Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition

Actin-based stress fiber formation is coupled to microtubule depolymerization through the local activation of RhoA. While the RhoGEF Lfc has been implicated in this cytoskeleton coupling process, it has remained elusive how Lfc is recruited to microtubules and how microtubule recruitment moderates Lfc activity. Here, we demonstrate that the dynein light chain protein Tctex-1 is required for localization of Lfc to microtubules. Lfc residues 139-161 interact with Tctex-1 at a site distinct from the cleft that binds dynein intermediate chain. An NMR-based GEF assay revealed that interaction with Tctex-1 represses Lfc nucleotide exchange activity in an indirect manner that requires both polymerized microtubules and phosphorylation of S885 by PKA. We show that inhibition of Lfc by Tctex-1 is dynein dependent. These studies demonstrate a pivotal role of Tctex-1 as a negative regulator of actin filament organization through its control of Lfc in the crosstalk between microtubule and actin cytoskeletons.     

Nothofagus dombeyi regeneration in declining Austrocedrus chilensis forests: Effects of overstory mortality and climatic events

This research examines the regeneration dynamics of Nothofagus dombeyi and Austrocedrus chilensis in A. chilensis-dominated forests growing near the eastern limit of N. dombeyi where precipitation is limiting. In these forests the widespread decline and mortality of overstory A. chilensis trees, known as ‘mal del ciprés’ (cypress sickness), generates large canopy gaps in which new individuals establish. Our objective was to study the population dynamics of N. dombeyi and A. chilensis in these forests to investigate the influences of overstory tree death and climatic variation on establishment. We sampled 6 symptomatic A. chilensis stands and used dendrochronological techniques to reconstruct basal area development and regeneration establishment over time. Bivariate event analysis was performed to examine the temporal relationships between tree establishment and mortality events and climatic variation. Overstory A. chilensis trees established as post-fire cohorts, with subsequent establishment of A. chilensis and N. dombeyi during the past 50–60 years. Regeneration in the past two decades was primarily N. dombeyi. The establishment of both A. chilensis and N. dombeyi was synchronous with overstory tree mortality events, but it was more consistent among stands and prolonged for N. dombeyi. Establishment of A. chilensis was not associated with climatic events but N. dombeyi establishment was synchronous with droughts, possibly related to climate-driven mortality creating canopy gaps or reducing competition within gaps. We have demonstrated that N. dombeyi has the ability to establish in post-fire A. chilensis-dominated forests resulting in mixed-species, uneven-aged forests. The ongoing increase in the abundance of N. dombeyi relative to A. chilensis represents a shift in composition and increased complexity in stand structure driven by ‘mal del ciprés’ and climatic variation.     

Cognitive Functions in Middle Aged Individuals Are Related to Metabolic Disturbances and Aerobic Capacity: A Cross-Sectional Study

Aims

Metabolic disturbances may contribute to cognitive dysfunction in patients with type 2 diabetes. We investigated the relation between cognitive impairment and metabolic deteriorations, low physical fitness, low-grade inflammation and abdominal obesity in middle aged individuals.

Methods

We conducted a cross-sectional study including 40 to 65 year-old patients with type 2 diabetes and limited co morbidity (N = 56), age-matched individuals with impaired glucose tolerance (N = 56) as well as age-matched controls with normal glucose tolerance (N = 72). Specific cognitive functions were assessed with focus on verbal memory, processing speed, executive functions, and a composite overall mean score. Oral glucose tolerance test, VO₂max test, systemic inflammation, DXA scanning and abdominal MRI were measured.

Results

Multiple linear regression analyses adjusting for age, gender and verbal intelligence demonstrated that a low score in processing speed, executive functions and overall cognitive function were related to high fasting C-peptide, as well as low insulin sensitivity, beta-cell function and VO₂max. Measurements of blood glucose, obesity and inflammation were not associated with cognitive function.

