The effects of irradiation on the biological and biomechanical properties of an acellular porcine superflexor tendon graft for cruciate ligament repair

Acellular xenogeneic tissues have the potential to provide ‘off‐the‐shelf’ grafts for anterior cruciate ligament (ACL) repair. To ensure that such grafts are sterile following packaging, it is desirable to use terminal sterilization methods. Here, the effects of gamma and electron beam irradiation on the biological and biomechanical properties of a previously developed acellular porcine superflexor tendon (pSFT) were investigated. Irradiation following treatment with peracetic acid was compared to peracetic acid treatment alone and the stability of grafts following long‐term storage assessed. Irradiation did not affect total collagen content or biocompatibility (determined using a contact cytotoxicity assay) of the grafts, but slightly increased the amount of denatured collagen in and decreased the thermal denaturation temperature of the tissue in a dose dependant fashion. Biomechanical properties of the grafts were altered by irradiation (reduced ultimate tensile strength and Young's modulus, increased failure strain), but remained superior to reported properties of the native human ACL. Long term storage at 4°C had no negative effects on the grafts. Of all the conditions tested, a dose of minimum 25 kGy of gamma irradiation had least effect on the grafts, suggesting that this dose produces a biocompatible pSFT graft with adequate mechanical properties for ACL repair

AtSPX1 affects the AtPHR1 -DNA binding equilibrium by binding monomeric AtPHR1 in solution

Phosphorus is an essential macronutrient for plant growth, and is deficient in about 50% of agricultural soils. The transcription factor Phosphate Starvation Response 1 (PHR1) plays a central role in regulating the expression of a subset of Phosphate Starvation Induced (PSI) genes through binding to a cis acting DNA element termed P1BS. In Arabidopsis and rice, activity of AtPHR1/OsPHR2 is regulated in part by their downstream target SPX proteins through protein-protein interaction. Here we provide kinetic and affinity data for interaction between AtPHR1 and P1BS sites. Using SPR, a tandem P1BS sequence showed ~50-fold higher affinity for MBPAtdPHR1 (a fusion protein comprising the DNA binding domain and coiled-coiled domain of AtPHR1 fused to maltose binding protein) than a single site. The affinity difference was largely reflected in a much slower dissociation rate from the 2x P1BS binding site, suggesting an important role for protein cooperativity. Injection of AtSPX1 in the presence of phosphate or inositol hexakisphosphate (InsP6) failed to alter the MBPAtdPHR1-P1BS dissociation rate, while pre-mixing of these two proteins in the presence of either 5 mM Pi or 500 µM InsP6 resulted in a much lower DNA binding signal from MBPAtdPHR1. These data suggest that in the Pi restored condition, AtSPX1 can bind to monomeric AtPHR1 in solution and therefore regulate PSI gene expression by tuning the AtPHR1-DNA binding equilibrium. This Pi-dependent regulation of AtPHR1-DNA binding equilibrium also generates a negative feedback loop on the expression of AtSPX1 itself, providing a tight control of PSI gene expression

Tenascin C upregulates interleukin-6 expression in human cardiac myofibroblasts via toll-like receptor 4.

AIM: To investigate the effect of Tenascin C (TNC) on the expression of pro-inflammatory cytokines and matrix metalloproteinases in human cardiac myofibroblasts (CMF). METHODS: CMF were isolated and cultured from patients undergoing coronary artery bypass grafting. Cultured cells were treated with either TNC (0.1 μmol/L, 24 h) or a recombinant protein corresponding to different domains of the TNC protein; fibrinogen-like globe (FBG) and fibronectin type III-like repeats (TNIII 5-7) (both 1 μmol/L, 24 h). The expression of the pro-inflammatory cytokines; interleukin (IL)-6, IL-1β, TNFα and the matrix metalloproteinases; MMPs (MMP1, 2, 3, 9, 10, MT1-MMP) was assessed using real time RT-PCR and western blot analysis. RESULTS: TNC increased both IL-6 and MMP3 (P < 0.01) mRNA levels in cultured human CMF but had no significant effect on the other markers studied. The increase in IL-6 mRNA expression was mirrored by an increase in protein secretion as assessed by enzyme-linked immunosorbant assay (P < 0.01). Treating CMF with the recombinant protein FBG increased IL-6 mRNA and protein (P < 0.01) whereas the recombinant protein TNIII 5-7 had no effect. Neither FBG nor TNIII 5-7 had any significant effect on MMP3 expression. The expression of toll-like receptor 4 (TLR4) in human CMF was confirmed by real time RT-PCR, western blot and immunohistochemistry. Pre-incubation of cells with TLR4 neutralising antisera attenuated the effect of both TNC and FBG on IL-6 mRNA and protein expression. CONCLUSION: TNC up-regulates IL-6 expression in human CMF, an effect mediated through the FBG domain of TNC and via the TLR4 receptor

Defining the remarkable structural malleability of a bacterial surface protein Rib domain implicated in infection

