Serum protein and imaging biomarkers after intermittent steroid treatment in muscular dystrophy.

Background: Weekly Steroids in Muscular Dystrophy (WSiMD) was a pilot study to evaluate once weekly prednisone in patients with Limb Girdle and Becker muscular dystrophy (LGMD and BMD, respectively). At study endpoint, there were trends towards increased lean mass, reduced fat mass, reduced creatine kinase and improved motor function. The investigation was motivated by studies in mouse muscular dystrophy models in which once weekly glucocorticoid exposure enhanced muscle strength and reduced fibrosis. Methods: WSiMD participants provided blood samples for aptamer serum profiling at baseline and after 6 months of weekly steroids. A subset completed magnetic resonance (MR) evaluation of muscle at study onset and endpoint. Results/Conclusions: At baseline compared to age and sex-matched healthy controls, the aggregate serum protein profile in the WSiMD cohort was dominated by muscle proteins, reflecting leak of muscle proteins into serum. Disease status produced more proteins differentially present in serum compared to steroid-treatment effect. Nonetheless, a response to prednisone was discernable in the WSiMD cohort, even at this low dose. Glucocorticoids downregulated muscle proteins and upregulated certain immune process- and matrix-associated proteins. Muscle MR fat fraction showed trends with functional status. The prednisone-responsive markers could be used in larger trial of prednisone efficacy.

The super-healing MRL strain promotes muscle growth in muscular dystrophy through a regenerative extracellular matrix.

The Murphy Roths Large (MRL) mouse strain has "super-healing" properties that enhance recovery from injury. In mice, the DBA/2J strain intensifies many aspects of muscular dystrophy, so we evaluated the ability of the MRL strain to suppress muscular dystrophy in the Sgcg-null mouse model of limb girdle muscular dystrophy. A comparative analysis of Sgcg-null mice in the DBA/2J versus MRL strains showed greater myofiber regeneration, with reduced structural degradation of muscle in the MRL strain. Transcriptomic profiling of dystrophic muscle indicated strain-dependent expression of extracellular matrix (ECM) and TGF-ß signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized myoscaffolds. Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-ß1 and TGF-ß3 throughout the matrix. Dystrophic myoscaffolds from the MRL background, but not the DBA/2J background, were enriched in myokines like IGF-1 and IL-6. C2C12 myoblasts seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J muscles showed the MRL background induced greater myoblast differentiation compared with dystrophic DBA/2J myoscaffolds. Thus, the MRL background imparts its effect through a highly regenerative ECM, which is active even in muscular dystrophy.

Physiological stress improves stem cell modeling of dystrophic cardiomyopathy.

Heart failure contributes to Duchenne muscular dystrophy (DMD), which arises from mutations that ablate dystrophin, rendering the plasma membrane prone to disruption. Cardiomyocyte membrane breakdown in patients with DMD yields a serum injury profile similar to other types of myocardial injury with the release of creatine kinase and troponin isoforms. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are highly useful but can be improved. We generated hiPSC-CMs from a patient with DMD and subjected these cells to equibiaxial mechanical strain to mimic in vivo stress. Compared to healthy cells, DMD hiPSC-CMs demonstrated greater susceptibility to equibiaxial strain after 2 h at 10% strain. We generated an aptamer-based profile of proteins released from hiPSC-CMs both at rest and subjected to strain and identified a strong correlation in the mechanical stress-induced proteome from hiPSC-CMs and serum from patients with DMD. We exposed hiPSC-CMs to recombinant annexin A6, a protein resealing agent, and found reduced biomarker release in DMD and control hiPSC-CMs subjected to strain. Thus, the application of mechanical strain to hiPSC-CMs produces a model that reflects an in vivo injury profile, providing a platform to assess pharmacologic intervention.

The super-healing MRL strain promotes muscle growth in muscular dystrophy through a regenerative extracellular matrix.

