Epitope mapping of SARS-CoV-2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls.

Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogen­deuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups. This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis. Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD. The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls.

Quantification of Thread Engagement in Screw-Plate Interface of Polyaxial Locking System Using X-ray Computed Tomography.

This study demonstrates a new method for quantifying thread engagement in mechanical connections and verifies its applicability using biomedical implants under push-out tests. The focus is on orthopedic plate implants employed for bone fracture fixation, which, by design, allow off-axis screw insertion to accommodate different contact conditions. Thread engagement is crucial in determining connection strength and stability. In medical practice, off-axis screw placement is usually necessary due to bone geometries and implant plate rigidity. To address this, the study proposes a quantification method using non-destructive testing with X-ray micro-computed tomography and automated image processing, although tuning the image processing parameters is vital for accurate and reliable results. This enables detailed 3D models of screw-plate interfaces for precise thread engagement measurement. The results show that thread engagement decreases with both, increased off-axis insertion angles and higher torque during insertion. Correlation analysis reveals a strong relationship (R2 > 0.6) between average thread engagement and push-out strength, underscoring the importance of proper screw placement for stable fixation.

Tibial Plateau Leveling Osteotomy in small and large breed dogs: a comparative retrospective study of complications and outcomes.

OBJECTIVE: To compare complication rates and outcomes of small and large breed dogs that had locking plate Tibial Plateau Leveling Osteotomy (TPLO) performed due to cranial cruciate ligament disease during the same time period at a single institution and identify potential influencing factors. MATERIAL AND METHODS: 136 cases with TPLO performed at a single institution between January 2013 and December 2015 were retrospectively reviewed. Dogs were grouped by plate sizes (2.0, 2.4, 2.7, 3.5 and 3.5 broad) and by small breeds (2.0-2.7 plate sizes) and large breeds (3.5 plates). Potential influencing factors on lameness and complications were recorded from the database and measured on radiographs and statistically compared. RESULTS: Small dogs experienced fewer complications than large dogs (10% vs. 22%) and not a single major complication. Small dogs were significantly less lame at recheck and at long-term follow-up. Progression of bone healing had an influence on the lameness grade of dogs at recheck after TPLO. Distance of the most proximal screw from the joint was identified as a risk factor for implant failure. The width of the patella ligament correlated with body weight and uniformly increased 2.4 times after TPLO. CONCLUSIONS: TPLO in small breed dogs has a lower overall complication rate than in large breed dogs. The TPLO plate should always be placed as close to the joint as possible to reduce the risk of implant failure. CLINICAL RELEVANCE: TPLO can be recommended as treatment for cranial cruciate ligament rupture (CCLR) in dogs of all sizes.

What Is the Cost of Off-Axis Insertion of Locking Screws? A Biomechanical Comparison of a 3.5 mm Fixed-Angle and 3.5 mm Variable-Angle Stainless Steel Locking Plate Systems.

OBJECTIVE: The aim of this study was to evaluate the effect of screw insertion angle and insertion torque on the mechanical properties of a 3.5 fixed-angle locking plate locking compression plate (LCP) and 3.5 variable-angle locking plate polyaxial locking system (PLS). METHODS: In the LCP group, screws were placed abaxially at 0, 5 and 10 degrees. In the PLS group, screws were placed at 0, 5, 10, 15 and 20 degrees abaxially. The insertion torque was set to 1.5 and 2.5 Nm in the LCP and PLS groups respectively. A load was applied parallel to the screw axis, and the screw push-out force was measured until the locking mechanism was loosened. RESULTS: The 3.5 LCP showed higher push-out strength than the 3.5 PLS when the screws were placed at 0 degree regardless of the insertion torque. The off-axis insertion of 3.5 LCP locking screws resulted in a significant decrease in push-out strength (p < 0.05). A higher insertion torque value significantly increased the screw holding strength for the 3.5 LCP (p < 0.05). The 3.5 PLS system had a significantly higher push-out force when the screws are at 0 degree than at 5, 10 and 15 degrees, and 20 degrees (p < 0.05) at any given insertion torque. An increase in the insertion torque did not have a significant effect on the push-out strength of the 3.5 PLS locking system. CONCLUSION: The 3.5 PLS is more sensitive to the screw insertion angle than to the insertion torque, whereas the 3.5 LCP is affected by both factors. Placing 3.5 LCP locking screws off-axis significantly reduces the screw holding strength; therefore, this approach has to be avoided. The findings of our research indicate that a 1.5 Nm torque can be used for a 3.5 PLS.

