Time-of-Flight MRA of Intracranial Aneurysms with Interval Surveillance, Clinical Segmentation and Annotations.

Intracranial aneurysms (IAs) are present in 2-6% of the global population and can be catastrophic upon rupture with a mortality rate of 30-50%. IAs are commonly detected through time-of-flight magnetic resonance angiography (TOF-MRA), however, this data is rarely available for research and training purposes. The provision of imaging resources such as TOF-MRA images is imperative to develop new strategies for IA detection, rupture prediction, and surgical training. To support efforts in addressing data availability bottlenecks, we provide an open-access TOF-MRA dataset comprising 63 patients, of which 24 underwent interval surveillance imaging by TOF-MRA. Patient scans were evaluated by a neuroradiologist, providing aneurysm and vessel segmentations, clinical annotations, 3D models, in addition to 3D Slicer software environments containing all this data for each patient. This dataset is the first to provide interval surveillance imaging for supporting the understanding of IA growth and stability. This dataset will support computational and experimental research into IA dynamics and assist surgical and radiology training in IA treatment.

Characterization of the dystrophin-associated protein complex by mass spectrometry.

The dystrophin-associated protein complex (DAPC) is a highly organized multiprotein complex that plays a pivotal role in muscle fiber structure integrity and cell signaling. The complex is composed of three distinct interacting subgroups, intracellular peripheral proteins, transmembrane glycoproteins, and extracellular glycoproteins subcomplexes. Dystrophin protein nucleates the DAPC and is important for connecting the intracellular actin cytoskeletal filaments to the sarcolemma glycoprotein complex that is connected to the extracellular matrix via laminin, thus stabilizing the sarcolemma during muscle fiber contraction and relaxation. Genetic mutations that lead to lack of expression or altered expression of any of the DAPC proteins are associated with different types of muscle diseases. Hence characterization of this complex in healthy and dystrophic muscle might bring insights into its role in muscle pathogenesis. This review highlights the role of mass spectrometry in characterizing the DAPC interactome as well as post-translational glycan modifications of some of its components such as α-dystroglycan. Detection and quantification of dystrophin using targeted mass spectrometry are also discussed in the context of healthy versus dystrophic skeletal muscle.

Altered muscle niche contributes to myogenic deficit in the D2-mdx model of severe DMD.

Lack of dystrophin expression is the underlying genetic basis for Duchenne muscular dystrophy (DMD). However, disease severity varies between patients, based on specific genetic modifiers. D2-mdx is a model for severe DMD that exhibits exacerbated muscle degeneration and failure to regenerate even in the juvenile stage of the disease. We show that poor regeneration of juvenile D2-mdx muscles is associated with an enhanced inflammatory response to muscle damage that fails to resolve efficiently and supports the excessive accumulation of fibroadipogenic progenitors (FAPs), leading to increased fibrosis. Unexpectedly, the extent of damage and degeneration in juvenile D2-mdx muscle is significantly reduced in adults, and is associated with the restoration of the inflammatory and FAP responses to muscle injury. These improvements enhance regenerative myogenesis in the adult D2-mdx muscle, reaching levels comparable to the milder B10-mdx model of DMD. Ex vivo co-culture of healthy satellite cells (SCs) with juvenile D2-mdx FAPs reduces their fusion efficacy. Wild-type juvenile D2 mice also manifest regenerative myogenic deficit and glucocorticoid treatment improves their muscle regeneration. Our findings indicate that aberrant stromal cell responses contribute to poor regenerative myogenesis and greater muscle degeneration in juvenile D2-mdx muscles and reversal of this reduces pathology in adult D2-mdx muscle, identifying these responses as a potential therapeutic target for the treatment of DMD.

Evaluation of 3D Printed Burr Hole Simulation Models Using 8 Different Materials.

