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Despite the popularity of surgical repair of rotator cuff tears, literature regarding the indications for and timing of surgery are sparse. We performed a systematic review of the literature to investigate factors influencing the decision to surgically repair symptomatic, full-thickness rotator cuff tears. Specifically, how do demographic variables, duration of symptoms, timing of surgery, physical examination findings, and size of tear affect treatment outcome and indications for surgery? We reviewed the best available evidence, which offers some guidelines for surgical decision making. Variables suggest earlier surgical intervention may be needed in the setting of weakness and substantial functional disability. With regard to demographic variables, the evidence is unclear regarding their association with treatment outcome. However, older chronological age does not seem to portend a worse outcome. Pending worker's compensation claims does seem to negatively affect treatment results. Further research is required to define the indications for surgery for full thickness rotator cuff tears. However, the design and conduct of an ethical study to obtain Level I evidence on this issue will be a major challenge. [ABSTRACT FROM AUTHOR]

Graft fixation in anterior cruciate ligament (ACL) reconstruction is commonly performed with bioabsorbable devices. This article presents a case of a broken bioabsorbable tibial interference screw (Gentle Threads; Biomet, Warsaw, Indiana) that presented as an intra-articular loose body 4 months after ACL reconstruction with posterior tibialis tendon allograft. A 19-year-old man presented with symptoms of pain and catching for 1 week but reported no history of trauma. The broken screw tip was identifi ed on magnetic resonance imaging examination, and the remaining screw appeared to be overinserted into the tibia. During arthroscopic removal, a 10-mm screw tip was found in the lateral gutter. The ACL graft was found to be well fixed, but small areas of chondral damage were found in the patellofemoral and medial compartment. The patient's symptoms resolved postoperatively.To our knowledge, this is the earliest report of a broken bioabsorbable interference screw and only the second report of subsequent chondral injury due to intra-articular migration. Although rare, late breakage and intra-articular migration of bioabsorbable interference screws should be considered during the postoperative evaluation of any patient with pain or mechanical symptoms, regardless of trauma. This case also supports the importance of both measurement of tibial tunnel length and inspection of the intercondylar notch following interference screw insertion. Orthopedic surgeons performing ACL reconstruction must be aware of this possible complication and its potential for devastating chondral injury. [ABSTRACT FROM AUTHOR]

Multimodality reporter gene imaging combines the sensitivity, resolution and translational potential of two or more signals. The approach has not been widely adopted by the animal imaging community, mainly because its utility in this area is unproven. We developed a new complementation-based reporter gene system where the large component of split NanoLuc luciferase (LgBiT) presented on the surface of cells (TM-LgBiT) interacts with a radiotracer consisting of the high-affinity complementary HiBiT peptide labeled with a radionuclide. Radiotracer uptake could be imaged in mice using SPECT/CT and bioluminescence within two hours of implanting reporter-gene-expressing cells. Imaging data were validated by ex vivo biodistribution studies. Following the demonstration of complementation between the TM-LgBiT protein and HiBiT radiotracer, we validated the use of the technology in the highly specific in vivo multimodal imaging of cells. These findings highlight the potential of this new approach to facilitate the advancement of cell and gene therapies from bench to clinic.

The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging., (© 2024. The Author(s).)

Bioluminescence imaging (BLI) allows non-invasive visualization of cells and biochemical events in vivo and thus has become an indispensable technique in biomedical research. However, BLI in the central nervous system remains challenging because luciferases show relatively poor performance in the brain with existing substrates. Here, we report the discovery of a NanoLuc substrate with improved brain performance, cephalofurimazine (CFz). CFz paired with Antares luciferase produces greater than 20-fold more signal from the brain than the standard combination of D-luciferin with firefly luciferase. At standard doses, Antares-CFz matches AkaLuc-AkaLumine/TokeOni in brightness, while occasional higher dosing of CFz can be performed to obtain threefold more signal. CFz should allow the growing number of NanoLuc-based indicators to be applied to the brain with high sensitivity. Using CFz, we achieve video-rate non-invasive imaging of Antares in brains of freely moving mice and demonstrate non-invasive calcium imaging of sensory-evoked activity in genetically defined neurons., (© 2023. The Author(s).)

Point-of-care tests are highly valuable in providing fast results for medical decisions for greater flexibility in patient care. Many diagnostic tests, such as ELISAs, that are commonly used within clinical laboratory settings require trained technicians, laborious workflows, and complex instrumentation hindering their translation into point-of-care applications. Herein, we demonstrate the use of a homogeneous, bioluminescent-based, split reporter platform that enables a simple, sensitive, and rapid method for analyte detection in clinical samples. We developed this point-of-care application using an optimized ternary, split-NanoLuc luciferase reporter system that consists of two small reporter peptides added as appendages to analyte-specific affinity reagents. A bright, stable bioluminescent signal is generated as the affinity reagents bind to the analyte, allowing for proximity-induced complementation between the two reporter peptides and the polypeptide protein, in addition to the furimazine substrate. Through lyophilization of the stabilized reporter system with the formulated substrate, we demonstrate a shelf-stable, all-in-one, add-and-read analyte-detection system for use in complex sample matrices at the point-of-care. We highlight the modularity of this platform using two distinct SARS-CoV-2 model systems: SARS-CoV-2 N-antigen detection for active infections and anti-SARS-CoV-2 antibodies for immunity status detection using chemically conjugated or genetically fused affinity reagents, respectively. This technology provides a simple and standardized method to develop rapid, robust, and sensitive analyte-detection assays with flexible assay formatting making this an ideal platform for research, clinical laboratory, as well as point-of-care applications utilizing a simple handheld luminometer., Competing Interests: ET, VR, VK, RH, MH, LE, KZ, SF, C-CH, TM, and MD were employed by the company Promega Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Torio, Ressler, Kincaid, Hurst, Hall, Encell, Zimmerman, Forsyth, Rehrauer, Accola, Hsu, Machleidt and Dart.)

