Modelling the genetic architecture of flowering time control in barley through nested association mapping

Background

Barley, globally the fourth most important cereal, provides food and beverages for humans and feed for animal husbandry. Maximizing grain yield under varying climate conditions largely depends on the optimal timing of flowering. Therefore, regulation of flowering time is of extraordinary importance to meet future food and feed demands. We developed the first barley nested association mapping (NAM) population, HEB-25, by crossing 25 wild barleys with one elite barley cultivar, and used it to dissect the genetic architecture of flowering time.

Results

Upon cultivation of 1,420 lines in multi-field trials and applying a genome-wide association study, eight major quantitative trait loci (QTL) were identified as main determinants to control flowering time in barley. These QTL accounted for 64% of the cross-validated proportion of explained genotypic variance (p_G). The strongest single QTL effect corresponded to the known photoperiod response gene Ppd-H1. After sequencing the causative part of Ppd-H1, we differentiated twelve haplotypes in HEB-25, whereof the strongest exotic haplotype accelerated flowering time by 11 days compared to the elite barley haplotype. Applying a whole genome prediction model including main effects and epistatic interactions allowed predicting flowering time with an unmatched accuracy of 77% of cross-validated p_G.

Conclusions

The elaborated causal models represent a fundamental step to explain flowering time in barley. In addition, our study confirms that the exotic biodiversity present in HEB-25 is a valuable toolbox to dissect the genetic architecture of important agronomic traits and to replenish the elite barley breeding pool with favorable, trait-improving exotic alleles.

     

In silico approach to inhibition of signaling pathways of Toll-like receptors 2 and 4 by ST2L

Toll-like receptors (TLRs) activate a potent immunostimulatory response. There is clear evidence that overactivation of TLRs leads to infectious and inflammatory diseases. Recent biochemical studies have shown that the membrane-bound form of ST2 (ST2L), a member of the Toll-like/IL-1 receptor superfamily, negatively regulates MyD88-dependent TLR signaling pathways by sequestrating the adapters MyD88 and Mal (TIRAP). Specifically, ST2L attenuates the recruitment of Mal and MyD88 adapters to their receptors through its intracellular TIR domain. Thus, ST2L is a potent molecule that acts as a key regulator of endotoxin tolerance and modulates innate immunity. So far, the inhibitory mechanism of ST2L at the molecular level remains elusive. To develop a working hypothesis for the interactions between ST2L, TLRs (TLR1, 2, 4, and 6), and adapter molecules (MyD88 and Mal), we constructed three-dimensional models of the TIR domains of TLR4, 6, Mal, and ST2L based on homology modeling. Since the crystal structures of the TIR domains of TLR1, 2 as well as the NMR solution structure of MyD88 are known, we utilized these structures in our analysis. The TIR domains of TLR1, 2, 4, 6, MyD88, Mal and ST2L were subjected to molecular dynamics (MD) simulations in an explicit solvent environment. The refined structures obtained from the MD simulations were subsequently used in molecular docking studies to probe for potential sites of interactions. Through protein-protein docking analysis, models of the essential complexes involved in TLR2 and 4 signaling and ST2L inhibiting processes were developed. Our results suggest that ST2L may exert its inhibitory effect by blocking the molecular interface of Mal and MyD88 adapters mainly through its BB-loop region. Our predicted oligomeric signaling models may provide a basis for the understanding of the assembly process of TIR domain interactions, which has thus far proven to be difficult via in vivo studies.     

Females with paired occurrence of cancers in the UADT and genital region have a higher frequency of either Glutathione S-transferase M1/T1 null genotype

Upper Aero digestive Tract (UADT) is the commonest site for the development of second cancer in females after primary cervical cancer. Glutathione S-transferase (GSTM1 and / or T1) null genotype modulates the risk of developing UADT cancer (primary as well as second cancer). The aim of this study was to evaluate the difference in GST null genotype frequencies in females with paired cancers in the UADT and genital region as compared to females with paired cancers in the UADT and non-genital region. Forty-nine females with a cancer in the UADT and another cancer (at all sites-genital and non-genital) were identified from a database of patients with multiple primary neoplasms and were analyzed for the GSTM1 and T1 genotype in addition to known factors such as age, tobacco habits, alcohol habits and family history of cancer. Frequencies of GSTM1 null, GSTT1 null, and either GSTM1/T1 null were higher in females with paired occurrence of cancer in the UADT and genital site (54%, 33% and 75% respectively) in comparison to females with paired occurrence of cancer in the UADT and non-genital sites (22%, 6% and 24% respectively). The significantly higher inherited frequency of either GSTM1/T1 null genotype in females with a paired occurrence of cancers in UADT and genital region (p = 0.01), suggests that these females are more susceptible to damage by carcinogens as compared to females who have UADT cancers in association with cancers at non-genital sites.     

Lecithin organogels as a potential phospholipid-structured system for topical drug delivery: A review

The purpose of this review is to give an insight into the considerable potential of lecithin organogels (LOs) in the applications meant for topical drug delivery. LOs are clear, thermodynamically stable, viscoelastic, and biocompatible jelly-like phases, chiefly composed of hydrated phospholipids and appropriate organic liquid. These systems are currently of interest to the pharmaceutical scientist because of their structural and functional benefits. Several therapeutic agents have been formulated as LOs for their facilitated transport through topical route (for dermal or transdermal effect), with some very encouraging results. The improved topical drug delivery has mainly been attributed to the biphasic drug solubility, the desired drug partitioning, and the modification of skin barrier function by the organogel components. Being thermodynamically stable, LOs are prepared by spontaneous emulsification and therefore posses prolonged shelf life. The utility of this novel matrix as a topical vehicle has further increased owing to its very low skin irritancy potential. Varied aspects of LOs viz formation, composition, phase behavior, and characterization have been elaborated, including a general discussion on the developmental background. Besides a comprehensive update on the topical applications of lecithin organogels, the review also includes a detailed account on the mechanistics of organogelling. Published: October 6, 2005     

Adult-derived stem cells and their potential for use in tissue repair and molecular medicine

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.     

Efficient refolding of aggregation-prone citrate synthase by polyol osmolytes: how well are protein folding and stability aspects coupled?

Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7 m glycerol and 10 mug/ml protein, a result superior to the action of the molecular chaperones GroEL and GroES in vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7 m. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance.     

Modeling amyloid beta-peptide insertion into lipid bilayers

Inspired by recent suggestions that the Alzheimer's amyloid beta peptide (Abeta) can insert into cell membranes and form harmful ion channels, we model insertion of the 40- and 42-residue forms of the peptide into cell membranes using a Monte Carlo code which is specific at the amino acid level. We examine insertion of the regular Abeta peptide as well as mutants causing familial Alzheimer's disease, and find that all but one of the mutants change the insertion behavior by causing the peptide to spend more simulation steps in only one leaflet of the bilayer. We also find that Abeta42, because of the extra hydrophobic residues relative to Abeta40, is more likely to adopt this conformation than Abeta40 in both wild-type and mutant forms. We argue qualitatively why these effects happen. Here, we present our results and develop the hypothesis that this partial insertion increases the probability of harmful channel formation. This hypothesis can partly explain why these mutations are neurotoxic simply due to peptide insertion behavior. We further apply this model to various artificial Abeta mutants which have been examined experimentally, and offer testable experimental predictions contrasting the roles of aggregation and insertion with regard to toxicity of Abeta mutants. These can be used through further experiments to test our hypothesis.