Botulinum neurotoxin type A blocks the morphological changes induced by chemical stimulation on the presynaptic membrane of Torpedo synaptosomes.

The action of botulinum neurotoxin on acetylcholine release, and on the structural changes at the presynaptic membrane associated with the transmitter release,was studied by using a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. Acetylcholine and ATP release were continuously monitored by chemiluminescent methods.To catch the membrane morphological changes, the quick-freezing method was applied. Our results show that botulinum neurotoxin inhibits the release of acetylcholine from these isolated nerve terminals in a dose-dependent manner, whereas ATP release is not affected. The maximal inhibition (70%) is achieved at neurotoxin concentrations as low as 125 pM with an incubation time of 6 min. This effect is not linked to an alteration of the integrity of the synaptosomes since, after poisoning by botulinum neurotoxin type A, they show a nonmodified occluded lactate dehydrogenase activity. Moreover, membrane potential is not altered by the toxin with respect to the control, either in resting condition or after potassium depolarization. In addition to acetylcholine release inhibition, botulinum neurotoxin blocks the rearrangement of the presynaptic intramembrane particles induced by potassium stimulation. The action of botulinum neurotoxin suggests that the intramembrane particle rearrangement is related to the acetylcholine secretion induced by potassium stimulation in synaptosomes isolated from the electric organ of Torpedo marmorata.

Predictors of developmental dyslexia in European orthographies with varying complexity.

Background:  The relationship between phoneme awareness, rapid automatized naming (RAN), verbal short-term/working memory (ST/WM) and diagnostic category is investigated in control and dyslexic children, and the extent to which this depends on orthographic complexity. Methods:  General cognitive, phonological and literacy skills were tested in 1,138 control and 1,114 dyslexic children speaking six different languages spanning a large range of orthographic complexity (Finnish, Hungarian, German, Dutch, French, English). Results:  Phoneme deletion and RAN were strong concurrent predictors of developmental dyslexia, while verbal ST/WM and general verbal abilities played a comparatively minor role. In logistic regression models, more participants were classified correctly when orthography was more complex. The impact of phoneme deletion and RAN-digits was stronger in complex than in less complex orthographies. Conclusions:  Findings are largely consistent with the literature on predictors of dyslexia and literacy skills, while uniquely demonstrating how orthographic complexity exacerbates some symptoms of dyslexia.

Care complexity, mood, and quality of life in liver pre-transplant patients.

RATIONALE: This study was intended to document the frequency of care complexity in liver transplant candidates, and its association with mood disturbance and poor health-related quality of life (HRQoL). METHODS: Consecutive patients fulfilling inclusion criteria, recruited in three European hospitals, were assessed with INTERMED, a reliable and valid method for the early assessment of bio-psychosocial health risks and needs. Blind to the results, they were also assessed with the Hospital Anxiety and Depression Scale (HADS). HRQoL was documented with the EuroQol and the SF36. Statistical analysis included multivariate and multilevel techniques. RESULTS: Among patients fulfilling inclusion criteria, 60 patients (75.9%) completed the protocol and 38.3% of them were identified as "complex" by INTERMED, but significant between-center differences were found. In support of the working hypothesis, INTERMED scores were significantly associated with all measures of both the SF36 and the EuroQol, and also with the HADS. A one point increase in the INTERMED score results in a reduction in 0.93 points in EuroQol and a 20% increase in HADS score. CONCLUSIONS: INTERMED-measured case complexity is frequent in liver transplant candidates but varies widely between centers. The use of this method captures in one instrument multiple domains of patient status, including mood disturbances and reduced HRQoL.

Morphological and molecular characterization of Pisolithus in soil under eucalyptus plantations in Brazil

Eighteen Pisolithus basidiomes were collected from Eucalyptus plantations in the state of Minas Gerais, Brazil. These basidiomes were characterized morphologically and molecularly. The basidiomes varied in shape, color and size. One of them was found underground, indicating a hypogeous fungus. The main morphological distinctive characteristic was spore ornamentation, which distinguished two groups. One group with short and erect spines was identified as Pisolithus microcarpus, and the other with long and curved spines as Pisolithus marmoratus, after analyzing the cladogram obtained by phylogenetic relationship based on internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA of these isolates.