OBJECTIVE: 3D printing is increasingly used to fabricate three-dimensional neurosurgical simulation models, making training more accessible and economical. 3D printing includes various technologies with different capabilities for reproducing human anatomy. This study evaluated different materials across a broad range of 3D printing technologies to identify the combination that most precisely represents the parietal region of the skull for burr hole simulation. METHODS: Eight different materials (polyethylene terephthalate glycol, Tough PLA, FibreTuff, White Resin, BoneSTN, SkullSTN, polymide [PA12], glass-filled polyamide [PA12-GF]) across 4 different 3D printing processes (fused filament fabrication, stereolithography, material jetting, selective laser sintering) were produced as skull samples that fit into a larger head model derived from computed tomography imaging. Five neurosurgeons conducted burr holes on each sample while blinded to the details of manufacturing method and cost. Qualities of mechanical drilling, visual appearance, skull exterior, and skull interior (i.e., diploë) and overall opinion were documented, and a final ranking activity was performed along with a semistructured interview. RESULTS: The study found that 3D printed polyethylene terephthalate glycol (using fused filament fabrication) and White Resin (using stereolithography) were the best models to replicate the skull, surpassing advanced multimaterial samples from a Stratasys J750 Digital Anatomy Printer. The interior (e.g., infill) and exterior structures strongly influenced the overall ranking of samples. All neurosurgeons agreed that practical simulation with 3D printed models can play a vital role in neurosurgical training. CONCLUSIONS: The study findings reveal that widely accessible desktop 3D printers and materials can play a valuable role in neurosurgical training.

Altered muscle niche contributes to myogenic deficit in the D2- mdx model of severe DMD.

Lack of dystrophin is the genetic basis for the Duchenne muscular dystrophy (DMD). However, disease severity varies between patients, based on specific genetic modifiers. D2- mdx is a model for severe DMD that exhibits exacerbated muscle degeneration and failure to regenerate even in the juvenile stage of the disease. We show that poor regeneration of juvenile D2- mdx muscles is associated with enhanced inflammatory response to muscle damage that fails to resolve efficiently and supports excessive accumulation of fibroadipogenic progenitors (FAPs). Unexpectedly, the extent of damage and degeneration of juvenile D2- mdx muscle is reduced in adults and is associated with the restoration of the inflammatory and FAP responses to muscle injury. These improvements enhance myogenesis in the adult D2- mdx muscle, reaching levels comparable to the milder (B10- mdx ) mouse model of DMD. Ex vivo co-culture of healthy satellite cells (SCs) with the juvenile D2- mdx FAPs reduced their fusion efficacy and in vivo glucocorticoid treatment of juvenile D2 mouse improved muscle regeneration. Our findings indicate that aberrant stromal cell response contributes to poor myogenesis and greater muscle degeneration in dystrophic juvenile D2- mdx muscles and reversal of this reduces pathology in adult D2- mdx mouse muscle, identifying these as therapeutic targets to treat dystrophic DMD muscles.

The X-linked Becker muscular dystrophy (bmx) mouse models Becker muscular dystrophy via deletion of murine dystrophin exons 45-47.

BACKGROUND: Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy (DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype. METHODS: We use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000 bp deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography. RESULTS: Overall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (-39%, P < 0.0001), wire hang (P = 0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (-25%, P = 0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P < 0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P = 0.0017) and centrally located nuclei indicating degeneration/regeneration (P < 0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P < 0.0001), collagen deposition (+1.4-fold, P = 0.0217), and sarcolemmal damage measured by intracellular IgM (P = 0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes. CONCLUSIONS: The bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.

Properties and Implementation of 3-Dimensionally Printed Models in Spine Surgery: A Mixed-Methods Review With Meta-Analysis.

OBJECTIVE: Spine surgery addresses a wide range of spinal pathologies. Potential applications of 3-dimensional (3D) printed in spine surgery are broad, encompassing education, planning, and simulation. The objective of this study was to explore how 3D-printed spine models are implemented in spine surgery and their clinical applications. METHODS: Methods were combined to create a scoping review with meta-analyses. PubMed, EMBASE, the Cochrane Library, and Scopus databases were searched from 2011 to 7 September 2021. Results were screened independently by 2 reviewers. Studies utilizing 3D-printed spine models in spine surgery were included. Articles describing drill guides, implants, or nonoriginal research were excluded. Data were extracted according to reporting guidelines in relation to study information, use of model, 3D printer and printing material, design features of the model, and clinical use/patient-related outcomes. Meta-analyses were performed using random-effects models. RESULTS: Forty articles were included in the review, 3 of which were included in the meta-analysis. Primary use of the spine models included preoperative planning, education, and simulation. Six printing technologies were utilized. A range of substrates were used to recreate the spine and regional pathology. Models used for preoperative and intraoperative planning showed reductions in key surgical performance indicators. Generally, feedback for the tactility, utility, and education use of models was favorable. CONCLUSIONS: Replicating realistic spine models for operative planning, education, and training is invaluable in a subspeciality where mistakes can have devastating repercussions. Future study should evaluate the cost-effectiveness and the impact spine models have of spine surgery outcomes.

