Protein phosphorylation plays a central role in controlling many diverse signal transduction pathways in all cells. Novel protein kinases are identified at a rapid rate using homology cloning methods and genetic screens or selections; however identification of the direct substrates of kinases has proven elusive to genetic methods because of the tremendous redundancy and overlapping of substrate specificities among protein kinases. We describe the development of a protein engineering-based method to identify the direct substrates of the prototypical protein tyrosine kinase v-Src, which controls fibroblast transformation by the Rous sarcoma virus. To differentiate the substrates of v-Src from all other kinase substrates, we mutated the ATP binding site of v-Src such that the engineered v-Src uniquely accepted an ATP analog. We show that the engineered v-Src kinase displayed catalytic efficiency with the ATP analog, N 6 -(cyclopentyl) ATP, which is similar to the wild-type kinase catalytic efficiency with ATP itself. However, the N 6 -(cyclopentyl) ATP analog was not accepted by the wild-type kinase. Furthermore, the engineered v-Src exhibited the same protein target specificity as wild-type v-Src despite the proximity of the reengineered nucleotide binding site to the phosphoacceptor binding site. The successful engineering of v-Src's active site to accept a unique nucleotide analog provides a unique handle by which the direct substrates of one kinase (v-Src) can be traced in the presence of any number of cellular kinases.

BackgroundThe diagnosis of periprosthetic joint infection (PJI) remains a serious clinical challenge. There is a pressing need for improved diagnostic testing methods; biomarkers offer one potentially promising approach.Questions/purposesWe evaluated the diagnostic characteristics of 16 promising synovial fluid biomarkers for the diagnosis of PJI.MethodsSynovial fluid was collected from 95 patients meeting the inclusion criteria of this prospective diagnostic study. All patients were being evaluated for a revision hip or knee arthroplasty, including patients with systemic inflammatory disease and those already receiving antibiotic treatment. The Musculoskeletal Infection Society (MSIS) definition was used to classify 29 PJIs and 66 aseptic joints. Synovial fluid samples were tested by immunoassay for 16 biomarkers optimized for use in synovial fluid. Sensitivity, specificity, and receiver operating characteristic curve analysis were performed to assess for diagnostic performance.ResultsFive biomarkers, including human α-defensin 1-3, neutrophil elastase 2, bactericidal/permeability-increasing protein, neutrophil gelatinase-associated lipocalin, and lactoferrin, correctly predicted the MSIS classification of all patients in this study, with 100% sensitivity and specificity for the diagnosis of PJI. An additional eight biomarkers demonstrated excellent diagnostic strength, with an area under the curve of greater than 0.9.ConclusionsSynovial fluid biomarkers exhibit a high accuracy in diagnosing PJI, even when including patients with systemic inflammatory disease and those receiving antibiotic treatment. Considering that these biomarkers match the results of the more complex MSIS definition of PJI, we believe that synovial fluid biomarkers can be a valuable addition to the methods utilized for the diagnosis of infection.Level of EvidenceLevel II, diagnostic study. See Instructions for Authors for a complete description of levels of evidence.

Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.

BackgroundSynovial fluid biomarkers have demonstrated diagnostic accuracy surpassing the currently used diagnostic tests for periprosthetic joint infection (PJI).Questions/purposesThe purpose of this study is to directly compare the sensitivity and specificity of the synovial fluid α-defensin immunoassay to the leukocyte esterase (LE) colorimetric test strip.MethodsSynovial fluid was collected from 46 patients meeting the inclusion criteria of this prospective diagnostic study. Synovial fluid samples were tested with both a novel synovial-fluid-optimized immunoassay for α-defensin and the LE colorimetric test strip. The Musculoskeletal Infection Society (MSIS) definition was used to classify 23 periprosthetic infections and 23 aseptic failures; this classification was used as the standard against which the two diagnostic tests were compared.ResultsThe synovial fluid α-defensin immunoassay correctly predicted the MSIS classification of all patients in the study, demonstrating a sensitivity and specificity of 100% for the diagnosis of PJI. The α-defensin assay could be read for all samples, including those with blood in the synovial fluid. The leukocyte esterase test strip could not be interpreted in eight of 46 samples (17%) as a result of blood interference. Analysis of the LE strips that could be interpreted yielded a sensitivity of 69% and a specificity of 100%.ConclusionsThe synovial fluid α-defensin immunoassay outperformed the LE colorimetric test strip in this study and provided reliable results even when the LE test strip failed as a result of blood interference. The simple analytic results provided by the α-defensin immunoassay, compared with the more complex and interpretive nature of both the MSIS criteria and LE colorimetric test strip, make it a highly attractive diagnostic tool.Level of EvidenceLevel II, diagnostic study. See Guidelines for Authors for a complete description of levels of evidence.

