Expression of exogenous fluorescent proteins in early freshwater pond snail embryos

We have for the first time succeeded in expressing in vitro-synthesized mRNAs in both the sinistral and the dextral Lymnaea stagnalis early embryos by microinjecting the mRNAs into the eggs before the first polar body stage. Translation of exogenous mRNA in developing embryos was confirmed by expressing various fluorescent proteins; mCherry, DsRed-Express, and enhanced green fluorescent protein. We have found that the protein expression derived from the introduced exogenous mRNA largely depends on the elapsed time after the microinjection and not on the developmental stage of injection, and also on the amount of injected mRNA. Developmental abnormalities were hardly observed. The first notable fluorescent signal was detected within 2–3 h after the injection while the embryos were still in uncleaved stage. Fluorescence gradually increased until 8–9 h and was stable up to 24 h. From these results, it is suggested that there is enough translation machinery necessary for early development and the translation of injected mRNA proceeds immediately and constantly in the early embryos. This is true for both the sinistral and dextral L. stagnalis embryos. Application of the developed method to other freshwater pond snails, dextral Lymnaea peregra, sinistral Physa acuta, and sinistral Indoplanorbis exustus revealed that their early expression mechanisms to be similar to that of L. stagnalis. Thus, in vitro-synthesized mRNA expression is expected to be important for the understanding of evolutional process and the molecular mechanism underlining the handedness determination in these freshwater snail embryos.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Role of two contrasting ecosystem engineers (<Emphasis Type="Italic">Zostera noltii</Emphasis> and <Emphasis Type="Italic">Cymodocea nodosa</Emphasis>) on the food intake rate of <Emphasis Type="Italic">Cerastoderma edule</Emphasis>

Seagrasses are well known ecosystem engineers that can significantly influence local hydrodynamics and the abundance and biodiversity of macrobenthic organisms. This study focuses on the potential role of the seagrass canopy structure in altering the abundance of filter-feeding organisms by modifying the hydrodynamic driven food supply. We quantified the effect of two ecosystem engineers with contrasting canopy properties (i.e. Zostera noltii and Cymodocea nodosa) on the food intake rate of a suspension-feeding bivalve Cerastoderma edule living in these seagrass meadows. Field experiments were carried out in two seagrass beds (Z. noltii and C. nodosa) and bare sediment, located on sandflat characterised by a relatively high hydrodynamic energy from waves and currents. Results demonstrated that the filter-feeding rate was almost twofold increased when C. edule was inhabiting Z. noltii meadows (1.10 ± 0.24 μg Chl g Fresh Weight⁻¹) when compared to cockles living on the bare sediment (0.65 ± 0.14 μg Chl g FW⁻¹). Intermediate values were found within C. nodosa canopy (0.97 ± 0.24 μg Chl g FW⁻¹), but filter feeding rate showed no significant differences with values for Z. noltii meadows. There were no apparent correlations between canopy properties and filter-feeding rates. Our results imply that food refreshment within the seagrass canopies was enough to avoid food depletion. We therefore expect that the ameliorated environmental conditions within vegetated areas (i.e. lower hydrodynamic conditions, higher sediment stability, lower predation pressure…) in combination with sufficient food supply to prevent depletion within both canopies are the main factors underlying our observations.     

Fluorescence changes accompanying short-term light adaptations in photosystem I and photosystem II of the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 and phycobiliprotein-impaired mutants: State 1/State 2 transitions and carotenoid-induced quenching of phycobilisomes