Genetic background shifts the severity of muscular dystrophy. In mice, the DBA/2J strain confers a more severe muscular dystrophy phenotype, whereas the Murphy's Roth Large (MRL) strain has "super-healing" properties that reduce fibrosis. A comparative analysis of the Sgcg null model of Limb Girdle Muscular Dystrophy in the DBA/2J versus MRL strain showed the MRL background was associated with greater myofiber regeneration and reduced structural degradation of muscle. Transcriptomic profiling of dystrophic muscle in the DBA/2J and MRL strains indicated strain-dependent expression of the extracellular matrix (ECM) and TGF-ß signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized "myoscaffolds". Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-ß1 and TGF-ß3 throughout the matrix, and dystrophic myoscaffolds from the MRL background were enriched in myokines. C2C12 myoblasts were seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J matrices. Acellular myoscaffolds from the dystrophic MRL background induced myoblast differentiation and growth compared to dystrophic myoscaffolds from the DBA/2J matrices. These studies establish that the MRL background also generates its effect through a highly regenerative ECM, which is active even in muscular dystrophy.

Nucleoli and the nucleoli-centromere association are dynamic during normal development and in cancer.

Centromeres are known to cluster around nucleoli in Drosophila and mammalian cells, but the significance of the nucleoli-centromere interaction remains underexplored. To determine whether the interaction is dynamic under different physiological and pathological conditions, we examined nucleolar structure and centromeres at various differentiation stages using cell culture models and the results showed dynamic changes in nucleolar characteristics and nucleoli-centromere interactions through differentiation and in cancer cells. Embryonic stem cells usually have a single large nucleolus, which is clustered with a high percentage of centromeres. As cells differentiate into intermediate states, the nucleolar number increases and the centromere association decreases. In terminally differentiated cells, including myotubes, neurons, and keratinocytes, the number of nucleoli and their association with centromeres are at the lowest. Cancer cells demonstrate the pattern of nucleoli number and nucleoli-centromere association that is akin to proliferative cell types, suggesting that nucleolar reorganization and changes in nucleoli-centromere interactions may play a role in facilitating malignant transformation. This idea is supported in a case of pediatric rhabdomyosarcoma, in which induced differentiation reduces the nucleolar number and centromere association. These findings suggest active roles of nucleolar structure in centromere function and genome organization critical for cellular function in both normal development and cancer.

Dynamin-2 deficiency causes age- and sex-dependent neutropenia and myelodysplasia in mice.

The dynamins are a family of ubiquitously expressed GTPase proteins, best known for their role in membrane remodeling. Their contribution to hematopoiesis is incompletely recognized. Individuals with Charcot-Marie-Tooth disease with dynamin-2 (DNM2) mutations often develop neutropenia. We previously reported that dynamin (DNM) inhibition impairs SDF1a-mediated migration in megakaryocytes. Here, we report on conditionally Dnm2 deleted mice in hematopoietic tissues using the Vav-Cre murine strain. Homozygous Dnm2 deletion in blood tissues is embryonic lethal. Dnm2het male mice only developed a slightly decreased hemoglobin level. Dnm2het female mice developed leukopenia by 40 weeks of age and neutropenia by 65 weeks of age. Flow cytometry revealed decreased lineage-negative cells and granulocyte-monocyte progenitors in Dnm2het female mice. Immunohistochemical staining of bone marrow (BM) for mature neutrophils with Ly6G was decreased and myelodysplastic features were present in the BM of Dnm2het female mice. A linear distribution of Ly6G+ BM cells along blood vessels was observed in fewer Dnm2het mice than in controls, suggesting that the migration pattern in the marrow is altered. Marrow neutrophils treated with dynamin inhibitor, dynasore, showed increased cell surface CXCR4, suggesting that abnormal migration results in marrow neutrophil retention. Dnm2het female mice also developed splenomegaly secondary to germinal center hyperplasia at younger ages, suggesting perturbed immunity. In summary, female mice with BM Dnm2 haploinsufficiency developed neutropenia as they aged with decreased granulocyte progenitor production and migration defects. Our studies indicate a potential mechanism for the development of chronic idiopathic neutropenia, a disease that predominantly presents in middle-aged women.

