Population genetic structure and connectivity of the harmful dinoflagellate Alexandrium minutum in the Mediterranean Sea

The toxin-producing microbial species Alexandrium minutum has a wide distribution in the Mediterranean Sea and causes high biomass blooms with consequences on the environment, human health and coastal-related economic activities. Comprehension of algal genetic differences and associated connectivity is fundamental to understand the geographical scale of adaptation and dispersal pathways of harmful microalgal species. In the present study, we combine A. minutum population genetic analyses based on microsatellites with indirect connectivity (C(i)) estimations derived from a general circulation model of the Mediterranean sea. Our results show that four major clusters of genetically homogeneous groups can be identified, loosely corresponding to four regional seas: Adriatic, Ionian, Tyrrhenian and Catalan. Each of the four clusters included a small fraction of mixed and allochthonous genotypes from other Mediterranean areas, but the assignment to one of the four clusters was sufficiently robust as proved by the high ancestry coefficient values displayed by most of the individuals (>84%). The population structure of A. minutum on this scale can be explained by microalgal dispersion following the main regional circulation patterns over successive generations. We hypothesize that limited connectivity among the A. minutum populations results in low gene flow but not in the erosion of variability within the population, as indicated by the high gene diversity values. This study represents a first and new integrated approach, combining both genetic and numerical methods, to characterize and interpret the population structure of a toxic microalgal species. This approach of characterizing genetic population structure and connectivity at a regional scale holds promise for the control and management of the harmful algal bloom events in the Mediterranean Sea.     

A metabonomic study of transgenic maize (Zea mays) seeds revealed variations in osmolytes and branched amino acids

The aim of the research was to investigate metabolic variations associated with genetic modifications in the grains of Zea mays using metabonomic techniques. With this in mind, the non-targeted characteristic of the technique is useful to identify metabolites peculiar to the genetic modification and initially undefined. The results obtained showed that the genetic modification, introducing Cry1Ab gene expression, induces metabolic variations involving the primary nitrogen pathway. Concerning the methodological aspects, the experimental protocol used has been applied in this field for the first time. It consists of a combination of partial least square-discriminant analysis and principal component analysis. The most important metabolites for discrimination were selected and the metabolic correlations linking them are identified. Principal component analysis on selected signals confirms metabolic variations, highlighting important details about the changes induced on the metabolic network by the presence of a Bt transgene in the maize genome.     

Control of membrane-attached biofilms using surfactants

The effect of surfactants on membrane-attached biofilms (MABs) was studied in a lab-scale extractive membrane bioreactor (EMB). Twenty-two surfactants were screened for their potential of increasing the cell wall negative charge (i.e. the electrostatic repulsion between bacteria) of Burkholderia sp. JS150 bacterial strain. Surfactants resulting in increased bacterial negative charge were further investigated for their effects on MAB population morphology and MAB attachment behaviour. Microscopic investigation of the bacterial population in MABs showed that surfactants affect the development of flagella, suggesting changes in the attachment capability of the JS150 strain in the presence of different surfactants. Among the screened surfactants, teepol showed the best characteristics in relation to the reduction of MAB accumulation, and it was tested in an EMB system for the extraction of monochlorobenzene from a synthetic wastewater. Comparison with a control EMB, operated without surfactants under the same conditions, proved that teepol effectively reduces MAB accumulation on the membrane walls. As a result, the overall mass transfer coefficient in the presence of teepol was 53% higher than in the control EMB.     

Marine reptiles (Thalattosuchia) from the Early Jurassic of Lombardy (northern Italy)

The fossil remains of two small reptiles recently discovered in the Sogno Formation (Lower Toarcian) near Cesana Brianza (Lecco Province), represent the first mesoeucrocodylians reported for Lombardy and some of the few Jurassic reptiles from Italy. Due to the absence of diagnostic skeletal elements (the skulls are lacking), it is not possible to refer the new specimens at genus level with confidence. Although the well developed dermal armour would characterise Toarcian thalattosuchians of the genera Steneosaurus (Teleosauridae) and Pelagosaurus (Metriorhynchidae), the peculiar morphology of the osteoderms allow to tentatively refer the remains to the latter taxon (cf. Pelagosaurus sp.). The small size, along with the opening of the neurocentral vertebral sutures and, possibly, the non sutured caudal pleurapophyses, indicate that the specimens were morphologically immature at death. These “marine crocodiles” confirm the affinities between the fauna of the Calcare di Sogno Formation and coeval outcrops of central Europe that also share the presence of similar fishes and crustaceans.     

