Kelly D. Garcia

Ethiodized oil uptake does not predict doxorubicin drug delivery after chemoembolization in VX2 liver tumors.

PURPOSE To investigate the accuracy of ethiodized oil as an imaging marker of chemotherapy drug delivery after liver tumor chemoembolization in an animal model of hepatocellular carcinoma. MATERIALS AND METHODS Eleven VX2 liver tumors (mean diameter, 1.9 cm ± 0.4) in six New Zealand White rabbits were treated with chemoembolization using ethiodized oil and doxorubicin emulsion, followed by immediate euthanasia. Postprocedure noncontrast computed tomography (CT) was used to evaluate intratumoral ethiodized oil distribution and calculate iodine content within four peripheral tumor quadrants and the tumor core at a central tumor slice (N = 55 total tumor sections). Liquid chromatography/tandem mass spectrometry (LC-MS/MS) was then used to directly measure doxorubicin concentration in the same tissue sections. Statistical correlation was performed between tissue iodine content and doxorubicin concentration by using linear regression. RESULTS Chemoembolization was successfully performed in all tumors via the left or proper hepatic artery. A mean of 0.9 mL ± 0.6 ethiodized oil and 1.8 mg ± 1.2 doxorubicin were injected. CT-calculated tissue iodine content averaged 335 mg/mL ± 218. Corresponding LC-MS/MS analysis yielded a mean doxorubicin concentration of 15.8 μg/mL ± 14.3 in each sample. Although iodine content (391 mg/mL vs 112 mg/mL; P = .000) and doxorubicin concentration (18.0 μg/mL vs 7.2 μg/mL; P = .023) were significantly greater along peripheral tumor sections compared with the tumor core, no significant predictable correlation was evident between these measures (R(2) = 0.0099). CONCLUSIONS Tissue ethiodized oil content is a poor quantitative predictor of local doxorubicin concentration after liver tumor chemoembolization. Future studies should aim to identify a better imaging marker for chemoembolization drug delivery.

The calcium channel α2/δ1 subunit is involved in extracellular signalling

The α2/δ1 subunit forms part of the dihydropyridine receptor, an essential protein complex for excitation–contraction (EC) coupling in skeletal muscle. Because of the lack of a viable knock‐out animal, little is known regarding the role of the α2/δ1 subunit in EC coupling or in other cell functions. Interestingly, the α2/δ1 appears before the α1 subunit in development and contains extracellular conserved domains known to be important in cell signalling and inter‐protein interactions. These facts raise the possibility that the α2/δ1 subunit performs vital functions not associated with EC coupling. Here, we tested the hypothesis that the α2/δ1 subunit is important for interactions of muscle cells with their environment. Using confocal microscopy, we followed the immunolocalization of α2/δ1 and α1 subunits with age. We found that in 2‐day‐old myotubes, the α2/δ1 subunit concentrated towards the ends of the cells, while the α1 subunit clustered near the centre. As myotubes aged (6–12 days), the α2/δ1 became evenly distributed along the myotubes and co‐localized with α1. When the expression of α2/δ1 was blocked with siRNA, migration, attachment and spreading of myoblasts were impaired while the L‐type calcium current remained unaffected. The results suggest a previously unidentified role of the α2/δ1 subunit in skeletal muscle and support the involvement of this protein in extracellular signalling. This new role of the α2/δ1 subunit may be crucial for muscle development, muscle repair and at times in which myoblast attachment and migration are fundamental.

Mechanism of Neuromuscular Dysfunction in Krabbe Disease

The atrophy of skeletal muscles in patients with Krabbe disease is a major debilitating manifestation that worsens their quality of life and limits the clinical efficacy of current therapies. The pathogenic mechanism triggering muscle wasting is unknown. This study examined structural, functional, and metabolic changes conducive to muscle degeneration in Krabbe disease using the murine (twitcher mouse) and canine [globoid cell leukodystrophy (GLD) dog] models. Muscle degeneration, denervation, neuromuscular [neuromuscular junction (NMJ)] abnormalities, and axonal death were investigated using the reporter transgenic twitcher–Thy1.1–yellow fluorescent protein mouse. We found that mutant muscles had significant numbers of smaller-sized muscle fibers, without signs of regeneration. Muscle growth was slow and weak in twitcher mice, with decreased maximum force. The NMJ had significant levels of activated caspase-3 but limited denervation. Mutant NMJ showed reduced surface areas and lower volumes of presynaptic terminals, with depressed nerve control, increased miniature endplate potential (MEPP) amplitude, decreased MEPP frequency, and increased rise and decay rate constants. Twitcher and GLD dog muscles had significant capacity to store psychosine, the neurotoxin that accumulates in Krabbe disease. Mechanistically, muscle defects involved the inactivation of the Akt pathway and activation of the proteasome pathway. Our work indicates that muscular dysfunction in Krabbe disease is compounded by a pathogenic mechanism involving at least the failure of NMJ function, activation of proteosome degradation, and a reduction of the Akt pathway. Akt, which is key for muscle function, may constitute a novel target to complement in therapies for Krabbe disease.

The calcium channel alpha2/delta1 subunit is involved in extracellular signalling.