Parallel Multichannel Assessment of Rotationally Manipulated Magnetic Nanoparticles.

Background: Rotational manipulation of chains or clusters of magnetic nanoparticles (MNPs) offers a means for directed translation and payload delivery that should be explored for clinical use. Multiple MNP types are available, yet few studies have performed side-by-side comparisons to evaluate characteristics such as velocity, movement at a distance, and capacity for drug conveyance or dispersion. Purpose: Our goal was to design, build, and study an electric device allowing simultaneous, multichannel testing (e.g., racing) of MNPs in response to a rotating magnetic field. We would then select the "best" MNP and use it with optimized device settings, to transport an unbound therapeutic agent. Methods: A magnetomotive system was constructed, with a Helmholtz pair of coils on either side of a single perpendicular coil, on top of which was placed an acrylic tray having multiple parallel lanes. Five different MNPs were tested: graphene-coated cobalt MNPs (TurboBeads™), nickel nanorods, gold-iron alloy MNPs, gold-coated Fe3O4 MNPs, and uncoated Fe3O4 MNPs. Velocities were determined in response to varying magnetic field frequencies (5-200 Hz) and heights (0-18 cm). Velocities were normalized to account for minor lane differences. Doxorubicin was chosen as the therapeutic agent, assayed using a CLARIOstar Plus microplate reader. Results: The MMS generated a maximal MNP velocity of 0.9 cm/s. All MNPs encountered a "critical" frequency at 20-30 Hz. Nickel nanorods had the optimal response based on tray height and were then shown to enable unbound doxorubicin dispersion along 10.5 cm in <30 sec. Conclusion: A rotating magnetic field can be conveniently generated using a three-coil electromagnetic device, and used to induce rotational and translational movement of MNP aggregates over mesoscale distances. The responses of various MNPs can be compared side-by-side using multichannel acrylic trays to assess suitability for drug delivery, highlighting their potential for further in vivo applications.

UBR7 acts as a histone chaperone for post-nucleosomal histone H3.

Histone chaperones modulate the stability of histones beginning from histone synthesis, through incorporation into DNA, and during recycling during transcription and replication. Following histone removal from DNA, chaperones regulate histone storage and degradation. Here, we demonstrate that UBR7 is a histone H3.1 chaperone that modulates the supply of pre-existing post-nucleosomal histone complexes. We demonstrate that UBR7 binds to post-nucleosomal H3K4me3 and H3K9me3 histones via its UBR box and PHD. UBR7 binds to the non-nucleosomal histone chaperone NASP. In the absence of UBR7, the pool of NASP-bound post-nucleosomal histones accumulate and chromatin is depleted of H3K4me3-modified histones. We propose that the interaction of UBR7 with NASP and histones opposes the histone storage functions of NASP and that UBR7 promotes reincorporation of post-nucleosomal H3 complexes.

Targeted sonodynamic destruction of glioblastoma cells using antibody-titanium dioxide nanoparticle conjugates.

Aim: We present data on sonodynamic therapy (SDT) against glioblastoma cells utilizing titanium dioxide (TiO2) nanoparticles conjugated to anti-EGFR antibody. Materials & methods: TiO2 nanoparticles were bound to anti-EGFR antibody to form antibody-nanoparticle conjugates (ANCs), then characterized by x-ray photoelectron spectroscopy and transmission electron microscopy. Cells underwent ultrasound and assessment on viability, reactive oxygen species and apoptosis were performed. Results: X-ray photoelectron spectroscopy analysis revealed the formation of an ANC. Transmission electron microscopy showed internalization of the ANCs by glioblastoma cells. With SDT, cell viabilities were reduced in the presence of ANCs, reactive oxygen species production was formed, but minimal effect on apoptosis was seen. Conclusion: For the first time, an ANC can be used with SDT to kill glioblastoma cells.

Etoposide-Bound Magnetic Nanoparticles Designed for Remote Targeting of Cancer Cells Disseminated Within Cerebrospinal Fluid Pathways.

