Whole cell, label free protein quantitation with data independent acquisition: quantitation at the MS2 level.

Label free quantitation by measurement of peptide fragment signal intensity (MS2 quantitation) is a technique that has seen limited use due to the stochastic nature of data dependent acquisition (DDA). However, data independent acquisition has the potential to make large scale MS2 quantitation a more viable technique. In this study we used an implementation of data independent acquisition--SWATH--to perform label free protein quantitation in a model bacterium Clostridium stercorarium. Four tryptic digests analyzed by SWATH were probed by an ion library containing information on peptide mass and retention time obtained from DDA experiments. Application of this ion library to SWATH data quantified 1030 proteins with at least two peptides quantified (∼ 40% of predicted proteins in the C. stercorarium genome) in each replicate. Quantitative results obtained were very consistent between biological replicates (R(2) ∼ 0.960). Protein quantitation by summation of peptide fragment signal intensities was also highly consistent between biological replicates (R(2) ∼ 0.930), indicating that this approach may have increased viability compared to recent applications in label free protein quantitation. SWATH based quantitation was able to consistently detect differences in relative protein quantity and it provided coverage for a number of proteins that were missed in some samples by DDA analysis.

Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma.

BACKGROUND: Neuropilin 2 (NRP2) isa multi-functional co-receptor to many receptors, including VEGF receptor, c-Met and others. NRP2 has recently been implicated in tumor angiogenesis, growth, and metastasis of many other cancers. However, its role in osteosarcoma remains poorly understood. RESULTS: NRP2 was overexpressed in osteosarcoma cell lines and tissues, and associated with poor survival of osteosarcoma patients. Knockdown of NRP2 expression by short-hairpin (Sh) RNA resulted in reduced tumor growth, metastasis, and blood vessel formation of osteosarcoma. Knockdown of NRP2 expression by ShRNA also inhibited the recruitment of HUVEC cells to osteosarcoma cells. Inhibition of Wnt signaling by overexpression of secreted Wnt antagonists soluble LRP5, Frzb, and WIF1 markedly down-regulated mRNA and protein expression of NRP2 in osteosarcoma cell lines. CONCLUSIONS: Regulation of NRP2 receptor expression may represent a novel approach for treatment of osteosarcoma through retarding osteosarcoma growth, metastasis and blood vessel formation. In addition, down-regulation of NRP2 expression can be achieved by expression of secreted Wnt antagonists.

Enhanced whole genome sequence and annotation of Clostridium stercorarium DSM8532T using RNA-seq transcriptomics and high-throughput proteomics.

BACKGROUND: Growing interest in cellulolytic clostridia with potential for consolidated biofuels production is mitigated by low conversion of raw substrates to desired end products. Strategies to improve conversion are likely to benefit from emerging techniques to define molecular systems biology of these organisms. Clostridium stercorarium DSM8532T is an anaerobic thermophile with demonstrated high ethanol production on cellulose and hemicellulose. Although several lignocellulolytic enzymes in this organism have been well-characterized, details concerning carbohydrate transporters and central metabolism have not been described. Therefore, the goal of this study is to define an improved whole genome sequence (WGS) for this organism using in-depth molecular profiling by RNA-seq transcriptomics and tandem mass spectrometry-based proteomics. RESULTS: A paired-end Roche/454 WGS assembly was closed through application of an in silico algorithm designed to resolve repetitive sequence regions, resulting in a circular replicon with one gap and a region of 2 kilobases with 10 ambiguous bases. RNA-seq transcriptomics resulted in nearly complete coverage of the genome, identifying errors in homopolymer length attributable to 454 sequencing. Peptide sequences resulting from high-throughput tandem mass spectrometry of trypsin-digested protein extracts were mapped to 1,755 annotated proteins (68% of all protein-coding regions). Proteogenomic analysis confirmed the quality of annotation and improvement pipelines, identifying a missing gene and an alternative reading frame. Peptide coverage of genes hypothetically involved in substrate hydrolysis, transport and utilization confirmed multiple pathways for glycolysis, pyruvate conversion and recycling of intermediates. No sequences homologous to transaldolase, a central enzyme in the pentose phosphate pathway, were observed by any method, despite demonstrated growth of this organism on xylose and xylan hemicellulose. CONCLUSIONS: Complementary omics techniques confirm the quality of genome sequence assembly, annotation and error-reporting. Nearly complete genome coverage by RNA-seq likely indicates background DNA in RNA extracts, however these preps resulted in WGS enhancement and transcriptome profiling in a single Illumina run. No detection of transaldolase by any method despite xylose utilization by this organism indicates an alternative pathway for sedoheptulose-7-phosphate degradation. This report combines next-generation omics techniques to elucidate previously undefined features of substrate transport and central metabolism for this organism and its potential for consolidated biofuels production from lignocellulose.