Radiographically confirmed outcomes after fracture repair with a PLS polyaxial locking system in 40 dogs and cats.

OBJECTIVE: To report radiographic findings and complications after fracture repair with a new polyaxial locking plate system (PLS polyaxial locking system; Aesculap/B Braun, Tuttlingen, Germany) in dogs and cats. STUDY DESIGN: Retrospective case review from four veterinary practices. SAMPLE POPULATION: Twenty-six dogs and 14 cats (40 long bone fractures). METHODS: Medical and radiographic records of dogs and cats with long bone fractures treated with the PLS were reviewed. Cases were included when operative records were complete and included documentation of radiographic union or complications. Phone interviews of owners were performed for long-term follow-up. Ancillary methods of fracture fixation and associated complications were recorded. RESULTS: Only two complications were recorded, one of which required a revision surgery. Radiographic follow-up was performed for all fractures. Radiographic union without complications was achieved in 38 of 40 (95%) fractures. Radiographic union was documented before 60 days in 19 of 40 (47.5%) fractures, between 61 and 90 days in 15 of 40 (37.5%) fractures, and after 90 days in six of 40 (15%) fractures. A functional union was observed at a mean time ± SD of 70.8 ± 38.9 days (range, 32-182). One or more ancillary fixation methods were used in 27 of 40 (67.5%) fractures. CONCLUSION: The PLS polyaxial locking system was often used with adjunct fixation in this series, and radiographically confirmed healing without complications was documented in most cases. CLINICAL SIGNIFICANCE: Use of the PLS can result in high success rates for fracture repair in dogs and cats, but ancillary fixation should be strongly considered.

Interband Cascade Laser Arrays for Simultaneous and Selective Analysis of C1-C5 Hydrocarbons in Petrochemical Industry.

The detection and measurement of hydrocarbons are of high interest for a variety of applications, for example within the oil and gas industry from extraction throughout the complete refining process, as well as for environmental monitoring and for portable safety devices. This paper presents a highly sensitive, selective, and robust tunable laser analyzer that has the capability to analyze several components in a gas sample stream. More specifically, a multi-gas system for simultaneous detection of C1 to iC5 hydrocarbons, using a room temperature distributed feedback interband cascade laser array, emitting in the 3.3 µm band has been realized. It combines all the advantages of the tunable laser spectroscopy method for a fast, sensitive, and selective in-line multicomponent tunable laser analyzer. Capable of continuous and milliseconds fast monitoring of C1-iC5 hydrocarbon compositions in a process stream, the analyzer requires no consumables (e.g., purging, carrier gas) and no in-field calibration, enabling a low cost of ownership for the analyzer. The system was built based on an industrial GasEye series platform and deployed for the first time in field at Preem refinery in Lysekil, Sweden, in autumn 2018. Results of the measurement campaign and comparison with gas chromatography instrumentation are presented.

How Do the Locking Screws Lock? A Micro-CT Study of 3.5-mm Locking Screw Mechanism.

OBJECTIVE: To quantify the amount of the screw head thread and the plate hole thread connection in two 3.5 mm locking plates: Locking Compression Plate (LCP) and Polyaxial Locking System (PLS). MATERIALS AND METHODS: A micro - CT scan of a screw head - plate hole connection was performed pre- and post destructive tests. Tests were performed on bone surrogates in a fracture gap model. The 3.5 LCP and 3.5 PLS plates, with 3 perpendicular screws per segment were used in a destructive static test. The 3.5 PLS plates with mono- and polyaxial screws were compared in a cyclic fatigue tests in two orthogonal directions. Pre - and post - test scan datasets were compared. Each dataset was converted into serial images depicting sections cut orthogonally to locking screw axis. The amount of engagement was detected through automated image postprocessing. RESULTS: The mean amount of the thread connection for the LCP was 28.85% before and 18.55% after destructive static test. The mean amount of the connection for the PLS was 16.20% before and 14.55% after destructive static test. When inserted monoaxially, the mean amount of the connection for the PLS screws was 14.4% before and 19.24% after destructive cyclic test. The mean amount of the connection for the polyaxial inserted PLS screws when loaded against plate thickness was 2.99% before and 2.08% after destructive cyclic test. The mean amount of the connection for the polyaxial inserted PLS screws when loaded against plate width was 3.36% before and 3.93% after destructive cyclic test. The 3D visualization of the thread connection showed that the initial interface points between screw head and plate hole are different for both LCP and PLS after the destructive testing. Depending on the type of applied force, there was either loss or increase of the contact. CLINICAL RELEVANCE: Micro-CT offers news possibilities in locking implant investigation. It might be helpful in better understanding the nature of locking mechanism and prediction of possible mode of failure in different systems.

