Clinical Practice Update: Achilles Tendon Rupture.

Achilles tendon rupture is a common injury. It most often occurs in middle aged men who participate in recreational sports. The injury classically presents with a loud popping noise and immediate pain and weakness of the lower extremity during actions such as jumping or running. The diagnosis is made clinically, but an MRI is often obtained for confirmation of rupture and to aid in surgical planning. Treatment is either operative, with open or minimally invasive approaches, or non-operative, with functional bracing or plaster casting. Surgical treatment was preferred for much of the 20th century, but non-operative treatment has gained significant favor in the past 15 years as new evidence has demonstrated similar long-term outcomes to surgery. Neither treatment option is currently considered superior to the other in all cases. Surgery is associated with a risk for surgical complications and is, therefore, often a poor option for the elderly and those with significant comorbidities. Non-operative management is associated with an increased risk for re-injury which is often undesirable for young and highly active patients. Ultimately, the goals and priorities of each individual patient should guide the decision of which treatment option to pursue.

Genetic Encoding of N6-(((Trimethylsilyl)methoxy)carbonyl)-l-lysine for NMR Studies of Protein-Protein and Protein-Ligand Interactions.

Trimethylsilyl (TMS) groups present outstanding NMR probes of biological macromolecules as they produce intense singlets in 1H NMR spectra near 0 ppm, where few other proton resonances occur. We report a system for genetic encoding of N6-(((trimethylsilyl)methoxy)carbonyl)-l-lysine (TMSK) for site-specific incorporation into proteins. The system is based on pyrrolysyl-tRNA synthetase mutants, which deliver proteins with high yield and purity in vivo and in cell-free protein synthesis. As the TMS signal can readily be identified in 1D 1H NMR spectra of high-molecular weight systems without the need of isotopic labeling, TMSK delivers an excellent site-specific NMR probe for the study of protein structure and function, which is both inexpensive and convenient. We demonstrate the utility of TMSK to detect ligand binding, measure the rate of conformational change, and assess protein dimerization by paramagnetic relaxation enhancement. In addition, we present a system for dual incorporation of two different unnatural amino acids (TMSK and O-tert-butyl-tyrosine) in the same protein in quantities sufficient for NMR spectroscopy. Close proximity of the TMS and tert-butyl groups was readily detected by nuclear Overhauser effects.

Genetically encoded amino acids with tert-butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy.

The amino acids 4-(tert-butyl)phenylalanine (Tbf) and 4-(trimethylsilyl)phenylalanine (TMSf), as well as a partially deuterated version of Tbf (dTbf), were chemically synthesized and site-specifically incorporated into different proteins, using an amber stop codon, suppressor tRNA and the broadband aminoacyl-tRNA synthetase originally evolved for the incorporation of p-cyano-phenylalanine. The 1H-NMR signals of the tert-butyl and TMS groups were compared to the 1H-NMR signal of tert-butyltyrosine (Tby) in protein systems with molecular weights ranging from 8 to 54 kDa. The 1H-NMR resonance of the TMS group appeared near 0 ppm in a spectral region with few protein resonances, facilitating the observation of signal changes in response to ligand binding. In all proteins, the R 2 relaxation rate of the tert-butyl group of Tbf was only little greater than that of Tby (less than two-fold). Deuteration of the phenyl ring of Tbf made only a relatively small difference. The effective T 2 relaxation time of the TMS signal was longer than 140 ms even in the 54 kDa system.

Trimethylsilyl tag for probing protein-ligand interactions by NMR.

Protein-ligand titrations can readily be monitored with a trimethylsilyl (TMS) tag. Owing to the intensity, narrow line shape and unique chemical shift of a TMS group, dissociation constants can be determined from straightforward 1D 1H-NMR spectra not only in the fast but also in the slow exchange limit. The tag is easily attached to cysteine residues and a sensitive reporter of ligand binding also at sites where it does not interfere with ligand binding or catalytic efficiency of the target protein. Its utility is demonstrated for the Zika virus NS2B-NS3 protease and the human prolyl isomerase FK506 binding protein.

Site-Specific Incorporation of Selenocysteine by Genetic Encoding as a Photocaged Unnatural Amino Acid.

