Effect of sediment elutriate on the growth of Gymnodinium sp. (Dinophyceae). I.

Abstract

Sediments from the Emilia Romagna coast, an area periodically subjected to eutrophication, were investigated to determine the organic carbon and nitrogen content from the solid phase and organic and inorganic phosphorus, nitrogen and N/P ratio from a liquid elutriate from these sediments. The elutriates were tested for growth using a bioassay with a Gymnodinium sp. which previously caused extensive blooms in the Adriatic. Cultures were grown in the elutriates either enriched or unenriched with F/2 medium. Comparisons with controls were made on the basis of the maximum standing stock. The results obtained with the unenriched elutriates demonstrated the primary role of phosphate, while enriched elutriates produced slighter enhancement than the control. In most cases, the chemical composition of the elutriate was characterized by a negligible phosphate concentration, with respect to ammonium: the nutrient balance did not agree with the nutritional algal requirement. However, all the elutriates showed large quantities of inorganic and organic nitrogen. This finding could indicate that sediment-bound nitrogen may be a ready source of nitrogen for algal growth.     

The Frequency of Granulocytes with Spontaneous Somatic Mutations: A Wide Distribution in a Normal Human Population

Germ-line mutation rate has been regarded classically as a fundamental biological parameter, as it affects the prevalence of genetic disorders and the rate of evolution. Somatic mutation rate is also an important biological parameter, as it may influence the development and/or the course of acquired diseases, particularly of cancer. Estimates of this parameter have been previously obtained in few instances from dermal fibroblasts and lymphoblastoid cells. However, the methodology required has been laborious and did not lend itself to the analysis of large numbers of samples. We have previously shown that the X-linked gene PIG-A, since its product is required for glycosyl-phosphatidylinositol-anchored proteins to become surface bound, is a good sentinel gene for studying somatic mutations. We now show that by this approach we can accurately measure the proportion of PIG-A mutant peripheral blood granulocytes, which we call mutant frequency, ƒ. We found that the results are reproducible, with a variation coefficient (CV) of 45%. Repeat samples from 32 subjects also had a CV of 44%, indicating that ƒ is a relatively stable individual characteristic. From a study of 142 normal subjects we found that log ƒ is a normally distributed variable; ƒ variability spans a 80-fold range, from less than 1×10⁻⁶ to 37.5×10⁻⁶, with a median of 4.9×10⁻⁶. Unlike other techniques commonly employed in population studies, such as comet assay, this method can detect any kind of mutation, including point mutation, as long as it causes functional inactivation of PIG-A gene. Since the test is rapid and requires only a small sample of peripheral blood, this methodology will lend itself to investigating genetic factors that underlie the variation in the somatic mutation rate, as well as environmental factors that may affect it. It will be also possible to test whether ƒ is a determinant of the risk of cancer.     

Influence of temperature, salinity and nutrient limitation on yessotoxin production and release by the dinoflagellate Protoceratium reticulatum in batch-cultures

The toxic dinoflagellate Protoceratium reticulatum (Claparède & Lachmann) Buetschli is recurrently present in the Adriatic sea. It is the producing organism of yessotoxin (YTX) and some of its analogues and thus its presence in seawater often results in shellfish farm closure for long periods. However, molluscs become highly toxic also at the presence of low cell concentrations, due to the high YTX content present in most algal strains. As no data were available on the environmental conditions favouring growth and YTX production by Adriatic P. reticulatum strains, in the present work, we investigated the effect of nutrient limitation, salinity and temperature on growth and YTX content in P. reticulatum cultures. Liquid chromatography–mass spectrometry (LC–MS) analyses were carried out to determine YTX production as well as the difference between the YTX amount retained in cells and that released in growth medium, in order to relate cell content to excretion mechanisms. The toxin content was determined in cells collected at the stationary phase, since both toxin production and release were found to be higher in this growth stage than in the exponential phase. As for nutrient-effect, a severe P-limitation strongly affected cell growth and favoured toxin accumulation, as consequences of both impaired cell division and lower toxin release. N-limited cultures, on the contrary, had a toxin content similar to controls and the highest percentage of release. P. reticulatum was confirmed to be tolerant towards salinity changes as it could grow at salinity values in the range of 22–42. The highest YTX production was observed at intermediate salinity values (32) whereas toxin release, expressed as percentage of the total amount produced, decreased as salinity increased. P. reticulatum growth was impaired in cultures kept at 26 °C in respect to those grown at 16 and 20 °C. YTX release decreased as temperature increased; however, cells kept at 26 °C displayed a very high YTX content. The environmental implications of these physiological behaviours highlight that farmed molluscs can become less toxic in colder waters at lower salinity values.     