Increased genital mucosal cytokines in Canadian women associate with higher antigen-presenting cells, inflammatory metabolites, epithelial barrier disruption, and the depletion of L. crispatus.

BACKGROUND: Cervicovaginal inflammation has been linked to negative reproductive health outcomes including the acquisition of HIV, other sexually transmitted infections, and cervical carcinogenesis. While changes to the vaginal microbiome have been linked to genital inflammation, the molecular relationships between the functional components of the microbiome with cervical immunology in the reproductive tract are understudied, limiting our understanding of mucosal biology that may be important for reproductive health. RESULTS: In this study, we used a multi'-omics approach to profile cervicovaginal samples collected from 43 Canadian women to characterize host, immune, functional microbiome, and metabolome features of cervicovaginal inflammation. We demonstrate that inflammation is associated with lower amounts of L. crispatus and higher levels of cervical antigen-presenting cells (APCs). Proteomic analysis showed an upregulation of pathways related to neutrophil degranulation, complement, and leukocyte migration, with lower levels of cornified envelope and cell-cell adherens junctions. Functional microbiome analysis showed reductions in carbohydrate metabolism and lactic acid, with increases in xanthine and other metabolites. Bayesian network analysis linked L. crispatus with glycolytic and nucleotide metabolism, succinate and xanthine, and epithelial proteins SCEL and IVL as major molecular features associated with pro-inflammatory cytokines and increased APCs. CONCLUSIONS: This study identified key molecular and immunological relationships with cervicovaginal inflammation, including higher APCs, bacterial metabolism, and proteome alterations that underlie inflammation. As APCs are involved in HIV transmission, parturition, and cervical cancer progression, further studies are needed to explore the interactions between these cells, bacterial metabolism, mucosal immunity, and their relationship to reproductive health. Video Abstract.

Information-dependent LC-MS/MS acquisition with exclusion lists potentially generated on-the-fly: case study using a whole cell digest of Clostridium thermocellum.

We have developed a real-time graphic-processor-unit-based search engine capable of high-quality peptide identifications in <500 µs per spectrum. The steps of peptide/protein identification, in-silico prediction of all possible tryptic peptides from these proteins, and the prediction of their expected retention times and m/z values take less than 5 s per cycle over ∼3000 MS/MS spectra. This lays the foundation for information-dependent acquisition with exclusion lists generated on-the-fly, as the instrument continues to acquire data. While a complete evaluation of the dynamic exclusion system requires the participation from instrument vendors, we conducted a series of model experiments using a whole cell tryptic digestion of the bacterium Clostridium thermocellum. We ran a series of five iterative LC-MS/MS runs, adding a new exclusion list at each of four chromatographic "tripping points" - the elution times of the four standard peptides spiked into the sample. Retention times of these standard peptides were also used for real-time "chromatographic calibration." The dynamic exclusion approach gave a ≈ 5% increase in confident protein identification (for typical 2 h LC-MS/MS run), and reduced the average number of identified peptides per protein from 4.7 to 2.9. Its application to a two-times shorter gradient gave a ≈ 17% increase in proteins identified. Further improvements are possible for instruments with better mass accuracy, by employing a more accurate retention prediction algorithm and by developing better understanding of the possible chemical modifications and fragmentations produced during electrospray ionization.

Optimal selection of 2D reversed-phase-reversed-phase HPLC separation techniques in bottom-up proteomics.

Recent developments in bottom-up proteomics have supplanted the use of gel-based approaches in favor of multidimensional chromatographic separations of peptide mixtures followed by mass spectrometry analysis. This trend is driven by the desire to eliminate labor-intensive in-gel digestion procedures and increase proteome coverage through better recovery of proteolytic fragments. Introduction of reversed-phase-reversed-phase 2D separation techniques is one of the major improvements that have made this possible. In this article, we review recent developments in 2D HPLC and highlight variations in reversed-phase HPLC separation selectivity that allow for superior peak capacity in peptide fractionation.

Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression.

BACKGROUND: Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. RESULTS: Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase. CONCLUSIONS: Relative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H2 synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.

Tat peptide-calmodulin binding studies and bioinformatics of HIV-1 protein-calmodulin interactions.

The human immunodeficiency virus type 1 (HIV-1) genome encodes 18 proteins and 2 peptides. Four of these proteins encode high-affinity calmodulin-binding sites for which direct interactions with calmodulin have already been described. In this study, the HIV-1 proteome is queried using an algorithm that predicts calmodulin-binding sites revealing seven new putative calmodulin-binding sites including residues 34-56 of the transactivator of transcription (Tat). Tat is a 101-residue intrinsically disordered RNA-binding protein that plays a central role in the regulation of HIV-1 replication. Interactions between a Tat peptide (residues 34-56), melittin, a well-characterized calmodulin-binding peptide, and calmodulin were examined by direct binding studies, mass spectrometry, and fluorescence. The Tat peptide binds to both calcium-saturated and apo-calmodulin with a low micromolar affinity. Conformational changes induced in the Tat peptide were determined by circular dichroism, and residues in calmodulin that interact with the peptide were identified by HSQC NMR spectroscopy. Multiple interactions between HIV-1 proteins and calmodulin, a highly promiscuous signal transduction hub protein, may be an important mechanism by which the virus controls cell physiology.

Development and characterization of SARS-CoV-2 variant-neutralizing monoclonal antibodies.

Vaccination and administration of monoclonal antibody cocktails are effective tools to control the progression of infectious diseases and to terminate pandemics such as COVID-19. However, the emergence of SARS-CoV-2 mutants with enhanced transmissibility and altered antigenicity requires broad-spectrum therapies. Here we developed a panel of SARS-CoV-2 specific mouse monoclonal antibodies (mAbs), and characterized them based on ELISA, Western immunoblot, isotyping, and virus neutralization. Six neutralizing mAbs that exhibited high-affinity binding to SARS-CoV-2 spike protein were identified, and their amino acid sequences were determined by mass spectrometry. Functional assays confirmed that three mAbs, F461G11, F461G15, and F461G16 neutralized four variants of concern (VOC): B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.2 (delta) These mAbs are promising candidates for COVID-19 therapy, and understanding their interactions with virus spike protein should support further vaccine and antibody development.

The Wnt inhibitory factor 1 restoration in prostate cancer cells was associated with reduced tumor growth, decreased capacity of cell migration and invasion and a reversal of epithelial to mesenchymal transition.

BACKGROUND: Aberrations in the Wnt pathway have been reported to be involved in the metastasis of prostate cancer (PCa) to bone. We investigated the effect and underlying mechanism of a naturally-occurring Wnt inhibitor, WIF1, on the growth and cellular invasiveness of a bone metastatic PCa cell line, PC3. RESULTS: The WIF1 gene promoter was hypermethylated and its expression down-regulated in the majority (7 of 8) of PCa cell lines. Restoration of WIF1 expression in PC-3 cells resulted in a decreased cell motility and invasiveness via up-regulation of epithelial markers (E-cadherin, Keratin-8 and-18), down-regulation of mesenchymal markers (N-cadherin, Fibronectin and Vimentin) and decreased activity of MMP-2 and -9. PC3 cells transfected with WIF1 consistently demonstrated reduced expression of Epithelial-to-Mesenchymal Transition (EMT) transcription factors, Slug and Twist, and a change in morphology from mesenchymal to epithelial. Moreover, WIF1 expression significantly reduced tumor growth by approximately 63% in a xenograft mouse model. This was accompanied by an increased expression of E-cadherin and Keratin-18 and a decreased expression of vimentin in tumor tissues. CONCLUSION: These data suggest that WIF1 regulates tumor invasion through EMT process and thus, may play an important role in controlling metastatic disease in PCa patients. Blocking Wnt signaling in PCa by WIF1 may represent a novel strategy in the future to reduce metastatic disease burden in PCa patients.

The important characteristics of soil organic matter affecting 2,4-dichlorophenoxyacetic acid sorption along a catenary sequence.

