Ornamental traits modification by Rol genes in Osteospermum ecklonis transformed with Agrobacterium tumefaciens

Summary Transgenic plants of Osteospermum ecklonis were produced by cocultivation of leaf fragments with Agrobacterium tumefaciens harboring rol genes from A. rhizogenes. The phenotypic alterations caused by the different transgenes were evaluated in field trials. The genetic manipulation produced transgenic plants characterized by the following features: 1) increased number of flowers (e.g., 35SrolC and rolABC); 2) early flowering (e.g., 35SrolC); 3) change of plant growth habit: erect (rolAB, rolABC and 35SrolC) with an increased number of branches (e.g., rolABC). The color of leaves was pale green in 35SrolC and dark green in rolAB transgenic plants. In conclusion this work reports: 1) genetic engineering of the ornamental species O. ecklonis, 2) modification of the main ornamental traits of this species by rol genes, and 3) segregation of the transgenes in the progeny.     

CiliateGEM: an open-project and a tool for predictions of ciliate metabolic variations and experimental condition design

Background

The study of cell metabolism is becoming central in several fields such as biotechnology, evolution/adaptation and human disease investigations. Here we present CiliateGEM, the first metabolic network reconstruction draft of the freshwater ciliate Tetrahymena thermophila. We also provide the tools and resources to simulate different growth conditions and to predict metabolic variations. CiliateGEM can be extended to other ciliates in order to set up a meta-model, i.e. a metabolic network reconstruction valid for all ciliates.Ciliates are complex unicellular eukaryotes of presumably monophyletic origin, with a phylogenetic position that is equal from plants and animals. These cells represent a new concept of unicellular system with a high degree of species, population biodiversity and cell complexity. Ciliates perform in a single cell all the functions of a pluricellular organism, including locomotion, feeding, digestion, and sexual processes.

Results

After generating the model, we performed an in-silico simulation with the presence and absence of glucose. The lack of this nutrient caused a 32.1% reduction rate in biomass synthesis. Despite the glucose starvation, the growth did not stop due to the use of alternative carbon sources such as amino acids.

Conclusions

The future models obtained from CiliateGEM may represent a new approach to describe the metabolism of ciliates. This tool will be a useful resource for the ciliate research community in order to extend these species as model organisms in different research fields. An improved understanding of ciliate metabolism could be relevant to elucidate the basis of biological phenomena like genotype-phenotype relationships, population genetics, and cilia-related disease mechanisms.

     

Habitat use at fine spatial scale: how does patch clustering criteria explain the use of meadows by red deer?

Large mammalian herbivores are keystone species in different ecosystems. To mediate the effects of large mammalian herbivores on ecosystems, it is crucial to understand their habitat selection pattern. At finer scales, herbivore patch selection depends strongly on plant community traits and therefore its understanding is constrained by patch definition criteria. Our aim was to assess which criteria for patch definition best explained use of meadows by wild, free-ranging, red deer (Cervus elaphus) in a study area in Northeast Portugal. We used two clustering criteria types based on floristic composition and gross forage classes, respectively. For the floristic criteria, phytosociological approach was used to classify plant communities, and its objectivity evaluated with a mathematical clustering of the floristic relevés. Cover of dominant plant species was tested as a proxy for the phytosociological method. For the gross forage classes, the graminoids/forbs ratio and the percentage cover of legumes were used. For assessing deer relative use of meadows we used faecal accumulation rates. Patches clustered according to floristic classification better explained selection of patches by deer. Plant community classifications based on phytosociology, or proxies of this, used for characterizing meadow patches resulted useful to understand herbivore selection pattern at fine scales and thus potentially suitable to assist wildlife management decisions.     

Cell proliferation in a peripheral target is required for the induction of central neurogenesis in the leech

Several days after the completion of the early phase of cell proliferation that generates most of the leech central nervous system, the pair of “sex ganglia” in the two reproductive segments of the midbody undergo a second period of neurogenesis that gives rise to several hundred peripherally induced central (PIC) neurons. This proliferative phase, which begins on embryonic day 17 (E17), is induced by the interaction of a few specific neurons in the sex ganglia with a peripheral target, the male genitalia, during a critical period that extends from E13 to E16. The central nervous system (CNS) determines the critical period, since the male genitalia have the capacity to induce PIC neurons beginning on E10 and continuing throughout embryogenesis. Here we first show, by injecting hydroxyurea into staged embryos to ablate dividing cells, that PIC neuron precursors begin to divide at a low rate before E17, during the critical period. Then, through a series of homochronic and heterochronic male organ transplantations combined with hydroxyurea treatment of hosts and/or donors, we show that cell proliferation is required in the target itself for it to be competent to induce PIC neurons. These observations demonstrate that a nerve connection can couple cell proliferation in a peripheral target to cell proliferation in the CNS, providing a novel means for size adjustment of a central neuronal population relative to a peripheral target. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 295–303, 1998     

Hepcidin assay in serum by SELDI-TOF-MS and other approaches

Hepcidin, a liver peptide hormone, is the central regulator of iron homeostasis. Hepcidin synthesis is modulated by iron stores, so that iron repletion increases its levels to prevent pathological overload, while iron deficiency strongly inhibits hepcidin to allow an increase in iron absorption from duodenal cells. The emerging pivotal role of hepcidin in iron homeostasis, along with its important links with basic pathways like inflammation, makes the availability of an accurate hepcidin assay as a potentially powerful investigative tool to improve our understanding as well as our diagnostic/prognostic capabilities in many human diseases. There has been a great interest worldwide in developing a reliable and widely applicable assay of the hormone in biological fluids. Being optimal for low-molecular-weight biomarkers, SELDI-TOF-MS has emerged as a valid tool for hepcidin assay. Here we review recent results obtained with this technique, as well as with other Mass Spectrometry-based and immunological methods.     