Sensitive and selective detection assays are essential for the accurate measurement of analytes in both clinical and research laboratories. Immunoassays that rely on nonoverlapping antibodies directed against the same target analyte (e.g., sandwich enzyme-linked immunosorbent assays (ELISAs)) are commonly used molecular detection technologies. Use of split enzyme reporters has simplified the workflow for these traditionally complex assays. However, identifying functional antibody pairs for a given target analyte can be cumbersome, as it generally involves generating and screening panels of antibodies conjugated to reporters. Accordingly, we sought a faster and easier reporter conjugation strategy to streamline antibody screening. We describe here the development of such a method that is based on an optimized ternary NanoLuc luciferase. This bioluminescence complementation system is comprised of a reagent-based thermally stable polypeptide (LgTrip) and two small peptide tags (β9 and β10) with lysine-reactive handles for direct conjugation onto antibodies. These reagents enable fast, single-step, wash-free antibody labeling and sensitive functional screening. Simplicity, speed, and utility of the one-pot labeling technology are demonstrated in screening antibody pairs for the analyte interleukin-4. The screen resulted in the rapid development of a sensitive homogeneous immunoassay for this clinically relevant cytokine.

Objective: Superior capsule reconstruction (SCR) is a treatment option for irreparable massive rotator cuff tears (MRCT). The purpose of this study is to describe a classification system for graft integrity and tear location., Methods: Patients who underwent SCR at a single institution were included. Pre-operative age, gender, prior surgery, Hamada grade, and Goutallier stage were recorded. An MRI was performed postoperatively to assess graft integrity and tear location., Results: 53 patients met inclusion criteria. Mean age was 60.1 ± 7.9 years. A post-operative MRI was performed in 42 (80%) patients at a mean of 14 ± 7 months (range, 6-40 months). MRIs demonstrated an intact graft in 16 (38%) shoulders. Of the 26 graft tears, 14 (54%) were from the glenoid, 5 (19%) mid-substance, 6 (23%) from the tuberosity, and 1 (3.8%) had complete graft absence., Conclusion: Graft tears are common following SCR. We describe four different graft tear locations and submit a classification system that can be used in future studies to better compare outcomes based on graft integrity and tear location. Clinical correlation with graft integrity and graft tear location needs to be further investigated., (Copyright © 2021. Published by Elsevier Inc.)

Gaining insight into the pharmacology of ligand engagement with G-protein coupled receptors (GPCRs) under biologically relevant conditions is vital to both drug discovery and basic research. NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a robust and sensitive method to quantify ligand engagement with specific GPCRs genetically fused to NanoLuc luciferase or the luminogenic HiBiT peptide. However, development of fluorescent tracers is often challenging and remains the principal bottleneck for this approach. One way to alleviate the burden of developing a specific tracer for each receptor is using promiscuous tracers, which is made possible by the intrinsic specificity of BRET. Here, we devised an integrated tracer discovery workflow that couples machine learning-guided in silico screening for scaffolds displaying promiscuous binding to GPCRs with a blend of synthetic strategies to rapidly generate multiple tracer candidates. Subsequently, these candidates were evaluated for binding in a NanoBRET ligand-engagement screen across a library of HiBiT-tagged GPCRs. Employing this workflow, we generated several promiscuous fluorescent tracers that can effectively engage multiple GPCRs, demonstrating the efficiency of this approach. We believe that this workflow has the potential to accelerate discovery of NanoBRET fluorescent tracers for GPCRs and other target classes.

Enzyme-linked immunosorbent assays (ELISAs) are used extensively for the detection and quantification of biomolecules in clinical diagnostics as well as in basic research. Although broadly used, the inherent complexities of ELISAs preclude their utility for straightforward point-of-need testing, where speed and simplicity are essential. With this in mind, we developed a bioluminescence-based immunoassay format that provides a sensitive and simple method for detecting biomolecules in clinical samples. We utilized a ternary, split-NanoLuc luciferase complementation reporter consisting of two small peptides (11mer, 13mer) and a 17 kDa polypeptide combined with a luminogenic substrate to create a complete, shelf-stable add-and-read assay detection reagent. Directed evolution was used to optimize reporter constituent sequences to impart chemical and thermal stability, as well as solubility, while formulation optimization was applied to stabilize an all-in-one reagent that can be reconstituted in aqueous buffers or sample matrices. The result of these efforts is a robust, first-generation bioluminescence-based homogenous immunoassay reporter platform where all assay components can be configured into a stable lyophilized cake, supporting homogeneous, rapid, and sensitive one-step biomolecule quantification in complex human samples. This technology represents a promising alternative immunoassay format with significant potential to bring critical diagnostic molecular detection testing closer to the point-of-need.

For in vivo multicolor bioluminescence applications, red and near-infrared signals are desirable over shorter wavelength signals because they are not as susceptible to light attenuation by blood and tissue. Herein, we describe the development of a new click beetle luciferase mutant, CBG2, with a red-shifted color emission. When paired with NH 2 -NpLH2 luciferin, CBG2 (λ = 660 nm) and CBR2 (λ = 730 nm) luciferases can be used for simultaneous dual-color bioluminescence imaging in deep tissue. Using a spectral unmixing algorithm tool it is possible to distinguish each spectral contribution. Ultimately, this enzyme pair can expand the near-infrared bioluminescent toolbox to enable rapid visualization of multiple biological processes in deep tissue using a single substrate., Competing Interests: Authors have no financial interests/commercial Conflict of Interest., (© 2020 The Author(s).)

Firefly luciferase-based ATP detection assays are frequently used as a sensitive, cost-efficient method for monitoring hygiene in many industrial settings. Solutions of detection reagent, containing a mixture of a substrate and luciferase enzyme that produces photons in the presence of ATP, are relatively unstable and maintain only a limited shelf life even under refrigerated conditions. It is therefore common for the individual performing a hygiene test to manually prepare fresh reagent at the time of monitoring. To simplify sample processing, a liquid detection reagent with improved thermal stability is needed. The engineered firefly luciferase, Ultra-Glo™, fulfills one aspect of this need and has been valuable for hygiene monitoring because of its high resistance to chemical and thermal inactivation. However, solutions containing both Ultra-Glo™ luciferase and its substrate luciferin gradually lose the ability to effectively detect ATP over time. We demonstrate here that dehydroluciferin, a prevalent oxidative breakdown product of luciferin, is a potent inhibitor of Ultra-Glo™ luciferase and that its formation in the detection reagent is responsible for the decreased ability to detect ATP. We subsequently found that dialkylation at the 5-position of luciferin (e.g., 5,5-dimethylluciferin) prevents degradation to dehydroluciferin and improves substrate thermostability in solution. However, since 5,5-dialkylluciferins are poorly utilized by Ultra-Glo™ luciferase as substrates, we used structural optimization of the luciferin dialkyl modification and protein engineering of Ultra-Glo™ to develop a luciferase/luciferin pair that shows improved total reagent stability in solution at ambient temperature. The results of our studies outline a novel luciferase/luciferin system that could serve as foundations for the next generation of bioluminescence ATP detection assays with desirable reagent stability., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Authors are paid employees of Promega Corporation. Promega Corporation manufactures and sells Ultra-Glo™ luciferase, luciferin, and ATP detection reagents. Ultra-Glo™ luciferase mutations are disclosed in the published patent application US20200071682A1 “Luciferase Enzymes For Use With Thermostable Luciferins In Bioluminescent Assays”, and the luciferin analogs are disclosed in granted patent US 10,400,264 and the published patent application US 20190338340A1, “5,5-disubstituted Luciferins And Their Use In Luciferase-based Assays” owned by Promega Corporation. The authors confirm that these competing interests do not alter their adherence to all the PLOS ONE policies on sharing data and materials.