Selenocysteine (Sec) is a naturally occurring amino acid that is also referred to as the 21st amino acid. Site-specific incorporation of Sec into proteins is attractive, because the reactivity of a selenol group exceeds that of a thiol group and thus allows site-specific protein modifications. It is incorporated into proteins by an unusual enzymatic mechanism which, in E. coli and other organisms, involves the recognition of a selenocysteine insertion sequence (SECIS) in the mRNA of the target protein. Reengineering of the natural machinery for Sec incorporation at arbitrary sites independent of SECIS elements, however, is challenging. Here we demonstrate an alternative route, whereby a photocaged selenocysteine (PSc) is incorporated as an unnatural amino acid in response to an amber stop codon, using a mutant Methanosarcina mazei pyrrolysyl-tRNA synthetase, Mm PCC2RS, and its cognate tRNACUA. Following decaging by UV irradiation, proteins synthesized with PSc are readily tagged, e.g., with NMR probes to study ligand binding by NMR spectroscopy. The approach provides a facile route for genetically encoded Sec incorporation. It allows the production of pure selenoproteins and the Sec residue enables site-specific covalent protein modification with reagents that would usually react first with naturally occurring cysteine residues. The much greater reactivity of Sec residues allows their selective alkylation in the presence of highly solvent-exposed cysteine residues.

Small neutral Gd(iii) tags for distance measurements in proteins by double electron-electron resonance experiments.

Spin labels containing a Gd(iii) ion have become important for measuring nanometer distances in proteins by double electron-electron resonance (DEER) experiments at high EPR frequencies. The distance resolution and sensitivity of these measurements strongly depend on the Gd(iii) tag used. Here we report the performance of two Gd(iii) tags, propargyl-DO3A and C11 in DEER experiments carried out at W-band (95 GHz). Both tags are small, uncharged and devoid of bulky hydrophobic pendants. The propargyl-DO3A tag is designed for conjugation to the azide-group of an unnatural amino acid. The C11 tag is a new tag designed for attachment to a single cysteine residue. The tags delivered narrower distance distributions in the E. coli aspartate/glutamate binding protein and the Zika virus NS2B-NS3 protease than previously established Gd(iii) tags. The improved performance is consistent with the absence of specific hydrophobic or charge-charge interactions with the protein. In the case of the Zika virus NS2B-NS3 protease, unexpectedly broad Gd(iii)-Gd(iii) distance distributions observed with the previously published charged C9 tag, but not the C11 tag, illustrate the potential of tags to perturb a labile protein structure and the importance of different tags. The results obtained with the C11 tag demonstrate the closed conformation in the commonly used linked construct of the Zika virus NS2B-NS3 protease, both in the presence and absence of an inhibitor.

Chemical Tagging with tert-Butyl and Trimethylsilyl Groups for Measuring Intermolecular Nuclear Overhauser Effects in a Large Protein-Ligand Complex.

Intermolecular 1 H-1 H nuclear Overhauser effects (NOE) present a powerful tool to assess contacts between proteins and binding partners, but are difficult to identify for complexes of high molecular weight. This report shows that intermolecular NOEs can readily be observed following chemical labeling with tert-butyl or trimethylsilyl (TMS) groups. Proteins can be furnished with tert-butyl or TMS groups site-specifically using genetically encoded unnatural amino acids or by chemical modification of single cysteine residues. No isotope labeling is required. The approach is demonstrated with the 95 kDa complex between tetrameric E. coli single-stranded DNA binding protein (SSB) and single-stranded DNA.

Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags.

Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δχ) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd(3+)-Gd(3+) distances measured by double electron-electron resonance (DEER) experiments to define the metal position, allowing Δχ-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs. Using this approach with the 32 kDa E. coli aspartate/glutamate binding protein (DEBP), we demonstrate a structural transition between substrate-bound and substrate-free DEBP, supported by PCSs generated by C3-Tm(3+) and C3-Tb(3+) tags attached to a genetically encoded p-azidophenylalanine residue. The significance of small PCSs was magnified by considering the difference between the chemical shifts measured with Tb(3+) and Tm(3+) rather than involving a diamagnetic reference. The integrative sparse data approach developed in this work makes poorly soluble proteins of limited stability amenable to structural studies in solution, without having to rely on cysteine mutations for tag attachment.

Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA.

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.

Mechanical Analysis of a Novel 3D-printed External Fixator Design Versus Industry-standard External Fixators.

