Local Application of Ibandronate/Gelatin Sponge Improves Osteotomy Healing in Rabbits

Delayed healing or non-union of skeletal fractures are common clinical complications. Ibandronate is a highly potent anti-catabolic reagent used for treatment of osteopenia and fracture prevention. We hypothesized that local application of ibandronate after fracture fixation may improve and sustain callus formation and therefore prevent delayed healing or non-union. This study tested the effect of local application of an ibandronate/gelatin sponge composite on osteotomy healing. A right-side distal-femoral osteotomy was created surgically, with fixation using a k-wire, in forty adult male rabbits. The animals were divided into four groups of ten animals and treated by: (i) intravenous injection of normal saline (Control); (ii) local implantation of absorbable gelatin sponge (GS); (iii) local implantation of absorbable GS containing ibandronate (IB+GS), and (iv) intravenous injection of ibandronate (IB i.v.). At two and four weeks the affected femora were harvested for X-ray photography, computed tomography (CT), biomechanical testing and histopathology. At both time-points the results showed that the calluses in both the ibandronate-treated groups, but especially in the IB+GS group, were significantly larger than in the control and GS groups. At four weeks the cross sectional area (CSA) and mechanical test results of ultimate load and energy in the IB+GS group were significantly higher than in other groups. Histological procedures showed a significant reduction in osteoclast numbers in the IB+GS and IB i.v. groups at day 14. The results indicate that local application of an ibandronate/gelatin sponge biomaterial improved early osteotomy healing after surgical fixation and suggest that such treatment may be a valuable local therapy to enhance fracture repair and potentially prevent delayed or non-union.     

Proteome dynamics and proteome function of cardiac 19S proteasomes

Myocardial proteasomes are comprised of 20S core particles and 19S regulatory particles, which together carry out targeted degradation of cardiac proteins. The 19S complex is unique among the regulators of proteasomes in that it affects both the capacity and specificity of protein degradation. However, a comprehensive molecular characterization of cardiac 19S complexes is lacking. In this investigation, we tailored a multidimensional chromatography-based purification strategy to isolate structurally intact and functionally viable 19S complexes from murine hearts. Two distinct subpopulations of 19S complexes were isolated based upon (1) potency of activating 20S proteolytic activity, and (2) molecular composition using a combination of immuno-detection, two-dimensional-differential gel electrophoresis, and MS-based approaches. Heat shock protein 90 (Hsp90) was identified to be characteristic to 19S subpopulation I. The physical interaction of Hsp90 with 19S complexes was demonstrated via multiple approaches. Inhibition of Hsp90 activity using geldanamycin or BIIB021 potentiated the ability of subpopulation I to activate 20S proteasomes in the murine heart, thus demonstrating functional specificity of Hsp90 in subpopulation I. This investigation has advanced our understanding of the molecular heterogeneity of cardiac proteasomes by identifying molecularly and functionally distinct cardiac 19S complexes. The preferential association of Hsp90 with 19S subpopulation I unveils novel targets for designing proteasome-based therapeutic interventions for combating cardiac disease.     

Structural analysis of alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5: implications for adaptation to alkaline conditions

Significant progress has been made in isolating novel alkaline β-mannanases, however, there is a paucity of information concerning the structural basis for alkaline tolerance displayed by these β-mannanases. We report the catalytic domain structure of an industrially important β-mannanase from the alkaliphilic Bacillus sp. N16-5 (BSP165 MAN) at a resolution of 1.6 Å. This enzyme, classified into subfamily 8 in glycosyl hydrolase family 5 (GH5), has a pH optimum of enzymatic activity at pH 9.5 and folds into a classic (β/α)₈-barrel. In order to gain insight into molecular features for alkaline adaptation, we compared BSP165 MAN with previously reported GH5 β-mannanases. It was revealed that BSP165 MAN and other subfamily 8 β-mannanases have significantly increased hydrophobic and Arg residues content and decreased polar residues, comparing to β-mannanases of subfamily 7 or 10 in GH5 which display optimum activities at lower pH. Further, extensive structural comparisons show alkaline β-mannanases possess a set of distinctive features. Position and length of some helices, strands and loops of the TIM barrel structures are changed, which contributes, to a certain degree, to the distinctly different shaped (β/α)₈-barrels, thus affecting the catalytic environment of these enzymes. The number of negatively charged residues is increased on the molecular surface, and fewer polar residues are exposed to the solvent. Two amino acid substitutions in the vicinity of the acid/base catalyst were proposed to be possibly responsible for the variation in pH optimum of these homologous enzymes in subfamily 8 of GH5, identified by sequence homology analysis and pK _a calculations of the active site residues. Mutational analysis has proved that Gln91 and Glu226 are important for BSP165 MAN to function at high pH. These findings are proposed to be possible factors implicated in the alkaline adaptation of GH5 β-mannanases and will help to further understanding of alkaline adaptation mechanism.     

Tissue proteomics using capillary isoelectric focusing-based multidimensional separations

The capabilities of capillary isoelectric focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of capillary isoelectric focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.     