Purpose: Currently, a variety of red and green beetle luciferase variants are available for bioluminescence imaging (BLI). In addition, new luciferin analogues providing longer wavelength luminescence have been developed that show promise for improved deep tissue imaging. However, a detailed assessment of these analogues (e.g., Akalumine-HCl, CycLuc1, and amino naphthyl luciferin (NH 2 -NpLH2)) combined with state of the art luciferases has not been performed. The aim of this study was to evaluate for the first time the in vivo brightness and spectral characteristics of firefly (Luc2), click beetle green (CBG99), click beetle red 2 (CBR2), and Akaluc luciferases when paired with different D-luciferin (D-LH2) analogues in vivo., Procedures: Transduced human embryonic kidney (HEK 293T) cells expressing individual luciferases were analyzed both in vitro and in mice (via subcutaneous injection). Following introduction of the luciferins to cells or animals, the resulting bioluminescence signal and photon emission spectrum were acquired using a sensitive charge-coupled device (CCD) camera equipped with a series of band pass filters and spectral unmixing software., Results: Our in vivo analysis resulted in four primary findings: (1) the best substrate for Luc2, CBG99, and CBR2 in terms of signal strength was D-luciferin; (2) the spectra for Luc2 and CBR2 were shifted to a longer wavelength when Akalumine-HCl was the substrate; (3) CBR2 gave the brightest signal with the near-infrared substrate, NH 2 -NpLH2; and (4) Akaluc was brighter when paired with either CycLuc1 or Akalumine-HCl when paired with D-LH2., Conclusion: We believe that the experimental results described here should provide valuable guidance to end users for choosing the correct luciferin/luciferase pairs for a variety of BLI applications.

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.

To what extent are research results influenced by subjective decisions that scientists make as they design studies? Fifteen research teams independently designed studies to answer five original research questions related to moral judgments, negotiations, and implicit cognition. Participants from 2 separate large samples (total N > 15,000) were then randomly assigned to complete 1 version of each study. Effect sizes varied dramatically across different sets of materials designed to test the same hypothesis: Materials from different teams rendered statistically significant effects in opposite directions for 4 of 5 hypotheses, with the narrowest range in estimates being d = -0.37 to + 0.26. Meta-analysis and a Bayesian perspective on the results revealed overall support for 2 hypotheses and a lack of support for 3 hypotheses. Overall, practically none of the variability in effect sizes was attributable to the skill of the research team in designing materials, whereas considerable variability was attributable to the hypothesis being tested. In a forecasting survey, predictions of other scientists were significantly correlated with study results, both across and within hypotheses. Crowdsourced testing of research hypotheses helps reveal the true consistency of empirical support for a scientific claim. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Accurate identification of tumor-derived somatic variants in plasma circulating cell-free DNA (cfDNA) requires understanding of the various biological compartments contributing to the cfDNA pool. We sought to define the technical feasibility of a high-intensity sequencing assay of cfDNA and matched white blood cell DNA covering a large genomic region (508 genes; 2 megabases; >60,000× raw depth) in a prospective study of 124 patients with metastatic cancer, with contemporaneous matched tumor tissue biopsies, and 47 controls without cancer. The assay displayed high sensitivity and specificity, allowing for de novo detection of tumor-derived mutations and inference of tumor mutational burden, microsatellite instability, mutational signatures and sources of somatic mutations identified in cfDNA. The vast majority of cfDNA mutations (81.6% in controls and 53.2% in patients with cancer) had features consistent with clonal hematopoiesis. This cfDNA sequencing approach revealed that clonal hematopoiesis constitutes a pervasive biological phenomenon, emphasizing the importance of matched cfDNA-white blood cell sequencing for accurate variant interpretation.

Background: Noninvasive genotyping using plasma cell-free DNA (cfDNA) has the potential to obviate the need for some invasive biopsies in cancer patients while also elucidating disease heterogeneity. We sought to develop an ultra-deep plasma next-generation sequencing (NGS) assay for patients with non-small-cell lung cancers (NSCLC) that could detect targetable oncogenic drivers and resistance mutations in patients where tissue biopsy failed to identify an actionable alteration., Patients and Methods: Plasma was prospectively collected from patients with advanced, progressive NSCLC. We carried out ultra-deep NGS using cfDNA extracted from plasma and matched white blood cells using a hybrid capture panel covering 37 lung cancer-related genes sequenced to 50 000× raw target coverage filtering somatic mutations attributable to clonal hematopoiesis. Clinical sensitivity and specificity for plasma detection of known oncogenic drivers were calculated and compared with tissue genotyping results. Orthogonal ddPCR validation was carried out in a subset of cases., Results: In 127 assessable patients, plasma NGS detected driver mutations with variant allele fractions ranging from 0.14% to 52%. Plasma ddPCR for EGFR or KRAS mutations revealed findings nearly identical to those of plasma NGS in 21 of 22 patients, with high concordance of variant allele fraction (r = 0.98). Blinded to tissue genotype, plasma NGS sensitivity for de novo plasma detection of known oncogenic drivers was 75% (68/91). Specificity of plasma NGS in those who were driver-negative by tissue NGS was 100% (19/19). In 17 patients with tumor tissue deemed insufficient for genotyping, plasma NGS identified four KRAS mutations. In 23 EGFR mutant cases with acquired resistance to targeted therapy, plasma NGS detected potential resistance mechanisms, including EGFR T790M and C797S mutations and ERBB2 amplification., Conclusions: Ultra-deep plasma NGS with clonal hematopoiesis filtering resulted in de novo detection of targetable oncogenic drivers and resistance mechanisms in patients with NSCLC, including when tissue biopsy was inadequate for genotyping., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: With the cost of health care rising, the potential to avoid costs from an unplanned return to the emergency department (ED) or urgent care center (UC) after elective outpatient rotator cuff repair (RCR) has been discussed but not extensively assessed., Methods: Outpatient RCR procedures were queried in a closed health care system, and all unplanned ED and UC visits within 7 days of procedures were collected and compared with other typical outpatient orthopedic procedures (knee arthroscopy, carpal tunnel release, and anterior cruciate ligament reconstruction). Avoidable diagnoses (ADs) for the unplanned visits were defined in advance as visits for (1) constipation, (2) nausea or vomiting, (3) pain, and (4) urinary retention. Final tallies of all visits versus visits with ADs were compared., Results: From June 2015 to May 2016, 1306 outpatient RCRs were performed (729 male and 577 female patients; average age, 60 years). Of the patients, 90 returned for ED or UC visits (6.9%), with 34 for ADs (2.6%). Pain was the most common AD. However, when RCR was compared with other case types, ED or UC visits for urinary retention were significantly more common (P = .007), whereas there was no significant difference with the other ADs. The 1306 RCRs led to a greater proportion of ED or UC visits than the combined 5825 other cases studied (P < .001)., Discussion and Conclusions: Unplanned ED visits within 7 days of outpatient RCR are measurable and in many cases, such as ED or UC visits for pain, are avoidable. Visits for urinary retention are seen more commonly after RCR. Outpatient RCR led to more unplanned ED and UC visits than other common outpatient orthopedic surgical procedures., (Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (K D = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.

