Life history patterns of parental and hybrid Daphnia differ between lakes

1. We investigated whether Daphnia galeata × hyalina hybrids of Lake Constance and Lake Greifensee show the same pattern of life history parameters as previously reported for D. galeata × cucullata hybrids and whether such a pattern is consistent between Daphnia populations from those two lakes. 2. Hybrids in Lake Constance were intermediate in size compared with the parental species. Hybrids in Lake Greifensee were smaller than D. galeata. The intrinsic growth rate (r) of hybrids from Lake Constance was not significantly different from the faster growing parental taxon D. galeata. However, r of hybrids from Lake Greifensee was significantly lower than that of D. galeata. 3. The observed juvenile body length differences between the taxa varied with the clutch number. The first clutch juvenile lengths of the three taxa did not differ for Lake Constance. First clutch juveniles of Lake Greifensee D. galeata were smaller than hybrid first clutch juveniles. The third clutch juvenile length did not differ between taxa from Lake Greifensee, but D. galeata juveniles from Lake Constance were bigger than those of D. hyalina. 4. The life history pattern found in Lake Constance corresponds to previous findings from other studies. The hybrids in this lake combine the faster population growth of one parental species with a relatively small size. In the case of Lake Greifensee hybrids, the relatively large size of first clutch juveniles and the small size of the adults could be interpreted as dual adaptations to invertebrate and fish predation. We speculate that the lower population growth rate of the hybrids is a trade‐off for this twofold protection.     

Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients

Background

Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation.

Methods

Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene.

Results

The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38–76 %, depending on the gene. The highest MI value was found for RASSF1A (52 %) and the lowest for NPRL2/G21 (5 %). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71 % tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = −0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75–92 % NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found.

Conclusions

The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn’t seem to be a critical determinate of its promoter hypermethylation.     

The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation

Plant cell suspension cultures represent good model systems applicable for both basic research and biotechnological purposes. Nevertheless, it is widely known that a prolonged in vitro cultivation of plant cells is associated with genetic and epigenetic instabilities, which may limit the usefulness of plant lines. In this study, the age-dependent epigenetic and physiological changes in an asynchronous Arabidopsis T87 cell culture were examined. A prolonged cultivation period was found to be correlated with a decrease in the proliferation rate and a simultaneous increase in the expression of senescence-associated genes, indicating that the aging process started at the late growth phase of the culture. In addition, increases in the heterochromatin-specific epigenetic markers, i.e., global DNA methylation, H3K9 dimethylation, and H3K27 trimethylation, were observed, suggesting the onset of chromatin condensation, a hallmark of the early stages of plant senescence. Although the number of live cells decreased with an increase in the age of the culture, the remaining viable cells retained a high potential to efficiently perform photosynthesis and did not exhibit any symptoms of photosystem II damage.     

Molecular Identification and Hidden Diversity of Novel Daphnia Parasites from European Lakes