Magnetic nanoparticles (MNPs) have potential for enhancing drug delivery in selected cancer patients, including those which have cells that have disseminated within cerebrospinal fluid (CSF) pathways. Here, we present data related to the creation and in vitro use of new two-part MNPs consisting of magnetic gold-iron alloy cores which have streptavidin binding sites, and are coated with biotinylated etoposide. Etoposide was chosen due to its previous use in the CSF and ease of biotinylation. Etoposide magnetic nanoparticles ("Etop-MNPs") were characterized by several different methods, and moved at a distance by surface-walking of MNP clusters, which occurs in response to a rotating permanent magnet. Human cell lines including D283 (medulloblastoma), U138 (glioblastoma), and H2122 (lung adenocarcinoma) were treated with direct application of Etop-MNPs (and control particles), and after remote particle movement. Cell viability was determined by MTT assay and trypan blue exclusion. Results indicated that the biotinylated etoposide was successfully bound to the base MNPs, with the hybrid particle attaining a maximum velocity of 0.13 ± 0.018 cm/sec. Etop-MNPs killed cancer cells in a dose-dependent fashion, with 50 ± 6.8% cell killing of D283 cells (for example) with 24 h of treatment after remote targeting. U138 and H2122 cells were found to be even more susceptible to the killing effect of Etop-MNPs than D283 cells. These findings indicate that the novel Etop-MNPs have a cytotoxic effect, and can be moved relatively rapidly at physiologic distances, using a rotating magnet. While further testing is needed, intrathecal administration of Etop-MNPs holds promise for magnetically-enhanced eradication of cancer cells distributed within CSF pathways, particularly if given early in the course of the disease.

Rotating Magnetic Nanoparticle Clusters as Microdevices for Drug Delivery.

BACKGROUND: Magnetic nanoparticles (MNPs) hold promise for enhancing delivery of therapeutic agents, either through direct binding or by functioning as miniature propellers. Fluid-filled conduits and reservoirs within the body offer avenues for MNP-enhanced drug delivery. MNP clusters can be rotated and moved across surfaces at clinically relevant distances in response to a rotating magnet. Limited data are available regarding issues affecting MNP delivery by this mechanism, such as adhesion to a cellular wall. Research reported here was initiated to better understand the fundamental principles important for successful implementation of rotational magnetic drug targeting (rMDT). METHODS: Translational movements of four different iron oxide MNPs were tested, in response to rotation (3 Hz) of a neodymium-boron-iron permanent magnet. MNP clusters moved along biomimetic channels of a custom-made acrylic tray, by surface walking. The effects of different distances and cellular coatings on MNP velocity were analyzed using videography. Dyes (as drug surrogates) and the drug etoposide were transported by rotating MNPs along channels over a 10 cm distance. RESULTS: MNP translational velocities could be predicted from magnetic separation times. Changes in distance or orientation from the magnet produced alterations in MNP velocities. Mean velocities of the fastest MNPs over HeLa, U251, U87, and E297 cells were 0.24 ± 0.02, 0.26 ± 0.02, 0.28 ± 0.01, and 0.18 ± 0.03 cm/sec, respectively. U138 cells showed marked MNP adherence and an 87.1% velocity reduction at 5.5 cm along the channel. Dye delivery helped visualize the effects of MNPs as microdevices for drug delivery. Dye delivery by MNP clusters was 21.7 times faster than by diffusion. MNPs successfully accelerated etoposide delivery, with retention of chemotherapeutic effect. CONCLUSION: The in vitro system described here facilitates side-by-side comparisons of drug delivery by rotating MNP clusters, on a human scale. Such microdevices have the potential for augmenting drug delivery in a variety of clinical settings, as proposed.

An in vitro Model System for Evaluating Remote Magnetic Nanoparticle Movement and Fibrinolysis.