Variations in the characteristics of soil organic matter (SOM) at the field-scale are largely unknown, particularly in relation to observed variations in herbicide sorption. For the herbicide 2,4-D [2,4-dichlorophenoxyacetic acid], we found that its organic carbon-normalized sorption coefficient, Koc, varied by four-fold, from 76 to 315 L kg(-1), in the Ap-horizon along a slope transect in an undulating agricultural field in Manitoba, Canada. In order to explain the relatively large in-field variation in 2,4-D Koc values, techniques ranging from conventional chemical fractionation methods to solid state Cross Polarization and Magic-Angle Spinning (13)C-Nuclear Magnetic Resonance applied on whole soils, were used to derive SOM chemical, physical and structural parameters for correlation analyses with the measured 2,4-D Koc values on whole soils. Out of the 15 parameters considered, the 2,4-D Koc was significantly positively correlated with 1) the carbon (C) content of sodium hydroxide-extracted humic acids (r = 0.83, P < 0.01), a chemical parameter indicative of free form C in soil; 2) the molar absorptivity of humic acids at wavelength 280 nm (r = 0.81, P < 0.01), a physical parameter indicative of greater SOM aromaticity; and 3) the relatively intensity of aryl C (r = 0.92, P < 0.01) and O-aryl C (r = 0.93, P < 0.01) in whole soil, both structural parameters indicative of aromatic C. Consequently, the results suggest that in-field variations in 2,4-D Koc values are induced by variations in SOM aromaticity. Koc values are among the most sensitive parameters in herbicide fate models used in regulatory and environmental assessments. Currently, these herbicide fate models do not consider associations between SOM characteristics and Koc and hence revising model equations to include these associations may improve estimates of herbicide persistence, bioavailability and transport at the field-scale.

Natural History of Radiographic First Metatarsophalangeal Joint Osteoarthritis: A Nineteen-Year Population-Based Cohort Study.

OBJECTIVE: To assess the long-term prevalence, natural history, progression, and incidence of radiographic first metatarsophalangeal (MTP) joint osteoarthritis (OA). METHODS: A longitudinal cohort design was used in which radiographic OA at the first MTP joint was investigated in participants from the Chingford 1,000 Women Study at year 6 (1995) and year 23 (2013-2015). Radiographic features of osteophytes (OPs) and/or joint space narrowing (JSN) at the first MTP joint were scored according to a validated foot atlas. Natural history was determined by the change in prevalence, incidence, progression, and worsening of OA in the first MTP joint. RESULTS: Complete case-matched foot radiographic data were available for 193 of the women currently enrolled in the study (mean ± SD age 75.7 ± 5.2 years [range 69-90 years]). At the level of the first MTP joint, prevalence of OA at year 6 was 21.76% in the left and 24.35% in the right; at year 23, it was 23.83% in the left and 32.64% in the right. Over the 19-year period, 13.5% of the women developed incident OA in the right first MTP joint and 8.3% in the left. Both progression and worsening of OA were more evident for OPs and in the right first MTP joints. CONCLUSION: In this study of the natural history of radiographic first MTP joint OA, which to our knowledge is the longest study to date, the prevalence and incidence of first MTP joint OA increased over a 19-year period. Progression and/or worsening of OA at the first MTP joint over time appears to be driven by OP development rather than JSN, which suggests a biomechanical cause.

Intestinal proteomic analysis of a novel non-human primate model of experimental colitis reveals signatures of mitochondrial and metabolic dysfunction.

Animal models recapitulating features of chronic colitis, such as ulcerative colitis, Crohn's disease, or HIV infection, are critical to study disease pathogenesis and test novel therapeutics. In this study, we used a proteomics approach to explore the molecular intestinal response in two rhesus macaque (RM) animal models of experimentally induced colitis using dextran sulfate sodium (DSS) and simian immunodeficiency virus (SIV) infection. Proteomic analysis detected more than 2500 proteins in colonic tissue collected from 30 RMs. Differential protein expression analysis revealed a protein expression pattern in DSS-treated RMs resembling the proteome of human ulcerative colitis. In a group of 12 DSS-treated RMs compared to 6 with no treatment, decrease in expression of proteins related to mitochondrial energy metabolism, including fatty acid metabolism was noted, while innate immune activation pathways, including complement and coagulation proteins were upregulated. SIV infection of RMs resulted in increased innate immune responses related to viral defense. Proteomic signatures of barrier damage were apparent in both DSS treatment or SIV infection. These results demonstrate that DSS treatment in a non-human primate model resembles features of human ulcerative colitis, making this a promising tool to study important immunological mechanisms in inflammatory bowel disease.