The features of the two types of short-term light-adaptations of photosynthetic apparatus, State 1/State 2 transitions, and non-photochemical fluorescence quenching of phycobilisomes (PBS) by orange carotene-protein (OCP) were compared in the cyanobacterium Synechocystis sp. PCC 6803 wild type, CK pigment mutant lacking phycocyanin, and PAL mutant totally devoid of phycobiliproteins. The permanent presence of PBS-specific peaks in the in situ action spectra of photosystem I (PSI) and photosystem II (PSII), as well as in the 77 K fluorescence excitation spectra for chlorophyll emission at 690 nm (PSII) and 725 nm (PSI) showed that PBS are constitutive antenna complexes of both photosystems. The mutant strains compensated the lack of phycobiliproteins by higher PSII content and by intensification of photosynthetic linear electron transfer. The detectable changes of energy migration from PBS to the PSI and PSII in the Synechocystis wild type and the CK mutant in State 1 and State 2 according to the fluorescence excitation spectra measurements were not registered. The constant level of fluorescence emission of PSI during State 1/State 2 transitions and simultaneous increase of chlorophyll fluorescence emission of PSII in State 1 in Synechocystis PAL mutant allowed to propose that spillover is an unlikely mechanism of state transitions. Blue–green light absorbed by OCP diminished the rout of energy from PBS to PSI while energy migration from PBS to PSII was less influenced. Therefore, the main role of OCP-induced quenching of PBS is the limitation of PSI activity and cyclic electron transport under relatively high light conditions.     

TEP on the tide in killifish (<Emphasis Type="Italic">Fundulus heteroclitus</Emphasis>): effects of progressively changing salinity and prior acclimation to intermediate or cycling salinity

Transepithelial potentials (TEP) were measured in killifish, acclimated to freshwater (FW), seawater (SW), 33% SW or cycling salinities relevant to tidal cycles in an estuary, and subsequently subjected to salinity changes in progressive or random order. Random compared to progressive salinity changes in an upward or downward direction in FW- and SW-acclimated fish, respectively, did not greatly influence responses to salinity change. Fish acclimated to SW or 33% SW as well as those acclimated to cycling salinities behaved similarly (TEP more positive than +15 mV in 100% SW, decreasing to ~0 mV at 20–40% SW, and more negative than −30 mV in FW). In contrast, FW-acclimated fish displayed a less pronounced TEP response to salinity (0 mV in FW through 20% SW, increasing thereafter to values more positive than +10 mV at 100% SW). We conclude that when evaluated under estuarine tidal conditions, the killifish gill exhibits adaptive electrical characteristics, opposing Na⁺ loss at low salinity and favouring Na⁺ extrusion at high salinity, changes explained at least in part by the Cl⁻ to Na⁺ permeability ratio. Thus animals living in the estuaries can move to lower and higher salinities for short periods with little physiological disturbance, but this ability is lost after acclimation to FW.     

Genetics of psychosis; insights from views across the genome

The major psychotic illnesses, schizophrenia and bipolar disorder (BD), are among the most heritable common disorders, but finding specific susceptibility genes for them has not been straightforward. The reasons are widely assumed to include lack of valid phenotypic definition, absence of good theories of pathophysiology for candidate gene studies, and the involvement of many genes, each making small contributions to population risk. Within the last year or so, a number of genome wide association (GWAS) of schizophrenia and BD have been published. These have produced stronger evidence for association to specific risk loci than have earlier studies, specifically for the zinc finger binding protein 804A (ZNF804A) locus in schizophrenia and for the calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNA1C) and ankyrin 3, node of Ranvier (ANK3) loci in bipolar disorder. The ZNF804A and CACNA1C loci appear to influence risk for both disorders, a finding that supports the hypothesis that schizophrenia and BD are not aetiologically distinct. In the case of schizophrenia, a number of rare copy number variants have also been detected that have fairly large effect sizes on disease risk, and that additionally influence risk of autism, mental retardation, and other neurodevelopmental disorders. The existing findings point to some likely pathophysiological mechanisms but also challenge current concepts of disease classification. They also provide grounds for optimism that larger studies will reveal more about the origins of these disorders, although currently, very little of the genetic risk of either disorder is explained.     