Large scale assessment of regulatory evolution and transcriptome complexity in mammals

AbstractIn addition to genetic changes affecting the function of gene products, changes in gene expression have been suggested to underlie many or even most of the phenotypic differences among mammals. However, detailed gene expression comparisons were, until recently, restricted to closely related species, owing to technological limitations. Thus, we took advantage of the latest technologies (RNA-Seq) to generate extensive qualitative and quantitative transcriptome data for a unique collection of somatic and germline tissues from representatives of all major mammalian lineages (placental mammals, marsupials and monotremes) and birds, the evolutionary outgroup.In the first major project of my thesis, we performed global comparative analyses of gene expression levels based on these data. Our analyses provided fundamental insights into the dynamics of transcriptome change during mammalian evolution (e.g., the rate of expression change across species, tissues and chromosomes) and allowed the exploration of the functional relevance and phenotypic implications of transcription changes at a genome-wide scale (e.g., we identified numerous potentially selectively driven expression switches).In a second project of my thesis, which was also based on the unique transcriptome data generated in the context of the first project we focused on the evolution of alternative splicing in mammals. Alternative splicing contributes to transcriptome complexity by generating several transcript isoforms from a single gene, which can, thus, perform various functions. To complete the global comparative analysis of gene expression changes, we explored patterns of alternative splicing evolution. This work uncovered several general and unexpected patterns of alternative splicing evolution (e.g., we found that alternative splicing evolves extremely rapidly) as well as a large number of conserved alternative isoforms that may be crucial for the functioning of mammalian organs.Finally, the third and final project of my PhD consisted in analyzing in detail the unique functional and evolutionary properties of the testis by exploring the extent of its transcriptome complexity. This organ was previously shown to evolve rapidly both at the phenotypic and molecular level, apparently because of the specific pressures that act on this organ and are associated with its reproductive function. Moreover, my analyses of the amniote tissue transcriptome data described above, revealed strikingly widespread transcriptional activity of both functional and nonfunctional genomic elements in the testis compared to the other organs. To elucidate the cellular source and mechanisms underlying this promiscuous transcription in the testis, we generated deep coverage RNA-Seq data for all major testis cell types as well as epigenetic data (DNA and histone methylation) using the mouse as model system. The integration of these complete dataset revealed that meiotic and especially post-meiotic germ cells are the major contributors to the widespread functional and nonfunctional transcriptome complexity of the testis, and that this "promiscuous" spermatogenic transcription is resulting, at least partially, from an overall transcriptionally permissive chromatin state. We hypothesize that this particular open state of the chromatin results from the extensive chromatin remodeling that occurs during spermatogenesis which ultimately leads to the replacement of histones by protamines in the mature spermatozoa. Our results have important functional and evolutionary implications (e.g., regarding new gene birth and testicular gene expression evolution).Generally, these three large-scale projects of my thesis provide complete and massive datasets that constitute valuables resources for further functional and evolutionary analyses of mammalian genomes.

Similarities and differences in altitudinal versus latitudinal variation for morphological traits in Drosophila melanogaster.

Understanding how natural environments shape phenotypic variation is a major aim in evolutionary biology. Here, we have examined clinal, likely genetically based variation in morphology among 19 populations of the fruit fly (Drosophila melanogaster) from Africa and Europe, spanning a range from sea level to 3000 m altitude and including locations approximating the southern and northern range limit. We were interested in testing whether latitude and altitude have similar phenotypic effects, as has often been postulated. Both latitude and altitude were positively correlated with wing area, ovariole number, and cell number. In contrast, latitude and altitude had opposite effects on the ratio between ovariole number and body size, which was negatively correlated with egg production rate per ovariole. We also used transgenic manipulation to examine how increased cell number affects morphology and found that larger transgenic flies, due to a higher number of cells, had more ovarioles, larger wings, and, unlike flies from natural populations, increased wing loading. Clinal patterns in morphology are thus not a simple function of changes in body size; instead, each trait might be subject to different selection pressures. Together, our results provide compelling evidence for profound similarities as well as differences between phenotypic effects of latitude and altitude.

Viscous fingering in liquid crystals: Anisotropy and morphological transitions

We show that a minimal model for viscous fingering with a nematic liquid crystal in which anisotropy is considered to enter through two different viscosities in two perpendicular directions can be mapped to a twofold anisotropy in the surface tension. We numerically integrate the dynamics of the resulting problem with the phase-field approach to find and characterize a transition between tip splitting and side branching as a function of both anisotropy and dimensionless surface tension. This anisotropy dependence could explain the experimentally observed (reentrant) transition as temperature and applied pressure are varied. Our observations are also consistent with previous experimental evidence in viscous fingering within an etched cell and simulations of solidification.

Aggregation of integrins and RhoA activation are required for Thy-1-induced morphological changes in astrocytes.