Nonoperative Treatment of Elbow Ulnar Collateral Ligament Injuries With and Without Platelet-Rich Plasma in Professional Baseball Players: A Comparative and Matched Cohort Analysis.

BACKGROUND: Recent studies evaluating nonoperative treatment of elbow ulnar collateral ligament (UCL) injuries augmented with platelet-rich plasma (PRP) have shown promising results. To date, no comparative studies have been performed on professional baseball players who have undergone nonoperative treatment with or without PRP injections for UCL injuries. HYPOTHESIS: Players who received PRP injections would have better outcomes than those who did not receive PRP. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: The Major League Baseball (MLB) Health and Injury Tracking System identified 544 professional baseball players who were treated nonoperatively for elbow UCL injuries between 2011 and 2015. Of these, 133 received PRP injections (PRP group) before starting their nonoperative treatment program, and 411 did not (no-PRP group). Player outcomes and a Kaplan-Meier survival analysis were compared between groups. In addition, to reduce selection bias, a 1:1 matched comparison of the PRP group versus the no-PRP group was performed. Players were matched by age, position, throwing side, and league status: major (MLB) and minor (Minor League Baseball [MiLB]). A single radiologist with extensive experience in magnetic resonance imaging (MRI) interpretation of elbow injuries in elite athletes analyzed 243 MRI scans for which images were accessible for tear location and grade interpretation. RESULTS: Nonoperative treatment of UCL injuries resulted in an overall 54% rate of return to play (RTP). Players who received PRP had a significantly longer delay in return to throwing (P < .001) and RTP (P = .012). The matched cohort analysis showed that MLB and MiLB pitchers in the no-PRP group had a significantly faster return to throwing (P < .05) and the MiLB pitchers in the no-PRP group had a significantly faster RTP (P = .045). The survival analysis did not reveal significant differences between groups over time. The use of PRP, MRI grade, and tear location were not statistically significant predictors for RTP or progression to surgery. CONCLUSION: In this retrospective matched comparison of MLB and MiLB pitchers and position players treated nonoperatively for a UCL tear, PRP did not improve RTP outcomes or ligament survivorship, although there was variability with respect to PRP preparations, injection protocols, time from injury to injection, and rehabilitation programs. MRI grade and tear location also did not significantly affect RTP outcomes or progression to surgery.

Methods and approaches to disease mechanisms using systems kinomics.

All cellular functions, ranging from regular cell maintenance and homeostasis, specialized functions specific to cellular types, or generating responses due to external stimulus, are mediated by proteins within the cell. Regulation of these proteins allows the cell to alter its behavior under different circumstances. A major mechanism of protein regulation is utilizing protein kinases and phosphatases; enzymes that catalyze the transfer of phosphates between substrates [1]. Proteins involved in phosphate signaling are well studied and include kinases and phosphatases that catalyze opposing reactions regulating both structure and function of the cell. Kinomics is the study of kinases, phosphatases and their targets, and has been used to study the functional changes in numerous diseases and infectious diseases with aims to delineate the cellular functions affected. Identifying the phosphate signaling pathways changed by certain diseases or infections can lead to novel therapeutic targets. However, a daunting 518 putative protein kinase genes have been identified [2], indicating that this protein family is very large and complex. Identifying which enzymes are specific to a particular disease can be a laborious task. In this review, we will provide information on large-scale systems biology methodologies that allow global screening of the kinome to more efficiently identify which kinase pathways are pertinent for further study.

Understanding mucosal and microbial functionality of the female reproductive tract by metaproteomics: Implications for HIV transmission.

The mucosal surface of the female genital tract contains physiological, immunological, and microbial components that collectively comprise a functioning "mucosal system" that is critical for reproductive health. Alterations or imbalances to any of these components can have significant consequences for susceptibility to sexually transmitted infections, such as HIV. In recent years the advent of advanced systems biology technologies, such as metaproteomics, has provided new toolsets to studying mucosal systems. Studies have linked an altered mucosal proteome to many HIV risk factors including mucosal inflammation, bacterial vaginosis, hormonal contraceptives, and reduced efficacy of antiretroviral drugs for HIV prevention. Herein we will discuss how metaproteomics has been used to study mucosal system components, including epithelial barriers, inflammation, and the microbiome, with a focus on what alterations may contribute to increased HIV transmission risk in women.