Solution structure of the bacterial frataxin ortholog, CyaY: mapping the iron binding sites

CyaY is the bacterial ortholog of frataxin, a small mitochondrial iron binding protein thought to be involved in iron sulphur cluster formation. Loss of frataxin function leads to the neurodegenerative disorder Friedreich's ataxia. We have solved the solution structure of CyaY and used the structural information to map iron binding onto the protein surface. Comparison of the behavior of wild-type CyaY with that of a mutant indicates that specific binding with a defined stoichiometry does not require aggregation and that the main binding site, which hosts both Fe(2+) and Fe(3+), occupies a highly anionic surface of the molecule. This function is conserved across species since the corresponding region of human frataxin is also able to bind iron, albeit with weaker affinity. The presence of secondary binding sites on CyaY, but not on frataxin, hints at a possible polymerization mechanism. We suggest mutations that may provide further insights into the frataxin function.     

The insulin receptor substrate 1 (IRS1) in intestinal epithelial differentiation and in colorectal cancer

Colorectal cancer (CRC) is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1) is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and in vitro polarizing Caco2 and HT29 cells. IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Analysis of IRS1 immunostaining in 24 cases of primary CRC with paired colonic epithelium and hepatic metastasis showed that staining intensity was significantly higher in metastases relative to both primary CRC (P<0.01) and colonic epithelium (P<0.01). Primary and metastatic CRCs, compared to colonic epithelium, contained significantly higher numbers of IRS1-positive cells (P = 0.013 and P = 0.014, respectively). Pathologic correlations in 163 primary CRCs revealed that diffuse IRS1 staining was associated with tumors combining differentiated phenotype and aggressive markers (high Ki67, p53, and ß-catenin). In Caco 2 IRS1 and InsR were maximally expressed after polarization, while IGF1R was highest in pre-polarized cells. No nuclear IRS1 was detected, while, with polarization, phosphorylated IRS1 (pIRS1) shifted from the lateral to the apical plasma membrane and was expressed in surface cells only. In HT29, that carry mutations constitutively activating survival signaling, IRS1 and IGF1R decreased with polarization, while pIRS1 localized in nuclear spots throughout the course. Overall, these data provide evidence that IRS1 is modulated according to CRC differentiation, and support a role of IRS1 in CRC progression and liver metastatization.     

Characterization of the structure and the amyloidogenic properties of the Josephin domain of the polyglutamine-containing protein ataxin-3

Expansion of the polyglutamine (polyQ) region in the protein ataxin-3 is associated with spinocerebellar ataxia type 3, an inherited neurodegenerative disorder that belongs to the family of polyQ diseases. Increasing evidence indicates that protein aggregation and fibre formation play an important role in these pathologies. In a previous study, we determined the domain architecture of ataxin-3, suggesting that it comprises a globular domain, named Josephin, and a more flexible C-terminal region, that includes the polyQ tract. Here, we have characterised for the first time the biophysical properties of the isolated Josephin motif, showing that it is an autonomously folded unit and that it has no significant interactions with the C-terminal region. Study of its thermodynamic stability indicates that Josephin has an intrinsic tendency to aggregate and forms temperature-induced fibrils similar to those described for expanded ataxin-3. We show that, under destabilising conditions, the behaviours of the isolated Josephin domain and ataxin-3 are extremely similar. Our data therefore strongly suggest that the stability and aggregation properties of non-expanded ataxin-3 are determined by those of the Josephin domain, which is sufficient to reproduce the behaviour of the full-length protein. Our data support a mechanism in which the thermodynamic stability of ataxin-3 is governed by the properties of the Josephin domain, but the presence of an expanded polyQ tract increases dramatically the protein's tendency to aggregate.     

Determination of the post mortem interval in skeletal remains by the comparative use of different physico-chemical methods: Are they reliable as an alternative to 14C?

The determination of the post-mortem interval (PMI) of skeletal remains is a challenging aspect in the forensic field. Previous studies focused their attention on different macroscopic and morphological aspects but a thorough and complete evaluation of the potential of chemical and physical analyses in this field of research has not been performed. In addition to luminol test and Oxford histology index (OHI) reported in a recent paper, widely spread and accessible methods based on physical aspect and chemical characteristics of skeletal remains have been investigated as potential alternatives to dating by determination of ¹⁴C.The investigation was performed on a total of 24 archeological and forensic bone samples with known PMI, with inductively coupled plasma optical emission spectrometer (ICP-OES), inductively coupled plasma quadruple mass spectrometry (ICP-MS), Fourier transform infrared (FT-IR) spectroscopy, energy dispersive X-ray analysis (EDX), powder X-ray diffraction analysis (XRPD) and scanning electron microscopy (SEM). Finally, the feasibility of such alternative methods was discussed. Some results such as carbonates/phosphates ratio from FT-IR, the amounts of organic and inorganic matter by EDX, crystallite sizes with XRPD, and surface morphology obtained by SEM, showed significant trends along with PMI. Though, from a chemical point of view cut-off values and gold-standard methods still present challenges, and rather different techniques together can provide useful information toward the assessment of the PMI of skeletal remains. It is however clear that in a hypothetical flowchart those methods may be placed practically at the same level and a choice should always consider the evaluation of results by each technique, execution times and a costs/benefits relationship.