Parasites play important roles in local population dynamics and genetic structure. However, due to insufficient diagnostic tools, detailed host-parasite interactions may remain concealed by hidden parasite diversity in natural systems. Microscopic examination of 19 European lake Daphnia populations revealed the presence of three groups of parasites: fungi, microsporidia, and oomycetes. For most of these parasites no genetic markers have been described so far. Based on sequence similarities of the nuclear small-subunit and internal transcribed spacer (ITS) rRNA gene regions, one fungus, four microsporidian, and nine oomycete taxa were discovered in 147 infected Daphnia (and/or three other zooplankton crustaceans). Additionally, cloning of rRNA gene regions revealed parasite sequence variation within host individuals. This was most pronounced in the ITS region of one microsporidian taxon, where the within-host sequence variation ranged from 1.7% to 5.3% polymorphic sites for parasite isolates from 14 different geographical locations. Interestingly, the parasite isolates from close locations grouped together based on sequence similarities, suggesting that there was parasite dispersal. Taken together, the data obtained in this study revealed hidden diversity of parasite communities in Daphnia lake populations. Moreover, a higher level of resolution for identifying parasite strains makes it possible to test new hypotheses with respect to parasite dispersal, transmission routes, and coinfection.During the last decade, microparasites of Daphnia species, which are small zooplankton crustaceans, have become a popular study system in ecological and evolutionary research (for a review, see reference 15). It has been shown both in the field and under controlled laboratory conditions that parasites have a substantial impact on Daphnia fitness (7, 21, 52). Parasite-induced reductions in Daphnia population density (11, 12) or even population crashes (17) might result in disruptions of aquatic food webs, as daphnids play important roles as main phytoplankton grazers and as a major food of planktivorous fish (27). Moreover, as infections are often genotype specific (6, 8), they can lead to changes in the gene pool of a Daphnia population (7, 14), sometimes significantly increasing the genetic diversity of the host population (12, 54). Thus, Daphnia parasites cause not only ecological but also evolutionary changes in aquatic systems.Conclusions regarding the importance of parasites in natural systems require powerful tools to detect and properly identify parasite taxa. Thus far, few species-specific molecular markers have been developed for Daphnia parasites (33, 38, 39, 41) and then used in experimental studies (3). In surveys of natural Daphnia populations, parasite identification has been based primarily on microscopic examination (4, 5, 29, 52), with only one exception (32). The parasites recorded in natural populations of Daphnia are thus considered members of certain taxa, or even species, without genetic confirmation. The fact that molecular markers are not used to characterize Daphnia infections makes it difficult to compare epidemic patterns across different habitats and/or various field surveys, as parasites cannot be unambiguously identified by microscopic examination alone. Even if microscopic identification is theoretically possible (for example, by examining ultrastructural morphology by electron microscopy [37]), this approach is not feasible for routine analysis. Consequently, classification of parasites that actually belong to different taxa in the same group might introduce noise into field surveys, as parasite taxa differ widely in virulence and host range (for a review, see reference 15).Most of the known Daphnia parasites that have been described were obtained from small temporary ponds and rock pools (4, 16, 43). In permanent lakes, lower parasite diversity was assumed, mainly because increased fish predation reduces the population density of potential hosts (18), whereas high host density is a crucial determinant of epidemic spread (1, 2, 45). In addition, infected Daphnia spp. are more vulnerable to fish that hunt visually due to loss of their transparent appearance (11, 13). On the other hand, it was recently shown that even if Daphnia host density was reduced by selective fish predation, the prevalence of infection did not decline, probably due to the very high rates of transmission of the parasite that was observed (11). In contrast, we expected that the highly heterogeneous biotic and abiotic conditions in permanent lakes (27) would provide a variety of niches (for a review, see reference 47), which also favor a high level of parasite diversity. Therefore, Czech canyon-shaped reservoirs were chosen as our main study systems, because in these lakes environmental gradients are particularly pronounced in both the horizontal and vertical dimensions (42). Moreover, the Daphnia communities of these reservoirs are dominated by members of the Daphnia longispina complex (35), taxa which have previously been shown to be infected by a variety of parasites (52).The results of our study revealed a high level of diversity of Daphnia parasites in permanent lakes. Fourteen different parasite taxa were detected using nuclear small-subunit (SSU) and internal transcribed spacer (ITS) rRNA gene sequence information. In addition, a high level of sequence variation was observed in the ITS region of one microsporidian taxon. Thus, molecular markers are now available which allow discrimination with high resolution among and within parasite taxa and provide tools to address more detailed questions concerning lake Daphnia-microparasite systems.     

