Protein-induced surface structuring in myelin membrane monolayers

Monolayers prepared from myelin conserve all the compositional complexity of the natural membrane when spread at the air-water interface. They show a complex pressure-dependent surface pattern that, on compression, changes from the coexistence of two liquid phases to a viscous fractal phase embedded in a liquid phase. We dissected the role of major myelin protein components, myelin basic protein (MBP), and Folch-Lees proteolipid protein (PLP) as crucial factors determining the structural dynamics of the interface. By analyzing mixtures of a single protein with the myelin lipids we found that MBP and PLP have different surface pressure-dependent behaviors. MBP stabilizes the segregation of two liquid phases at low pressures and becomes excluded from the film under compression, remaining adjacent to the interface. PLP, on the contrary, organizes a fractal-like pattern at all surface pressures when included in a monolayer of the protein-free myelin lipids but it remains mixed in the MBP-induced liquid phase. The resultant surface topography and dynamics is regulated by combined near to equilibrium and out-of-equilibrium effects. PLP appears to act as a surface skeleton for the whole components whereas MBP couples the structuring to surface pressure-dependent extrusion and adsorption processes.     

Engineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA

One of the most severe diseases of cultivated tomato worldwide is caused by tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci. Here we describe the application of antisense RNAs to interfere with the disease caused by TYLCV. The target of the antisense RNA is the rare messenger RNA of the Rep protein, encoded by the C1 gene. Transgenic Nicotiana benthamiana plants expressing C1 antisense RNA were obtained and shown to resist infection by TYLCV. Some of the resistant lines are symptomless, and the replication of challenge TYLCV almost completely suppressed. The transgenes mediating resistance were shown to be effective through at least two generations of progeny.     

Modulation of septin and molecular motor recruitment in the microtubule environment of the Taxol-resistant human breast cancer cell line MDA-MB-231

Cell resistance to low doses of paclitaxel (Taxol) involves a modulation of microtubule (MT) dynamics. We applied a proteomic approach based on 2-DE coupled with MS to identify changes in the MT environment of Taxol-resistant breast cancer cells. Having established a proteomic pattern of the microtubular proteins extracted from MDA-MB-231 cells, we verified by Western blotting that in resistant cells, α- and β-tubulins (more specifically the βIII and βIV isotypes) increased. Interestingly, four septins (SEPT2, 8, 9 and 11), which are GTPases involved in cytokinesis and in MT/actin cytoskeleton organization, were overexpressed and enriched in the MT environment of Taxol-resistant cells compared to their sensitive counterpart. Changes in the MT proteome of resistant cells also comprised increased kinesin-1 heavy chain expression and recruitment on MTs while dynein light chain-1 was downregulated. Modulation of motor protein recruitment around MTs might reflect their important role in controlling MT dynamics via the organization of signaling pathways. The identification of proteins previously unknown to be linked to taxane-resistance could also be valuable to identify new biological markers of resistance.     

Phylogenetics, species boundaries and timing of resource tracking in a highly specialized group of seed beetles (Coleoptera: Chrysomelidae: Bruchinae)

Though for a long time it was hypothesized that the extraordinary diversity of phytophagous insects was better explained by a synchronous pattern of co-diversification with plants, the results of recent studies have led to question this theory, suggesting that the diversification of insects occurred well after that of their hosts. In this study we address this issue by investigating the timing of diversification of a highly specialized group of seed beetles, which mostly feeds on legume plants from the tribe Indigofereae. To that purpose, a total of 130 specimens were sequenced for six genes and analyzed under a Bayesian phylogenetic framework. Based on the resulting trees we performed several analyses that allowed a better definition of the group boundaries and to investigate the status of several taxa through the use of molecular species delimitation analyses in combination with morphological evidences. In addition the evolution of host plant use was reconstructed and different molecular-dating approaches were carried out in order to assess the ages of several clades of interest. The resulting framework suggests a more ancient than previously thought origin for seed beetles, and a pattern of rapid host plant colonization. These findings call for further similar studies in other highly specialized groups of phytophagous insects.     

Milk ejection in mice LG/J x SM/J

In mammals, milk provision is crucial to offspring survival and growth from birth to weaning. Milk deficiency early in life may cause death or changes in the progeny metabolism that later may lead to obesity and metabolic disorders. This study investigates milk ejection (ME) the first day after birth (D1) in F₂ females from the intercross of LG/J and SM/J inbred mice strains. The absence of milk in F₃ pups?? stomach at D1 is directly associated with their survival (p?<?0.001) and growth pattern (p?<?0.001) in the early stages of life. Furthermore, late growth pattern is also affected by this lack of nutrients at D1 because pups that survive this absence, mostly males, are heavier at weaning (p?<?0.001) which, after necropsy, is shown to be due to significant higher total fat deposition (p?<?0.01). We performed QTL analysis for ME at D1 in these F₂ females. Maternal performance of ME revealed a complex genetic architecture which even though it contains only a single QTL (accounting for 8?% of the variation in ME), it is totally context-dependent on the genetic background. We discovered many regions involved in epistatic interactions that together with the single QTL explain 19?% of the genetic variation for this trait. Milk ejection is an important component of maternal care, and understanding the mechanisms modulating its variation, along with other maternal features, may help to disentangle the complexity that is the mother/offspring relationship.     

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.     

Auxin metabolism and rooting in young and mature clones of Sequoia sempervirens

The capacity of young and mature Sequoia sempervirens clones to produce roots in vitro was studied after wounding and indole-3-butyric acid (IBA) treatments. Rooting was not observed in mature or in young cuttings cultivated for 30 days in medium without IBA. The presence of 25 μ M IBA in the medium resulted in the appearance of roots at the base of the cuttings. More roots appeared and grew faster on cuttings of the young than on the mature clone. This difference in rooting capacity between young and mature cuttings may be related to differences in the hormone levels at the base of the 5 mm long cuttings during the first 4 days of the root inductive period. After HPLC fractionation. IAA. IBA and related compounds, including indole-3-aspartic acid (IAAsp) and IBA-glucose ester (IBA-GE), were determined by MS and MS-MS and their levels measured by ELISA. Another immunoreactive compound was also found and determined to be N,N-dimethyltryptophan (DMT), a compound previously reported to inhibit auxin-enhanced ethylene production. Wounding of the stem without IBA treatment revealed a transient increase in IAA, IAAsp and DMT levels in young cuttings while a dramatic increase in the levels of DMT was observed in mature cuttings. Following IBA treatment. IAA levels increased in both clones, but higher levels were measured in the young than in the mature clone. IBA and IBA-GE were also found but in higher levels in the mature clone. Thus, the difficult-to-root mature clone differs from the young clone in its auxin metabolism.