Relevance of Rheological Properties of Sodium Alginate in Solution to Calcium Alginate Gel Properties

The purpose of this study is to determine whether sodium alginate solutions’ rheological parameters are meaningful relative to sodium alginate’s use in the formulation of calcium alginate gels. Calcium alginate gels were prepared from six different grades of sodium alginate (FMC Biopolymer), one of which was available in ten batches. Cylindrical gel samples were prepared from each of the gels and subjected to compression to fracture on an Instron Universal Testing Machine, equipped with a 1-kN load cell, at a cross-head speed of 120 mm/min. Among the grades with similar % G, (grades 1, 3, and 4), there is a significant correlation between deformation work (L _E) and apparent viscosity (η _app). However, the results for the partial correlation analysis for all six grades of sodium alginate show that L _E is significantly correlated with % G, but not with the rheological properties of the sodium alginate solutions. Studies of the ten batches of one grade of sodium alginate show that η _app of their solutions did not correlate with L _E while tan δ was significantly, but minimally, correlated to L _E. These results suggest that other factors—polydispersity and the randomness of guluronic acid sequencing—are likely to influence the mechanical properties of the resultant gels. In summary, the rheological properties of solutions for different grades of sodium alginate are not indicative of the resultant gel properties. Inter-batch differences in the rheological behavior for one specific grade of sodium alginate were insufficient to predict the corresponding calcium alginate gel’s mechanical properties.     

Combined high-resolution genotyping and geospatial analysis reveals modes of endemic urban typhoid fever transmission

Typhoid is a systemic infection caused by Salmonella Typhi and Salmonella Paratyphi A, human-restricted bacteria that are transmitted faeco-orally. Salmonella Typhi and S. Paratyphi A are clonal, and their limited genetic diversity has precluded the identification of long-term transmission networks in areas with a high disease burden. To improve our understanding of typhoid transmission we have taken a novel approach, performing a longitudinal spatial case-control study for typhoid in Nepal, combining single-nucleotide polymorphism genotyping and case localization via global positioning. We show extensive clustering of typhoid occurring independent of population size and density. For the first time, we demonstrate an extensive range of genotypes existing within typhoid clusters, and even within individual households, including some resulting from clonal expansion. Furthermore, although the data provide evidence for direct human-to-human transmission, we demonstrate an overwhelming contribution of indirect transmission, potentially via contaminated water. Consistent with this, we detected S. Typhi and S. Paratyphi A in water supplies and found that typhoid was spatially associated with public water sources and low elevation. These findings have implications for typhoid-control strategies, and our innovative approach may be applied to other diseases caused by other monophyletic or emerging pathogens.     

Hybridization between two oil-secreting orchids in South Africa

Natural hybrids among the specialized terrestrial oil-secreting orchids of South Africa are extremely rare even where multiple closely related species co-occur. We found putative hybrids between Pterygodium catholicum Sw. and P. acutifolium Lindl., two closely related oil-secreting orchids that lack morphological barriers to inter-breeding. The purpose of this study was to confirm the parentage of the putative hybrids using molecular data from one nuclear (ITS) and two plastid (matK-trnK and trnL/F introns) DNA regions. Phylogenetic analyses of sequences as well as nucleotide substitution patterns of the putative hybrids, putative parents, and their closest relatives were consistent with a hypothesis of hybridization. We suggest that the hybrids were the result of visits to both orchids by a single species of oil-collecting bee during a brief period of flowering overlap. These results suggest that the rarity of hybrids between these orchids is due to factors other than genetic incompatibility.     

Measles virus glycoprotein-pseudotyped lentiviral vectors are highly superior to vesicular stomatitis virus G pseudotypes for genetic modification of monocyte-derived dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells capable of promoting or regulating innate and adaptive immune responses against non-self antigens. To better understand the DC biology or to use them for immune intervention, a tremendous effort has been made to improve gene transfer in these cells. Lentiviral vectors (LVs) have conferred a huge advantage in that they can transduce nondividing cells such as human monocyte-derived DCs (MDDCs) but required high amounts of viral particles and/or accessory proteins such as Vpx or Vpr to achieve sufficient transduction rates. As a consequence, these LVs have been shown to cause dramatic functional modifications, such as the activation or maturation of transduced MDDCs. Taking advantage of new pseudotyped LVs, i.e., with envelope glycoproteins from the measles virus (MV), we demonstrate that MDDCs are transduced very efficiently with these new LVs compared to the classically used vesicular stomatitis virus G-pseudotyped LVs and thus allowed to achieve high transduction rates at relatively low multiplicities of infection. Moreover, in this experimental setting, no activation or maturation markers were upregulated, while MV-LV-transduced cells remained able to mature after an appropriate Toll-like receptor stimulation. We then demonstrate that our MV-pseudotyped LVs use DC-SIGN, CD46, and CD150/SLAM as receptors to transduce MDDCs. Altogether, our results show that MV-pseudotyped LVs provide the most accurate and simple viral method for efficiently transferring genes into MDDCs without affecting their activation and/or maturation status.     

