Cytokinin vectors mediate marker-free and backbone-free plant transformation

Conventional Agrobacterium-mediated transformation methods rely on complex and genotype-specific tissue culture media for selection, proliferation, and regeneration of genetically modified cells. Resulting transgenic plants may not only contain selectable marker genes but also carry fragments of the vector backbone. Here, we describe a new method for the production of transgenic plants that lack such foreign DNA. This method employs vectors containing the bacterial isopentenyltransferase (ipt) gene as backbone integration marker. Agrobacterium strains carrying the resulting ipt gene-containing "cytokinin" vectors were used to infect explants of various Solanaceous plant species as well as canola (Brassica napus). Upon transfer to hormone-free media, 1.8% to 9.9% of the infected explants produced shoots that contained a marker-free T-DNA while lacking the backbone integration marker. These frequencies often equal or exceed those for backbone-free conventional transformation.     

Evaluation of preseparator performance for the 8-stage nonviable Andersen impactor

The preseparator of an Andersen impactor with different coating treatments for a range of particle-size distributions was evaluated. Limited theoretical simulations constrained by simplifying assumptions of the airflow fields in the preseparator and upper stages of an 8-stage Andersen impactor were used to reveal low-velocity and high-pressure regions for potential deposition. These regions were then sampled in subsequent particle deposition experiments. Disodium fluorescein aerosols were sampled with different coating treatments of the preseparator floor. Particles collected at impactor stages determined particle size distributions. Stage deposition was compared between different preseparator treatments (buffer and silicon oil). Collection efficiency in the preseparator followed the pattern buffer >silicon oil >untreated. Statistical differences (P>0.05) were noted in collection efficiency of large particles (45 μm-75 μm) in the preseparator. The mass median aerodynamic diameters and geometric standard deviations showed some statistical differences when different preseparator treatments for large particles were used; therefore, preseparator coating was shown to influence performance and thereby estimates of particle size by intertial impaction.     

Folding of a designed simple ankyrin repeat protein

Ankyrin repeats (AR) are 33-residue motifs containing a beta-turn, followed by two alpha-helices connected by a loop. AR occur in tandem arrangements and stack side-by-side to form elongated domains involved in very different cellular tasks. Recently, consensus libraries of AR repeats were constructed. Protein E1_5 represents a member of the shortest library, and consists of only a single consensus repeat flanked by designed N- and C-terminal capping repeats. Here we present a biophysical characterization of this AR domain. The protein is compactly folded, as judged from the heat capacity of the native state and from the specific unfolding enthalpy and entropy. From spectroscopic data, thermal and urea-induced unfolding can be modeled by a two-state transition. However, scanning calorimetry experiments reveal a deviation from the two-state behavior at elevated temperatures. Folding and unfolding at 5 degrees C both follow monoexponential kinetics with k(folding) = 28 sec(-1) and k(unfolding) = 0.9 sec(-1). Kinetic and equilibrium unfolding parameters at 5 degrees C agree very well. We conclude that E1_5 folds in a simple two-state manner at low temperatures while equilibrium intermediates become populated at higher temperatures. A chevron-plot analysis indicates that the protein traverses a very compact transition state along the folding/unfolding pathway. This work demonstrates that a designed minimal ankyrin repeat protein has the thermodynamic and kinetic properties of a compactly folded protein, and explains the favorable properties of the consensus framework.     

BEST: a novel computational approach for comparing gene expression patterns from early stages of Drosophila melanogaster development

Embryonic gene expression patterns are an indispensable part of modern developmental biology. Currently, investigators must visually inspect numerous images containing embryonic expression patterns to identify spatially similar patterns for inferring potential genetic interactions. The lack of a computational approach to identify pattern similarities is an impediment to advancement in developmental biology research because of the rapidly increasing amount of available embryonic gene expression data. Therefore, we have developed computational approaches to automate the comparison of gene expression patterns contained in images of early stage Drosophila melanogaster embryos (prior to the beginning of germ-band elongation); similarities and differences in gene expression patterns in these early stages have extensive developmental effects. Here we describe a basic expression search tool (BEST) to retrieve best matching expression patterns for a given query expression pattern and a computational device for gene interaction inference using gene expression pattern images and information on the associated genotypes and probes. Analysis of a prototype collection of Drosophila gene expression pattern images is presented to demonstrate the utility of these methods in identifying biologically meaningful matches and inferring gene interactions by direct image content analysis. In particular, the use of BEST searches for gene expression patterns is akin to that of BLAST searches for finding similar sequences. These computational developmental biology methodologies are likely to make the great wealth of embryonic gene expression pattern data easily accessible and to accelerate the discovery of developmental networks.     