The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system's peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice.

We report the synthesis and characterization of novel coelenterazine analogues that demonstrate a red-shift in their bioluminescent emission with NanoLuc luciferase. These coelenterazines can be tuned to shift the bioluminescent emission from blue light in the native system. In particular, direct attachment of an aryl moiety to the imidazopyrazinone core of furimazine at the C8 position provides a significant red-shift while maintaining reasonable light output. In addition, modification of the C6 aryl moiety provided additive red-shifts, and by combining the most promising modifications we report a coelenterazine with a maximum emission near 600 nm with NanoLuc. Finally, we show that this new bioluminescent system is capable of efficient BRET to far-red fluorophores. We anticipate these new principles of NanoLuc substrate design will impact applications that depend on shifting the colour of emission to the red, most notably in vivo bioluminescent imaging.

Novel engineered NanoLuc (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or Renilla (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate 1, a hit from a 42 000 PubChem compound library with a low micromolar IC 50 against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nanomolar, cell permeable inhibitors. Further elaboration revealed a channel that allowed access to the external Nluc surface, resulting in a series of highly potent cell impermeable Nluc inhibitors with negatively charged groups likely extending to the protein surface. The permeability was evaluated by comparing EC 50 shifts calculated from both live and lysed cells expressing Nluc cytosolically. Luminescence imaging further confirmed that cell permeable compounds inhibit both intracellular and extracellular Nluc, whereas less permeable compounds differentially inhibit extracellular Nluc and Nluc on the cell surface. The compounds displayed little to no toxicity to cells and high luciferase specificity, showing no activity against firefly luciferase or even the closely related NanoBit system. Looking forward, the structural motifs used to gain access to the Nluc surface can also be appended with other functional groups, and therefore interesting opportunities for developing assays based on relief-of-inhibition can be envisioned.

A potential risk factor for immunogenicity of a biotherapeutic is the low levels of host cell protein (HCP) impurities that remain in the product following the purification process. During process development, significant attention has been devoted to removing HCPs due to their potential safety risk. Samples from different purification steps of several monoclonal antibodies (mAbs) purified by one type of platform were evaluated for their residual Chinese Hamster Ovary (CHO) cell-derived HCP content. HCPs in both in-process (high levels of HCP) and highly purified (low levels of HCP) samples were identified and quantitated by proteomic analysis via mass spectrometry. The responses to HCPs were evaluated in an in vitro assay using PBMC from a population of healthy and disease state individuals. Results indicated that samples with up to 4000 ppm HCP content (levels 200 times greater than the drug substance) did not pose a higher immunogenicity risk than highly purified mAb samples. As an orthogonal method to predict immunogenicity risk, in silico algorithms that probe amino acid sequence for foreign epitope content were used to evaluate over 20 common HCPs (identified in the different mAb samples). Only a few HCPs were identified as high risk by the algorithms; however, the in vitro assay results indicated that the concentration of these HCPs from in-process biotherapeutic mAb samples was not sufficient to stimulate an immune response. This suggests that high levels of HCP in mAb biotherapeutics purified by this type of platform do not increase the potential risk of immunogenicity of these molecules. Insights from these studies can be applied to HCP control and risk assessment strategies.

Background: X&Y chromosomal aneuploidies are among the most common human whole-chromosomal copy number changes, but the population-based incidence and prevalence in the child-bearing population is unclear., Methods: This retrospective analysis of prospectively collected data leveraged a routine non-invasive prenatal test (NIPT) using parental genotyping to estimate the population-based incidence of X&Y chromosome variations in this population referred for NIPT (generally due to advanced maternal age)., Results: From 141,916 women and 29,336 men, 119 X&Y chromosomal abnormalities (prevalence: 1 in 1,439) were identified. Maternal findings include: 43 cases of 45,X (40 mosaic); 30 cases of 47,XXX (12 mosaic); 3 cases of 46,XX uniparental disomy; 2 cases of 46,XY/46,XX; 23 cases of mosaicism of unknown type; 2 cases of 47,XX,i(X)(q10). Paternal findings include: 2 cases of 47,XXY (1 mosaic); 10 cases of 47,XYY (1 mosaic); 4 partial Y deletions., Conclusions: Single chromosome aneuploidy was present in one of every 1,439 individuals considered in this study, showing 47,XXX; 47,XX,i(X)(q10); 47,XYY; 47,XXY, partial Y deletions, and a high level of mosaicism for 45,X. This expands significantly our understanding of X&Y chromosomal variations and fertility issues, and is critical for families and adults affected by these disorders. This current and extensive information on fertility will be beneficial for genetic counseling on prenatal diagnoses as well as for newly diagnosed postnatal cases.