Conclusion

Low cognitive scores are seen in middle aged individuals with hyperinsulinemia, low insulin sensitivity, beta-cell function and low aerobic capacity. These findings emphasize the importance of appropriate lifestyle and not only blood glucose control in prevention of cognitive disability.     

Further Evidence of Subphenotype Association with Systemic Lupus Erythematosus Susceptibility Loci: A European Cases Only Study

Introduction

Systemic Lupus Erythematosus (SLE) shows a spectrum of clinical manifestations that complicate its diagnosis, treatment and research. This variability is likely related with environmental exposures and genetic factors among which known SLE susceptibility loci are prime candidates. The first published analyses seem to indicate that this is the case for some of them, but results are still inconclusive and we aimed to further explore this question.

Methods

European SLE patients, 1444, recruited at 17 centres from 10 countries were analyzed. Genotypes for 26 SLE associated SNPs were compared between patients with and without each of 11 clinical features: ten of the American College of Rheumatology (ACR) classification criteria (except ANAs) and age of disease onset. These analyses were adjusted for centre of recruitment, top ancestry informative markers, gender and time of follow-up. Overlap of samples with previous studies was excluded for assessing replication.

Results

There were three new associations: the SNPs in XKR6 and in FAM167A-BLK were associated with lupus nephritis (OR = 0.76 and 1.30, P_corr = 0.007 and 0.03, respectively) and the SNP of MECP2, which is in chromosome X, with earlier age of disease onset in men. The previously reported association of STAT4 with early age of disease onset was replicated. Some other results were suggestive of the presence of additional associations. Together, the association signals provided support to some previous findings and to the characterization of lupus nephritis, autoantibodies and age of disease onset as the clinical features more associated with SLE loci.

Conclusion

Some of the SLE loci shape the disease phenotype in addition to increase susceptibility to SLE. This influence is more prominent for some clinical features than for others. However, results are only partially consistent between studies and subphenotype specific GWAS are needed to unravel their genetic component.     

Transient transfection of purified Babesia bovis merozoites

Transient transfection of intraerythrocytic Babesia bovis parasites has been previously reported. In this study, we describe the development and optimization of methods for transfection of purified B. bovis merozoites using either nucleofection (Amaxa) or conventional electroporation (Gene Pulser II, BioRad). Initially, the optimal buffer ("Plasmodium 88A6") and program (v-24) for nucleofection of free merozoites with a plasmid containing the luciferase gene as a reporter were determined. Using the same reporter plasmid, optimal voltage, capacitance and resistance for transfecting free merozoites by electroporation were defined to be 1.2 kV/25 microF/200 Omega. Using these optimal parameters, analysis of the time course of luciferase expression using either system to transfect free B. bovis merozoites showed high enzyme activity at 24h, with a rapid decline thereafter. Nucleofection was approximately five times more effective than electroporation when using a small quantity (2 microg) of DNA, while electroporation was twice as effective as nucleofection when a larger quantity of plasmid DNA (100 microg) was used. Parasite viability was significantly higher when using nucleofection when compared to electroporation regardless of the amount of DNA used. Comparison of luciferase expression after transfection of merozoites with circular, linearized, or double digested plasmid indicated that intact, circular plasmid was necessary for optimal luciferase expression. Overall, the results provide a basis for optimal transfection of purified B. bovis merozoites using either nucleofection or conventional electroporation. However, nucleofection is significantly more efficient when transfecting either circular or restriction digested DNA in the 2-10 microg range.     

