MSKI-RADS: An MRI-based Musculoskeletal Infection Reporting and Data System for the Diagnosis of Extremity Infections.

Background Current terms used to describe the MRI findings for musculoskeletal infections are nonspecific and inconsistent. Purpose To develop and validate an MRI-based musculoskeletal infection classification and scoring system. Materials and Methods In this retrospective cross-sectional internal validation study, a Musculoskeletal Infection Reporting and Data System (MSKI-RADS) was designed. Adult patients with radiographs and MRI scans of suspected extremity infections with a known reference standard obtained between June 2015 and May 2019 were included. The scoring categories were as follows: 0, incomplete imaging; I, negative for infection; II, superficial soft-tissue infection; III, deeper soft-tissue infection; IV, possible osteomyelitis (OM); V, highly suggestive of OM and/or septic arthritis; VI, known OM; and NOS (not otherwise specified), nonspecific bone lesions. Interreader agreement for 20 radiologists from 13 institutions (intraclass correlation coefficient [ICC]) and true-positive rates of MSKI-RADS were calculated and the accuracy of final diagnoses rendered by the readers was compared using generalized estimating equations for clustered data. Results Among paired radiographs and MRI scans from 208 patients (133 male, 75 female; mean age, 55 years ± 13 [SD]), 20 were category I; 34, II; 35, III; 30, IV; 35, V; 18, VI; and 36, NOS. Moderate interreader agreement was observed among the 20 readers (ICC, 0.70; 95% CI: 0.66, 0.75). There was no evidence of correlation between reader experience and overall accuracy (P = .94). The highest true-positive rate was for MSKI-RADS I and NOS at 88.7% (95% CI: 84.6, 91.7). The true-positive rate was 73% (95% CI: 63, 80) for MSKI-RADS V. Overall reader accuracy using MSKI-RADS across all patients was 65% ± 5, higher than final reader diagnoses at 55% ± 7 (P < .001). Conclusion MSKI-RADS is a valid system for standardized terminology and recommended management of imaging findings of peripheral extremity infections across various musculoskeletal-fellowship-trained reader experience levels. © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Schweitzer in this issue.

Phosphoproteomic Identification of Vasopressin/cAMP/Protein Kinase A-Dependent Signaling in Kidney.

Water excretion by the kidney is regulated by the neurohypophyseal peptide hormone vasopressin through actions in renal collecting duct cells to regulate the water channel protein aquaporin-2. Vasopressin signaling is initiated by binding to a G-protein-coupled receptor called V2R, which signals through heterotrimeric G-protein subunit Gs α, adenylyl cyclase 6, and activation of the cAMP-regulated protein kinase (PKA). Signaling events coupling PKA activation and aquaporin-2 regulation were largely unknown until the advent of modern protein mass spectrometry techniques that allow proteome-wide quantification of protein phosphorylation changes (phosphoproteomics). This short review documents phosphoproteomic findings in collecting duct cells describing the response to V2R-selective vasopressin agonists and antagonists, the response to CRISPR-mediated deletion of PKA, results from in vitro phosphorylation studies using recombinant PKA, the response to the broad-spectrum kinase inhibitor H89 (N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulphonamide), and the responses underlying lithium-induced nephrogenic diabetes insipidus. These phosphoproteomic data sets have been made available online for modeling vasopressin signaling and signaling downstream from other G-protein-coupled receptors. SIGNIFICANCE STATEMENT: New developments in protein mass spectrometry are facilitating progress in identification of signaling networks. Using mass spectrometry, it is now possible to identify and quantify thousands of phosphorylation sites in a given cell type (phosphoproteomics). The authors describe the use of phosphoproteomics technology to identify signaling mechanisms downstream from a G-protein-coupled receptor, the vasopressin V2 subtype receptor, and its role of the regulation and dysregulation of water excretion in the kidney. Data from multiple phosphoproteomic data sets are provided as web-based resources.

Protein kinase A catalytic-α and catalytic-ß proteins have nonredundant regulatory functions.