The growing popularity of bioluminescent assays has highlighted the need for coelenterazine analogues possessing properties tuned for specific applications. However, the structural diversity of known coelenterazine analogues has been limited by current syntheses. Known routes for the preparation of coelenterazine analogues employ harsh reaction conditions that limit access to many substituents and functional groups. Novel synthetic routes reported here establish simple and robust methods for synthesis and investigation of structurally diverse marine luciferase substrates. Specifically, these new routes allow synthesis of coelenterazine analogues containing various heterocyclic motifs and substituted aromatic groups with diverse electronic substituents at the R(2) position. Interesting analogues described herein were characterized by their physicochemical properties, bioluminescent half-life, light output, polarity and cytotoxicity. Some of the analogues represent leads that can be utilized in the development of improved bioluminescent systems., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein A chromatography is commonly used as the initial step for purifying monoclonal antibody biotherapeutics expressed in mammalian tissue culture cells. The purpose of this step, as well as later chromatography steps, is, in part, to remove host cell proteins (HCPs) and other related impurities. Understanding the retention mechanism for the subset of HCPs retained during this step is of great interest to monoclonal antibody (mAb) process developers because it allows formation of a guided HCP clearance strategy. However, only limited information is available about the specific HCPs that co-purify with mAbs at this step. In this study, a comprehensive comparison of HCP subpopulations that associated with 15 different mAbs during protein A chromatography was conducted by a 2D-LC-HDMS(E) approach. We found that a majority of CHO HCPs binding to and eluting with the mAbs were common among the mAbs studied, with only a small percentage (∼10% on average) of a mAb's total HCP content in the protein A (PrA) eluate specific for a particular antibody. The abundance of these HCPs in cell culture fluids and their ability to interact with mAbs were the two main factors determining their prevalence in protein A eluates. Potential binding segments for HCPs to associate with mAbs were also studied through their co-purification with individual Fc and (Fab')2 antibody fragments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:708-717, 2016., (© 2016 American Institute of Chemical Engineers.)

Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 μM) and association constants (kon = 500 M(-1) s(-1), koff = 0.2 s(-1)) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction between SME-1 β-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and β-arrestin-2, were rapid, reversible, and robust to temperature (21-37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.

Rationale and Objectives: Coronary artery calcium (CAC) scoring is an excellent imaging tool for subclinical atherosclerosis detection and risk stratification. We hypothesize that although CAC has been underreported in the past on computed tomography (CT) scans of the abdomen, specialized resident educational intervention can improve on this underreporting., Materials and Methods: Beginning July 2009, a dedicated radiology resident cardiac imaging rotation and curriculum was initiated. A retrospective review of the first 500 abdominal CT reports from January 2009, 2011, and 2013 was performed including studies originally interpreted by a resident and primary attending physician interpretations. Each scan was reevaluated for presence or absence of CAC and coronary artery disease (CAD) by a cardiovascular CT expert reader. These data were then correlated to determine if the presence of CAC had been properly reported initially. The results of the three time periods were compared to assess for improved rates of CAC and CAD reporting after initiation of a resident cardiac imaging curriculum., Results: Statistically significant improvements in the reporting of CAC and CAD on CT scans of the abdomen occurred after the initiation of formal resident cardiac imaging training which included two rotations (4 weeks each) of dedicated cardiac CT and cardiac magnetic resonance imaging interpretation during the resident's second, third, or fourth radiology training years. The improvement was persistent and increased over time, improving from 1% to 72% after 2 years and to 90% after 4 years., Conclusions: This single-center retrospective analysis shows association between implementation of formal cardiac imaging training into radiology resident education and improved CAC detection and CAD reporting on abdominal CT scans., (Copyright © 2015 AUR. Published by Elsevier Inc. All rights reserved.)

Background: Criteria for return to unrestricted activity after anterior cruciate ligament (ACL) reconstruction varies, with some using time after surgery as the sole criterion-most often at 6 months. Patients may have residual neuromuscular deficits, which may increase the risk of ACL injury. A single-leg squat test (SLST) can dynamically assess for many of these deficits prior to return to unrestricted activity., Hypothesis: A significant number of patients will continue to exhibit neuromuscular deficits with SLST at 6 months after ACL reconstruction., Study Design: Cross-sectional study; Level of evidence, 3., Methods: Patients using a standardized accelerated rehabilitation protocol at their 6-month follow-up after primary ACL reconstruction were enrolled. Evaluation included bilateral SLST, single-leg hop distance, hip abduction strength, and the subjective International Knee Documentation Committee (IKDC) score., Results: Thirty-three patients were enrolled. Poor performance of the operative leg SLST was found in 15 of 33 patients (45%). Of those 15 patients, 7 (45%) had concomitant poor performance of the nonoperative leg compared with 2 of 18 patients (11%) in those who demonstrated good performance in the operative leg. The poor performers were significantly older (33.6 years) than the good performers (24.2 years) (P = .007). Those with poor performance demonstrated decreased hip abduction strength (17.6 kg operative leg vs 20.5 kg nonoperative leg) (P = .024), decreased single-leg hop distance (83.3 cm operative leg vs 112.3 cm nonoperative leg) (P = .036), and lower IKDC scores (67.9 vs 82.3) (P = .001)., Conclusion: Nearly half of patients demonstrated persistent neuromuscular deficits on SLST at 6 months, which is when many patients return to unrestricted activity. Those with poor performance were of a significantly older age, decreased hip abduction strength, decreased single-leg hop distance, and lower IKDC subjective scores., Clinical Relevance: The SLST can be used to identify neuromuscular risk factors for ACL rupture. Many patients at 6 months have persistent neuromuscular deficits on SLST. Caution should be used when using time alone to determine when patients can return to unrestricted activity.

