Agrobacterium rhizogenes transformation of the Phaseolus spp.: a tool for functional genomics

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.     

A biomechanics-based method for the quantification of muscle selectivity in a musculoskeletal system

In this paper, we have developed a novel and simple method to quantify the ability to selectively activate our muscles in an effective pattern to achieve a particular task. In the context of this study, we define an effective pattern as that in which muscles whose mechanical contribution to the task is greatest, are mostly active, while the antagonist muscles are mostly silent. This new method uses biomechanical parameters to project the multi-channel EMGs into a three-dimensional artificial torque space, where the EMGs are represented as muscle activation vectors. Using the muscle activation vectors we defined a simple scalar, the muscle selection index, to quantify muscle selectivity. We demonstrate that by using this index we are able to quantify the muscle selectivity during the generation of isometric shoulder or elbow torques in brain-injured and able-bodied subjects. This method can be used during both static and dynamic motor tasks in a multi-articular musculoskeletal system.     

Fluorescent silica particles for monitoring oxygen levels in three-dimensional heterogeneous cellular structures

Bacterial biofilms are a major obstacle challenging the development of more effective therapies to treat implant infections. Oxygen availability to bacterial cells has been implicated in biofilm formation and planktonic cell detachment; however, there are insufficient tools available to measure oxygen concentrations within complex three‐dimensional structures with ～1 µm resolution. Such measurements may complement measures of biofilm structure and cell activity to provide a more comprehensive understanding of biofilm biology. Thus, we developed oxygen‐sensing microparticles specifically designed to characterize oxygen transport through the volume of bacterial biofilms. The Stöber method was used to synthesize monodisperse silica microparticles of approximately the same size as a bacterium (～1 µm). Two fluorophores, oxygen‐sensitive Ru(Ph₂phen₃)Cl₂, and the reference fluorophore Nile blue chloride were immobilized on the surface of the particles. We demonstrate application of the microparticles toward measuring the oxygen concentration profiles within a live Staphylococcus aureus biofilm. Biotechnol. Bioeng. 2012; 109: 2663–2670. © 2012 Wiley Periodicals, Inc.     

S-layer proteins of Lactobacillus acidophilus inhibits JUNV infection

We investigated the regulation of Hsp27 phosphorylation by protein kinase C δ (PKCδ) during etoposide-induced apoptosis. The phosphorylation of Hsp27 at Ser78 was temporally correlated with the proteolytic activation of PKCδ during apoptosis. Hsp27 phosphorylation was dependent on the activity of PKCδ since treatment with rottlerin, a chemical inhibitor of PKCδ, or overexpression of a PKCδ dominant negative mutant abolished the phosphorylation. In addition, recombinant PKCδ phosphorylated Hsp27 at Ser78 in vitro. Moreover, caspase-3 was specifically activated following Hsp27 phosphorylation at Ser78. Pull-down assays using a phosphomimetic Hsp27 mutant revealed that binding between Hsp27 and cytochrome c was abolished by the phosphorylation. These results suggest that Hsp27 dissociates from cytochrome c following PKCδ-mediated phosphorylation at Ser78, which allows formation of the apoptosome and stimulates apoptotic progression.     

Accelerating the domestication of trees using genomic selection: accuracy of prediction models across ages and environments

? Genomic selection is increasingly considered vital to accelerate genetic improvement. However, it is unknown how accurate genomic selection prediction models remain when used across environments and ages. This knowledge is critical for breeders to apply this strategy in genetic improvement. ? Here, we evaluated the utility of genomic selection in a Pinus taeda population of c. 800 individuals clonally replicated and grown on four sites, and genotyped for 4825 single-nucleotide polymorphism (SNP) markers. Prediction models were estimated for diameter and height at multiple ages using genomic random regression best linear unbiased predictor (BLUP). ? Accuracies of prediction models ranged from 0.65 to 0.75 for diameter, and 0.63 to 0.74 for height. The selection efficiency per unit time was estimated as 53-112% higher using genomic selection compared with phenotypic selection, assuming a reduction of 50% in the breeding cycle. Accuracies remained high across environments as long as they were used within the same breeding zone. However, models generated at early ages did not perform well to predict phenotypes at age 6 yr. ? These results demonstrate the feasibility and remarkable gain that can be achieved by incorporating genomic selection in breeding programs, as long as models are used at the relevant selection age and within the breeding zone in which they were estimated.     

