximate and ultimate factors driving its evolution remain uncertain. Polymorphic species show variation in behavioural responses to selective forces. Therefore, we estimated effects of various environmental factors (temperature, humidity, food availability, (micro)habitat structure and predatory pressure) on behavioural response (frequency of locomotion, climbing and hiding) of C. nemoralis morphs, in experimental and natural conditions. In the experimental part of study, the frequency of locomotion was negatively affected by temperature and the presence of food and positively influenced by the presence of light. Morphs significantly differed in behavioural responses to environmental variability. Pink mid-banded and yellow five-banded morphs climbed less often and hide in shelter more often than yellow and pink unbanded individuals when temperature was low and food was absent. Snails fed most often at moderate temperature compared to low and high temperatures. Field investigations partially confirmed differences among morphs in frequency of climbing, but not in terms of probability of hiding in sheltered sites. In natural colonies, temperature and (micro)habitat structure significantly affected frequency of climbing as well as hiding in shelter. Snails more often hid in sheltered sites where thrushes preyed on Cepaea. Tendency of unbanded morphs to climb trees may have evolved under avian predatory pressure as thrushes forage on a ground. Tendency of banded morphs to hide in sheltered sites may reflect prey preferences for cryptic background. The results implicate that differential behaviour of C. nemoralis morphs compensate for their morphological and physiological limitations of adaptation to habitat.

Nicotine affects protein complex rearrangement in Caenorhabditis elegans cells.

Nicotine may affect cell function by rearranging protein complexes. We aimed to determine nicotine-induced alterations of protein complexes in Caenorhabditis elegans (C. elegans) cells, thereby revealing links between nicotine exposure and protein complex modulation. We compared the proteomic alterations induced by low and high nicotine concentrations (0.01 mM and 1 mM) with the control (no nicotine) in vivo by using mass spectrometry (MS)-based techniques, specifically the cetyltrimethylammonium bromide (CTAB) discontinuous gel electrophoresis coupled with liquid chromatography (LC)-MS/MS and spectral counting. As a result, we identified dozens of C. elegans proteins that are present exclusively or in higher abundance in either nicotine-treated or untreated worms. Based on these results, we report a possible network that captures the key protein components of nicotine-induced protein complexes and speculate how the different protein modules relate to their distinct physiological roles. Using functional annotation of detected proteins, we hypothesize that the identified complexes can modulate the energy metabolism and level of oxidative stress. These proteins can also be involved in modulation of gene expression and may be crucial in Alzheimer's disease. The findings reported in our study reveal putative intracellular interactions of many proteins with the cytoskeleton and may contribute to the understanding of the mechanisms of nicotinic acetylcholine receptor (nAChR) signaling and trafficking in cells.

Putative new groups of invertebrate water channels based on the snail Helix pomatia L. (Helicidae) MIP protein identification and phylogenetic analysis.

Water channel proteins, classified as a family of Membrane Intrinsic Proteins (MIPs) superfamily, enable rapid movement of water and small uncharged molecules through biological membranes. Although water channel proteins are required in several important processes characteristic for the animals, such as osmoregulation, mucus secretion, or defense against desiccation, molluscs, until now, have been very poorly explored in this aspect. Therefore, we decided to study MIPs in Helix pomatia L. applied as a model in studies on terrestrial snail physiology. Our studies consisted in: the snail organ transcriptome sequencing and consecutive bioinformatic analysis of the predicted protein, estimation of the encoding transcript expression (qPCR), investigation of the predicted protein function in the yeast Saccharomyces cerevisiae cells, and the phylogenetic analysis. We identified six water channel proteins, named HpAQP1 to HpAQP6. All of them were proven to transport water, two of them (HpAQP3 and HpAQP4) were also shown to be able to transport glycerol, and other two (HpAQP5 and HpAQP6) to transport H2O2. Phylogenetic analysis indicated that the proteins either fell into aquaporins (HpAQP1, HpAQP2 and HpAQP5) or formed new groups of invertebrate water channel proteins, not described until now, that we suggest to term malacoglyceroporins (HpAQP3 and HpAQP4) and malacoaquaporins (HpAQP6). Thus, the classification of animal water channels based on the vertebrate proteins and including aquaporin, aquaglyceroporin, S-aquaporin and AQP8-type grades does not reflect diversity of these proteins in invertebrates. The obtained results provide important data concerning diversity of water channel protein repertoire in aquatic and terrestrial invertebrates and should also contribute to the improvement of animal water channel classification system.