INTRODUCTION: External fixation is a critical component of orthopaedic fracture management and is used for various conditions, including trauma and pediatric orthopaedics. However, the availability and high cost of external fixation devices are a concern, especially in rural and developing countries. 3D printing technology has shown promise in reducing manufacturing costs and improving accessibility to external fixation devices. The purpose of this study was to evaluate the mechanical properties of a fully 3D-printed desktop external fixation device and compare the results with the mechanical properties of commonly used, clinically available external fixators. METHODS: A fully 3D printable external fixator was designed and printed in polylactic acid at two infill densities, 20% and 100%. The mechanical properties of the 3D-printed external fixators and several commercially available fixators were tested according to applicable sections of the American Society for Testing and Materials F1541 standard protocol in axial, medial-lateral, and anterior-posterior orientations. The primary outcomes measured included failure load, safe load, rigidity, and yield load. The mean differences between experimental and control groups were calculated using one-way analysis of variance and Tukey tests. RESULTS: The 20% infill 3D-printed construct showed poor performance compared with commercially available external fixators in all testing conditions and across most variables. The 100% infill 3D-printed construct was comparable with or superior to all commercially available devices in most testing conditions. The cost for printing a single 3D-printed 100% infill external fixator was $14.49 (United States Dollar). DISCUSSION: This study demonstrates that a low-cost desktop 3D printer can create an entirely 3D-printed external fixator that resists clinically relevant forces similar to medical-grade industry-standard external fixators. Therefore, there is potential for customizable and low-cost external fixators to be manufactured with desktop 3D printing for use in remote areas and other resource-constrained environments for fracture care.

Recommendations for success in biomechanics outreach.

Student engagement in science, technology, engineering, and math (STEM) through informal outreach events is critical to the current educational pipeline. National Biomechanics Day (NBD) is a STEM outreach event that is an international celebration of the science of biomechanics with the goal of introducing high school students to the field. While NBD has experienced global success and substantial growth in recent years, it is an equally rewarding and challenging endeavor to host an NBD event. In this paper, we provide recommendations and mechanisms for biomechanics professionals to support their success in hosting biomechanics outreach events. Although these guidelines are framed around hosting an NBD event, the underlying principles can apply to hosting any STEM outreach event.

Arthroscopic Repair of the Acetabular Labrum Using an Anchor-First Modified Toggle Suture Technique.

The goal of acetabular labral repair is to restore stable contact between the labrum and acetabular rim while maintaining the anatomic suction seal. One of the challenges of labral repair is achieving proper in-round repair, so that the labrum contacts the femoral head in the native position. This technique article presents a repair method that allows for enhanced inversion of the labrum to assist with anatomic repair. Our modified toggle suture technique utilizes an anchor-first method and has various distinct technical advantages. We present an efficient and vendor-agnostic technique that allows for straight or curved guides. Similarly, the anchors may be all-suture or hard-anchor designs that accommodate suture sliding. This technique also utilizes a self-retaining hand-tied knot construct to facilitate preventing knots from migrating toward the femoral head or joint space.

Clinical shoulder measurements related to joint loads in collegiate pitchers.

Background: Pitchers are prone to upper extremity injury due to repetitive high joint loads. Clinical measures of shoulder strength and range of motion (ROM) have shown links to injury risk in pitchers, however, these factors have rarely been studied in relation to throwing joint loads. The purpose of this study was to identify which clinical ROM and isokinetic strength variables were related to peak shoulder and elbow joint torques in collegiate pitchers. Methods: Thirty-three healthy collegiate pitchers participated in this study. Fastball velocity, shoulder concentric and eccentric strength, and passive shoulder ROM variables were analyzed using a Lasso regression to determine what factors influenced shoulder internal rotation torque and elbow varus torque. Results: Fastball velocity was selected by the Lasso as indicator of increased shoulder and elbow torque. Passive shoulder external rotation ROM was also selected as an important factor in joint loading with increased shoulder external rotation ROM being related to lower joint loads. The bilateral ratio of shoulder internal rotator concentric strength was related to peak shoulder and elbow torques with an increase in the bilateral ratio of shoulder strength leading to reduced joint torques. Increases in the eccentric external rotator to concentric internal rotator strength (functional ratio) of the dominant arm and increases in dominant arm eccentric internal rotator strength were both related to increases in each joint torque. Conclusion: Results from the study indicate that pitch speed, passive shoulder external rotation ROM, and the isokinetic shoulder strength profile including internal rotator strength and functional strength ratio of pitchers are related to joint loading during the pitch and may be important to monitor in relation to injury risk and/or during rehabilitation. These results provide insight into the role that both shoulder ROM and rotator cuff strength play in the dynamic stabilization of the elbow and shoulder during pitching.