BACKGROUND: Thrombotic events continue to be a major cause of morbidity and mortality worldwide. Tissue plasminogen activator (tPA) is used for the treatment of acute ischemic stroke and other thrombotic disorders. Use of tPA is limited by its narrow therapeutic time window, hemorrhagic complications, and insufficient delivery to the location of the thrombus. Magnetic nanoparticles (MNPs) have been proposed for targeting tPA delivery. It would be advantageous to develop an improved in vitro model of clot formation, to screen thrombolytic therapies that could be enhanced by addition of MNPs, and to test magnetic drug targeting at human-sized distances. METHODS: We utilized commercially available blood and endothelial cells to construct 1/8th inch (and larger) biomimetic vascular channels in acrylic trays. MNP clusters were moved at a distance by a rotating permanent magnet and moved along the channels by surface walking. The effect of different transport media on MNP velocity was studied using video photography. MNPs with and without tPA were analyzed to determine their velocities in the channels, and their fibrinolytic effect in wells and the trays. RESULTS: MNP clusters could be moved through fluids including blood, at human-sized distances, down straight or branched channels, using the rotating permanent magnet. The greatest MNP velocity was closest to the magnet: 0.76 ± 0.03 cm/sec. In serum, the average MNP velocity was 0.10 ± 0.02 cm/sec. MNPs were found to enhance tPA delivery, and cause fibrinolysis in both static and dynamic studies. Fibrinolysis was observed to occur in 85% of the dynamic MNP + tPA experiments. CONCLUSION: MNPs hold great promise for use in augmenting delivery of tPA for the treatment of stroke and other thrombotic conditions. This model system facilitates side by side comparisons of MNP-facilitated drug delivery, at a human scale.

Cell Biology: Hacking Alpha Satellites out of the HAC.

Human artificial chromosomes (HACs) are a potentially powerful technique for genomic engineering, but their use is limited by the repetitive centromeric alpha-satellite DNA needed to form a centromere. A new study presents a method to induce HAC centromere formation on non-repetitive templates through sequence-directed CENP-A nucleosome seeding.

Intraoperative external iliac venogram and intravenous ultrasound to assist retrieval of intrapelvic acetabular screw.

Intrapelvic acetabular screw placement is a known complication of total hip arthroplasty and is associated with risks including damage to neurovascular and intrapelvic structures such as the external iliac vessels, obturator vessels, and iliopsoas muscles. Retrieval of intrapelvic acetabular screws is similarly fraught with risk, and appropriate imaging should be performed preoperatively. We report on a case of intrapelvic acetabular screw placement with abutment of the external iliac vein, which was managed with intraoperative venogram and intravenous ultrasound to maintain access to the external iliac vein and successfully show no vascular tear before and after screw removal.

Complications following allograft reconstruction for primary bone tumors: Considerations for management.

INTRODUCTION: The aim of this study was to investigate complication rates and types following allograft reconstruction and discuss unique considerations for management. METHODS: Seventy-four consecutive patients underwent large segment allograft reconstruction following resection of primary musculoskeletal tumors from 1991 to 2016. Mean patient age was 32 ±â€¯20 years (range, 5-71 years). Minimum follow-up was 2 years unless patients were lost to disease prior. Mean follow-up was 105 months. RESULTS: Thirty-five patients had complications requiring subsequent surgery at a mean of 30 months (range, 1-146 months) post-operatively. Individual complication rates were 29%, 50%, and 42% for Allograft Prosthetic Composite, Intercalary, and Osteoarticular allograft reconstruction, respectively. Risk factors for complication included age less than 30 (OR 4.5; p = 0.002), male gender (OR 2.8; p = 0.031), chemotherapy (OR 4.4; p = 0.003), lower extremity disease (OR 3.4; p = 0.025). In patients with complications, limb-retention rate was 91% and mean MSTS scores were 23.6. CONCLUSION: Despite considerable complication rates, management with a systematic approach results in successful outcomes with limb-retention greater than 90% and mean MSTS scores of 79%. In carefully selected patients, allografts provide a reliable method of reconstruction with treatable complications occurring at a mean of 30 months.

Customizable orthopaedic oncology implants: one institution's experience with meeting current IRB and FDA requirements.

