Astaxanthin from Haematococcus pluvialis Prevents Oxidative Stress on Human Endothelial Cells without Toxicity

Astaxanthin, a powerful antioxidant, is a good candidate for the prevention of intracellular oxidative stress. The aim of the study was to compare the antioxidant activity of astaxanthin present in two natural extracts from Haematococcus pluvialis, a microalgae strain, with that of synthetic astaxanthin. Natural extracts were obtained either by solvent or supercritical extraction methods. UV, HPLC-DAD and (HPLC-(atmospheric pressure chemical ionization (APCI)+)/ion trap-MS) characterizations of both natural extracts showed similar compositions of carotenoids, but different percentages in free astaxanthin and its ester derivatives. The Trolox equivalent antioxidant capacity (TEAC) assay showed that natural extracts containing esters displayed stronger antioxidant activities than free astaxanthin. Their antioxidant capacities to inhibit intracellular oxidative stress were then evaluated on HUVEC cells. The intracellular antioxidant activity in natural extracts was approximately 90-times higher than synthetic astaxanthin (5 µM). No modification, neither in the morphology nor in the viability, of vascular human cells was observed by in vitro biocompatibility study up to 10 µM astaxanthin concentrations. Therefore, these results revealed the therapeutic potential of the natural extracts in vascular human cell protection against oxidative stress without toxicity, which could be exploited in prevention and/or treatment of cardiovascular diseases.     

Butterfly dispersal in farmland: a replicated landscape genetics study on the meadow brown butterfly (<Emphasis Type="Italic">Maniola jurtina</Emphasis>)

Context

Anthropogenic activities readily result in the fragmentation of habitats such that species persistence increasingly depends on their ability to disperse. However, landscape features that enhance or limit individual dispersal are often poorly understood. Landscape genetics has recently provided innovative solutions to evaluate landscape resistance to dispersal.

Objectives

We studied the dispersal of the common meadow brown butterfly, Maniola jurtina, in agricultural landscapes, using a replicated study design and rigorous statistical analyses. Based on existing behavioral and life history research, we hypothesized that the meadow brown would preferentially disperse through its preferred grassy habitats (meadows and road verges) and avoid dispersing through woodlands and the agricultural matrix.

Methods

Samples were collected in 18 study landscapes of 5 × 5 km in three contrasting agricultural French regions. Using circuit theory, least cost path and transect-based methods, we analyzed the effect of the landscape on gene flow separately for each sex.

Results

Analysis of 1681 samples with 6 microsatellites loci revealed that landscape features weakly influence meadow brown butterfly gene flow. Gene flow in both sexes appeared to be weakly limited by forests and arable lands, whereas grasslands and grassy linear elements (road verges) were more likely to enhance gene flow.

Conclusion

Our results are consistent with the hypothesis of greater dispersal through landscape elements that are most similar to suitable habitat. Our spatially replicated landscape genetics study allowed us to detect subtle landscape effects on butterfly gene flow, and these findings were reinforced by consistent results across analytical methods.

     

Non‐spherical pearl layers in the Polynesian ‘black‐lipped’ Pinctada margaritifera: The non‐nacreous deposits compared to microstructure of the shell growing edge

Cultivated pearls frequently exhibit morphological irregularities making obvious that mineral deposition was irregularly distributed onto nucleus surface. Taking advantage of experimental cultivations with short durations (from 10 days to few months), these irregular deposits predating occurrence of the nacre were investigated in Polynesian pearls by biochemical characterizations and a series of physical methods. Diversity in the resulting data suggests that various in‐depth alterations of the biomineralization mechanism may have occurred during the grafting process, leading to diversity in the biochemical pathways to nacreous deposition. This allows a precise discussion of current views about pearl formation. The “reversed shell theory” is formally disproved through point to point comparison with development of the shell growing edge. Similarity of pearl formation with “regeneration” or “shell repair” is also discussed, emphasizing the differences between these concepts.     

Identification of a Pro-Angiogenic Potential and Cellular Uptake Mechanism of a LMW Highly Sulfated Fraction of Fucoidan from Ascophyllum nodosum

Herein we investigate the structure/function relationships of fucoidans from Ascophyllum nodosum to analyze their pro-angiogenic effect and cellular uptake in native and glycosaminoglycan-free (GAG-free) human endothelial cells (HUVECs). Fucoidans are marine sulfated polysaccharides, which act as glycosaminoglycans mimetics. We hypothesized that the size and sulfation rate of fucoidans influence their ability to induce pro-angiogenic processes independently of GAGs. We collected two fractions of fucoidans, Low and Medium Molecular Weight Fucoidan (LMWF and MMWF, respectively) by size exclusion chromatography and characterized their composition (sulfate, fucose and uronic acid) by colorimetric measurement and Raman and FT-IR spectroscopy. The high affinities of fractionated fucoidans to heparin binding proteins were confirmed by Surface Plasmon Resonance. We evidenced that LMWF has a higher pro-angiogenic (2D-angiogenesis on Matrigel) and pro-migratory (Boyden chamber) potential on HUVECs, compared to MMWF. Interestingly, in a GAG-free HUVECs model, LMWF kept a pro-angiogenic potential. Finally, to evaluate the association of LMWF-induced biological effects and its cellular uptake, we analyzed by confocal microscopy the GAGs involvement in the internalization of a fluorescent LMWF. The fluorescent LMWF was mainly internalized through HUVEC clathrin-dependent endocytosis in which GAGs were partially involved. In conclusion, a better characterization of the relationships between the fucoidan structure and its pro-angiogenic potential in GAG-free endothelial cells was required to identify an adapted fucoidan to enhance vascular repair in ischemia.     