Multi-jet fusion for additive manufacturing of radiotherapy immobilization devices: Effects of color, thickness, and orientation on surface dose and tensile strength.

Immobilization devices are used to obtain reproducible patient setup during radiotherapy treatment, improving accuracy, and reducing damage to surrounding healthy tissue. Additive manufacturing is emerging as a viable method for manufacturing and personalizing such devices. The goal of this study was to investigate the dosimetric and mechanical properties of a recent additive technology called multi-jet fusion (MJF) for radiotherapy applications, including the ability for this process to produce full color parts. Skin dose testing included 50 samples with dimensions 100 mm × 100 mm with five different thicknesses (1 mm, 2 mm, 3 mm, 4 mm, and 5 mm) and grouped into colored (cyan, magenta, yellow, and black (CMYK) additives) and non-colored (white) samples. Results using a 6 MV beam found that surface dose readings were predominantly independent of the colored additives. However, for an 18 MV beam, the additives affected the surface dose, with black recording significantly lower surface dose readings compare to other colors. The accompanying tensile testing of 175 samples designed to ASTM D638 type I standards found that the black agent resulted in the lowest ultimate tensile strength (UTS) for each thickness of 1-5 mm. It was also found that the print orientation had influence on the skin dose and mechanical properties of the samples. When all data were combined and analyzed using a multiple-criteria decision-making technique, magenta was found to offer the best balance between high UTS and low surface dose across different thicknesses and orientations, making it an optimal choice for immobilization devices. This is the first study to consider the use of color MJF for radiotherapy immobilization devices, and suggests that color additives can affect both dosimetry and mechanical performance. This is important as industrial additive technologies like MJF become increasingly adopted in the health and medical sectors.

Diuretics in States of Volume Overload: Core Curriculum 2022.

Volume overload, defined as excess total body sodium and water with expansion of extracellular fluid volume, characterizes common disorders such as congestive heart failure, end-stage liver disease, chronic kidney disease, and nephrotic syndrome. Diuretics are the cornerstone of therapy for volume overload and comprise several classes whose mechanisms of action, pharmacokinetics, indications, and adverse effects are essential principles of nephrology. Loop diuretics are typically the first-line treatment in the management of hypervolemia, with additional drug classes indicated in cases of diuretic resistance and electrolyte or acid-base disorders. Separately, clinical trials highlight improved outcomes in some states of volume overload, such as loop diuretics and sodium/glucose cotransporter 2 inhibitors in patients with congestive heart failure. Resistance to diuretics is a frequent, multifactorial clinical challenge that requires creative and physiology-based solutions. In this installment of AJKD's Core Curriculum in Nephrology, we discuss the pharmacology and therapeutic use of diuretics in states of volume overload and strategies to overcome diuretic resistance.

Anoctamin 5 Knockout Mouse Model Recapitulates LGMD2L Muscle Pathology and Offers Insight Into in vivo Functional Deficits.

Mutations in the Anoctamin 5 (Ano5) gene that result in the lack of expression or function of ANO5 protein, cause Limb Girdle Muscular Dystrophy (LGMD) 2L/R12, and Miyoshi Muscular Dystrophy (MMD3). However, the dystrophic phenotype observed in patient muscles is not uniformly recapitulated by ANO5 knockout in animal models of LGMD2L. Here we describe the generation of a mouse model of LGMD2L generated by targeted out-of-frame deletion of the Ano5 gene. This model shows progressive muscle loss, increased muscle weakness, and persistent bouts of myofiber regeneration without chronic muscle inflammation, which recapitulates the mild to moderate skeletal muscle dystrophy reported in the LGMD2L patients. We show that these features of ANO5 deficient muscle are not associated with a change in the calcium-activated sarcolemmal chloride channel activity or compromised in vivo regenerative myogenesis. Use of this mouse model allows conducting in vivo investigations into the functional role of ANO5 in muscle health and for preclinical therapeutic development for LGMD2L.

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy.

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies -Eteplirsen, Golodirsen, Viltolarsen, and Casimersen -for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

Effects of Chronic, Maximal Phosphorodiamidate Morpholino Oligomer (PMO) Dosing on Muscle Function and Dystrophin Restoration in a Mouse Model of Duchenne Muscular Dystrophy.