The Restoration of Degraded Mountain Woodlands: Effects of Seed Provenance and Microsite Characteristics on Polylepis australis Seedling Survival and Growth in Central Argentina

South American high‐mountain ecosystems are greatly influenced by human disturbance. In the mountains of Córdoba, Argentina, Polylepis australis (Rosaceae) woodlands are currently highly fragmented and subject to extensive burning and livestock grazing, resulting in severe changes of habitat characteristics, which hamper natural regeneration. In order to find out how to achieve successful reforestation, we compared P. australis seedling survival and growth and the development of a shrubby habit for two seed provenances and different planting microsites. Survival of planted seedlings after 5 years was 70%, with most deaths (19%) in the first year and declining mortality with ongoing establishment. Survival did not show any relationship with seed provenance or microsite characteristics. Height growth averaged 34.6 ± 1.2 cm in 5 years. Seedlings produced from seeds collected in a well‐preserved woodland grew taller and showed a higher tendency for development of shrubby habit than those produced from seeds collected in a degraded woodland. Seedlings planted in more degraded microsites with exposed soil or rock due to past grazing pressure grew less and developed a more shrubby habit than those planted in better preserved microsites. Our results show that restoration of degraded areas with P. australis is possible and that there is potential to improve restoration success with a careful selection of seed provenance and planting microsites.     

Mechanisms of mutagenesis in mammalian cells. Application to human thyroid tumours

Mutations are defined as stable and irreversible modifications of the normal genetic message due to small changes in the number or type of bases, or to large modifications of the genome such as deletions, insertions or chromosome rearrangements. These lesions are due to either polymerase errors during normal DNA replication or unrepaired DNA lesions, which will give rise to mutations through a mutagenic pathway. The molecular process leading to mutagenesis depends largely on the type of DNA lesions. Base modifications, such as 8-oxo-guanine or thymine glycol, both induced by ionizing radiations (IR), are readily replicated leading to direct mutations, usually base-pair substitutions. The 8-oxo-G gives rise predominantly to G to T transversions, the type of mutations found in ras or p53 gene from IR-induced tumors. Bulky adducts produced by chemical carcinogens or UV-irradiation are usually repaired by the nucleotide excision repair (NER) pathway which is able to detect structural distortion in the normal double-strand DNA backbone. These lesions represent a blockage to DNA and RNA polymerases as well as some signal for p53 accumulation in the damaged cell. In the absence of repair, these lesions could be eventually replicated owing to the induction of specific proteins at least in bacteria during the SOS process. The precise nature of the error-prone replication across an unexcised DNA lesion in the template is not fully understood in detailed biochemical terms, in mammalian cells. IR basically produce a very large number of DNA lesions from unique base modifications to single- or double-strand breaks and even complex DNA lesions due to the passage of very high energy particles or to a local re-emission of numerous radicals. The breakage of the double-helix is a difficult lesion to repair. Either it will result in cell death or, after an incorrect recombinational pathway, it will induce frameshifts, large deletions or chromosomal rearrangements. Most of the IR-induced mutations are recessive ones, requiring therefore a second genetic event in order to exhibit any harmful effect and a long latency period before the development of a radiation-induced tumor. The fact that IR essentially induced deletions and chromosomal translocations renders very difficult the use of the p53 gene as a marker for mutation analysis. In agreement with the type of lesions induced by IR, it is interesting to point out that the presence has been observed, in a vast majority of radiation-induced papillary thyroid carcinomas (PTC), of an activated ret proto-oncogene originated by the fusion of the tyrosine kinase 3' domain of this gene with the 5' domain of four different genes. These ret chimeric genes which are due to intra- or inter-chromosomal translocations, were called RET/PTC1 to PTC5. The RET/PTC rearrangements were found in PTC from children contaminated by the Chernobyl fall-out as well as in tumours from patients with a history of therapeutic external radiation, with a frequency of 60-84%. This frequency was only 15% in 'spontaneous' PTC. The type of ret chimeric gene predominantly originated by the accidental or therapeutic IR was different. Indeed, PTC1 was present in 75% of the tumours linked to a therapeutic radiation and PTC3 in 75% of the Chernobyl ones. The other forms of RET/PTC were observed in only a minority of the post-Chernobyl PTC (< 20%). The difference in the frequency of PTC1 and PTC3 in both types of PTC, is statistically significant (P < 10(-5), Fischer's exact test). In two of the post-therapeutic radiation PTC, RET/PTC1 and PTC3 were simultaneously present. A PTC1 gene was also observed in 45% of the adenomas appearing after therapeutic radiation. The long-period of latency between exposure to IR and the appearance of thyroid tumours is probably due to the conversion of a heterozygote genotype of IR-induced mutations to a homozygote one. It will be interesting to use this time lag in accidental or therapeutic-irradiated p     