Objective: The purpose of this study was to estimate the performance of a single-nucleotide polymorphism (SNP)-based noninvasive prenatal test for 5 microdeletion syndromes., Study Design: Four hundred sixty-nine samples (358 plasma samples from pregnant women, 111 artificial plasma mixtures) were amplified with the use of a massively multiplexed polymerase chain reaction, sequenced, and analyzed with the use of the Next-generation Aneuploidy Test Using SNPs algorithm for the presence or absence of deletions of 22q11.2, 1p36, distal 5p, and the Prader-Willi/Angelman region., Results: Detection rates were 97.8% for a 22q11.2 deletion (45/46) and 100% for Prader-Willi (15/15), Angelman (21/21), 1p36 deletion (1/1), and cri-du-chat syndromes (24/24). False-positive rates were 0.76% for 22q11.2 deletion syndrome (3/397) and 0.24% for cri-du-chat syndrome (1/419). No false positives occurred for Prader-Willi (0/428), Angelman (0/442), or 1p36 deletion syndromes (0/422)., Conclusion: SNP-based noninvasive prenatal microdeletion screening is highly accurate. Because clinically relevant microdeletions and duplications occur in >1% of pregnancies, regardless of maternal age, noninvasive screening for the general pregnant population should be considered., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: We sought to determine the ability of single-nucleotide polymorphism-based noninvasive prenatal testing (NIPT) to identify triploid, unrecognized twin, and vanishing twin pregnancies., Study Design: The study included 30,795 consecutive reported clinical cases received for NIPT for fetal whole-chromosome aneuploidies; known multiple gestations were excluded. Cell-free DNA was isolated from maternal blood samples, amplified via 19,488-plex polymerase chain reaction, and sequenced. Sequencing results were analyzed to determine fetal chromosome copy number and to identify the presence of additional fetal haplotypes., Results: Additional fetal haplotypes, indicative of fetal triploidy, vanishing twin, or undetected twin pregnancy, were identified in 130 (0.42%) cases. Clinical confirmation (karyotype for singleton pregnancies, ultrasound for multifetal pregnancies) was available for 58.5% (76/130) of cases. Of the 76 cases with confirmation, 42.1% were vanishing twin, 48.7% were viable twin, 5.3% were diandric triploids, and 3.9% were nontriploid pregnancies that lacked evidence of co-twin demise. One pregnancy had other indications suggesting triploidy but lacked karyotype confirmation. Of the 5 vanishing twin cases with a known date of demise, 100% of losses occurred in the first trimester; up to 8 weeks elapsed between loss and detection by NIPT., Conclusion: This single-nucleotide polymorphism-based NIPT successfully identified vanished twin, previously unrecognized twin, and triploid pregnancies. As vanishing twins are more likely to be aneuploid, and undetected residual cell-free DNA could bias NIPT results, the ability of this method to identify additional fetal haplotypes is expected to result in fewer false-positive calls and prevent incorrect fetal sex calls., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Historically, since the metabolism of administered peptide/protein drugs ("biotherapeutics") has been expected to undergo predictable pathways similar to endogenous proteins, comprehensive biotherapeutic metabolism studies have not been widely reported in the literature. However, since biotherapeutics have rapidly evolved into an impressive array of eclectic modalities, there has been a shift toward understanding the impact of metabolism on biotherapeutic development. For biotherapeutics containing non-native chemical linkers and other moieties besides natural amino acids, metabolism studies are critical as these moieties may impart undesired toxicology. For biotherapeutics that are composed solely of natural amino acids, where end-stage peptide and amino acid catabolites do not generally pose toxicity concerns, the understanding of biotherapeutic biotransformation, defined as in vivo modifications such as peripherally generated intermediate circulating catabolites prior to end-stage degradation or elimination, may impact in vivo stability and potency/clearance. As of yet, there are no harmonized methodologies for understanding biotherapeutic biotransformation and its impact on drug development, nor is there clear guidance from regulatory agencies on how and when these studies should be conducted. This review provides an update on biotherapeutic biotransformation studies and an overview of lessons learned, tools that have been developed, and suggestions of approaches to address issues. Biotherapeutic biotransformation studies, especially for certain modalities, should be implemented at an early stage of development to 1) understand the impact on potency/clearance, 2) select the most stable candidates or direct protein re-engineering efforts, and 3) select the best bioanalytical technique(s) for proper drug quantification and subsequent pharmacokinetic profiling and exposure/response assessment., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Objective: We sought to report on laboratory and clinical experience following 6 months of clinical implementation of a single-nucleotide polymorphism-based noninvasive prenatal aneuploidy test in high- and low-risk women., Study Design: All samples received from March through September 2013 and drawn ≥9 weeks' gestation were included. Samples that passed quality control were analyzed for trisomy 21, trisomy 18, trisomy 13, and monosomy X. Results were reported as high or low risk for fetal aneuploidy for each interrogated chromosome. Relationships between fetal fraction and gestational age and maternal weight were analyzed. Follow-up on outcome was sought for a subset of high-risk cases. False-negative results were reported voluntarily by providers. Positive predictive value (PPV) was calculated from cases with an available prenatal or postnatal karyotype or clinical evaluation at birth., Results: Samples were received from 31,030 patients, 30,705 met study criteria, and 28,739 passed quality-control metrics and received a report detailing aneuploidy risk. Fetal fraction correlated positively with gestational age, and negatively with maternal weight. In all, 507 patients received a high-risk result for any of the 4 tested conditions (324 trisomy 21, 82 trisomy 18, 41 trisomy 13, 61 monosomy X; including 1 double aneuploidy case). Within the 17,885 cases included in follow-up analysis, 356 were high risk, and outcome information revealed 184 (51.7%) true positives, 38 (10.7%) false positives, 19 (5.3%) with ultrasound findings suggestive of aneuploidy, 36 (10.1%) spontaneous abortions without karyotype confirmation, 22 (6.2%) terminations without karyotype confirmation, and 57 (16.0%) lost to follow-up. This yielded an 82.9% PPV for all aneuploidies, and a 90.9% PPV for trisomy 21. The overall PPV for women aged ≥35 years was similar to the PPV for women aged <35 years. Two patients were reported as false negatives., Conclusion: The data from this large-scale report on clinical application of a commercially available noninvasive prenatal test suggest that the clinical performance of this single-nucleotide polymorphism-based noninvasive prenatal test in a mixed high- and low-risk population is consistent with performance in validation studies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: To report the full cohort of identifiable anomalies, regardless of known clinical significance, in a large-scale cohort of postmiscarriage products-of-conception samples analyzed using a high-resolution single-nucleotide polymorphism (SNP)-based microarray platform. High-resolution chromosomal microarray analysis allows for the identification of visible and submicroscopic cytogenomic imbalances; the specific use of SNPs permits detection of maternal cell contamination, triploidy, and uniparental disomy., Methods: Miscarriage specimens were sent to a single laboratory for cytogenomic analysis. Chromosomal microarray analysis was performed using a SNP-based genotyping microarray platform. Results were evaluated at the cytogenetic and microscopic (greater than 10 Mb) and submicroscopic (less than 10 Mb) levels. Maternal cell contamination was assessed using information derived from fetal and maternal SNPs., Results: Results were obtained on 2,389 of 2,392 specimens (99.9%) that were less than 20 weeks of gestation. Maternal cell contamination was identified in 528 (22.0%) specimens. The remaining 1,861 specimens were considered to be of true fetal origin. Of these, 1,106 (59.4%) showed classical cytogenetic abnormalities: aneuploidy accounted for 945 (85.4%), triploidy for 114 (10.3%), and structural anomalies or tetraploidy for the remaining 47 (4.2%). Of the 755 (40.6%) cases considered normal at the cytogenetic level, SNP chromosomal microarray analysis revealed a clinically significant copy number change or whole-genome uniparental disomy in 12 (1.6%) and three (0.4%) cases, respectively., Conclusion: Chromosomal microarray analysis of products-of-conception specimens yields a high diagnostic return. Using SNPs extends the scope of detectable genomic abnormalities and facilitates reporting "true" fetal results. This supports the use of SNP chromosomal microarray analysis for cytogenomic evaluation of miscarriage specimens when clinically indicated., Level of Evidence: III.