BACKGROUND: Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is currently used in clinical development to treat Duchenne muscular dystrophy (DMD), with four exon-skipping drugs achieving regulatory approval. Exon skipping elicits a truncated, but semi-functional dystrophin protein, similar to the truncated dystrophin expressed in patients with Becker Muscular dystrophy (BMD) where the disease phenotype is less severe than DMD. Despite promising results in both dystrophic animal models and DMD boys, restoration of dystrophin by exon skipping is highly variable, leading to contradictory functional outcomes in clinical trials. OBJECTIVE: To develop optimal PMO dosing protocols that result in increased dystrophin and improved outcome measures in preclinical models of DMD. METHODS: Tested effectiveness of multiple chronic, high dose PMO regimens using biochemical, histological, molecular, and imaging techniques in mdx mice. RESULTS: A chronic, monthly regimen of high dose PMO increased dystrophin rescue in mdx mice and improved specific force in the extensor digitorum longus (EDL) muscle. However, monthly high dose PMO administration still results in variable dystrophin expression localized throughout various muscles. CONCLUSIONS: High dose monthly PMO administration restores dystrophin expression and increases muscle force; however, the variability of dystrophin expression at both the inter-and intramuscular level remains. Additional strategies to optimize PMO uptake including increased dosing frequencies or combination treatments with other yet-to-be-defined therapies may be necessary to achieve uniform dystrophin restoration and increases in muscle function.

Properties and Characteristics of Three-Dimensional Printed Head Models Used in Simulation of Neurosurgical Procedures: A Scoping Review.

BACKGROUND: Intracranial surgery can be complex and high risk. Safety, ethical and financial factors make training in the area challenging. Head model 3-dimensional (3D) printing is a realistic training alternative to patient and traditional means of cadaver and animal model simulation. OBJECTIVE: To describe important factors relating to the 3D printing of human head models and how such models perform as simulators. METHODS: Searches were performed in PubMed, the Cochrane Library, Scopus, and Web of Science. Articles were screened independently by 3 reviewers using Covidence software. Data items were collected under 5 categories: study information; printers and processes; head model specifics; simulation and evaluations; and costs and production times. RESULTS: Forty articles published over the last 10 years were included in the review. A range of printers, printing methods, and substrates were used to create head models and tissue types. Complexity of the models ranged from sections of single tissue type (e.g., bone) to high-fidelity integration of multiple tissue types. Some models incorporated disease (e.g., tumors and aneurysms) and artificial physiology (e.g., pulsatile circulation). Aneurysm clipping, bone drilling, craniotomy, endonasal surgery, and tumor resection were the most commonly practiced procedures. Evaluations completed by those using the models were generally favorable. CONCLUSIONS: The findings of this review indicate that those who practice surgery and surgical techniques on 3D-printed head models deem them to be valuable assets in cranial surgery training. Understanding how surgical simulation on such models affects surgical performance and patient outcomes, and considering cost-effectiveness, are important future research endeavors.

Validation of Chemokine Biomarkers in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease involving complex skeletal muscle pathogenesis. The pathogenesis is triggered by sarcolemma instability due to the lack of dystrophin protein expression, leading to Ca2+ influx, muscle fiber apoptosis, inflammation, muscle necrosis, and fibrosis. Our lab recently used two high-throughput multiplexing techniques (e.g., SomaScan® aptamer assay and tandem mass tag-(TMT) approach) and identified a series of serum protein biomarkers tied to different pathobiochemical pathways. In this study, we focused on validating the circulating levels of three proinflammatory chemokines (CCL2, CXCL10, and CCL18) that are believed to be involved in an early stage of muscle pathogenesis. We used highly specific and reproducible MSD ELISA assays and examined the association of these chemokines with DMD pathogenesis, age, disease severity, and response to glucocorticoid treatment. As expected, we confirmed that these three chemokines were significantly elevated in serum and muscle samples of DMD patients relative to age-matched healthy controls (p-value < 0.05, CCL18 was not significantly altered in muscle samples). These three chemokines were not significantly elevated in Becker muscular dystrophy (BMD) patients, a milder form of dystrophinopathy, when compared in a one-way ANOVA to a control group but remained significantly elevated in the age-matched DMD group (p < 0.05). CCL2 and CCL18 but not CXCL10 declined with age in DMD patients, whereas all three chemokines remained unchanged with age in BMD and controls. Only CCL2 showed significant association with time to climb four steps in the DMD group (r = 0.48, p = 0.038) and neared significant association with patients' reported outcome in the BMD group (r = 0.39, p = 0.058). Furthermore, CCL2 was found to be elevated in a serum of the mdx mouse model of DMD, relative to wild-type mouse model. This study suggests that CCL2 might be a suitable candidate biomarker for follow-up studies to demonstrate its physiological significance and clinical utility in DMD.