Vasopressin regulates osmotic water transport in the renal collecting duct by protein kinase A (PKA)-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA-Cα) and PKA catalytic ß (PKA-Cß). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-Cα or PKA-Cß was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4,635 phosphopeptides that were quantified, 67 phosphopeptides were significantly altered in abundance with PKA-Cα deletion, whereas 21 phosphopeptides were significantly altered in abundance with PKA-Cß deletion. However, only four sites were changed in both. The target proteins identified in PKA-Cα-null cells were largely associated with cell membranes and membrane vesicles, whereas target proteins in PKA-Cß-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-Cα and PKA-Cß resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in in vivo samples showed that PKA-Cα deletion resulted in a near disappearance of AQP2 protein, whereas PKA-Cß deletion did not decrease AQP2 abundance. We conclude that PKA-Cα and PKA-Cß serve substantially different regulatory functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g., mediated by A-kinase anchor proteins, C-kinase anchoring proteins, or PDZ binding.

Hydroxyapatite nanoparticle-modified porous bone grafts with improved cell attachment.

Emulsion-templated foams have displayed promise as injectable bone grafts; however, the use of a surfactant as an emulsifier resulted in relatively small pores and impedes cell attachment. Hydroxyapatite nanoparticles were explored as an alternative stabilizer to address these limitations. To this end, hydroxyapatite nanoparticles were first modified with myristic acid to generate the appropriate balance of hydrophobicity to stabilize a water-in-oil emulsion of neopentyl glycol diacrylate and 1,4-butanedithiol. In situ surface modification of the resulting foam with hydroxyapatite was confirmed with elemental mapping and transmission electron microscopy. Nanoparticle-stabilized foams displayed improved human mesenchymal stem cell viability (91 ± 5%) over surfactant-stabilized foams (23 ± 11%). Although the pore size was appropriate for bone grafting applications (115 ± 71 µm), the foams lacked the interconnected architecture necessary for cell infiltration. We hypothesized that a co-stabilization approach with both surfactant and nanoparticles could be used to achieve interconnected pores while maintaining improved cell attachment and larger pore sizes. A range of hydroxyapatite nanoparticle and surfactant concentrations were investigated to determine the effects on microarchitecture and cell behavior. By balancing these interactions, a co-stabilized foam was identified that possessed large, interconnected pores (108 ± 67 µm) and improved cell viability and attachment. The co-stabilized foam was then evaluated as an injectable bone graft including network formation, microscale integration with bone, push out strength, and compressive properties. Overall, this work demonstrated that in situ surface modification with nHA improved cell attachment while retaining desirable bone grafting features and injectability.

Associations of Cumulative Perceived Stress with Cardiovascular Risk Factors and Outcomes: Findings from The Dallas Heart Study.

