Assessment of formalin preserved and fresh frozen quadriceps tendon graft-suture constructs for load to failure testing: a biomechanical cadaveric study.

PURPOSE: Fresh-frozen specimen availability and cost may be a barrier for initiation of biomechanical studies where soft tissue is used in a construct with other medical devices. The impact of soft tissue preservation method on the outcomes of biomechanical studies in the specific case of graft-suture constructs is relatively unexplored. This study aimed to observe peak loads and failure modes in biomechanical testing of fresh-frozen (FF) versus formalin embalmed (FE) quadriceps tendon (QT) graft-suture constructs for soft tissue fixation in ACLR and assess suitability of FE QT graft constructs for load-to-fail testing. METHODS: Twenty QT grafts were harvested from human cadaver specimens. Ten grafts came from fresh-frozen donors and 10 from embalmed donors. All grafts were prepared with the modified Prusik knot using a braided composite suture and subjected to tensile loading. Comparisons between the biomechanical properties of the graft-suture constructs were made with unpaired t tests with α = 0.05. RESULTS: FE and FF constructs displayed similar peak loads and failure modes. FF constructs had greater elongation after pre-tensioning than FE (7.3 vs. 5.5 mm, p = 0.02) and greater elongation after cyclic loading than FE constructs (17.5 vs. 10.5 mm, p = 0.01). Hysteresis was greater for FF constructs at the 50th, 100th, 150th, and 200th cycle (p = 0.02, p = 0.07, p < 0.001, p = 0.004, respectively). FE constructs were stiffer than fresh-frozen (103 vs. 84 N/mm, p < 0.001). CONCLUSION: FE constructs were significantly stiffer but displayed similar peak load and failure mode to FF which was reflective of the strength of the suture material. FE grafts can offer an alternative to FF grafts in graft-suture constructs for biomechanical studies where load at failure and knot security and strength is of main interest.

Biomechanics of hamstring tendon, quadriceps tendon, and bone-patellar tendon-bone grafts for anterior cruciate ligament reconstruction: a cadaveric study.

PURPOSE: The three most commonly used autografts for anterior cruciate ligament reconstruction (ACL) are: bone-patellar tendon-bone (BTB), hamstring tendons (HT), and quadriceps tendon (QT). A cadaveric study was performed to determine if there were any differences in mechanical and structural properties under biomechanical testing. METHODS: Twenty-seven graft specimens were harvested from 9 human cadaveric legs. Mean donor age was 75.2 years (range 53-85 years). Twenty-two specimens (8 HT, 7 QT, and 7 BTB) completed cyclic preconditioning from 50 to 800 N for 200 cycles and a load to failure test at an extension rate of 1 mm/s. Structural and mechanical properties of BTB, HT, and QT grafts were compared using a one-way ANOVA and Tukey's honest significant difference. RESULTS: There was no difference in the ultimate load to failure (N) across all 3 graft types (p = 0.951). Quadriceps tendon demonstrated greater cross-sectional area (mm2) when compared to both HT and BTB (p = 0.001) and was significantly stiffer (N/mm) than HT but not BTB (p = 0.004). Stress (N/mm2) of the HT at ultimate load was greater than QT but not BTB (p = 0.036). Elastic modulus (MPa) of HT was greater than both QT and BTB (p = 0.016). CONCLUSION: There was no difference in the ultimate load to failure of BTB, HT, and QT grafts harvested from the same specimens. All 3 grafts had similar loads to failure with a significant increase in stiffness when compared to the native ACL. Furthermore, QT demonstrated more favourable structural properties compared to HT and BTB with greater cross-sectional area to both HT and BTB and greater stiffness compared to HT.

ORF-selector ESPRIT: a second generation library screen for soluble protein expression employing precise open reading frame selection.

Here we present ORF-selector ESPRIT, a 9-fold enhanced version of our technology for screening incremental truncation libraries to identify soluble high yielding constructs of challenging proteins. Gene fragments are truncated at both termini to access internal domains and the resulting reading frame problem is addressed by an unbiased, intein-based open reading frame selection yielding only in-frame DNA inserts. This enriched library is then subcloned into a standard high-level expression plasmid where tens of thousands of constructs can be assayed in a two-step process using colony- and liquid-handling robots to isolate rare highly expressing clones useful for production of multi milligram quantities of purifiable proteins. The p85α protein was used to benchmark the system resulting in isolation of all known domains, either alone or in tandem. The human kinase IKK1 was then screened resulting in purification of a predicted internal domain. This strategy provides an integrated, facile route to produce soluble proteins from challenging and poorly understood target genes at quantities compatible with structural biology, screening applications and immunisation studies. The high genetic diversity that can be sampled opens the way to study more diverse systems including multisubunit complexes.

CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes.

Structural and biophysical studies of protein complexes require multi-milligram quantities of soluble material. Subunits are often unstable when expressed separately so co-expression strategies are commonly employed since in vivo complex formation can provide stabilising effects. Defining constructs for subunit co-expression experiments is difficult if the proteins are poorly understood. Even more problematic is when subunit polypeptide chains co-fold since individually they do not have predictable domains. We have developed CoESPRIT, a modified version of the ESPRIT random library construct screen used previously on single proteins, to express soluble protein complexes. A random library of target constructs is screened against a fixed bait protein to identify stable complexes. In a proof-of-principle study, C-terminal fragments of the influenza polymerase PB2 subunit containing folded domains were isolated using importin alpha as bait. Separately, a C-terminal fragment of the PB1 subunit was used as bait to trap N-terminal fragments of PB2 resulting in co-folded complexes. Subsequent expression of the target protein without the bait indicates whether the target is independently stable, or co-folds with its partner. This highly automated method provides an efficient strategy for obtaining recombinant protein complexes at yields compatible with structural, biophysical and functional studies.

Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen.

SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity.

Biological and biophysical properties of the histone deacetylase inhibitor suberoylanilide hydroxamic acid are affected by the presence of short alkyl groups on the phenyl ring.

Inhibition of histone deacetylases (HDACs) leads to growth arrest, differentiation, or apoptosis of tumor cell lines, suggesting HDACs as promising targets for cancer therapy. At present, only one HDAC inhibitor (HDACi) is used in therapy: suberoylanilide hydroxamic acid (SAHA). Here, we describe the synthesis and biological evaluation of a new series of compounds derived from SAHA by substituting short alkyl chains at various positions of the phenyl ring. Such modifications induced variable effects ranging from partial loss of activity to increased potency. Through molecular modeling, we describe a possible interaction between HDAC7 proline 809, a residue that is strictly conserved within class 2 enzymes only, and the amide group of HDACi, while nuclear magnetic resonance experiments indicated that dimethyl m-substitution may stabilize the inhibitor in the active site. Our data provide novel information on the structure-activity relationship of HDACi and suggest new ways for developing second generation SAHA-like molecules.

Solubility survey of fragments of the neurofibromatosis type 1 protein neurofibromin.

The protein giant neurofibromin (320kDa) is the protein product of the NF1 tumor suppressor gene, alterations of which are responsible for the pathogenesis of neurofibromatosis type 1 (NF1). Neurofibromin is a Ras-specific GTPase activating protein (RasGAP) that, 15 years after the cloning of the gene, remains the only clearly defined function of the protein. In a structural proteomics approach, we aimed at defining functions beyond RasGAP activity based on the discovery of structural modules. Given the poor outcome of domain prediction tools, we have undertaken a fragment solubility survey covering the full protein sequence, with the aim of defining new domain boundaries or fragments that could be investigated by biochemical methods including structural analysis. More than 200 constructs have been expressed and tested for solubility in small scale assays. Boundaries were chosen based upon secondary structure predictions, sequence conservation among neurofibromin orthologues and chemical properties of amino acids. Using this strategy we recently discovered a novel bipartite module in neurofibromin. We have expanded our study to include ESPRIT, a library-based construct screen, to perform fragment testing at a finer level with respect to the choice of terminal residues. Our study confirms earlier notions about the challenges neurofibromin presents to the biochemist and points to strategies whereby the success rate may be enhanced in the future.

Fabrication of protein function microarrays for systems-oriented proteomic analysis.

Protein microarrays have many potential applications in high-throughput analysis of protein function. However, simple, reproducible, and robust methods for array fabrication are required. Here we discuss the background to different routes to array fabrication and describe in detail one approach in which the purification and immobilization procedures are combined into a single step, dramatically simplifying the array fabrication process. We illustrate this approach by reference to the creation of an array of p53 variants, and discuss methods for assay and data analysis on such arrays.

Involvement of human intracisternal A-type retroviral particles in autoimmunity.

Prior studies have linked retroviruses to various arthropathies and autoimmune diseases. Sjögren's syndrome (SS), a systemic autoimmune disease, is characterized by aggressive infiltration of lymphocytes into the salivary and lacrimal glands, resulting in destruction of the glands and dry mouth and eyes (sicca syndrome). The infiltrating lymphocytes in SS may become overtly malignant, and thus, the incidence of lymphoma is greatly increased in SS patients. A human intracisternal A-type retroviral particle type I (HIAP-I) has been isolated from persons with SS. HIAP-I shares a limited number of antigenic epitopes with human immunodeficiency virus (HIV), but is distinguishable from HIV by morphological, physical, and biochemical criteria. A substantial majority of patients with SS or systemic lupus erythematosus (SLE) have serum antibodies to the proteins of this human retrovirus. Fewer than 3% of the normal blood donor population have antibodies to any HIAP-associated proteins. A second type of a human intracisternal A-type retrovirus, HIAP-II, was detected in a subset of patients with idiopathic CD4 lymphocytopenia (ICL), an AIDS-like immunodeficiency disease. Most HIAP-II positive ICL patients were also antinuclear antibody positive. Reviewed here are additional studies from several laboratories suggesting that HIAP or related viruses may be involved in SLE and other autoimmune conditions. Additionally, results of comprehensive surveys of autoimmune patients to determine seroreactivity to HIAP, and other human retroviruses, including HIV and human T-lymphotropic virus type I, are reported.

Functional protein microarrays for parallel characterisation of p53 mutants.

Understanding the way in which single nucleotide polymorphisms and mutations in the human genome result in individual susceptibility to disease is a major goal in the postgenomic era. Such knowledge should accelerate the development of personalised medicine in which drug treatment can specifically match an individual's genotype. High-throughput DNA sequencing is generating the initial information required, but new technologies are required that can rapidly characterise the phenotypic effects of the identified polymorphisms. For example, many thousands of allelic variants of the p53 gene have been described and are responsible for more than 50% of cancers, however few of the protein products have been functionally characterised. Here we have quantified in parallel the effects of mutations and polymorphisms on the DNA-binding function of the p53 oncoprotein using a protein microarray, allowing their subclassification according to functional effect. Protein-protein interactions between p53 variants and (i) a regulatory oncoprotein, (ii) a regulatory kinase resulting in on-chip phosphorylation, are also described, suggesting the more general utility of this high-throughput assay format.