The α2/δ1 subunit forms part of the dihydropyridine receptor, an essential protein complex for excitation–contraction (EC) coupling in skeletal muscle. Because of the lack of a viable knock‐out animal, little is known regarding the role of the α2/δ1 subunit in EC coupling or in other cell functions. Interestingly, the α2/δ1 appears before the α1 subunit in development and contains extracellular conserved domains known to be important in cell signalling and inter‐protein interactions. These facts raise the possibility that the α2/δ1 subunit performs vital functions not associated with EC coupling. Here, we tested the hypothesis that the α2/δ1 subunit is important for interactions of muscle cells with their environment. Using confocal microscopy, we followed the immunolocalization of α2/δ1 and α1 subunits with age. We found that in 2‐day‐old myotubes, the α2/δ1 subunit concentrated towards the ends of the cells, while the α1 subunit clustered near the centre. As myotubes aged (6–12 days), the α2/δ1 became evenly distributed along the myotubes and co‐localized with α1. When the expression of α2/δ1 was blocked with siRNA, migration, attachment and spreading of myoblasts were impaired while the L‐type calcium current remained unaffected. The results suggest a previously unidentified role of the α2/δ1 subunit in skeletal muscle and support the involvement of this protein in extracellular signalling. This new role of the α2/δ1 subunit may be crucial for muscle development, muscle repair and at times in which myoblast attachment and migration are fundamental.

The Oncopig Cancer Model: An Innovative Large Animal Translational Oncology Platform

Despite an improved understanding of cancer molecular biology, immune landscapes, and advancements in cytotoxic, biologic, and immunologic anti-cancer therapeutics, cancer remains a leading cause of death worldwide. More than 8.2 million deaths were attributed to cancer in 2012, and it is anticipated that cancer incidence will continue to rise, with 19.3 million cases expected by 2025. The development and investigation of new diagnostic modalities and innovative therapeutic tools is critical for reducing the global cancer burden. Toward this end, transitional animal models serve a crucial role in bridging the gap between fundamental diagnostic and therapeutic discoveries and human clinical trials. Such animal models offer insights into all aspects of the basic science-clinical translational cancer research continuum (screening, detection, oncogenesis, tumor biology, immunogenicity, therapeutics, and outcomes). To date, however, cancer research progress has been markedly hampered by lack of a genotypically, anatomically, and physiologically relevant large animal model. Without progressive cancer models, discoveries are hindered and cures are improbable. Herein, we describe a transgenic porcine model—the Oncopig Cancer Model (OCM)—as a next-generation large animal platform for the study of hematologic and solid tumor oncology. With mutations in key tumor suppressor and oncogenes, TP53R167H and KRASG12D, the OCM recapitulates transcriptional hallmarks of human disease while also exhibiting clinically relevant histologic and genotypic tumor phenotypes. Moreover, as obesity rates increase across the global population, cancer patients commonly present clinically with multiple comorbid conditions. Due to the effects of these comorbidities on patient management, therapeutic strategies, and clinical outcomes, an ideal animal model should develop cancer on the background of representative comorbid conditions (tumor macro- and microenvironments). As observed in clinical practice, liver cirrhosis frequently precedes development of primary liver cancer or hepatocellular carcinoma. The OCM has the capacity to develop tumors in combination with such relevant comorbidities. Furthermore, studies on the tumor microenvironment demonstrate similarities between OCM and human cancer genomic landscapes. This review highlights the potential of this and other large animal platforms as transitional models to bridge the gap between basic research and clinical practice.

Functional in situ Assessment of Muscle Contraction in Wild Type and mdx Mice

Introduction: Reports of muscle testing are frequently limited to maximal force alone. The experiments reported here show that force generation and relaxation rates can be obtained from the same experiments and provide a more complete functional characterization. Methods: Partial in situ testing was performed on the tibialis anterior of young wild‐type (WT) mice, young mdx mice, and old mdx mice. Force, force generation rate, and relaxation rates were measured during a fatigue test, 2 frequency–force tests, and a passive tension test. Results: We measured increased force but decreased force generation rate in WT compared with mdx muscles, and increased force but decreased relaxation rate of old compared with young mdx muscles. Young mdx muscles were the most sensitive to increases in passive tension. Conclusions: These measurements offer an improved understanding of muscle capability and are readily acquired by further analysis of the same tests used to obtain force measurements. Muscle Nerve 53: 260–268, 2016

The calcium channel α2/δ1 subunit is involved in extracellular signalling: α2/δ1 subunit and cell adhesion

The α2/δ1 subunit forms part of the dihydropyridine receptor, an essential protein complex for excitation–contraction (EC) coupling in skeletal muscle. Because of the lack of a viable knock‐out animal, little is known regarding the role of the α2/δ1 subunit in EC coupling or in other cell functions. Interestingly, the α2/δ1 appears before the α1 subunit in development and contains extracellular conserved domains known to be important in cell signalling and inter‐protein interactions. These facts raise the possibility that the α2/δ1 subunit performs vital functions not associated with EC coupling. Here, we tested the hypothesis that the α2/δ1 subunit is important for interactions of muscle cells with their environment. Using confocal microscopy, we followed the immunolocalization of α2/δ1 and α1 subunits with age. We found that in 2‐day‐old myotubes, the α2/δ1 subunit concentrated towards the ends of the cells, while the α1 subunit clustered near the centre. As myotubes aged (6–12 days), the α2/δ1 became evenly distributed along the myotubes and co‐localized with α1. When the expression of α2/δ1 was blocked with siRNA, migration, attachment and spreading of myoblasts were impaired while the L‐type calcium current remained unaffected. The results suggest a previously unidentified role of the α2/δ1 subunit in skeletal muscle and support the involvement of this protein in extracellular signalling. This new role of the α2/δ1 subunit may be crucial for muscle development, muscle repair and at times in which myoblast attachment and migration are fundamental.