Human muscle stem cells are refractory to aging.

Age-related loss of muscle mass and strength is widely attributed to limitation in the capacity of muscle resident satellite cells to perform their myogenic function. This idea contains two notions that have not been comprehensively evaluated by experiment. First, it entails the idea that we damage and lose substantial amounts of muscle in the course of our normal daily activities. Second, it suggests that mechanisms of muscle repair are in some way exhausted, thus limiting muscle regeneration. A third potential option is that the aged environment becomes inimical to the conduct of muscle regeneration. In the present study, we used our established model of human muscle xenografting to test whether muscle samples taken from cadavers, of a range of ages, maintained their myogenic potential after being transplanted into immunodeficient mice. We find no measurable difference in regeneration across the range of ages investigated up to 78 years of age. Moreover, we report that satellite cells maintained their myogenic capacity even when muscles were grafted 11 days postmortem in our model. We conclude that the loss of muscle mass with increasing age is not attributable to any intrinsic loss of myogenicity and is most likely a reflection of progressive and detrimental changes in the muscle microenvironment such as to disfavor the myogenic function of these cells.

Ecological half-life of radiocesium in white-tailed deer on the Department of Energy's Savannah River Site: What can a half century of field monitoring tell us?

This paper estimates the 137Cs ecological half-life of white-tailed deer inhabiting the Department of Energy's Savannah River Site (SRS) based on sex and age using data collected over a 51-year time-period. With a physical half-life of 30.2 yr, the biological half-life for the deer herd is considerably shorter because of the isotope's biochemical mimicry of K+ inside the body. Leveraging this long-term dataset and robust sample size, we compared the long-term half-century estimates to shorter decadal increments. The simple exponential decay model for the entire 51-year sampling period predicted an ecological half-life of 23.15 years. When breaking the sample data into decadal increments 137Cs body burden had complex temporal dynamics with predicted half-lives ranging from 9.25 to 32.33 years. Exponential decay for the entire 51-year sampling period for models evaluated by sex, age, sex*age to determine how these variables influence the predictability in the mean depuration rate, the ecological half-lives were between 21 and 23 years for all permutations, except for fawns that had a half-life no different than the physical half-life of the isotope itself. Differential habitat use and competition most likely explains why both yearling and adult females consistently had higher body burdens than males over the 51-year time period, showing how dynamic this radioisotope is in biological systems. This study is one of the most robust long-term datasets in the world (n = 42,412) that is specifically focused on monitoring the uptake and depuration of 137Cs in a wild species.

3D Printing: An Alternative Microfabrication Approach with Unprecedented Opportunities in Design.

In the past decade, 3D printing technologies have been adopted for the fabrication of microfluidic devices. Extrusion-based approaches including fused filament fabrication (FFF), jetting technologies including inkjet 3D printing, and vat photopolymerization techniques including stereolithography (SLA) and digital light projection (DLP) are the 3D printing methods most frequently adopted by the microfluidic community. Each printing technique has merits toward the fabrication of microfluidic devices. Inkjet printing offers a good selection of materials and multimaterial printing, and the large build space provides manufacturing throughput, while FFF offers a great selection of materials and multimaterial printing but at lower throughput compared to inkjet 3D printing. Technical and material developments adopted from adjacent research fields and developed by the microfluidic community underpin the printing of sub-100 µm enclosed microchannels by DLP, but challenges remain in multimaterial printing throughput. With the feasibility of 3D printed microfluidics established, we look ahead at trends in 3D printing to gain insights toward the future of this technology beyond the sole prism of being an alternative fabrication approach. A shift in emphasis from using 3D printing for prototyping, to mimic conventionally manufactured outputs, toward integrated approaches from a design perspective is critically developed.

Assessment of Interobserver Reliability of Nephrologist Examination of Urine Sediment.