Reaching the hard-to-reach: a probability sampling method for assessing prevalence of driving under the influence after drinking in alcohol outlets

Drinking alcoholic beverages in places such as bars and clubs may be associated with harmful consequences such as violence and impaired driving. However, methods for obtaining probabilistic samples of drivers who drink at these places remain a challenge--since there is no a priori information on this mobile population--and must be continually improved. This paper describes the procedures adopted in the selection of a population-based sample of drivers who drank at alcohol selling outlets in Porto Alegre, Brazil, which we used to estimate the prevalence of intention to drive under the influence of alcohol. The sampling strategy comprises a stratified three-stage cluster sampling: 1) census enumeration areas (CEA) were stratified by alcohol outlets (AO) density and sampled with probability proportional to the number of AOs in each CEA; 2) combinations of outlets and shifts (COS) were stratified by prevalence of alcohol-related traffic crashes and sampled with probability proportional to their squared duration in hours; and, 3) drivers who drank at the selected COS were stratified by their intention to drive and sampled using inverse sampling. Sample weights were calibrated using a post-stratification estimator. 3,118 individuals were approached and 683 drivers interviewed, leading to an estimate that 56.3% (SE = 3,5%) of the drivers intended to drive after drinking in less than one hour after the interview. Prevalence was also estimated by sex and broad age groups. The combined use of stratification and inverse sampling enabled a good trade-off between resource and time allocation, while preserving the ability to generalize the findings. The current strategy can be viewed as a step forward in the efforts to improve surveys and estimation for hard-to-reach, mobile populations.     

Sulphonamide-based small molecule VLA-4 antagonists

The discovery of a sulphonamide by-product with VLA-4 antagonistic activity led to a series of potent, small molecule VLA-4 antagonists. Synthesis, SAR and in vivo evaluation of the selected compound will be presented.     

Ca2+]i rise at in vitro maturation in bovine cumulus-oocyte complexes

An intracellular calcium ([Ca(2+)]i) rise has been described in cumulus-oocyte complexes (COCs) following luteinizing hormone (LH) exposure. Together with cAMP, Ca(2+) is a candidate signal for resumption of meiosis. Here, we analyzed if the most common hormones involved in oocyte maturation can induce the same Ca(2+) signal. In addition, we characterized the source of this signal. Immature, in vitro-matured, and roscovitine-meiotically arrested COCs were loaded with Fluo-4 AM, stimulated with hormones/growth factors, and tested for [Ca(2+)](i) variations in cumulus cells. Reagents known to inhibit or stimulate [Ca(2+)](i) rises were used to characterize these [Ca(2+)](i) dynamics. Finally, expression of LH receptors (LHRs) in COCs was analyzed by immunofluorescence. In immature COCs, follicle-stimulating hormone (FSH) elicited a single [Ca(2+)](i) rise that was higher than those induced by LH and growth hormone (GH), whereas epithelial growth factor failed to induce any changes in [Ca(2+)](i). The [Ca(2+)](i) rise induced by FSH was higher in immature COCs; was reduced in roscovitine-arrested, immature COCs; and was negligible in gonadotropin-induced, in vitro-matured COCs. In the case of spontaneous- and GH-matured COCs, however, FSH stimulation caused a lower [Ca(2+)](i) rise. The hormone-induced [Ca(2+)](i) rise was due to: (i) external Ca(2+) entry; (ii) intercellular communication; and (iii) intracellular Ca(2+) stores. Immunofluorescence revealed that LHRs were expressed throughout the cumulus cells. The above results show that: (i) gonadotropins and GH cause a [Ca(2+)](i) rise in cumulus cells; (ii) this [Ca(2+)](i) rise results from extra-, inter-, and intra-cellular cumulative Ca(2+) fluxes; and (iii) LHRs are distributed on either outer or inner cumulus cells.