Cleavage of Zearalenone by Trichosporon mycotoxinivorans to a Novel Nonestrogenic Metabolite

Zearalenone (ZON) is a potent estrogenic mycotoxin produced by several Fusarium species most frequently on maize and therefore can be found in food and animal feed. Since animal production performance is negatively affected by the presence of ZON, its detoxification in contaminated plant material or by-products of bioethanol production would be advantageous. Microbial biotransformation into nontoxic metabolites is one promising approach. In this study the main transformation product of ZON formed by the yeast Trichosporon mycotoxinivorans was identified and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-diode array detector (DAD) analysis. The metabolite, named ZOM-1, was purified, and its molecular formula, C₁₈H₂₄O₇, was established by time of flight MS (TOF MS) from the ions observed at m/z 351.1445 [M-H]⁻ and at m/z 375.1416 [M+Na]⁺. Employing nuclear magnetic resonance (NMR) spectroscopy, the novel ZON metabolite was finally identified as (5S)-5-({2,4-dihydroxy-6-[(1E)-5-hydroxypent-1-en-1-yl]benzoyl}oxy)hexanoic acid. The structure of ZOM-1 is characterized by an opening of the macrocyclic ring of ZON at the ketone group at C6′. ZOM-1 did not show estrogenic activity in a sensitive yeast bioassay, even at a concentration 1,000-fold higher than that of ZON and did not interact with the human estrogen receptor in an in vitro competitive binding assay.Zearalenone (ZON) is the main member of a growing family of biologically important “resorcylic acid lactones” (RALs), which have been found in nature. ZON is produced by several Fusarium species, which colonize maize, barley, oat, wheat, and sorghum and tend to develop ZON during prolonged cool, wet growing and harvest seasons (38). Maize is the most frequently contaminated crop plant, and therefore, ZON can be found frequently in animal feeding stuff. Occurrence, toxicity, and metabolism data of ZON were summarized by the European Food Safety Authority (EFSA) (5) and in recent reviews (12, 38).The potent xenohormone ZON leads to hyperestrogenism symptoms and in extreme cases to infertility problems, especially in pigs (15). Ovarian changes in pigs have been noted with toxin levels as low as of 50 μg/kg in the diet (1). Ruminants are more tolerant to ZON ingestion; however, hyperestrogenic syndrome, including restlessness, diarrhea, infertility, decreased milk yields, and abortion, have been well documented with cattle and sheep (4, 29).Because widespread ZON contamination in feed can occur in problematic years, efficient ways to detoxify are desirable. The transformation of mycotoxins to nontoxic metabolites by pure cultures of microorganisms or by cell-free enzyme preparations (3) is an attractive possibility. Microbial metabolization of ZON to alpha-ZOL and beta-ZOL cannot be regarded as detoxification, because both ZOL products are still estrogenic (14). Also, formation of ZON-glucosides and -diglucosides (8, 17) and ZON-sulfate (7) cannot be considered true detoxification but rather formation of masked mycotoxins, because the conjugates may be hydrolyzed during digestion (11, 23), releasing ZON again (2).As the estrogenic activity of ZON and its derivates can be explained by its chemical structure, which resembles natural estrogens (20), it can be expected that cleavage of the lactone undecyl ring system of ZON results in permanent detoxification.El-Sharkawy and Abul-Hajj (9) were the first to report inactivation of ZON after opening of the lactone ring by Gliocladium roseum. This filamentous fungus was capable of metabolizing ZON in yields of 80 to 90%. Also Takahashi-Ando et al. (31) described the degradation reaction of ZON with Clonostachys rosea (synonym of G. roseum). A hydrolase (encoded by a gene designated ZHD101) cleaves the lactone ring, and as recently proved (37; unpublished data) by subsequent decarboxylation of the intermediate acid, the compound 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1′E-undecene-6′-one is formed. In contrast to ZON and 17β-estradiol, which showed potent estrogenic activity, this cleavage product did not show any estrogenic activity in the human breast cancer MCF-7 cell proliferation assay (16). Further details, e.g., on the conditions of the maximum activity of ZHD101 and its exploitation in genetically modified grains, can be found in later published work of this research group (32, 33).Only a few authors reported the loss of estrogenicity in microbial metabolites of ZON, which are based on reactions other than cleavage of the lactone undecyl ring system. El-Sharkawy and Abul-Hajj demonstrated (10) that binding to rat uterine estrogen receptors requires a free 4-OH phenolic group (devoid of methylation or glycosylation). Loss of estrogenicity was, for instance, observed with 2,4-dimethoxy-ZON, one of the metabolites produced by Cunninghamella bainieri ATCC 9244B. Nevertheless, this rule cannot be generalized, as 8′-hydroxyzearalenone formed by Streptomyces rimosus NRRL 2234, despite having a free 4-phenolic hydroxyl group, did not bind to the estrogen receptor. Also, other authors reported that 8′-hydroxyzearalenone and 8′-epi-hydroxyzearalenone are nonestrogenic (13). However, so far, no practical application in feed or food detoxification has been found for the microorganisms producing these compounds.It has been shown previously that the yeast Trichosporon mycotoxinivorans has a very high capability to degrade both ochratoxin A (OTA) and ZON (22, 26, 27). When T. mycotoxinivorans is used as a feed additive preparation, microbial degradation of the mycotoxins is assumed to take place in the gastrointestinal tract of the animal after consumption of contaminated feed. The protective effect of T. mycotoxinivorans against OTA toxicity has already been shown with broiler chicken (24).In the present study we report the isolation, analytical characterization, and structure elucidation, as well as the evaluation, of the estrogenic activity of the main degradation product of ZON produced by T. mycotoxinivorans.     

