Self assembly of human septin 2 into amyloid filaments

Septins are a conserved group of GTP-binding proteins that form hetero-oligomeric complexes which assemble into filaments. These are essential for septin function, including their role in cytokinesis, cell division, exocytosis and membrane trafficking. Septin 2 (SEPT2) is a member of the septin family and has been associated with neurofibrillary tangles and other pathological features of senile plaques in Alzheimer's disease. An in silico analysis of the amino acid sequence of SEPT2 identified regions with a significant tendency to aggregate and/or form amyloid. These were all observed within the GTP-binding domain. This was consistent with the experimental identification of a structure rich in β-sheet during temperature induced unfolding transitions observed for both the full length protein and the GTP-binding domain alone. This intermediate state is characterized by irreversible aggregation and has the ability to bind Thioflavin-T, suggesting its amyloid nature. Under electron microscopy, fibers extending for several micrometers in length could be visualized. The results shown in this study support the hypothesis that single septins, when present in excess or with unbalanced stoichiometries, may be unstable and assemble into amyloid-like structures.     

Cryopreservation of some useful microalgae species for biotechnological exploitation

Culture collections of microalgae represent a biological resource for scientific research and biotechnological applications. When compared to the current methods of maintenance and sub-culturing, cryopreservation minimizes labor costs and is an effective method for maintaining a large range of species over long periods with high stability. In order to determine the best cryopreservation method for microalgae species with great biotechnological potential, three freezing protocols were employed using different cryoprotectants (dimethyl sulfoxide—Me₂SO; methanol—MeOH). Three marine microalgae species (Thalassiosira weissflogii; Nannochloropsis oculata, and Skeletonema sp.) were cooled by directly plunging into liquid nitrogen (?196°C) and with two-step controlled cooling protocols (?18°C and ?80°C pre-treatments). After storage periods ranging from 10 to 120 days, viability was determined by the ability of cells to actively grow again. Results obtained for T. weissflogii showed that this species could be preserved at ultra-low temperature (?196°C) for 10 and 30 days with 10?% Me₂SO and 5?% MeOH when employed a controlled cooling protocol (?80°C). N. oculata was successfully cryopreserved either by direct freezing or with controlled cooling protocols. N. oculata samples presented good responses when treated with 5?% Me₂SO, 10?% Me₂SO, 5?% MeOH and even without any cryoprotectant. Skeletonema sp. did not survive cryopreservation in any of the tested conditions. The results indicate the difficulty in establishing common protocols for different microalgae species, being necessary further studies for a better understanding of cell damages during freezing and thawing conditions for each species.     

Climatic impacts and drought control of radial growth and seasonal wood formation in Pinus halepensis

Short- and long-term growth responses to drought and climatic influences still remain poorly understood. In this study, we investigated the impact of climatic drivers (temperature, precipitation) and drought, using the Standardized Precipitation Index (SPI) calculated at different time scales (1–48?months), on earlywood (EW) and latewood (LW) widths in Pinus halepensis. Nine forests subjected to dry summer conditions were sampled in Mediterranean semi-arid areas from north-eastern Spain. In addition, we explored the seasonal dynamics of cambial activity and wood formation in relation to short-term climate variability. We found two peaks of tracheid cell production corresponding to EW (May–June) and LW (mid-July–August) growth phases, associated with a sharp decrease in enlarging cells in early July in response to low water availability. In the period of analysis (1970–2005), EW growth was positively correlated with precipitation in previous December and current January, April, May and June, while it was negatively correlated with temperature in June and July. LW was correlated positively with minimum temperatures in January. Probably this was an indirect relationship as a consequence of increased EW width at higher January temperatures. Drought affected more negatively EW than LW formation as evidenced the higher SPI-EW correlation (r?=?0.72) than the SPI-LW one (r?=?0.54). The strongest EW response to drought was observed in July, whereas the highest LW response to drought occurred in September; and this seasonal pattern matched the phases of lowest EW and LW tracheid production. Under a future reduction of winter and spring precipitation, the studied forests may show a decrease in tracheid cell production, causing a decline of radial growth, a reduction in hydraulic conductivity and, indirectly, a hampered carbon uptake in such semi-arid woodlands.     