Mechanical Performance of a Polyaxial Locking Plate and the Influence of Screw Angulation in a Fracture Gap Model.

OBJECTIVE: The aim of this study was to compare the locking compression plate (LCP) with polyaxial locking system (PLS) using single cycle to failure 4-point bending test and to investigate the behaviour of PLS with screws inserted mono- and polyaxially using cyclic fatigue test in two bending directions. MATERIALS AND METHODS: Tests were performed on bone surrogates in a fracture gap model. The 3.5 LCP and 3.5 PLS plates were tested in single cycle to failure. The 3.5 PLS plates with mono- and polyaxial screws were compared in a cyclic fatigue tests in two orthogonal directions. For both experiments, micro-computed tomography (CT) scans were performed pre- and post-testing to investigate the connections between the screw head and the plate hole. Means of forces and cycles needed to failure were statistically compared. RESULTS: The PLS plates were on average 30% weaker than LCP plates. Mode of failure was plate bending in the single cycle to failure tests, and plate breakage in the cyclic fatigue tests. Neither screw breakage nor loss of the screw-plate interface occurred. Mono- and polyaxial constructs performed similarly when loaded in the same direction. Micro-CT revealed no additional internal cracks in the plates or screws after testing. It also showed for both PLS and LCP that there was only partial contact of the screw head with the plate hole. CLINICAL RELEVANCE: PLS offers a durable locking system, even when the screws are placed polyaxially. The weaker bending properties of the PLS compared with LCP should be considered during preoperative planning.

Citrullinome of Porphyromonas gingivalis Outer Membrane Vesicles: Confident Identification of Citrullinated Peptides.

Porphyromonas gingivalis is a key pathogen in chronic periodontitis and has recently been mechanistically linked to the development of rheumatoid arthritis via the activity of peptidyl arginine deiminase generating citrullinated epitopes in the periodontium. In this project the outer membrane vesicles (OMV) from P. gingivalis W83 wild-type (WT), a W83 knock-out mutant of peptidyl arginine deiminase (ΔPPAD), and a mutant strain expressing PPAD with the active site cysteine mutated to alanine (C351A), have been analyzed using a two-dimensional HFBA-based separation system combined with LC-MS. For optimal and positive identification and validation of citrullinated peptides and proteins, high resolution mass spectrometers and strict MS search criteria were utilized. This may have compromised the total number of identified citrullinations but increased the confidence of the validation. A new two-dimensional separation system proved to increase the strength of validation, and along with the use of an in-house build program, Citrullia, we establish a fast and easy semi-automatic (manual) validation of citrullinated peptides. For the WT OMV we identified 78 citrullinated proteins having a total of 161 citrullination sites. Notably, in keeping with the mechanism of OMV formation, the majority (51 out of 78) of citrullinated proteins were predicted to be exported via the inner membrane and to reside in the periplasm or being translocated to the bacterial surface. Citrullinated surface proteins may contribute to the pathogenesis of rheumatoid arthritis. For the C351A-OMV a single citrullination site was found and no citrullinations were identified for the ΔPPAD-OMV, thus validating the unbiased character of our method of citrullinated peptide identification.

An IgY-based immunoassay to evaluate the biomarker potential of the Tannerella forsythia virulence factor karilysin in human saliva.