Extrinsic and intrinsic sources of calcitonin gene-related peptide immunoreactivity in the lamb ileum: a morphometric and neurochemical investigation

To investigate extrinsic origins of calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibres in the sheep ileum, the retrograde fluorescent tracer Fast Blue (FB) was injected into the ileum wall. Sections of thoraco-lumbar dorsal root ganglia (DRG) and distal (nodose) vagal ganglia showing FB-labelled neurons were processed for CGRP immunohistochemistry. The distribution of CGRP-IR in fibres and nerve cell bodies in the ileum was also studied. CGRP-IR enteric neurons were morphometrically analysed in myenteric (MP) and submucosal plexuses (SMP) of lambs (2–4 months). Sensory neurons retrogradely labelled with FB were scattered in T5-L4 DRG but most were located at the upper lumbar levels (L1-L3); only a minor component of the extrinsic afferent innervation of the ileum was derived from nodose ganglia. In the DRG, 57% of retrogradely labelled neurons were also CGRP-IR. In cryostat sections, a dense network of CGRP-IR fibres was observed in the lamina propria beneath the epithelium, around the lacteals and lymphatic follicles (Peyer's platches), and along and around enteric blood vessels. Rare CGRP-IR fibres were also present in both muscle layers. Dense pericellular baskets of CGRP-IR fibres were observed around CGRP-negative somata. The only CGRP-IR nerve cells were well-defined Dogiel type II neurons localised in the MP and in the external and internal components of the SMP. CGRP-IR neurons in the myenteric ganglia were significantly larger than those in the submucosal ganglia (mean profile areas: about 1,400 μm² for myenteric neurons, 750 μm² for submucosal neurons). About 6% of myenteric neurons and 25% of submucosal neurons were CGRP-IR Dogiel type II neurons. The percentages of CGRP-IR neurons that were also tachykinin-IR were about 9% (MP) and 42% (SMP), whereas no CGRP-IR neurons exhibited immunoreactivity for vasoactive intestinal peptide, nitric oxide synthase or tyrosine hydroxylase in either plexus. Thus, CGRP immunoreactivity occurs in the enteric nervous system of the sheep ileum (as in human small intestine and MP of pig ileum) in only one morphologically defined type of neuron, Dogiel type II cells. These are probably intrinsic primary afferent neurons. This work was supported by grants from the Ricerca Fondamentale Orientata (RFO) and Fondazione Del Monte di Bo e Ra.     

Object segmentation and recovery via neural oscillators implementing the similarity and prior knowledge gestalt rules

Object recognition requires the solution of the binding and segmentation problems, i.e., grouping different features to achieve a coherent representation. Synchronization of neural activity in the gamma-band, associated with gestalt perception, has often been proposed as a putative mechanism to solve these problems, not only as to low-level processing, but also in higher cortical functions. In the present work, a network of Wilson-Cowan oscillators is used to segment simultaneous objects, and recover an object from partial or corrupted information, by implementing two gestalt rules: similarity and prior knowledge. The network consists of H different areas, each devoted to representation of a particular feature of the object, according to a topological organization. The similarity law is realized via lateral intra-area connections, arranged as a "Mexican-hat". Prior knowledge is realized via inter-area connections, which link properties belonging to a previously memorized object. A global inhibitor allows segmentation of several objects avoiding interference. Simulation results, performed using three simultaneous input objects, show that the network is able to detect an object even in difficult conditions (i.e., when some features are absent or shifted with respect to the original one). Moreover, the trade-off between sensitivity (capacity to detect true positives) and specificity (capacity to reject false positives) can be controlled acting on the extension of lateral synapses (i.e., on the level of accepted similarity). Finally, the network can also deal with correlated objects, i.e., objects which have some common features. Simulations performed using a different number of objects (2, 3, 4 or 5) suggest that the network is able to segment and recall up to four objects, but the oscillation frequency must increase, the lower the number of objects simultaneously present. The model, although quite simpler compared with neurophysiology, may represent a theoretical framework for the analysis of the relationships between object representation, memory, learning, and gamma-band activity. In particular, it extends previous studies on autoassociative memory since it exploits not only oscillatory dynamics, but also a topological organization of features.     

A new member of the aldo-keto reductase family from the plant pathogen Xylella fastidiosa

The Xylella fastidiosa genome program generated a large number of gene sequences that belong to pathogenicity, virulence and adaptation categories from this important plant pathogen. One of these genes (XF1729) encodes a protein similar to a superfamily of aldo-keto reductase together with a number of structurally and functionally related NADPH-dependent oxidoreductases. In this work, the similar sequence XF1729 from X. fastidiosa was cloned onto the pET32Xa/LIC vector in order to overexpress a recombinant His-tag fusion protein in Escherichia coli BL21(DE3). The expressed protein in the soluble fraction was purified by immobilized metal affinity chromatography (agarose-IDA-Ni resin). Secondary structure contents were verified by circular dichroism spectroscopy. Small angle X-ray scattering (SAXS) measurements furnish general structural parameters and provide a strong indication that the protein has a monomeric form in solution. Also, ab initio calculations show that the protein has some similarities with a previously crystallized aldo-keto reductase protein. The recombinant XF1729 purified to homogeneity catalyzed the reduction of dl-glyceraldehyde (K(cat) 2.26s(-1), Km 8.20+/-0.98 mM) and 2-nitrobenzaldehyde (K(cat) 11.74 s(-1), Km 0.14+/-0.04 mM) in the presence of NADPH. The amino acid sequence deduced from XF1729 showed the highest identity (40% or higher) with several functional unknown proteins. Among the identified AKRs, we found approximately 29% of identity with YakC (AKR13), 30 and 28% with AKR11A and AKR11B, respectively. The results establish XF1729 as the new member of AKR family, AKR13B1. Finally, the first characterization by gel filtration chromatography assays indicates that the protein has an elongated shape, which generates an apparent higher molecular weight. The study of this protein is an effort to fight X. fastidiosa, which causes tremendous losses in many economically important plants.     

