Large, long range tensile forces drive convergence during

Indirect evidence suggests that blastopore closure during gastrulation of anamniotes, including amphibians such as Xenopus laevis, depends on circumblastoporal convergence forces generated by the marginal zone (MZ), but direct evidence is lacking. We show that explanted MZs generate tensile convergence forces up to 1.5 mN during gastrulation and over 4 mN thereafter. These forces are generated by convergent thickening (CT) until the midgastrula and increasingly by convergent extension (CE) thereafter. Explants from ventralized embryos, which lack tissues expressing CE but close their blastopores, produce up to 2 mN of tensile force, showing that CT alone generates forces sufficient to close the blastopore. Uniaxial tensile stress relaxation assays show stiffening of mesodermal and ectodermal tissues around the onset of neurulation, potentially enhancing long-range transmission of convergence forces. These results illuminate the mechanobiology of early vertebrate morphogenic mechanisms, aid interpretation of phenotypes, and give insight into the evolution of blastopore closure mechanisms. © Shook et al

Integrin α5β1 and Fibronectin Regulate Polarized Cell Protrusions Required for Xenopus Convergence and Extension

SummaryBackgroundIntegrin recognition of fibronectin is required for normal gastrulation including the mediolateral cell intercalation behaviors that drive convergent extension and the elongation of the frog dorsal axis; however, the cellular and molecular mechanisms involved are unclear.ResultsWe report that depletion of fibronectin with antisense morpholinos blocks both convergent extension and mediolateral protrusive behaviors in explant preparations. Both chronic depletion of fibronectin and acute disruptions of integrin α5β1 binding to fibronectin increases the frequency and randomizes the orientation of polarized cellular protrusions, suggesting that integrin-fibronectin interactions normally repress frequent random protrusions in favor of fewer mediolaterally oriented ones. In the absence of integrin α5β1 binding to fibronectin, convergence movements still occur but result in convergent thickening instead of convergent extension.ConclusionsThese findings support a role for integrin signaling in regulating the protrusive activity that drives axial extension. We hypothesize that the planar spatial arrangement of the fibrillar fibronectin matrix, which delineates tissue compartments within the embryo, is critical for promoting productive oriented protrusions in intercalating cells

Gene Composer in a structural genomics environment

For structural biology applications, protein-construct engineering is guided by comparative sequence analysis and structural information, which allow the researcher to better define domain boundaries for terminal deletions and nonconserved regions for surface mutants. A database software application called Gene Composer has been developed to facilitate construct design

Gene Composer: database software for protein construct design, codon engineering, and gene synthesis

<p>Abstract</p> <p>Background</p> <p>To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering.</p> <p>Results</p> <p>An interactive <b>Alignment Viewer </b>allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The <b>Construct Design Module </b>enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned <it>in silico </it>into defined expression vectors. The <b>Gene Design Module </b>uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies.</p> <p>Conclusion</p> <p>We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease error correction in combination with PIPE cloning. In a sister manuscript we present data on how Gene Composer designed genes and protein constructs can result in improved protein production for structural studies.</p

Antiproton catalyzed microfission/fusion propulsion

Inertial confinement fusion (ICF) utilizing an antiproton catalyzed hybrid fission/fusion target is discussed as a potential energy source for interplanetary propulsion. A proof-of-principle experiment underway at Phillips Laboratory, Kirtland AFB and antiproton trapping experiments at CERN, Geneva, Switzerland, are presented. The ICAN propulsion concept is described and results of performance analyses are reviewed. Future work to further define the ICAN concept is outlined

Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

<p>Abstract</p> <p>Background</p> <p>With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript.</p> <p>Results</p> <p>In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both <it>E. coli </it>and a cell-free wheat germ translation system. We also compare the protein expression levels in <it>E. coli </it>for a set of 11 different proteins with greatly varied G:C content and codon bias.</p> <p>Conclusion</p> <p>The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from <it>Bacillus subtilis</it>. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.</p