3D Printed Orthopaedic External Fixation Devices: A Systematic Review.

BACKGROUND: External fixators are complex, expensive orthopaedic devices used to stabilize high-energy and complex fractures of the extremities. Although the technology has advanced dramatically over the last several decades, the mechanical goals for fracture stabilization of these devices have remained unchanged. Three-dimensional (3D) printing technology has the potential to advance the practice and access to external fixation devices in orthopaedics. This publication aims to systematically review and synthesize the current literature on 3D printed external fixation devices for managing orthopaedic trauma fractures. METHODS: The Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) protocols were followed for this manuscript with minor exceptions. PubMed, Embase, Cochrane Review, Google Scholar, and Scopus online databases were systematically searched. Two independent reviewers screened the search results based on predetermined inclusion and exclusion criteria related to 3D printing and external fixation of fractures. RESULTS: Nine studies met the inclusion criteria. These included one mechanical testing study, two computational simulation studies, three feasibility studies, and three clinical case studies. Fixator designs and materials varied significantly between authors. Mechanical testing revealed similar strength to traditional metal external fixators. Across all clinical studies, five patients underwent definitive treatment with 3D printed external fixators. They all had satisfactory reduction and healing with no reported complications. CONCLUSIONS: The current literature on this topic is heterogeneous, with highly variable external fixator designs and testing techniques. A small and limited number of studies in the scientific literature have analyzed the use of 3D printing in this area of orthopaedic surgery. 3D printed external fixation design advancements have yielded promising results in several small clinical case studies. However, additional studies on a larger scale with standardized testing and reporting techniques are needed.

Rapid Elaboration of Fragments into Leads Applied to Bromodomain-3 Extra-Terminal Domain.

The development of low-affinity fragment hits into higher-affinity leads is a major hurdle in fragment-based drug design. Here, we demonstrate the Rapid Elaboration of Fragments into Leads (REFiL) by applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure-activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical diversity. After a fragment screen against bromodomain-3 extra-terminal (BRD3-ET) domain, we applied the REFiL workflow, which allowed us to develop a series of ligands that bind to BRD3-ET. With REFiL, we were able to rapidly improve binding affinity > 30-fold. REFiL can be applied readily to a broad range of proteins without the need for a structure, allowing the efficient evolution of low-affinity fragments into higher-affinity leads and chemical probes.

Delivery of toll-like receptor agonists by complement C3-targeted liposomes activates immune cells and reduces tumour growth.

Activation of antigen presenting cells (APCs) is necessary for immune recognition and elimination of cancer. Our lab has developed a liposome nanoparticle that binds to complement C3 proteins present in serum. These C3-liposomes are specifically internalised by APCs and other myeloid cells, which express complement C3-binding receptors. Known immune stimulating compounds, toll-like receptor (TLR) agonists, were encapsulated within the C3-liposomes, including monophosphoryl lipid A (MPLA), R848, and CpG 1826, specific for TLR4, TLR7/8, and TLR9 respectively. When recognised by their respective TLRs within the myeloid cells, these compounds trigger signal cascades that ultimately lead to increased expression of inflammatory cytokines and activation markers (CD80, CD83, CD86 and CD40). RT-PCR analysis of murine bone marrow cells treated with C3-liposomes revealed a significant increase in gene expression of pro-inflammatory cytokines and factors (IL-1ß, IL-6, IL-12, TNF-α, IRF7, and IP-10). Furthermore, treatment of 4T1 tumour-bearing mice with C3-liposomes containing TLR agonists resulted in reduced tumour growth, compared to PBS treated mice. Collectively, these results demonstrate that C3-liposome delivery of TLR agonists activates APCs and induces tumour-specific adaptive immune responses, leading to reduced tumour growth in a breast cancer model.

Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme.

Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron-electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.