Methods of staining and visualization of sphingolipid enriched and non-enriched plasma membrane regions of Arabidopsis thaliana with fluorescent dyes and lipid analogues

ABSTRACT: BACKGROUND: Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. RESULTS: Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan. CONCLUSION: Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested.     

Differences in the activity and bacterial community structure of drained grassland and forest peat soils

The microbial activity and bacterial community structure were investigated in two types of peat soil in a temperate marsh. The first, a drained grassland fen soil, has a neutral pH with partially degraded peat in the upper oxic soil horizons (16% soil organic carbon). The second, a bog soil, was sampled in a swampy forest and has a very high soil organic carbon content (45%), a low pH (4.5), and has occasional anoxic conditions in the upper soil horizons due to the high water table level. The microbial activity in the two soils was measured as the basal and substrate-induced respiration (SIR). Unexpectedly, the SIR (μl CO₂ g⁻¹ dry soil) was higher in the bog than in the fen soil, but lower when CO₂ production was expressed per volume of soil. This may be explained by the notable difference in the bulk densities of the two soils. The bacterial communities were assessed by terminal restriction fragment length polymorphism (T-RFLP) profiling of 16S rRNA genes and indicated differences between the two soils. The differences were determined by the soil characteristics rather than the season in which the soil was sampled. The 16S rRNA gene libraries, constructed from the two soils, revealed high proportions of sequences assigned to the Acidobacteria phylum. Each library contained a distinct set of phylogenetic subgroups of this important group of bacteria.     

Hydrocarbons in Seawater and Water Extract of Jarzouna-Bizerte Coastal of Tunisia (Mediterranean Sea): Petroleum Origin Investigation Around Refinery Rejection Place

In this study, the occurrence of toxic heavy metals (As, Cd, Cr, Cu, Pb, and Zn) and relative bioaccumulation in biota samples were investigated in a freshwater ecosystem, the Basento river, one of the main aquatic systems in the south of Italy, which over the last years has been transformed into a sink of urban and industrial wastes. Therefore, the levels of arsenic, cadmium, chromium, copper, lead, and zinc were determined in water, sediments, and tissues of some macroinvertebrate—which are natural assessment endpoints for the evaluation of ecological risk in aquatic systems. Accumulation factors, as a ratio between the concentration of a given contaminant in biota and the one in an abiotic medium, were considered in order to estimate heavy metal contamination loads in biota. Statistical analysis was performed for a comparative evaluation of bioaccumulation among various macroinvertebrates, according to different feeding guilds. The Tukey honestly significantly different test showed significant differences in the bioaccumulation of As, Cd, and Cr among the considered biological receptors (collector–gatherer, predator, and filterer), suggesting that the biological uptake from immediate contact with the sediment or solid substratum (collector–gatherer), instead of the bioconcentration from water (filterer) or biomagnification along the biotic food webs (predators), is the more effective biological sequestering pathway for these metals. Biota–sediment accumulation factors, commonly used for the evaluation of sediment’s role in aquatic systems contamination, were determined for the considered metals. A linear correlation between the concentrations of As, Cd, Cr, and Zn in macroinvertebrates and those in the sediments suggested that the metal uptake data in macroinvertebrates can provide useful information for the estimation of heavy metal exposure risk or bioavailability when making assessments of sediment toxicity in freshwater ecosystems.     

Meta-analysis of the effects of endoparasites on pig performance

A meta-analysis was carried out in order to study the effects of endoparasites on the performance of growing pigs. Criteria that should be considered for the publication selection were: (1) the health challenge caused by parasites; (2) pig in growing phase; (3) presentation of the nutritional composition of the diets and (4) animal performance. Meta-analysis followed three sequential analysis: graphical, correlation and variance-covariance. The group that were infected with parasites had an average daily feed intake 5% lower than that the control group (2044 vs. 2147 g d(-1); P<0.001), their average daily weight gain was also 31% lower (665 vs. 987 g d(-1); P<0.001) and their feed conversion ratio was 17% superior than that of the control group (3.07 vs. 2.62; P<0.001). The variance decomposition demonstrated that 59% of the reduction in weight gain was explained by the reduction in their feed intake, as well as a 6% reduction being due to parasites.     

Potential of Exopolysaccharide from Porphyridium marinum to Contend with Bacterial Proliferation,Biofilm Formation,and Breast Cancer

Exopolysaccharide (EPS) from marine microalgae are promising sources of a new generation of drugs. However, lot of them remain to be discovered and tested. In this study, EPS produced by Porphyridium marinum and its oligomers prepared by High Pressure Homogenizer have been tested for different biological activities, i.e., antibacterial, anti-fungal and antibiofilm activities on Candida albicans, as well as for their effects on the viability of murine breast cancer cells. Results have shown that all EPS samples present some biological activity. For antibacterial and antibiofilm activities, the native EPS exhibited a better efficiency with Minimum Inhibitory Concentration (MIC) from 62.5 µg/mL to 1000 µg/mL depending on the bacterial strain. For Candida albicans, the biofilm formation was reduced by about 90% by using only a 31.3 µg/mL concentration. Concerning breast cancer cells, lower molar masses fractions appeared to be more efficient, with a reduction of viability of up to 55%. Finally, analyses of polymers composition and viscosity measurements were conducted on all samples, in order to propose hypotheses involving the activities caused by the intrinsic properties of polymers.     