Tannerella forsythia is a gram-negative anaerobic bacterium that is associated with the development of destructive periodontal disease. T. forsythia secretes the metalloprotease-like enzyme karilysin. Using in vitro systems karilysin has been shown to modulate the host immune response by degradation of complement system proteins and by inactivation of the antimicrobial peptide LL-37 by proteolytic cleavage. This makes karilysin a highly interesting virulence factor to study in the framework of drug development and diagnostics. However, to date the presence of karilysin in clinical samples has not been demonstrated due to the lack of specific probes. In the present work, a high titer and stable affinity-purified avian IgY antibody against karilysin was developed. By surface plasmon resonance imaging the IgY affinity was found to be in the low nanomolar range. The antibody could be used to detect karilysin in saliva samples by immuno-blotting and was specific when tested towards human MMP-3. Furthermore, an avian IgY-based immunoassay was developed, which demonstrated low intra- and interday assay variability (CV's below 10%). Application of the immunoassay on a well-characterized set of saliva samples from adolescents with or without signs of periodontitis showed that it was possible to detect karilysin in saliva. A significant difference in karilysin concentration was found between saliva from participants with signs of periodontitis and saliva from healthy controls (p = .0024). The median of karilysin levels among periodontitis cases was 957 pg/ml (IQR, 499-2132 pg/ml) and the median for controls was 569 pg/ml (IQR, 210-1343 pg/ml). Collectively our data confirm the presence of karilysin in clinical samples. The described IgY-based immunoassay may prove useful as part of protein-based biomarker screenings in the clinic or in point-of care settings.

Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator.

Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a vital role in extracellular conversion of inactive plasminogen into catalytically active plasmin. Activated plasmin facilitates the release of several proteolytic enzymes, which control processes like pericellular proteolysis and remodeling of ECM. uPA and the receptor uPAR, are overexpressed in a number of malignant tumours and uPA/uPAR play major roles in adhesion, migration, invasion and metastasis of cancer cells. Elevated levels of uPA have been reported as a risk biomarker for disease relapse, increased cancer malignancy and poor survival prognosis. For these reasons uPA is considered an important target for anticancer drug therapy. In this study we isolated two camel single domain antibodies (nanobodies) from a naïve library by phage display. The nanobody sequences were sequence-optimized for Escherichia coli expression, cloned into the pET22-B(+) vector system, expressed in BL-21 cells and purified from the periplasmic fraction by IMAC. ELISA tests demonstrated that the purified nanobodies were specific for uPA when tested towards other trypsin-like serine proteases. The apparent affinities of the nanobodies were determined by competitive ELISA to 80 nM and 522 nM, respectively. The best binder did not inhibit uPA (nAb-C3), however the lowest affinity binder (nAb-C8) was able to inhibit the uPA-mediated cleavage of the substrate S-2444. The results validate the naïve library as a resource for retrieval of relevant lead molecules and the novel uPA-nanobodies can be useful pharmacological tools to study uPA structure-function relationships.

Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis.

Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases termed gingipains, and in this study we have used the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naive camel nanobody library and used phage display to select one nanobody toward RgpB with picomolar affinity. The nanobody was used in an inhibition assay for detection of RgpB in buffer as well as in saliva. The nanobody was highly specific for RgpB given that it did not bind to the homologous gingipain HRgpA. This indicated the presence of a binding epitope within the immunoglobulin-like domain of RgpB. A subtractive inhibition assay was used to demonstrate that the nanobody could bind native RgpB in the context of intact cells. The nanobody bound exclusively to the P. gingivalis membrane-bound RgpB isoform (mt-RgpB) and to secreted soluble RgpB. Further cross-reactivity studies with P. gingivalis gingipain deletion mutants showed that the nanobody could discriminate between native RgpB and native Kgp and RgpA in complex bacterial samples. This study demonstrates that RgpB can be used as a specific biomarker for P. gingivalis detection and that the presented nanobody-based assay could supplement existing methods for P. gingivalis detection.

Cystic nephroma in adults. A report of two cases and review of the literature.

We present two cases of cystic nephroma in a 55-year old and a 61-year old women. In both patients the results of ultrasound and clinical examinations were not characteristic enough to establish the precise preoperative diagnosis. Due to the age of the patients and the location of the lesions, possibility of clear cell carcinoma with cystic changes was considered. However, microscopic examination of postoperative specimens revealed benign nature of the tumors.