Mesozoic tectono-sedimentary evolution of Rocca Busambra in western Sicily

The Rocca Busambra ridge in western Sicily is a shallow to pelagic Meso-Cenozoic carbonate structural unit of the Sicilian Chain with a variety of tectono-sedimentary features. Palaeofaults, unconformities (buttress unconformity, onlap, downlap), a network of neptunian dykes with several infilling generations, several large hiatuses, different facies and lateral facies changes, and erosional submarine and subaerial surfaces are observed. Detailed fieldwork and structural analyses have indicated the occurrence of fault planes with different orientations. These data, combined with facies studies and physical-stratigraphy analyses, allow for the distinction of different depositional regions. A lateral change from an open-marine carbonate platform with a stepped fault margin (located in the westernmost sector) to a deeper basinal depositional setting in the east, in the context of an upper slope scalloped margin and base-of-slope systems with talus breccias, is envisaged here. Extensional to transtensional tectonic pulses punctuated the sedimentary evolution during Early Toarcian, Late Jurassic, Early Cretaceous, Late Cretaceous, and Early Miocene times. The collected data show that most fault planes have preserved their original orientations throughout the reactivation processes. The reconstructed Meso-Cenozoic tectono-sedimentary evolution is closely related to the late syn-rift and post-rift tectonic evolution of the Tethyan continental margin.     

Non-Invasive Separation of Alcoholic and Non-Alcoholic Liver Disease with Predictive Modeling

Background & Objective

Currently, a major clinical challenge is to distinguish between chronic liver disease caused by metabolic syndrome (non-alcoholic fatty liver disease, NAFLD) from that caused by long term or excessive alcohol consumption (ALD). The etiology of severe liver disease affects treatment options and priorities for liver transplantation and organ allocation. Thus we compared physiologically similar NAFLD and ALD patients to detect biochemical differences for improved separation of these mechanistically overlapping etiologies.

Methods

In a cohort of 31 NAFLD patients with BMI below 30 and a cohort of ALD patient with (ALDC n = 51) or without cirrhosis (ALDNC n = 51) serum transaminases, cell death markers and (adipo-)cytokines were assessed. Groups were compared with One-way ANOVA and Tukey''s correction. Predictive models were built by machine learning techniques.

Results

NAFLD, ALDNC or ALDC patients did not differ in demographic parameters. The ratio of alanine aminotransferase/aspartate aminotransferase - common serum parameters for liver damage - was significantly higher in the NAFLD group compared to both ALD groups (each p<0.0001). Adiponectin and tumor necrosis factor(TNF)-alpha were significantly lower in NAFLD than in ALDNC (p<0.05) or ALDC patients (p<0.0001). Significantly higher serum concentrations of cell death markers, hyaluronic acid, adiponectin, and TNF-alpha (each p<0.0001) were found in ALDC compared to ALDNC. Using machine learning techniques we were able to discern NAFLD and ALDNC (up to an AUC of 0.9118±0.0056) or ALDC and ALDNC (up to an AUC of 0.9846±0.0018), respectively.

Conclusions

Machine learning techniques relying on ALT/AST ratio, adipokines and cytokines distinguish NAFLD and ALD. In addition, severity of ALD may be non-invasively diagnosed via serum cytokine concentrations.     

Molecular identification and dynamics of microbial communities in reactor treating organic household waste

The prokaryotic diversity associated with organic household waste (OHW), leachate (start-up inoculum), and mesophilic anaerobic digestion processes in the degradation of OHW for 44 and 90 days was investigated using a culture-independent approach. Bacterial and archaeal 16S rRNA and mcrA gene clone libraries were constructed from community DNA preparations. Bacterial clones were affiliated with 13 phyla, of which Firmicutes, Proteobacteria, and Bacteroidetes were represented in all libraries, whereas Actinobacteria, Thermotogae, Lentisphaerae, Acidobacteria, Chloroflexi, Cyanobacteria, Synergistetes, Spirochaetes, Deferribacteres, and Deinococcus-Thermus were exclusively identified in a single library. Within the Archaea domain, the Euryarchaeota phylum was the only one represented. Corresponding sequences were associated with the following orders of hydrogenotrophic methanogens: Methanomicrobiales (Methanoculleus genus) and Methanobacteriales (Methanosphaera and Methanobacterium genera). One archaeal clone was not affiliated with any order and may represent a novel taxon. Diversity indices showed greater diversity of Bacteria when compared to methanogenic Archaea.