The Static Fertilization Experiment at the Start and the End of the 20th Century

Changes of yields, nitrogen uptake on the zero plots and nitrogen balances are discussed in the course of the decades at the Static Fertilization Experiment exemplary. 1991-2000 yields of cereals on the fertilized plots were nearly 200% in comparison to 1906-1915. At potatoes and sugar beets increase of yields was smaller. In the middle of all crops 52kg/ha nitrogen were taken up on the unfertilized plots in the years 1959-2000. Since there is a balance in the soil this nitrogen can only derived from the air. On the PK plots the atmogenic nitrogen input is 6kg/ha still higher. Crops with a long vegetation period like winter wheat and sugar beets take up more atmogenic nitrogen than such with a shorter vegetation period like summer barley and potatoes. The atmogenic nitrogen input per day of vegetation period is between 0,24 (winter wheat) and 0,38 (sugar beet)kg/ha and day. Nitrogen balances of the more important variants show that nitrogen uptake is higher than nitrogen input for a long time. In comparison of the decades 1961-1970 and 1991-2000 the best effect is in the case of mineral fertilization alone, also at increasing nitrogen input at the end of the century. The reason is the optimal control of nitrogen amount and time of application. In contrast organic fertilization and nitrogen release from the organic matter you can't control with the result of more unfavourable balances.     

Novel application of square-wave adsorptive-stripping voltammetry for the determination of xanthohumol in spent hops

This paper reports the development of a novel electrochemical assay for xanthohumol (XN) by square-wave adsorptive-stripping voltammetry (SWAdSV) with a hanging mercury drop electrode. The method showed good repeatability (CV < 2%) and linearity (between 10 and 250 μg L(-1)), as well as suitable limits of detection (2.6 μg L(-1)) and quantification (8.8 μg L(-1)). The method was applied for the quantification of this compound in spent hops, and the results obtained were compared with the HPLC-UV method. XN contents determined by the SWAdSV method were 16 ± 1 and 100 ± 4 μg L(-1) for aqueous and methanolic extracts, respectively. The developed new methodology considerably reduces the analysis time, approximately from 25 min (HPLC-UV method) to 7 min, enabling a high sample throughput. In addition, the detection and quantification limits were approximately 5-fold lower than those obtained with the chromatographic method.     

Characterization of the LH peak after short and long fixed‐time artificial insemination protocols in sheep raised in the tropics

The aim of this study was to evaluate the peak in luteinizing hormone (LH) and the pregnancy rate of sheep (Texel × Santa Inês) in the tropics using short‐ (6 days) and long‐term (12 days) progesterone protocols followed by artificial insemination (AI) both in and out of the breeding season. Experiment 1 was conducted within (IN) the breeding season (autumn, n = 36), and experiment 2 was conducted outside (OUT) of the breeding season (spring, n = 43). In each experiment, the sheep were divided into two groups (6 or 12 days) according to the duration of treatment with a single‐use progesterone release vaginal device (CIDR^®, Pfizer, São Paulo, SP, Brazil), and blood samples were collected from 10 animals per group every 4 hr to measure the LH and progesterone concentrations. In the spring, the characteristics of the LH peak did not differ between groups; but in the autumn, there were differences between groups at the beginning (G‐6 IN: 36.44 ± 5.46 hr; G‐12 IN: 26.57 ± 4.99 hr) and end of the LH peak (G‐6 IN: 46.22 ± 7.51 hr; G‐12 IN: 34.86 ± 8.86 hr). The results showed alterations in the LH peak during the breeding season only in the sheep undergoing the short‐term protocol.     

Broodstock spawning and larviculture of whiting (Merlangius merlangus L.) reared in captivity

In recent years, an increasing interest in the farming of marine ‘white’ fish, such as cod, has been observed in the aquaculture industry. One species being considered for aquaculture development is whiting (Merlangius merlangus L.). With little data being available on the domestication of this species, our study aimed to characterize spawning behaviour and rhythmicity in captive broodstock and provide original data on egg incubation, embryonic development and larval rearing. The results observed through video recording showed that spawning mainly occurred at night with active courtship behaviour. Daily egg batches were collected from two separate spawning seasons, with an average fertilization success declining from 77% to 53%. Time to hatch was inversely related to the incubation temperature and occurred after 78 degree‐days (9 days at 8 °C), with emerging larvae ranging in size from 3.3 to 3.46 mm total length and hatch success ranging from 48.3% to 99.9%. In comparison with a clear water environment, a green water regime during the first days of larval rearing significantly improved larval growth and survival. These results are the first to describe spawning activity, early development and larval performances in whiting and are promising for the potential future development of whiting aquaculture.