Importance: Urine sediment microscopy is commonly performed during the evaluation of kidney disease. Interobserver reliability of nephrologists' urine sediment examination has not been well studied. Objective: Assess interobserver reliability of the urine sediment examination. Design, Setting, and Participants: In this diagnostic test study, urine samples were prospectively collected from a convenience sample of adult patients from an academic hospital in the United States undergoing kidney biopsy from July 11, 2018, to March 20, 2019. Digital images and videos of urine sediment findings were captured using a bright-field microscope. These images and videos along with urine dipstick results were incorporated in online surveys and sent to expert nephrologists at 15 US teaching hospitals. They were asked to identify individual sediment findings and the most likely underlying disease process. Exposures: Urine dipstick results and urine sediment images from patients undergoing native kidney biopsy. Main Outcomes and Measures: Interobserver reliability of urine sediment microscopy findings estimated by overall percent agreement and Fleiss κ coefficients. Secondary outcomes included concordance of diagnoses suspected by nephrologists with corresponding kidney biopsy results. Results: In total, 10 surveys from 10 patients containing 76 study questions on individual features were sent to 21 nephrologists, 14 (67%) of whom completed them all. Their combined 1064 responses were analyzed. Overall percent agreement for casts was an estimated 59% (95% CI, 50%-69%), κ = 0.52 (95% CI, 0.42-0.62). For other sediment findings, overall percent agreement was an estimated 69% (95% CI, 61%-77%), κ = 0.65 (95% CI, 0.56-0.73). The κ estimates ranged from 0.13 (95% CI, 0.10-0.17) for mixed cellular casts to 0.90 (95% CI, 0.87-0.94) for squamous epithelial cells. Conclusions and Relevance: In this study, substantial variability occurred in the interpretation of urine sediment findings, even among expert nephrologists. Educational or technological innovations may help improve the urine sediment as a diagnostic tool.

Membrane Repair Deficit in Facioscapulohumeral Muscular Dystrophy.

Deficits in plasma membrane repair have been identified in dysferlinopathy and Duchenne Muscular Dystrophy, and contribute to progressive myopathy. Although Facioscapulohumeral Muscular Dystrophy (FSHD) shares clinicopathological features with these muscular dystrophies, it is unknown if FSHD is characterized by plasma membrane repair deficits. Therefore, we exposed immortalized human FSHD myoblasts, immortalized myoblasts from unaffected siblings, and myofibers from a murine model of FSHD (FLExDUX4) to focal, pulsed laser ablation of the sarcolemma. Repair kinetics and success were determined from the accumulation of intracellular FM1-43 dye post-injury. We subsequently treated FSHD myoblasts with a DUX4-targeting antisense oligonucleotide (AON) to reduce DUX4 expression, and with the antioxidant Trolox to determine the role of DUX4 expression and oxidative stress in membrane repair. Compared to unaffected myoblasts, FSHD myoblasts demonstrate poor repair and a greater percentage of cells that failed to repair, which was mitigated by AON and Trolox treatments. Similar repair deficits were identified in FLExDUX4 myofibers. This is the first study to identify plasma membrane repair deficits in myoblasts from individuals with FSHD, and in myofibers from a murine model of FSHD. Our results suggest that DUX4 expression and oxidative stress may be important targets for future membrane-repair therapies.

TGF-ß-driven muscle degeneration and failed regeneration underlie disease onset in a DMD mouse model.

Duchenne muscular dystrophy (DMD) is a chronic muscle disease characterized by poor myogenesis and replacement of muscle by extracellular matrix. Despite the shared genetic basis, severity of these deficits varies among patients. One source of these variations is the genetic modifier that leads to increased TGF-ß activity. While anti-TGF-ß therapies are being developed to target muscle fibrosis, their effect on the myogenic deficit is underexplored. Our analysis of in vivo myogenesis in mild (C57BL/10ScSn-mdx/J and C57BL/6J-mdxΔ52) and severe DBA/2J-mdx (D2-mdx) dystrophic models reveals no defects in developmental myogenesis in these mice. However, muscle damage at the onset of disease pathology, or by experimental injury, drives up TGF-ß activity in the severe, but not in the mild, dystrophic models. Increased TGF-ß activity is accompanied by increased accumulation of fibroadipogenic progenitors (FAPs) leading to fibro-calcification of muscle, together with failure of regenerative myogenesis. Inhibition of TGF-ß signaling reduces muscle degeneration by blocking FAP accumulation without rescuing regenerative myogenesis. These findings provide in vivo evidence of early-stage deficit in regenerative myogenesis in D2-mdx mice and implicates TGF-ß as a major component of a pathogenic positive feedback loop in this model, identifying this feedback loop as a therapeutic target.