Low resolution structure of the human alpha4 protein (IgBP1) and studies on the stability of alpha4 and of its yeast ortholog Tap42

The yeast Tap42 and mammalian alpha4 proteins belong to a highly conserved family of regulators of the type 2A phosphatases, which participate in the rapamycin-sensitive signaling pathway, connecting nutrient availability to cell growth. The mechanism of regulation involves binding of Tap42 to Sit4 and PPH21/22 in yeast and binding of alpha4 to the catalytic subunits of type 2A-related phosphatases PP2A, PP4 and PP6 in mammals. Both recombinant proteins undergo partial proteolysis, generating stable N-terminal fragments. The full-length proteins and alpha4 C-terminal deletion mutants at amino acids 222 (alpha4Delta222), 236 (alpha4Delta236) and 254 (alpha4Delta254) were expressed in E. coli. alpha4Delta254 undergoes proteolysis, producing a fragment similar to the one generated by full-length alpha4, whereas alpha4Delta222 and alpha4Delta236 are highly stable proteins. alpha4 and Tap42 show alpha-helical circular dichroism spectra, as do their respective N-terminal proteolysis resistant products. The cloned truncated proteins alpha4Delta222 and alpha4Delta236, however, possess a higher content of alpha-helix, indicating that the C-terminal region is less structured, which is consistent with its higher sensitivity to proteolysis. In spite of their higher secondary structure content, alpha4Delta222 and alpha4Delta236 showed thermal unfolding kinetics similar to the full-length alpha4. Based on small angle X-ray scattering (SAXS), the calculated radius of gyration for alpha4 and Tap42 were 41.2 +/- 0.8 A and 42.8 +/- 0.7 A and their maximum dimension approximately 142 A and approximately 147 A, respectively. The radii of gyration for alpha4Delta222 and alpha4Delta236 were 21.6 +/- 0.3 A and 25.7 +/- 0.2 A, respectively. Kratky plots show that all studied proteins show variable degree of compactness. Calculation of model structures based on SAXS data showed that alpha4Delta222 and alpha4Delta236 proteins have globular conformation, whereas alpha4 and Tap42 exhibit elongated shapes.     

Performance and behaviour of rabbit does in a group-housing system with natural mating or artificial insemination

This study compared reproductive performance and behaviour of does raised in a group-housing system and in a regular cage system. The group-housing pen was divided into different functional areas for suckling, resting, and eating and special hiding areas for kits when they had left the nest-boxes and does to favour the species specific behavioural traits. Does had access to their nest-box by means of an individual Electronic Nest-box Recognition System (ENRS) activated by a coded transponder placed in their eartags. Eight does were housed in each pen. Natural mating (NM, with a buck in the group) or artificial inseminations (AI) were applied. Litter size, kit mortality and kit weight at 14 d of age were similar for group-housing and cages when NM were applied. With a natural reproduction rhythm group-housing led to an increase of +38% of litters. However, from a management point of view, a cycled production system with AI is preferred. With AI and group-housing, a lower kindling rate and a lower kit weight at weaning were found. The lower kindling rate was partly caused by pseudo-pregnancies that were found in 23% (P < 0.01) of the does in the group-housing system against 0% in the control group. Sixteen to 20% of the does in the group-housing system had skin injuries, which is an indicator for aggression among does. Most of the injuries were seen on the body and most of them were superficial bites. Based on the results of this study, it can be concluded that group-housing of rabbit does seems possible, but more research is needed to solve the problems of the decreased kindling rate and occurrence of pseudo-pregnancies, the lower weight at weaning and aggressiveness among does.     

Synthesis and alpha4beta2 nicotinic affinity of unichiral 5-(2-pyrrolidinyl)oxazolidinones and 2-(2-pyrrolidinyl)benzodioxanes

The RS and SR enantiomers of 2-oxazolidinone and 1,4-benzodioxane bearing a 2-pyrrolidinyl substituent at the 5- and 2-position, respectively, were synthesized as candidate nicotinoids. One of the two benzodioxane stereoisomers reasonably fits the pharmacophore elements of (S)-nicotine and binds at alpha4beta2 nicotinic acetylcholine receptor with submicromolar affinity. Interestingly, both the synthesized pyrrolidinylbenzodioxanes exhibit analogous affinity at alpha(2) adrenergic receptor resembling the behaviour of some known alpha(2)-AR ligands recently proved to possess neuronal nicotinic affinity.