Phylogeographically concordant chloroplast DNA divergence in sympatric Nothofagus s.s. How deep can it be?

? Here, we performed phylogenetic analyses and estimated the divergence times on mostly sympatric populations of five species within subgenus Nothofagus. We aimed to investigate whether phylogenetic relationships by nuclear internal transcribed spacer (ITS) and phylogeographic patterns by chloroplast DNA (cpDNA) mirror an ancient evolutionary history that was not erased by glacial eras. Extant species are restricted to Patagonia and share a pollen type that was formerly widespread in all southern land masses. Weak reproductive barriers exist among them. ? Fifteen cpDNA haplotypes resulted from the analysis of three noncoding regions on 330 individuals with a total alignment of 1794 bp. Nuclear ITS data consisted of 822 bp. We found a deep cpDNA divergence dated 32 Ma at mid-latitudes of Patagonia that predates the phylogenetic divergence of extant taxa. Other more recent breaks by cpDNA occurred towards the north. ? Complex paleogeographic features explain the genetic discontinuities. Long-lasting paleobasins and marine ingressions have impeded transoceanic dispersal during range expansion towards lower latitudes under cooler trends since the Oligocene. ? Cycles of hybridization-introgression among extant and extinct taxa have resulted in widespread chloroplast capture events. Our data suggest that Nothofagus biogeography will be resolved only if thorough phylogeographic analyses and molecular dating methods are applied using distinct genetic markers.     

Cellular associations of pituitary gonadotrophs in a rodent (Lagostomus maximus maximus) with photoperiod-dependent reproduction

The morphological characteristics and percentage of the cellular associations between gonadotrophs (LH- and FSH-secreting cells) and other cellular types were studied in pituitary pars distalis of adult male viscachas (Lagostomus maximus maximus) by double immunohistochemistry using specific antibodies to LH, FSH, PRL, GH, ACTH, TSH and S-100 protein (by folliculostellate cells; FSC), during long and short photoperiods. Bihormonal gonadotrophs were observed in ventro-medial and dorsal regions, interspersed between monohormonal gonadotrophs, and their number increased in short photoperiod. LH- and FSH-gonadotrophs were found around lactotrophs, enclosed by somatotrophs in the dorsal region, and associated with irregular corticotrophs. Gonadotrophs and thyrotrophs were associated along blood vessels and follicular structures. The cytoplasmic prolongations of FSC were in contact with both gonadotrophs. The percentage of LH–FSH, LH–ACTH, LH–FSC, FSH–LH, FSH–PRL, FSH–GH, FSH–ACTH, FSH–TSH and FSH–FSC associations decreased, whereas LH–PRL increased in short as compared to long photoperiod. The most abundant associations were LH–GH and LH–TSH during long photoperiod, but LH–GH and LH–PRL during short photoperiod. FSH–GH and FSH–PRL were the most numerous associations, and LH–FSC and FSH–FSC were the less abundant ones in both photoperiods. These results provide the morphological evidence for specific cellular associations between gonadotrophs and other cellular types of viscacha pituitary.     

Enhancement of the inhibitory effect of an IL‐15 antagonist peptide by alanine scanning

IL‐15 is a proinflammatory cytokine that acts early in the inflammatory response and has been associated with several autoimmune diseases including rheumatoid arthritis, where it had been proposed as a therapeutic target. We recently reported an IL‐15 antagonist peptide corresponding to sequence 36–45 of IL‐15 (KVTAMKCFLL) named P8, which specifically binds to IL‐15Rα and inhibits IL‐15 biological activity with a half maximal inhibitory concentration (IC50) of 130 µ m in CTLL‐2 proliferation assay. In order to improve binding of peptide P8 to the receptor IL‐15Rα, we used an Ala scan strategy to study contribution of each individual amino acid to the peptide's antagonist effect. Here, we found that Phe and Cys are important for peptide binding to IL‐15Rα. We also investigated other single site mutations and replaced the second Lys in the sequence by the polar non‐charged amino acid threonine. The resulting peptide [K6T]P8 exhibited a higher activity than P8 with an IC50 of 24 µm . We also found that this peptide was more active than peptide P8 in the inhibition of TNFα secretion by synovial cells from rheumatoid arthritis patients. The peptide [K6T]P8 described in this work is a new type of IL‐15 antagonist and constitutes a potential therapeutic agent for rheumatoid arthritis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.