Molecular identification of first putative aquaporins in snails.

Aquaporins (AQPs), also known as water channel proteins, are members of a large protein family termed Major Intrinsic Proteins (MIP). The mammalian AQPs have been most comprehensively described, while knowledge about AQPs in invertebrates is limited mainly to insects. Not a single AQP protein has been described in snails to date. Consequently, we decided to search for the proteins in gastropod representatives, namely Lymnaea stagnalis, Catascopia occulta, and Stagnicola palustris (Mollusca; Gastropoda; Pulmonata; Lymnaeidae). Using the molecular approach, we identified L. stagnalis, C. occulta, and S. palustris open reading frames (ORFs) showing homology to AQP genes available in GenBank database, and characterized the encoded proteins, referred to as LsAQP1, CoAQP1, and SpAQP1, respectively. The putative snail aquaporins contain 299 amino acids, have a molecular mass of about 32 kDa, display the general AQP topology and three-dimensional structure congruent with orthodox AQPs, i.e., water-specific ones. Due to high levels of similarity in their characteristics, LsAQP1 was chosen for further studies, as the obtained results were supposed to be applicable for CoAQP1 and SpAQP1. Expression analysis revealed the presence of LsAQP1 transcript in the digestive tract, the cerebral ganglia, the kidney, the reproductive system, and the foot, suggesting that LsAQP1 as well as CoAQP1 and SpAQP1 are ubiquitous proteins and may play important roles in many essential water transport processes. The role appears to be confirmed by results of the yeast growth complementation assay pointing at functionality of LsAQP1. Thus, the obtained results support the AQP expression in gastropod tissues for the first time.

In vitro genoprotective and genotoxic effect of nicotine on human leukocytes evaluated by the comet assay.

The comet assay was used to measure the DNA damage induced in vitro by nicotine in human leukocytes as the extent of DNA migration in the comet head area, tail length, percent DNA in the tail, and Olive tail moment. Samples of whole blood were collected and blood cells were challenged with acute doses of 0.1, 1 and 10 µM of (-)-nicotine for 60 minutes. We found that nicotine treatment had dose-dependent effects on the level of DNA damage. At 1 and 10 µM of nicotine, both Olive tail moment and percent DNA in the tail significantly increased (p < 0.001), compared to the control. In the presence of 10 µM of nicotine, the shortest tail length and the smallest head area were detected. At a concentration of 0.1 µM, surprisingly, DNA damage detected by the comet assay was lower than in the control, which was proved by the observed significantly (p < 0.001) lower Olive tail moment and percent DNA in the tail as well as larger head area. The results suggest that nicotine, at a reasonably low concentration (0.1 µM), comparable to those found in the blood of habitual smokers, may have a protective effect, whereas higher doses of nicotine (1 and 10 µM) are genotoxic. The possible participation of reactive oxygen species in the DNA-damaging potential of nicotine is discussed.

Differential shell strength of Cepaea nemoralis colour morphs--implications for their anti-predator defence.

One of the most spectacular evolutionary forces is predation, evidenced to stimulate polymorphism in many prey species. Shell colour polymorphism of the land snail Cepaea nemoralis is a well-known model in evolutionary research. Nevertheless, the knowledge on the ecological causes driving its evolution remains incomplete and proximal factors shaping predatory pressure on C. nemoralis morphs are unknown. We evaluated shell crushing resistance and thickness, constituting crucial snail anti-predator defences in two shell areas (the apex and labium) of eight C. nemoralis morphotypes differing in shell colour and banding pattern. A GLM showed a significant effect of shell colour, banding pattern and shell thickness on shell strength. Pink shells were stronger than yellow ones, and banded forms had stronger shells than unbanded snails. The labium (usually attacked by mice) was generally thicker and more resistant than the apex (usually crushed by birds). Thicker shells were more resistant to crushing, and the rate of shell strength increase per unit of shell thickness was greater in pink and banded individuals compared to yellow and unbanded ones. Yellow and unbanded morphs have been found to be preferred by mice in the previous studies, which suggests that shell strength may be an important trait used in prey selection by these shell-crushing predators. The differences in potential anti-predator defences among snail morphs, found in the present study, justify future research on direct effect of C. nemoralis morphs shell strength on predator selectivity.