CRISPR/Cas9-Targeted Knockout of Rice Susceptibility Genes OsDjA2 and OsERF104 Reveals Alternative Sources of Resistance to Pyricularia oryzae

Rice genes OsDjA2 and OsERF104, encoding a chaperone protein and an APETELA2/ ethylene-responsive factor, respectively, are strongly induced in a compatible interaction with blast fungus, and also have function in plant susceptibility validated through gene silencing. Here, we reported the CRISPR/Cas9 knockout of OsDjA2 and OsERF104 genes resulting in considerable improvement of blast resistance. A total of 15 OsDjA2 (62.5%) and 17 OsERF104 (70.8%) T₀ transformed lines were identified from 24 regenerated plants for each target and used in downstream experiments. Phenotyping of homozygous T₁ mutant lines revealed not only a significant decrease in the number of blast lesions but also a reduction in the percentage of diseased leaf area, compared with the infected control plants. Our results supported CRISPR/Cas9-mediated target mutation in rice susceptibility genes as a potential and alternative breeding strategy for building resistance to blast disease.     

Two major evolutionary lineages revealed by molecular phylogeny in the parthenogenetic collembola species Folsomia candida

In order to measure the genetic variability and determine the evolutionary relationships among strains of the parthenogenetic “standard” springtail Folsomia candida, we used Random Amplified Polymorphic DNA (RAPD-PCR) markers and determined the nucleotide sequence of the 18S and 28S rDNA genes. Both types of molecular characters were found to be polymorphic. We obtained phylogenetic trees using Direct Optimization in the dynamic homology paradigm. The trees were polarized with Isotoma viridis as an outgroup. All the trees based on one or the other type of molecular characters or based on all characters pooled together, support the hypothesis of an early divergence of two distinct lineages among the 11 strains of F. candida under study. Our results also suggest that these lineages differ in their rate of evolution and mode of diversification. The geographical origin of the studied strains was examined but we found no clear relation between the phylogenetic relationships and probable geographical origins. The early divergence of several lineages in this species should be taken into account when comparing studies on genetically different strains of this model organism. RAPD-PCR typing is an easy and efficient tool for doing such a task.     

Root cone angle is enlarged in <Emphasis Type="Italic">docs1</Emphasis> LRR-RLK mutants in rice

Background

The DEFECTIVE IN OUTER CELL LAYER SPECIFICATION 1 (DOCS1) gene belongs to the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) subfamily. It has been discovered few years ago in Oryza sativa (rice) in a screen to isolate mutants with defects in sensitivity to aluminum. The c68 (docs1–1) mutant possessed a nonsense mutation in the C-terminal part of the DOCS1 kinase domain.

Findings

We have generated a new loss-of-function mutation in the DOCS1 gene (docs1–2) using the CRISPR-Cas9 technology. This new loss-of-function mutant and docs1–1 present similar phenotypes suggesting the original docs1–1 was a null allele. Besides the aluminum sensitivity phenotype, both docs1 mutants shared also several root phenotypes described previously: less root hairs and mixed identities of the outer cell layers. Moreover, our new results suggest that DOCS1 could also play a role in root cap development. We hypothesized these docs1 root phenotypes may affect gravity responses. As expected, in seedlings, the early gravitropic response was delayed. Furthermore, at adult stage, the root gravitropic set angle of docs1 mutants was also affected since docs1 mutant plants displayed larger root cone angles.

Conclusions

All these observations add new insights into the DOCS1 gene function in gravitropic responses at several stages of plant development.

     

Antinociceptive activity of pre- versus post-operative intra-articular bupivacaine in goats undergoing stifle arthrotomy

ObjectiveTo evaluate the peri-operative analgesic efficacy of intra-articular bupivacaine administered before or after stifle arthrotomy.Study designProspective, randomized, blind, placebo-controlled experimental trial.AnimalsThirty-nine healthy goats.MethodsThe goats were allocated randomly to one of three intra-articular treatment groups: group PRE (bupivacaine before and saline after surgery), group POST (saline before and bupivacaine after surgery) and group CON (saline before and after surgery). Anaesthesia was maintained with a constant end-tidal sevoflurane of 2.5%. Intra-operatively heart rate (HR), respiratory rate and mean arterial blood pressure (MAP) after critical surgical events (CSE) were recorded and compared with pre-incision values. Propofol requirements to maintain surgical anaesthesia were recorded. Flunixin was administered for 5 days. Post-operative pain assessment at 20 minutes, 2 hours, 4 hours after recovery and on day 2 and 3 included a multidimensional pain score (MPS), a lameness score and mechanical nociceptive threshold (MNT) testing. Rescue analgesia consisted of systemic opioids. Data were analysed using Kruskal–Wallis, Mann–Whitney, Friedman or chi-square tests as appropriate.ResultsIntra-operatively, group PRE had lower HR and MAP at several CSEs than groups POST/CON and required less propofol [0 mg kg^?1 (0–0 mg kg^?1)] than group POST/CON [0.3 mg kg^?1 (0–0.6 mg kg^?1)]. Post-operatively, group POST had significantly higher peri-articular MNTs than groups PRE and CON up to 4 hours after recovery. No treatment effect was detected for MPS, lameness scores and rescue analgesic consumption at any time point.Conclusions and clinical relevancePre-operative intra-articular bupivacaine provided notable intra-operative analgesia in goats undergoing stifle arthrotomy but did not reduce post-operative pain. Post-operative intra-articular bupivacaine provided a short lasting reduction of peri-articular hyperalgesia without affecting the requirements for systemic analgesia. Multimodal perioperative pain therapy is recommended to provide adequate analgesia for stifle arthrotomy in goats.