The effect of variation in interpretation of the La Trobe radiographic foot atlas on the prevalence of foot osteoarthritis in older women: the Chingford general population cohort.

BACKGROUND: The prevalence of foot osteoarthritis (OA) is much less understood than hip, knee and hand OA. The foot is anatomically complex and different researchers have investigated different joints with lack of methodological standardisation across studies. The La Trobe Foot Atlas (LFA) is the first to address these issues in providing quantitative assessment of radiographic foot OA, but has not been tested externally. The aim of this study was to evaluate three different interpretive approaches to using the LFA for grading OA when scoring is difficult due to indistinct views of interosseous space and joint contour. METHODS: Foot radiographs of all remaining participants (n = 218) assessed in the Chingford Women Study 23 year visit (mean (SD) for age: 75.5 years (5.1)) were scored using the LFA defined protocol (Technique 1). Two revised scoring strategies were applied to the radiographs in addition to the standard LFA analyses. Technique 2 categorised joints that were difficult to grade as 'missing'. Technique 3 included joints that were difficult to grade as an over estimated score. Radiographic OA prevalence was defined for the foot both collectively and separately for individual joints. RESULTS: When radiographs were scored using the LFA (Technique 1), radiographic foot OA was present in 89.9%. For Technique 2 the presence of radiographic foot OA was 83.5% and for Technique 3 it was 97.2%. At the individual joint level, using Technique 1, the presence of radiographic foot OA was higher with a wider range (18.3-74.3%) than Technique 2 (17.9-46.3%) and lower with a wider range (18.3-74.3%) than Technique 3 (39.9-79.4%). CONCLUSION: The three different ways of interpreting the LFA scoring system when grading of individual joints is technically difficult and result in very different estimates of foot OA prevalence at both the individual joint and global foot level. Agreement on the best strategy is required to improve comparability between studies.

Anterior Cruciate Ligament Reconstruction With Bone-Patellar Tendon-Bone Autograft and a Medial Parapatellar Portal.

Anterior cruciate ligament (ACL) reconstruction is one of the most extensively studied surgical procedures in orthopaedics. The importance of this ligament for knee function and stability has been widely studied. For athletes who participate in activities involving cutting, twisting, and running, surgical reconstruction of the ACL has become the standard of care. However, there is much debate regarding the techniques involved in ACL reconstruction, including graft choice, technique of drilling the femoral tunnel, and single- versus double-bundle reconstruction. In recent studies, ACL femoral tunnel drilling via a medial parapatellar or accessory anteromedial portal provides a more anatomic graft placement than transtibial femoral drilling. Long-term outcomes of these techniques have not been widely studied. This article details our technique for ACL reconstruction with bone-patellar tendon-bone autograft and a medial parapatellar portal.

Bioabsorbable interference screw failure in anterior cruciate ligament reconstruction: A case series and review of the literature.

BACKGROUND: To report a case series of failures of bioabsorbable interference screws with possible identification of a novel failure mechanism. METHODS: A retrospective review of ACL reconstructions by the senior author utilizing BioComposite™ Interference Screws (Arthrex, Inc., Naples, FL) was performed. Complications related to screw placement, including fracture, breakage or bending were examined. Our rate and methods of failure were compared to those quoted in the current literature. RESULTS: Eighty-seven patients of average age 23.8 years met inclusion criteria. There were eight screw failures in six patients, with femoral failure in seven and tibial failure in one. The femoral screw fractured halfway between the tip and head in five, while the head of the screw broke in one and the screw bent in another. In the case of tibial interference screw fracture, failure occurred halfway between the tip and head. The insertion device that was used was replaced after recognition of material deformation and considered a potential contributor to the breakage risk as no further screw failures have occurred since. CONCLUSIONS: We demonstrate a unique failure mechanism of bio-absorbable interference screws. In each case, the reconstruction was salvaged. Regular inspection of materials and implants can ensure optimal outcomes and decrease complications intra-operatively.