N-glycan biosynthesis inhibitors induce in vitro anticancer activity in colorectal cancer cells

During malignant transformation, changes in the expression profile of glycans may be involved in a variety of events, including the loss of cell-cell and cell-matrix adhesion, migration, invasion, and evasion of apoptosis. Therefore, modulation of glycan expression with drugs has promising therapeutic potential for various cancer types. In this study, we investigated the in vitro anticancer activity of the N-glycan biosynthesis inhibitors (swainsonine and tunicamycin) in cells derived from colorectal cancer (CRC). We also examined whether these inhibitors are able to induce radiosensitization and toxicity when used in combination with cisplatin or irinotecan, two current anticancer drugs. Our results show that treatment with tunicamycin inhibits cellular mechanisms related to the malignant phenotype, such as anchorage-dependent and anchorage-independent colony formation, migration and invasion, in undifferentiated HCT-116 colon cancer cells, whereas swainsonine only inhibits cell migration. We also observed that tunicamycin, but not swainsonine, caused radiosensitivity in HCT-116 cells. Moreover, the combination of swainsonine with cisplatin or irinotecan enhanced their toxicity in HCT-116 cells, while the combination of tunicamycin with these drugs had no effect. Given these results, we suggest that the modulation of N-glycan biosynthesis appears to be a potential therapeutic tool for CRC treatment because inhibition of this process induced anticancer activity in vitro. Additionally, the inhibition of the N-glycan biosynthesis in combination with chemotherapic drugs is a promising therapeutic strategy for enhancing radiation therapy.     

Molecular population genetics of the insulin/TOR signal transduction pathway: a network-level analysis in Drosophila melanogaster

The IT-insulin/target of rapamycin (TOR)-signal transduction pathway is a relatively well-characterized pathway that plays a central role in fundamental biological processes. Network-level analyses of DNA divergence in Drosophila and vertebrates have revealed a clear gradient in the levels of purifying selection along this pathway, with the downstream genes being the most constrained. Remarkably, this feature does not result from factors known to affect selective constraint such as gene expression, codon bias, protein length, and connectivity. The present work aims to establish whether the selective constraint gradient detected along the IT pathway at the between-species level can also be observed at a shorter time scale. With this purpose, we have surveyed DNA polymorphism in Drosophila melanogaster and divergence from D. simulans along the IT pathway. Our network-level analysis shows that DNA polymorphism exhibits the same polarity in the strength of purifying selection as previously detected at the divergence level. This equivalent feature detected both within species and between closely and distantly related species points to the action of a general mechanism, whose action is neither organism specific nor evolutionary time dependent. The detected polarity would be, therefore, intrinsic to the IT pathway architecture and function.     

Mechanical unfolding pathways of the enhanced yellow fluorescent protein revealed by single molecule force spectroscopy

We used single molecule force spectroscopy to characterize the mechanical stability of the enhanced yellow fluorescent protein (EYFP) (a mutant form of the green fluorescent protein (GFP)) and two of its circularly permutated variants. In all three constructs, we found two main unfolding peaks; the first corresponds to a transition state placed close to the termini and the second to a transition state placed halfway through the molecule. We attribute the second transition state to the shear rupture of the beta1- and beta6-strands, which we verified by introducing a point mutation in this region. Although both unfolding peaks were observed in all three EYFP variants, their relative frequency of occurrence varied. Our results demonstrated that the mechanical unfolding pathways in EYFP could be deciphered through the use of circular permutation.     

Stepwise unfolding of ankyrin repeats in a single protein revealed by atomic force microscopy

Using single-molecule atomic force microscopy, we find that a protein consisting of six identical ankyrin repeat units flanked by N- and C-terminal modules (N6C) unfolds in a stepwise, unit-by-unit fashion under a mechanical force. Stretching a N6C molecule results in a sawtooth pattern fingerprint, with as many as six peaks separated by approximately 10 nm and an average unfolding force of 50 +/- 20 pN. Our results demonstrate that a stretching force can unfold multiple repeat units individually in a single protein molecule, despite extensive hydrophobic interactions between adjacent units.     

Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.