Background: Data remain sparse regarding the impact of chronic stress on cardiovascular disease (CVD) risk factors and outcomes. Prior work has been limited by incomplete assessments of perceived stress and focus on single stress domains. We evaluated the association between a composite measure of perceived stress and CVD risk factors and outcomes. Methods: Participants from the Dallas Heart Study phase 2 (2007-2009) without prevalent CVD who completed questionnaire assessments of perceived stress were included (n=2685). Individual perceived stress subcomponents (generalized stress, psychosocial, financial, and neighborhood stress) were standardized and integrated into a single cumulative stress score (CSS) with equal weighting for each component. Associations between CSS and demographics, psychosocial variables and cardiac risk factors were assessed in univariable and multivariable analyses. Cox proportional hazards models were used to determine associations of the CSS with atherosclerotic CVD (ASCVD) and Global CVD (ASCVD, heart failure, and atrial fibrillation) after adjustment for demographics and traditional risk factors. Results: Median age of the study population was 48 years, 55% were female, 49% Black and 15% Hispanic/Latinx. CSS was higher among participants who were younger, female, Black or Hispanic, and those with lower income and educational attainment (p<.0001 for each). Higher CSS was associated with self-report of racial/ethnic discrimination, lack of health insurance and last medical contact > one year previously (p<.0001 for each). In multivariable regression models adjusting for age, gender, race/ethnicity, income and education, higher CSS associated with hypertension, smoking, and higher body mass index, waist circumference Hemoglobin A1C, hs-CRP and sedentary time (p< 0.01 for each). Over a median follow-up of 12.4 years, higher CSS associated with ASCVD (adjusted HR 1.22 per SD, 95% CI 1.01-1.47) and Global CVD (HR 1.20, 95% CI 1.03-1.40). No interactions were seen between CSS, demographic factors, and outcomes. Conclusion: Composite multidimensional assessments of perceived stress may help to identify individuals at risk for CVD who may be targeted for stress mitigation or enhanced prevention strategies. These approaches may be best focused on vulnerable populations, given the higher burden of stress in women, Black and Hispanic individuals, and those with lower income and education. WHAT IS NEW?: A novel measure of cumulative stress was created that integrates generalized, psychosocial, financial, and neighborhood perceived stress.Cumulative stress was higher among women, Black and Hispanic participants, younger individuals and persons with lower income and educational attainment and was associated with adverse health behaviors and increased burden of cardiovascular disease (CVD) risk factors.In a diverse cohort, higher cumulative stress associated with incident CVD after adjustment for demographics and traditional risk factors. No interactions were seen based on demographic factors. CLINICAL IMPLICATIONS: Although associations of chronic stress with CVD were similar across demographic subgroups, the higher burden of stress among younger individuals, women, Black and Hispanic participants, and those with lower SES suggests that CVD risk associated with higher stress affects marginalized groups disproportionately.Cumulative Stress is associated with modifiable risk factors and health behaviors. Future studies should explore targeting behavioral modification and risk factor reduction programs, as well as stress reduction strategies, to individuals with high cumulative stress.Additional research is needed to uncover mechanisms that underly the association between chronic stress and cardiovascular disease.

PoreScript: Semi-automated pore size algorithm for scaffold characterization.

The scaffold pore size influences many critical physical aspects of tissue engineering, including tissue infiltration, biodegradation rate, and mechanical properties. Manual measurements of pore sizes from scanning electron micrographs using ImageJ/FIJI are commonly used to characterize scaffolds, but these methods are both time-consuming and subject to user bias. Current semi-automated analysis tools are limited by a lack of accessibility or limited sample size in their verification process. The work here describes the development of a new MATLAB algorithm, PoreScript, to address these limitations. The algorithm was verified using three common scaffold fabrication methods (e.g., salt leaching, gas foaming, emulsion templating) with varying pore sizes and shapes to demonstrate the versatility of this new tool. Our results demonstrate that the pore size characterization using PoreScript is comparable to manual pore size measurements. The PoreScript algorithm was further evaluated to determine the effect of user-input and image parameters (relative image magnification, pixel intensity threshold, and pore structure). Overall, this work validates the accuracy of the PoreScript algorithm across several fabrication methods and provides user-guidance for semi-automated image analysis and increased throughput of scaffold characterization.

Engineering Toolbox for Systematic Design of PolyHIPE Architecture.

Polymerization of high internal phase emulsions (polyHIPEs) is a well-established method for the production of high porosity foams. Researchers are often regulated to using a time-intensive trial and error approach to achieve target pore architectures. In this work, we performed a systematic study to identify the relative effects of common emulsion parameters on pore architecture (mixing speed, surfactant concentration, organic phase viscosity, molecular hydrophobicity). Across different macromer chemistries, the largest magnitude of change in pore size was observed across surfactant concentration (~6 fold, 5-20 wt%), whereas changing mixing speeds (~4 fold, 500-2000 RPM) displayed a reduced effect. Furthermore, it was observed that organic phase viscosity had a marked effect on pore size (~4 fold, 6-170 cP) with no clear trend observed with molecular hydrophobicity in this range (logP = 1.9-4.4). The efficacy of 1,4-butanedithiol as a reactive diluent was demonstrated and provides a means to reduce organic phase viscosity and increase pore size without affecting polymer fraction of the resulting foam. Overall, this systematic study of the microarchitectural effects of these macromers and processing variables provides a framework for the rational design of polyHIPE architectures that can be used to accelerate design and meet application needs across many sectors.

Phosphoproteomic identification of vasopressin-regulated protein kinases in collecting duct cells.