Objective: To estimate performance of a single-nucleotide polymorphism-based noninvasive prenatal screen for fetal aneuploidy in high-risk and low-risk populations on single venopuncture., Methods: One thousand sixty-four maternal blood samples from 7 weeks of gestation and beyond were included; 1,051 were within specifications and 518 (49.3%) were low risk. Cell-free DNA was amplified, sequenced, and analyzed using the Next-generation Aneuploidy Test Using SNPs algorithm. Samples were called as trisomies 21, 18, 13, or monosomy X, or euploid, and male or female., Results: Nine hundred sixty-six samples (91.9%) successfully generated a cell-free DNA result. Among these, sensitivity was 100% for trisomy 21 (58/58, confidence interval [CI] 93.8-100%), trisomy 13 (12/12, CI 73.5-100%), and fetal sex (358/358 female, CI 99.0-100%; 418/418 male, CI 99.1-100%), 96.0% for trisomy 18 (24/25, CI 79.7-99.9%), and 90% for monosomy X (9/10, CI 55.5-99.8%). Specificity for trisomies 21 and 13 was 100% (905/905, CI 99.6-100%; and 953/953, CI 99.6-100%, respectively) and for trisomy 18 and monosomy X was 99.9% (938/939, CI 99.4-100%; and 953/954, CI 99.4-100%, respectively). However, 16% (20/125) of aneuploid samples did not return a result; 50% (10/20) had a fetal fraction below the 1.5th percentile of euploid pregnancies. Aneuploidy rate was significantly higher in these samples (P<.001, odds ratio 9.2, CI 4.4-19.0). Sensitivity and specificity did not differ in low-risk and high-risk populations., Conclusions: This noninvasive prenatal screen performed with high sensitivity and specificity in high-risk and low-risk cohorts. Aneuploid samples were significantly more likely to not return a result; the number of aneuploidy samples was especially increased among samples with low fetal fraction. This underscores the importance of redraws or, in rare cases, invasive procedures based on low fetal fraction., Level of Evidence: II.

Purpose: To determine how a single nucleotide polymorphism (SNP)- and informatics-based non-invasive prenatal aneuploidy test performs in detecting trisomy 13., Methods: Seventeen trisomy 13 and 51 age-matched euploid samples, randomly selected from a larger cohort, were analyzed. Cell-free DNA was isolated from maternal plasma, amplified in a single multiplex polymerase chain reaction assay that interrogated 19,488 SNPs covering chromosomes 13, 18, 21, X, and Y, and sequenced. Analysis and copy number identification involved a Bayesian-based maximum likelihood statistical method that generated chromosome- and sample-specific calculated accuracies., Results: Of the samples that passed a stringent DNA quality threshold (94.1%), the algorithm correctly identified 15/15 trisomy 13 and 49/49 euploid samples, for 320/320 correct copy number calls., Conclusions: This informatics- and SNP-based method accurately detects trisomy 13-affected fetuses non-invasively and with high calculated accuracy.

Femoral tunnel intersection in combined anterior cruciate ligament and posterolateral corner reconstruction has been reported to be high. The purpose of this study was to examine the risk of intersection between an anatomic femoral anterior cruciate ligament tunnel created with a retrograde reaming device and femoral lateral collateral ligament reconstruction tunnels of varying trajectory in a synthetic femur model., (Copyright 2013, SLACK Incorporated.)

Objective: This study aimed to develop a single-nucleotide polymorphism-based and informatics-based non-invasive prenatal test that detects sex chromosome aneuploidies early in pregnancy., Methods: Sixteen aneuploid samples, including thirteen 45,X, two 47,XXY, and one 47,XYY, along with 185 euploid controls, were analyzed. Cell-free DNA was isolated from maternal plasma, amplified in a single multiplex polymerase chain reaction assay that targeted 19,488 polymorphic loci covering chromosomes 13, 18, 21, X, and Y, and sequenced. Sequencing results were analyzed using a Bayesian-based maximum likelihood statistical method to determine copy number of interrogated chromosomes, calculating sample-specific accuracies., Results: Of the samples that passed a stringent quality control metric (93%), the algorithm correctly identified copy number at all five chromosomes in all but one of the 187 samples, for 934/935 correct calls as early as 9.4 weeks of gestation. We detected 45,X with 91.7% sensitivity (CI: 61.5-99.8%) and 100% specificity (CI: 97.9-100%), and 47,XXY and 47,XYY. The average calculated accuracy was 99.78%., Conclusion: This method non-invasively detected 45,X, 47,XXY, and 47,XYY fetuses from cell-free DNA isolated from maternal plasma with high calculated accuracies and thus offers a non-invasive method with the potential to function as a routine screen allowing for early prenatal detection of rarely diagnosed yet commonly occurring sex aneuploidies., (© 2013 John Wiley & Sons, Ltd.)