BACKGROUND: Customizable orthopaedic implants are often needed for patients with primary malignant bone tumors due to unique anatomy or complex mechanical problems. Currently, obtaining customizable orthopaedic implants for orthopaedic oncology patients can be an arduous task involving submitting approval requests to the Institutional Review Board (IRB) and the Food and Drug Administration (FDA). There is great potential for the delay of a patient's surgery and unnecessary paperwork if the submission pathways are misunderstood or a streamlined protocol is not in place. PURPOSE: The objective of this study was to review the existing FDA custom implant approval pathways and to determine whether this process was improved with an institutional protocol. METHODS: An institutional protocol for obtaining IRB and FDA approval for customizable orthopaedic implants was established with the IRB at our institution in 2013. This protocol was approved by the IRB, such that new patients only require submission of a modification to the existing protocol with individualized patient information. During the two-year period of 2013-2014, eight patients were retrospectively identified as having required customizable implants for various orthopaedic oncology surgeries. The dates of request for IRB approval, request for FDA approval, and total time to surgery were recorded, along with the specific pathway utilized for FDA approval. RESULTS: The average patient age was 12 years old (7-21 years old). The average time to IRB approval of a modification to the pre-approved protocol was 14 days (7-21 days). Average time to FDA approval after submission of the IRB approval to the manufacturer was 12.5 days (7-19 days). FDA approval was obtained for all implants as compassionate use requests in accordance with Section 561 of the Federal Food Drug and Cosmetic Act's expanded access provisions. CONCLUSIONS: Establishment of an institutional protocol with pre-approval by the IRB can expedite the otherwise time-consuming and complicated process of obtaining customizable orthopaedic implants for orthopaedic oncology patients. LEVEL OF EVIDENCE: Retrospective case series, Level IV. See the Guidelines for authors for a complete description of levels of evidence.

Heterotopic Ossification and Entrapment of the Tibial Nerve Within the Tarsal Tunnel: A Case Report.

Heterotopic ossification has been reported to occur after musculoskeletal trauma (including orthopedic procedures). This has been known to cause nerve entrapment syndromes and persistent pain, limiting joint mobility. We present a case of a 19-year old female collegiate athlete who had previously undergone ankle arthroscopy and arthrotomy to remove 2 ossicles. At approximately 1 year postoperatively, the patient developed pain when planting and pivoting her foot. Imaging revealed a radiodense lesion at the posteromedial ankle consistent with heterotopic ossification and entrapment of the tibial nerve within the tarsal tunnel. The patient underwent surgical resection and postoperative indomethacin prophylaxis. At the 1-year follow-up visit, the patient remained asymptomatic, without evidence of recurrence of the heterotopic ossification. In our review of the published data, we found no previously reported cases of heterotopic ossification causing entrapment of the tibial nerve within the tarsal tunnel. In the present case report, we describe this rare case and the postulated etiologies and pathophysiology of this disease process. In addition, we discuss the clinical signs and symptoms and recommended imaging modalities and treatment.

Synthesis of well-defined bicapped octahedral iron clusters [((tren) L)2 Fe8 (PMe2 Ph)2 ](n) (n=0, -1).

The synthesis of polynuclear clusters with control over size and cluster geometry remains an unsolved challenge. Herein, we report the synthesis and characterization of open-shell octairon clusters supported by two heptaamine ligands [o-H2 NC6 H4 NH(CH2 )2 ]3 N ((tren) LH9 ). The crystal structure of the all-ferrous species ([(tren) L)2 Fe8 (PMe2 Ph)2 ] (1) displays a bicapped octahedral geometry with FeFe distances ranging from 2.4071(6) to 2.8236(5) Å, where the ligand amine units are formally in amine, amide, and imide oxidation states. Several redox states of the octairon cluster are accessible, as ascertained using cyclic voltammetry. The one-electron-reduced clusters [M](+) [((tren) L)2 Fe8 (PMe2 Ph)2 ](-) (M=Bu4 N (2 a); (15-crown-5)Na(thf) (2 b)) were isolated and characterized. Variable-temperature magnetic susceptibility data indicates that the exchange coupling within the [Fe8 ] core is antiferromagnetic which is attenuated upon reduction to the mixed valent anion.