BACKGROUND AND PURPOSE: The peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V2 receptor, which triggers a cAMP-mediated activation of a PKA-dependent signalling network. The protein kinases downstream from PKA have not been fully identified or mapped to regulated phosphoproteins. EXPERIMENTAL APPROACH: We carried out systems-level analysis of large-scale phosphoproteomic data quantifying vasopressin-induced changes in phosphorylation in aquaporin-2-expressing cultured collecting duct (mpkCCD) cells. Quantification was done using stable isotope labelling (SILAC method). KEY RESULTS: Six hundred forty phosphopeptides were quantified. Stringent statistical analysis identified significant changes in response to vasopressin in 429 of these phosphopeptides. The corresponding phosphoproteins were mapped to known vasopressin-regulated cellular processes. The vasopressin-regulated sites were classified according to the sequences surrounding the phosphorylated amino acids giving 11 groups. Among the vasopressin-regulated phosphoproteins were 25 distinct protein kinases. Among these, six plus PKA appeared to account for phosphorylation of about 81% of the 313 vasopressin-regulated phosphorylation sites. The six downstream kinases were salt-inducible kinase 2 (Sik2), cyclin-dependent kinase 18 (Cdk18), calmodulin-dependent kinase kinase 2 (Camkk2), protein kinase D2 (Prkd2), mitogen-activated kinase 3 (Mapk3) and myosin light chain kinase (Mylk). CONCLUSION AND IMPLICATIONS: In V2 receptor-mediated signalling, PKA is at the head of a complex network that includes at least six downstream vasopressin-regulated protein kinases that are prime targets for future study. The extensive phosphoproteomic data reported in this study are provided as a web-based data resource for future studies of GPCRs.

Review of Integrin-Targeting Biomaterials in Tissue Engineering.

The ability to direct cell behavior has been central to the success of numerous therapeutics to regenerate tissue or facilitate device integration. Biomaterial scientists are challenged to understand and modulate the interactions of biomaterials with biological systems in order to achieve effective tissue repair. One key area of research investigates the use of extracellular matrix-derived ligands to target specific integrin interactions and induce cellular responses, such as increased cell migration, proliferation, and differentiation of mesenchymal stem cells. These integrin-targeting proteins and peptides have been implemented in a variety of different polymeric scaffolds and devices to enhance tissue regeneration and integration. This review first presents an overview of integrin-mediated cellular processes that have been identified in angiogenesis, wound healing, and bone regeneration. Then, research utilizing biomaterials are highlighted with integrin-targeting motifs as a means to direct these cellular processes to enhance tissue regeneration. In addition to providing improved materials for tissue repair and device integration, these innovative biomaterials provide new tools to probe the complex processes of tissue remodeling in order to enhance the rational design of biomaterial scaffolds and guide tissue regeneration strategies.

Improved in situ seeding of 3D printed scaffolds using cell-releasing hydrogels.

The design of tissue engineered scaffolds based on polymerized high internal phase emulsions (polyHIPEs) has emerged as a promising bone grafting strategy. We previously reported the ability to 3D print emulsion inks to better mimic the structure and mechanical properties of native bone while precisely matching defect geometry. In the current study, redox-initiated hydrogel carriers were investigated for in situ delivery of human mesenchymal stem cells (hMSCs) utilizing the biodegradable macromer, poly(ethylene glycol)-dithiothreitol. Hydrogel carrier properties including network formation time, sol-gel fraction, and swelling ratio were modulated to achieve rapid cure without external stimuli and a target cell-release period of 5-7 days. These in situ carriers enabled improved distribution of hMSCs in 3D printed polyHIPE grafts over standard suspension seeding. Additionally, carrier-loaded polyHIPEs supported sustained cell viability and osteogenic differentiation of hMSCs post-release. In summary, these findings demonstrate the potential of this in situ curing hydrogel carrier to enhance the cell distribution and retention of hMSCs in bone grafts. Although initially focused on improving bone regeneration, the ability to encapsulate cells in a hydrogel carrier without relying on external stimuli that can be attenuated in large grafts or tissues is expected to have a wide range of applications in tissue engineering.