Thy-1, a cell adhesion molecule abundantly expressed in mammalian neurons, binds to a beta(3)-containing integrin on astrocytes and thereby stimulates the assembly of focal adhesions and stress fibers. Such events lead to morphological changes in astrocytes that resemble those occurring upon injury in the brain. Extracellular matrix proteins, typical integrin ligands, bind to integrins and promote receptor clustering as well as signal transduction events that involve small G proteins and cytoskeletal changes. Here we investigated the possibility that the cell surface protein Thy-1, when interacting with a beta(3)-containing integrin on astrocytes, could trigger signaling events similar to those generated by extracellular matrix proteins. DI-TNC(1) astrocytes were stimulated with Thy-1-Fc immobilized on beads, and increased RhoA activity was confirmed using an affinity precipitation assay. The effect of various inhibitors on the cellular response was also studied. The presence of Y-27632, an inhibitor of Rho kinase (p160ROCK), a key downstream effector of RhoA, significantly reduced focal adhesion and stress fiber formation induced by Thy-1. Similar effects were obtained when astrocytes were treated with C3 transferase, an inhibitor of RhoA. Alternatively, astrocytes were transfected with an expression vector encoding fusion proteins of enhanced green fluorescent protein with either the Rho-binding domain of Rhotekin, which blocks RhoA function, or the dominant-negative N19RhoA mutant. In both cases, Thy-1-induced focal adhesion formation was inhibited. Furthermore, we observed that RhoA activity after stimulation with soluble Thy-1-Fc molecule was augmented upon further cross-linking using protein A-Sepharose beads. The same was shown by cross-linking beta(3)-containing integrin with anti-beta(3) antibodies. Together, these results indicate that Thy-1-mediated astrocyte stimulation depended on beta(3) integrin clustering and the resulting increase in RhoA activity.

Animal Toxins: How is Complexity Represented in Databases?

Peptide toxins synthesized by venomous animals have been extensively studied in the last decades. To be useful to the scientific community, this knowledge has been stored, annotated and made easy to retrieve by several databases. The aim of this article is to present what type of information users can access from each database. ArachnoServer and ConoServer focus on spider toxins and cone snail toxins, respectively. UniProtKB, a generalist protein knowledgebase, has an animal toxin-dedicated annotation program that includes toxins from all venomous animals. Finally, the ATDB metadatabase compiles data and annotations from other databases and provides toxin ontology.

MORPHOLOGICAL AND MICROMORPHOLOGICAL CHANGES IN THE STRUCTURE OF A RHODIC HAPLUDOX AS A RESULT OF AGRICULTURAL MANAGEMENT

In evaluation of soil quality for agricultural use, soil structure is one of the most important properties, which is influenced not only by climate, biological activity, and management practices but also by mechanical and physico-chemical forces acting in the soil. The purpose of this study was to evaluate the influence of conventional agricultural management on the structure and microstructure of a Latossolo Vermelho distroférrico típico (Rhodic Hapludox) in an experimental area planted to maize. Soil morphology was described using the crop profile method by identifying the distinct structural volumes called Morphologically Homogeneous Units (MHUs). For comparison, we also described a profile in an adjacent area without agricultural use and under natural regrowth referred to as Memory. We took undisturbed samples from the main MHUs so as to form thin sections and blocks of soil for micromorphological and micromorphometrical analyses. Results from the application of the crop profile method showed the occurrence of the following structural types: loose (L), fragmented (F) and continuous (C) in both profiles analyzed. In the Memory soil profile, the fragmented structures were classified as Fptμ∆+tf and Fmt∆μ, whose micromorphology shows an enaulic-porphyric (porous) relative distribution with a great deal of biological activity as indicated by the presence of vughs and channels. Lower down, from 0.20 to 0.35 m, there was a continuous soil volume (sub-type C∆μ), with a subangular block microstructure and an enaulic-porphyric relative distribution, though in this case more compact and with aggregate coalescence and less biological activity. The micromorphometrical study of the soil of the Memory Plot showed the predominance of complex pores in NAM (15.03 %), Fmt∆μ (11.72 %), and Fptμ∆+tf (7.73 %), and rounded pores in C∆μ (8.21 %). In the soil under conventional agricultural management, we observed fragmented structures similar to the Memory Plot from 0.02 to 0.20 m, followed by a volume with a compact continuous structure (C∆μ), without visible porosity and with few roots. In the MHUs under conventional management, reduction in the packing pores (40 %) was observed, mainly in the continuous units (C). The microstructure had well-defined blocks, with the occurrence of planar pores and less evidence of biological activity. In conclusion, the morphological and micromorphological analyses of the soil profiles studied offered complementary information regarding soil structural quality, especially concerning the changes in pore types as result of mechanical stress undergone by the soil.

Serum-free aggregate cultures of rat CNS glial cells: biochemical, immunocytochemical and morphological characterization.

Aggregate cultures of mixed glial cells, as well as of enriched astrocytes and oligodendrocytes were prepared, and maintained in serum-free medium for up to 25 days. Biochemical measurements of both neuron-specific and glia-specific enzyme activities showed that these three types of aggregate cultures were virtually devoid of neurons. Astrocyte-enriched cultures were greater than 95% pure, with oligodendrocytes as the only apparent contaminant, whereas oligodendrocyte-enriched cultures still contained a considerable proportion of astrocytes. In all these neuron-free aggregate cultures both astrocytes and oligodendrocytes attained a high degree of maturation. These findings were confirmed by morphological examinations, and by immunofluorescence studies. Furthermore, ultrastructural as well as immunocytochemical investigations using antibodies to myelin basic protein revealed that all three types of glial cell aggregate cultures contained myelin membranes, indicating that the presence of axons is not a prerequisite for the formation of myelin.