[Absorption, metabolism and excretion of nicotine in humans]. / Wchlanianie, przemiany metaboliczne i wydalanie nikotyny u czlowieka.

Nicotine is an alkaloid present in many plants of Solanaceae family. The levorotatory enantiomer (S) is a naturally occurring form. Nicotine enters the human body as a component of tobacco smoke. In alkaline environment the rate of nicotine permeation through biological membranes is increased. Almost 90% of nicotine absorbed by the body is metabolized in the liver. Nicotine may also be metabolized in the kidneys, lungs, brain, and respiratory epithelium membranes. The nicotine undergoes many transformations. Key role in the metabolism of nicotine is played by cytochrome P450 oxidases (mainly CYP2A6). Apart from them, UDP-glucuronosyltransferases, cytosolic aldehyde oxidase, amine N-methyltransferase, and flavin-containing monooxygenase 3 are involved in the decomposition of nicotine. Six major metabolites of nicotine have been identified. One of the most important metabolite is cotinine, from which is formed of trans-3'-hydroxycotinine--the compound which is excreted in the largest amount within the urine. The rate of nicotine metabolism is affected by diversified activity of polymorphic enzymes involved in this process, diet, gender and physiological condition of the organism.

Concentration- and time-dependent behavioral changes in Caenorhabditis elegans after exposure to nicotine.

Nicotine induces profound behavioral responses in the model organism Caenorhabditis elegans. We tested the effect of a broad range of concentrations of nicotine (from 0.001 mM to 30 mM in nematode growth medium) on C. elegans locomotor behavior. We also followed the time-course influence on the sensitivity of C. elegans to nicotine (from 0 min to 300 min). A low concentration (0.001 mM) of this alkaloid causes a reduction of the speed of movement. By contrast, moderate concentrations (0.01 and 0.1 mM) induced acceleration of the mean speed of locomotion of C. elegans. High doses of nicotine (above 1 mM) induced slowing down of the movements and, finally, paralysis. Time-dependent analysis revealed that the stimulating effect of nicotine abolished the slowing down of C. elegans in control experiments after 30 min in the presence of 0.001, 0.1 and 10 mM nicotine. In the presence of 0.1 mM nicotine, the stimulation phase lasted up to 70 min. The evidence indicates that nicotine can have dual effects on the speed of locomotion, which is dependent on differences in its dosage and treatment time.

Genotoxicity of nicotine in cell culture of Caenorhabditis elegans evaluated by the comet assay.

To assess the genotoxicity of nicotine, its DNA-damaging effect on Caenorhabditis elegans cells was tested with the alkaline single-cell microgel electrophoresis (comet) assay. The degree of DNA migration (a measure of possible DNA single-strand breaks, alkali-labile sites, and incomplete excision repair sites) was expressed as the head DNA%, tail length, and Olive tail moment. Large differences were found between experimental variants: 0, 1, 10, and 100 microM (-)-nicotine. At concentrations of 1 and 10 microM, no damages were detected by the comet assay, and the Olive tail moment and tail length were significantly lower than in the control (P < 0.001). The highest head DNA% and the lowest tail length and Olive tail moment were observed in the presence of 1 microM of nicotine. At 100 microM of nicotine, a significant increase (P < 0.001) was observed in Olive tail moment and tail length (up to 2.7- and 3-fold, respectively, compared to the control). The results are consistent with the lowest head DNA% among the three tested variants. This study demonstrated that nicotine treatment had dose-dependent effects on the level of DNA damage. Generally, a high dose of nicotine (100 microM) is genotoxic, while a reasonably low concentration has a protective effect. The possible participation of reactive oxygen species in the DNA-damaging potential of nicotine in C. elegans is discussed.