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Background We have previously described a unique gene expression signature exhibited by synovial fluid leukocytes in response to bacterial infection, identifying a number of potential biomarkers for infection. However, the diagnostic performance of these potential biomarkers in an immunoassay format is unknown.Questions/purposes We therefore evaluated the sensitivity, specificity, and accuracy of several potential synovial fluid biomarkers for infection, and compared them to current standards of testing for periprosthetic infection. Methods We prospectively collected synovial fluid from 14 patients classified as having a periprosthetic infection and 37 patients classified as having an aseptic failure. The synovial fluid samples were tested for 23 potential biomarkers for periprosthetic infection. We then determined differences in biomarker levels between infected and aseptic groups, then computed the sensitivity, specificity, positive predictive value, negative predictive value and accuracy for select biomarkers, and finally compared those to current standard tests for infection. Results Twelve synovial fluid biomarkers had substantially higher average levels in the synovial fluid of infected versus aseptic patients. Synovial fluid levels of IL-1 were a mean of 258 times higher in patients with a periprosthetic infection compared to patients having revision for aseptic diagnoses. Synovial fluid IL-1 and IL-6 levels correctly classified all patients in this study with a sensitivity, specificity, positive predictive value, negative predictive value and accuracy equal to 1. Several markers tested in this study outperformed the ESR and CRP tests. Conclusions Patients with a periprosthetic infection have elevated levels of numerous synovial fluid biomarkers, when compared to patients with aseptic diagnoses. Several of these biomarkers exhibited nearly ideal sensitivity, specificity, and accuracy in this study, suggesting that synovial fluid biomarkers could be a valuable tool for diagnosing periprosthetic infection.

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BackgroundThe alpha-defensin test has been previously demonstrated to be highly accurate in the diagnosis of prosthetic joint infection (PJI), nearly matching the Musculoskeletal Infection Society definition for PJI. However, the relationship between alpha-defensin levels and differing infecting organism has not yet been investigated.Questions/purposesThe purpose of this study is to describe the breadth of organisms that can trigger a positive synovial fluid alpha-defensin test result in the setting of PJI and also to assess the magnitude of the alpha-defensin result in terms of various pathogen characteristics.MethodsBetween December 2012 and March 2014, one laboratory processed 2319 synovial fluid samples for alpha-defensin testing. The present study reviewed the results of the 1937 samples that simultaneously had a synovial fluid culture performed; these came from 418 surgeons in 42 states. The overall culture-positive rate was 49% (244 of 498) among alpha-defensin-positive synovial fluids and 1% (19 of 1439) among alpha-defensin-negative synovial fluids. The organisms recovered from 244 alpha-defensin-positive, culture-positive fluids were recorded and grouped based on various characteristics, including Gram type, species, virulence, oral pathogenicity, and source joint. Alpha-defensin-negative samples served as uninfected controls. Median alpha-defensin levels were calculated for each group, and Dunn’s multiple comparison test for nonparametric data was used to identify any statistically significant (p < 0.05) organism-specific differences in the alpha-defensin level.ResultsThe alpha-defensin test for PJI was positive in the setting of a wide spectrum of organisms typically causing PJI. The median alpha-defensin level for all 244 alpha-defensin-positive, culture-positive samples (4.7 [interquartile range {IQR}, 3.7–5.3]) was higher than negative controls (0.26 [IQR, 0.22–0.33]) with a median difference of 4.4 (p < 0.001). There were no differences in the median alpha-defensin levels when performing a multiple comparison test among Gram-positive organisms (4.7 [IQR, 3.6–5.3]), Gram-negative organisms (4.8 [IQR, 4.2–5.3]), yeast (4.1 [IQR, 2.2–5.1]), virulent organisms (4.7 [IQR, 3.8–5.2]), less virulent organisms (4.8 [IQR, 3.6–5.4]), oral pathogens (4.5 [IQR, 3.2–5.2]), knees (4.7 [IQR, 3.7–5.3]), hips (4.9 [IQR, 4.1–5.8]), or shoulders (5.3 [IQR, 4.0–10.7]) with all comparisons having a p > 0.999.ConclusionsThe alpha-defensin test provides consistent results regardless of the organism type, Gram type, species, or virulence of the organism and should be seriously considered to be a standard diagnostic tool in the evaluation for PJI. Future research should focus on the performance of this test in specific clinical scenarios such as the immediate postoperative period in the setting of severe immunocompromise and in the setting of a native joint.Level of EvidenceLevel III, diagnostic study.