Irradiation and second cancers. The thyroid as a case in point

The thyroid gland is highly sensitive to radiation during childhood: the risk of thyroid tumours is increased for mean doses as low as 100 mGy and for higher doses, the risk increases linearly with the dose. Excess relative risk is important, being 7.7 for 1 Gy delivered to the thyroid gland during childhood. The risk of thyroid tumours is modified by several factors: a) age at exposure: in childhood, the risk decreases with increasing age at exposure and is not significant after 20 years; b) gender: females are two times more likely than males to develop thyroid tumours; c) genetic predisposition due to a defect in DNA repair mechanisms, and dietary and hormonal factors may modify the risk; d) the influence of fractionation and dose rate is not well established. Radioiodine 131 (1311) used for medical purposes has almost no tumourigenic effect on the adult thyroid gland. The consequences of the Chernobyl accident have clearly shown that the risk of thyroid cancer after exposure to 1311 in childhood is important, and that such exposure should be prevented by potassium iodine prophylaxis. RET/PTC rearrangements are found in 60-80% of papillary carcinomas and in 45% of adenomas occurring after radiation exposure. They are found in 5-15% of papillary carcinoma and in no follicular adenomas that occurred in the absence of radiation exposure.     

Effect of high boron application on boron content and growth of melons

Synthetic chelates, such as ethylene diamine tetraacetic acid (EDTA), have been shown to enhance phytoextraction of Pb from contaminated soil but also cause leaching of heavy metal-chelate complexes, posing a groundwater contamination threat. In a soil column study, we examined the effect of EDTA and a biodegradable chelate [S,S] isomere of ethylene diamine disuccinate ([S,S]-EDDS), newly introduced in phytoextraction research, on the uptake of Pb by the Chinese cabbage (Brassica rapa) and Pb leaching through the soil profile. Soil water sorption characteristics were modified by acrylamide hydrogel. The addition of 0.1 and 0.2% (w/w) of hydrogel amendments increased soil field water capacity from initial 24.6% to 28.5% and 31.3%, respectively. The additions of 2.5, 5 and 10 mmol EDTA kg^–1 soil were more effective in enhancing Pb plant uptake than comparable [S,S]-EDDS treatments, but caused (as also 10 mmol kg^–1 [S,S]-EDDS additions) unacceptably high Pb leaching in treatments with any soil water sorption conditions tested. The most efficient level of EDTA (10 mmol kg^–1) enhanced plant Pb uptake by 97 times compared to the control. Shoots Pb concentrations reached 500 mg kg^–1 of dry biomass. However, in this treatment 36.2% of total initial Pb was leached from the soil during the first four weeks after chelate addition. Hydrogel soil amendments were more effective in treatments with [S,S]-EDDS than with EDTA. In treatments with 10 mmol kg^–1[S,S]-EDDS hydrogel amended soils, plant Pb uptake was significantly reduced and Pb leach was as high as 44.2% of total initial soil Pb. At lower [S,S]-EDDS concentrations, the effect of hydrogel soil amendment on Pb leaching was the opposite. The addition of 5 mmol kg^–1 [S,S]-EDDS soil to the soil amended with 0.2% hydrogel increased Pb uptake by 18 times while only 0.2% of total initial Pb was leached. In all treatments, the concentrations of Pb in dry plant biomass were far from concentrations required for efficient soil remediation within a reasonable time span.