No established guidelines currently exist to assist orthopedic surgeons in determining when a patient may safely control a motor vehicle after undergoing simple right knee arthroscopy. Despite this lack of concrete evidence, premature postoperative driving could expose orthopedic surgeons to legal liability and, more importantly, patients to danger and further injury. Through questionnaires directed at physicians, patients, and insurance companies, the authors attempted to identify common postoperative management trends among orthopedic surgeons in an effort to better identify patterns that could help direct practice for the optimized treatment of patients after right knee arthroscopy.Although 29.7% of physicians always incorporated postoperative driving instructions during routine preoperative consultation, 57% of physicians brought up these conversations half of the time or less. In addition, when the preoperative discussions were conducted, approximately 23.6% of physicians never initiated the conversation. The majority of physicians recommended driving after narcotics were discontinued (70%), when the patient felt they could subjectively control their vehicle (57.1%), and when postoperative symptoms would allow safe driving (38.8%); these achievements were most commonly reached at 1 week postoperatively. After simple right knee arthroscopy, the common consensus indicates that patients may safely return to driving 1 week postoperatively when they are narcotic-free and feel safe to control their vehicle., (Copyright 2013, SLACK Incorporated.)

Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA ("STAR" motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3' UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation.

Unlabelled: The outcomes of ACL reconstructions in terms of patient satisfaction and function are well known. Most orthopaedic surgeons feel that Medicare and other payors do not reimburse enough for this surgery. The purpose of this study is to determine how much patients are willing to pay for this surgery and compare it to reimbursement rates., Methods: We constructed a survey which described the function and limitations of an ACL deficient knee and the expected function of that knee after an ACL reconstruction. We then asked the volunteers how much they would be willing to pay for an ACL reconstruction if it were their knee. We also gathered data on the yearly earnings and Tegner activity level of the volunteers. In all, 143 volunteers completed the survey. We computed correlation coefficients between willingness to pay and both yearly earnings and Tegner activity level., Results: The average amount that the volunteers were willing to pay for an ACL reconstruction was $4,867.00. There was no correlation between yearly earnings and willingness to pay. The correlation coefficient was 0.34. There was a weak correlation between Tegner activity level and willingness to pay. This correlation coefficient was 0.81. The Medicare allowable rate for ACL reconstruction (CPT 29888) in the geographic area of the study was $1,132.00., Conclusion: The data demonstrates that patients are willing to pay much more than traditional payors for ACL reconstruction. These payors undervalue the benefit of this surgery to the patient. There is increasing pressure on orthopaedic surgeons to not participate in insurance plans that reimburse poorly. This places an increasing financial burden on the patient. This study suggests that patients may be willing to pay more for their surgery than their insurance plan and accept more of this burden.

Platelet rich plasma (PRP), an autologous sample of blood with a platelet concentration above baseline values, is hypothesized to augment soft tissue healing. Its use in sports medicine has risen dramatically, with common applications including the treatment of refractory tendinopathy and augmenting tendon repair. Many commercial preparation systems are available, but the optimal preparation remains unknown. Increasing numbers of clinical studies evaluating PRP have been reported and have provided both positive and negative evidence for its effectiveness. Well-designed, controlled studies are still lacking, but PRP may have a benefit for patients with tendinopathy that is refractory to other non-surgical treatments. Its use in tendon repair is currently not supported. Randomized, controlled studies with documentation of platelet, white blood cell, and growth factor concentration in the PRP preparation are necessary for future comparative research. Use of PRP should be approached judiciously until further evidence is available.

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

The knowledge of in vivo biotransformation (e.g., proteolysis) of protein therapeutic candidates reveals structural liabilities that impact stability. This information aids the development and confirmation of ligand-binding assays with the required specificity for bioactive moieties (including intact molecule and metabolites) for appropriate PK profiling. Furthermore, the information can be used for re-engineering of constructs to remove in vivo liabilities in order to design the most stable candidates. We have developed a strategic approach of ligand-binding mass spectrometry (LBMS) to study biotransformation of fusion proteins of peptides fused to human Fc ("peptibodies") using anti-human Fc immunoaffinity capture followed by tiered mass spectrometric interrogation. LBMS offers the combined power of selectivity of ligand capture with the specificity and detailed molecular-level information of mass spectrometry. In this paper, we demonstrate the preclinical application of LBMS to three peptibodies, AMG531 (romiplostim), AMG195(linear), and AMG195(loop), that target the thrombopoietin receptor. The data show that ligand capture offers excellent sample cleanup and concentration of intact peptibodies and metabolites for subsequent query by matrix-assisted laser desorption ionization time-of-flight mass spectrometry for identification of in vivo proteolytic points. Additional higher-resolution analysis by nanoscale liquid chromatography interfaced with electrospray ionization mass spectrometry is required for identification of heterogeneous metabolites. Five proteolytic points are accurately identified for AMG531 and two for AMG195(linear), while AMG195(loop) is the most stable construct in rats. We recommend the use of LBMS to assess biotransformation and in vivo stability during early preclinical phase development for all novel fusion proteins.

Osteochondritis dissecans (OCD) of the capitellum is an uncommon disorder seen primarily in the adolescent overhead athlete. Unlike Panner disease, a self-limiting condition of the immature capitellum, OCD is multifactorial and likely results from microtrauma in the setting of cartilage mismatch and vascular susceptibility. The natural history of OCD is poorly understood, and degenerative joint disease may develop over time. Multiple modalities aid in diagnosis, including radiography, MRI, and magnetic resonance arthrography. Lesion size, location, and grade determine management, which should attempt to address subchondral bone loss and articular cartilage damage. Early, stable lesions are managed with rest. Surgery should be considered for unstable lesions. Most investigators advocate arthroscopic débridement with marrow stimulation. Fragment fixation and bone grafting also have provided good short-term results, but concerns persist regarding the healing potential of advanced lesions. Osteochondral autograft transplantation appears to be promising and should be reserved for larger, higher grade lesions. Clinical outcomes and return to sport are variable. Longer-term follow-up studies are necessary to fully assess surgical management, and patients must be counseled appropriately.

The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUG(exp) RNA). To test whether CUG(exp) RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTG(exp) transgene and another homozygous for a defective muscleblind 1 (Mbnl1) gene. Strong correlation in splicing changes for approximately 100 new Mbnl1-regulated exons indicates that loss of Mbnl1 explains >80% of the splicing pathology due to CUG(exp) RNA. In contrast, only about half of mRNA-level changes can be attributed to loss of Mbnl1, indicating that CUG(exp) RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUG(exp) RNA causes two separate effects: loss of Mbnl1 function (disrupting splicing) and loss of another function that disrupts extracellular matrix mRNA regulation, possibly mediated by Mbnl2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.