Integrated capillary isoelectric focusing/nano-reversed phase liquid chromatography coupled with ESI-MS for characterization of intact yeast proteins

An integrated protein concentration/separation platform, combining capillary isoelectric focusing (CIEF) with nano-reversed phase liquid chromatography (nano-RPLC), is developed to provide significant protein concentration and high resolving power for the analysis of complex protein mixtures. Upon completion of protein focusing, the proteins are sequentially and hydrodynamically loaded into individual trap columns using a group of microinjection and microselection valves. Repeated pro-tein loadings and injections into trap columns are carried out automatically until the entire CIEF cap-illary content is sampled and fractionated. Each CIEF fraction "parked" in separate trap columns is further resolved using nano-RPLC, and the eluants are analyzed using electrospray ionization-mass spectrometry.     

Transient Inability to Manage Proteobacteria Promotes Chronic Gut Inflammation in TLR5-Deficient Mice

Colitis results from breakdown of homeostasis between intestinal microbiota and the mucosal immune system, with both environmental and genetic influencing factors. Flagellin receptor TLR5-deficient mice (T5KO) display elevated intestinal proinflammatory gene expression and colitis with incomplete penetrance, providing a genetically sensitized system to study the contribution of microbiota to driving colitis. Both colitic and noncolitic T5KO exhibited transiently unstable microbiotas, with lasting differences in colitic T5KO, while their noncolitic siblings stabilized their microbiotas to resemble wild-type mice. Transient high levels of proteobacteria, especially enterobacteria species including E.?coli, observed in close proximity to the gut epithelium were a striking feature of colitic microbiota. A Crohn's disease-associated E.?coli strain induced chronic colitis in T5KO, which persisted well after the exogenously introduced bacterial species had been eliminated. Thus, an innate immune deficiency can result in unstable gut microbiota associated with low-grade inflammation, and harboring proteobacteria can drive and/or instigate chronic colitis.     

Cloning,Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

A lumbrokinase gene encoding a blood-clot dissolving protein was cloned from earthworm (Eisenia fetida) by RT-PCR amplification. The gene designated as CST1 (GenBank No. {"type":"entrez-nucleotide","attrs":{"text":"AY840996","term_id":"56718408","term_text":"AY840996"}}AY840996) was sequence analyzed. The cDNA consists of 888 bp with an open reading frame of 729 bp, which encodes 242 amino acid residues. Multiple sequence alignments revealed that CST1 shares similarities and conserved amino acids with other reported lumbrokinases. The amino acid sequence of CST1 exhibits structural features similar to those found in other serine proteases, including human tissue-type (tPA), urokinase (uPA), and vampire bat (DSPAα1) plasminogen activators. CST1 has a conserved catalytic triad, found in the active sites of protease enzymes, which are important residues involved in polypeptide catalysis. CST1 was expressed as inclusion bodies in Escherichia coli BL21(DE3). The molecular mass of recombinant CST1 (rCST) was 25 kDa as estimated by SDS–PAGE, and further confirmed by Western Blot analysis. His-tagged rCST1 was purified and renatured using nickel-chelating resin with a recovery rate of 50% and a purity of 95%. The purified, renatured rCST1 showed fibrinolytic activity evaluated by both a fibrin plate and a blood clot lysis assay. rCST1 degraded fibrin on the fibrin plate. A significant percentage (65.7%) of blood clot lysis was observed when blood clot was treated with 80 mg/mL of rCST1 in vitro. The antithrombotic activity of rCST1 was 912 units/mg calculated by comparison with the activity of a lumbrokinase standard. These findings indicate that rCST1 has potential as a potent blood-clot treatment. Therefore, the expression and purification of a single lumbrokinase represents an important improvement in the use of lumbrokinases.     

A defensin from tomato with dual function in defense and development

Defensins are antimicrobial peptides that are part of the innate immune system, contributing to the first line of defense against invading pathogens. Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling. Here we show that the tomato defensin DEF2 is expressed during early flower development. Defensin mRNA abundance, peptide expression and processing are differentially regulated in developing flowers. Antisense suppression or constitutive overexpression of DEF2 reduces pollen viability and seed production. Furthermore, overexpression of DEF2 pleiotropically alters the growth of various organs and enhances foliar resistance to the fungal pathogen Botrytis cinerea. Partially purified extracts from leaves of a DEF2-overexpressing line inhibited tip growth of B. cinerea. Besides providing insights into regulation of defensin expression, these data demonstrate that plant defensins, like their animal counterparts, can assume multiple functions related to defense and development.     

Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes.     

Molecular characterization of atoxigenic Aspergillus flavus isolates collected in China

Aspergillus flavus strains were isolated frompeanut fields of Liaoning, Shandong, Hubei and Guangdong Provinces in China, and identified through phenotypic and molecular approaches. Of the 323 A. flavus strains isolated, 76 strains did not produce aflatoxins detectable by UPLC. The incidence of atoxigenic A. flavus strains decreased with increase in temperature and increased with increase in latitude in different geographical locations. Amplification of all the aflatoxin genes in the aflatoxin gene cluster in the atoxigenic isolates showed that there were 25 deletion patterns (A–Y), with 22 deletion patterns identified for the first time. Most of the atoxigenic A. flavus isolates with gene deletions (97%) had deletions in at least one of the four genes (aflT, nor-1, aflR, and hypB), indicating that these four genes could be targeted for rapid identification of atoxigenic strains. The atoxigenic isolates with gene deletions, especially the isolates with large deletions, are potential candidates for aflatoxin control.