Accuracy of marker-assisted selection with single markers and marker haplotypes in cattle

A key question for the implementation of marker-assisted selection (MAS) using markers in linkage disequilibrium with quantitative trait loci (QTLs) is how many markers surrounding each QTL should be used to ensure the marker or marker haplotypes are in sufficient linkage disequilibrium (LD) with the QTL. In this paper we compare the accuracy of MAS using either single markers or marker haplotypes in an Angus cattle data set consisting of 9323 genome-wide single nucleotide polymorphisms (SNPs) genotyped in 379 Angus cattle. The extent of LD in the data set was such that the average marker-marker r2 was 0.2 at 200 kb. The accuracy of MAS increased as the number of markers in the haplotype surrounding the QTL increased, although only when the number of markers in the haplotype was 4 or greater did the accuracy exceed that achieved when the SNP in the highest LD with the QTL was used. A large number of phenotypic records (>1000) were required to accurately estimate the effects of the haplotypes.     

Polymer‐coated viral vectors: hybrid nanosystems for gene therapy

The advantages and critical aspects of nanodimensional polymer‐coated viral vector systems potentially applicable for gene delivery are reviewed. Various viral and nonviral vectors have been explored for gene therapy. Viral gene transfer methods, although highly efficient, are limited by their immunogenicity. Nonviral vectors have a lower transfection efficiency as a result of their inability to escape from the endosome. To overcome these drawbacks, novel nanotechnology‐mediated interventions that involve the coating or modification of virus using polymers have emerged as a new paradigm in gene therapy. These alterations not only modify the tropism of the virus, but also reduce their undesirable interactions with the biological system. Also, co‐encapsulation of other therapeutic agents in the polymeric coating may serve to augment the treatment efficacy. The viral particles can aid endosomal escape, as well as nuclear targeting, thereby enhancing the transfection efficiency. The integration of the desirable properties of both viral and nonviral vectors has been found beneficial for gene therapy by enhancing the transduction efficiency and minimizing the immune response. However, it is essential to ensure that these attempts should not compromise on the inherent ability of viruses to target and internalize into the cells and escape the endosomes.     

Immune modulation by dendritic-cell-based cancer vaccines

The interplay between host immunity and tumour cells has opened the possibility of targeting tumour cells by modulation of the human immune system. Cancer immunotherapy involves the treatment of a tumour by utilizing the recombinant human immune system components to target the pro-tumour microenvironment or by revitalizing the immune system with the ability to kill tumour cells by priming the immune cells with tumour antigens. In this review, current immunotherapy approaches to cancer with special focus on dendritic cell (DC)-based cancer vaccines are discussed. Some of the DC-based vaccines under clinical trials for various cancer types are highlighted. Establishing tumour immunity involves a plethora of immune components and pathways; hence, combining chemotherapy, radiation therapy and various arms of immunotherapy, after analysing the benefits of individual therapeutic agents, might be beneficial to the patient.     

Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration

Neural tissue repair and regeneration strategies have received a great deal of attention because it directly affects the quality of the patient's life. There are many scientific challenges to regenerate nerve while using conventional autologous nerve grafts and from the newly developed therapeutic strategies for the reconstruction of damaged nerves. Recent advancements in nerve regeneration have involved the application of tissue engineering principles and this has evolved a new perspective to neural therapy. The success of neural tissue engineering is mainly based on the regulation of cell behavior and tissue progression through the development of a synthetic scaffold that is analogous to the natural extracellular matrix and can support three-dimensional cell cultures. As the natural extracellular matrix provides an ideal environment for topographical, electrical and chemical cues to the adhesion and proliferation of neural cells, there exists a need to develop a synthetic scaffold that would be biocompatible, immunologically inert, conducting, biodegradable, and infection-resistant biomaterial to support neurite outgrowth. This review outlines the rationale for effective neural tissue engineering through the use of suitable biomaterials and scaffolding techniques for fabrication of a construct that would allow the neurons to adhere, proliferate and eventually form nerves.     

Simple Signaling Molecules for Inductive Bone Regenerative Engineering

With greater than 500,000 orthopaedic procedures performed in the United States each year requiring a bone graft, the development of novel graft materials is necessary. We report that some porous polymer/ceramic composite scaffolds possess intrinsic osteoinductivity as shown through their capacity to induce in vivo host osteoid mineralization and in vitro stem cell osteogenesis making them attractive synthetic bone graft substitutes. It was discovered that certain low crystallinity ceramics partially dissociate into simple signaling molecules (i.e., calcium and phosphate ions) that induce stem cells to endogenously produce their own osteoinductive proteins. Review of the literature has uncovered a variety of simple signaling molecules (i.e., gases, ions, and redox reagents) capable of inducing other desirable stem cell differentiation through endogenous growth factor production. Inductive simple signaling molecules, which we have termed inducerons, represent a paradigm shift in the field of regenerative engineering where they can be utilized in place of recombinant protein growth factors.