Functions of Cholesterol and the Cholesterol Bilayer Domain Specific to the Fiber-Cell Plasma Membrane of the Eye Lens

The most unique feature of the eye lens fiber-cell plasma membrane is its extremely high cholesterol content. Cholesterol saturates the bulk phospholipid bilayer and induces formation of immiscible cholesterol bilayer domains (CBDs) within the membrane. Our results (based on EPR spin-labeling experiments with lens-lipid membranes), along with a literature search, have allowed us to identify the significant functions of cholesterol specific to the fiber-cell plasma membrane, which are manifest through cholesterol–membrane interactions. The crucial role is played by the CBD. The presence of the CBD ensures that the surrounding phospholipid bilayer is saturated with cholesterol. The saturating cholesterol content in fiber-cell membranes keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, the CBD helps to maintain lens-membrane homeostasis when the membrane phospholipid composition changes significantly. The CBD raises the barrier for oxygen transport across the fiber-cell membrane, which should help to maintain a low oxygen concentration in the lens interior. It is hypothesized that the appearance of the CBD in the fiber-cell membrane is controlled by the phospholipid composition of the membrane. Saturation with cholesterol smoothes the phospholipid-bilayer surface, which should decrease light scattering and help to maintain lens transparency. Other functions of cholesterol include formation of hydrophobic and rigidity barriers across the bulk phospholipid-cholesterol domain and formation of hydrophobic channels in the central region of the membrane for transport of small, nonpolar molecules parallel to the membrane surface. In this review, we provide data supporting these hypotheses.     

Synthesis,receptor binding studies,optical spectroscopic and in silico structural characterization of morphiceptin analogs with cis‐4‐amino‐L‐proline residues

Three novel morphiceptin analogs, in which Pro in position 2 and/or 4 was replaced by cis‐4‐aminoproline connected with the preceding amino acid through the primary amino group, were synthesized. The opioid receptor affinities, functional assay results, enzymatic degradation studies and experimental and in silico structural analysis of such analogs are presented. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of MMP-9 inhibition by doxycycline on proteome of lungs in high tidal volume mechanical ventilation-induced acute lung injury

Background  

Although mechanical ventilation (MV) is a major supportive therapy for patients with acute respiratory distress syndrome, it may result in side effects including lung injury. In this study we hypothesize that MMP-9 inhibition by doxycycline might reduce MV-related lung damage. Using a proteomic approach we identified the pulmonary proteins altered in high volume ventilation-induced lung injury (VILI). Forty Wistar rats were randomized to an orally pretreated with doxycycline group (n = 20) or to a placebo group (n = 20) each of which was followed by instrumentation prior to either low or high tidal volume mechanical ventilation. Afterwards, animals were euthanized and lungs were harvested for subsequent analyses.