Streptococcus groups A and B cause serious infections, including early onset sepsis and meningitis in newborns. Rib domain-containing surface proteins are found associated with invasive strains and elicit protective immunity in animal models. Yet, despite their apparent importance in infection, the structure of the Rib domain was previously unknown. Structures of single Rib domains of differing length reveal a rare case of domain atrophy through deletion of 2 core antiparallel strands, resulting in the loss of an entire sheet of the β-sandwich from an immunoglobulin-like fold. Previously, observed variation in the number of Rib domains within these bacterial cell wall-attached proteins has been suggested as a mechanism of immune evasion. Here, the structure of tandem domains, combined with molecular dynamics simulations and small angle X-ray scattering, suggests that variability in Rib domain number would result in differential projection of an N-terminal host-colonization domain from the bacterial surface. The identification of 2 further structures where the typical B-D-E immunoglobulin β-sheet is replaced with an α-helix further confirms the extensive structural malleability of the Rib domain

GPVI (Glycoprotein VI) Interaction With Fibrinogen Is Mediated by Avidity and the Fibrinogen αC-Region

Objective: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. Conclusions: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth

Trivalent Gd-DOTA reagents for modification of proteins

The development of novel protein-targeted MRI contrast agents crucially depends on the ability to derivatise suitable targeting moieties with a high payload of relaxation enhancer (e.g., gadolinium(III) complexes such as Gd-DOTA), without losing affinity for the target proteins. Here, we report robust synthetic procedures for the preparation of trivalent Gd-DOTA reagents with various chemical handles for site-specific modification of biomolecules. The reagents were shown to successfully label proteins through isothiocyanate ligation or through site-specific thiol–maleimide ligation and strain-promoted azide–alkyne cycloaddition

Heat Stress Enhances the Accumulation of Polyadenylated Mitochondrial Transcripts in Arabidopsis thaliana

Background: Polyadenylation of RNA has a decisive influence on RNA stability. Depending on the organisms or subcellular compartment, it either enhances transcript stability or targets RNAs for degradation. In plant mitochondria, polyadenylation promotes RNA degradation, and polyadenylated mitochondrial transcripts are therefore widely considered to be rare and unstable. We followed up a surprising observation that a large number of mitochondrial transcripts are detectable in microarray experiments that used poly(A)-specific RNA probes, and that these transcript levels are significantly enhanced after heat treatment. Methodology/Principal Findings: As the Columbia genome contains a complete set of mitochondrial genes, we had to identify polymorphisms to differentiate between nuclear and mitochondrial copies of a mitochondrial transcript. We found that the affected transcripts were uncapped transcripts of mitochondrial origin, which were polyadenylated at multiple sites within their 39region. Heat-induced enhancement of these transcripts was quickly restored during a short recovery period. Conclusions/Significance: Our results show that polyadenylated transcripts of mitochondrial origin are more stable than previously suggested, and that their steady-state levels can even be significantly enhanced under certain conditions. As many microarrays contain mitochondrial probes, due to the frequent transfer of mitochondrial genes into the genome

Genetic architecture and evolution of the S locus supergene in Primula vulgaris

Darwin’s studies on heterostyly in Primula described two floral morphs, pin and thrum, with reciprocal anther and stigma heights that promote insect-mediated cross-pollination. This key innovation evolved independently in several angiosperm families. Subsequent studies on heterostyly in Primula contributed to the foundation of modern genetic theory and the neo-Darwinian synthesis. The established genetic model for Primula heterostyly involves a diallelic S locus comprising several genes, with rare recombination events that result in self-fertile homostyle flowers with anthers and stigma at the same height. Here we reveal the S locus supergene as a tightly-linked cluster of thrum-specific genes that are absent in pins. We show that thrums are hemizygous not heterozygous for the S locus, which suggests that homostyles do not arise by recombination between S locus haplotypes as previously proposed. Duplication of a floral homeotic gene 51.7 MYA, followed by its neofunctionalisation, created the current S locus assemblage which led to floral heteromorphy in Primula. Our findings provide new insights into the structure, function and evolution of this archetypal supergene

A composite transcriptional signature differentiates responses towards closely related herbicides in Arabidopsis thaliana and Brassica napus

In this study, genome-wide expression profiling based on Affymetrix ATH1 arrays was used to identify discriminating responses of Arabidopsis thaliana to five herbicides, which contain active ingredients targeting two different branches of amino acid biosynthesis. One herbicide contained glyphosate, which targets 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), while the other four herbicides contain different acetolactate synthase (ALS) inhibiting compounds. In contrast to the herbicide containing glyphosate, which affected only a few transcripts, many effects of the ALS inhibiting herbicides were revealed based on transcriptional changes related to ribosome biogenesis and translation, secondary metabolism, cell wall modification and growth. The expression pattern of a set of 101 genes provided a specific, composite signature that was distinct from other major stress responses and differentiated among herbicides targeting the same enzyme (ALS) or containing the same chemical class of active ingredient (sulfonylurea). A set of homologous genes could be identified in Brassica napus that exhibited a similar expression pattern and correctly distinguished exposure to the five herbicides. Our results show the ability of a limited number of genes to classify and differentiate responses to closely related herbicides in A. thaliana and B. napus and the transferability of a complex transcriptional signature across species