• Automated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish

      Walker, Steven L.; Ariga, Junko; Mathias, Jonathan R.; Coothankandaswamy, Veena; Xie, Xiayang; Distel, Martin; Koster, Reinhard W.; Parsons, Michael J.; Bhalla, Kapil N.; Saxena, Meera T.; et al. (2012-01-4)
      Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed,
    • A Cost-Effective Transparency-Based Digital Imaging for Efficient and Accurate Wound Area Measurement

      Li, Pei-Nan; Li, Hong; Wu, Mo-Li; Wang, Shou-Yu; Kong, Qing-You; Zhang, Zhen; Sun, Yuan; Liu, Jia; Lv, De-Cheng; McNeil, Paul L.; et al. (2012-05-30)
      Wound measurement is an objective and direct way to trace the course of wound healing and to evaluate therapeutic efficacy. Nevertheless, the accuracy and efficiency of the current measurement methods need to be improved. Taking the advantages of reliability of transparency tracing and the accuracy of computer-aided digital imaging, a transparency-based digital imaging approach is established, by which data from 340 wound tracing were collected from 6 experimental groups (8 rats/group) at 8 experimental time points (Day 1, 3, 5, 7, 10, 12, 14 and 16) and orderly archived onto a transparency model sheet. This sheet was scanned and its image was saved in JPG form. Since a set of standard area units from 1 mm2 to 1 cm2 was integrated into the sheet, the tracing areas in JPG image were measured directly, using the â Magnetic lasso toolâ in Adobe Photoshop program. The pixel values/PVs of individual outlined regions were obtained and recorded in an average speed of 27 second/region. All PV data were saved in an excel form and their corresponding areas were calculated simultaneously by the formula of Y (PV of the outlined region)/X (PV of standard area unit) Ã Z (area of standard unit). It took a researcher less than 3 hours to finish area calculation of 340 regions. In contrast, over 3 hours were expended by three skillful researchers to accomplish the above work with traditional transparency-based method. Moreover, unlike the results obtained traditionally, little variation was found among the data calculated by different persons and the standard area units in different sizes and shapes. Given its accurate, reproductive and efficient properties, this transparency-based digital imaging approach would be of significant values in basic wound healing research and clinical practice.
    • Ex Vivo Stretch Reveals Altered Mechanical Properties of Isolated Dystrophin-Deficient Hearts

      Barnabei, Matthew S.; Metzger, Joseph M.; Department of Cellular Biology and Anatomy (2012-03-9)
      Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV) compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from b-sarcoglycan-null and laminin-a2 mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.
    • Genetic Ablation of CD68 Results in Mice with Increased Bone and Dysfunctional Osteoclasts

      Ashley, Jason W.; Shi, Zhenqi; Zhao, Haibo; Li, Xingsheng; Kesterson, Robert A.; Feng, Xu; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2011-10-3)
      CD68 is a member of the lysosome associated membrane protein (LAMP) family that is restricted in its expression to cells of the monocyte/macrophage lineage. This lineage restriction includes osteoclasts, and, while previous studies of CD68 in macrophages and dendritic cells have proposed roles in lipid metabolism, phagocytosis, and antigen presentation, the expression and function of CD68 in osteoclasts have not been explored. In this study, we investigated the expression and localization of CD68 in macrophages and osteoclasts in response to the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). We found that M-CSF stimulates CD68 expression and RANKL alters the apparent molecular weight of CD68 as measured by Western immunoblotting. In addition, we explored the significance of CD68 expression in osteoclasts by generating mice that lack expression of CD68. These mice have increased trabecular bone, and in vitro assessment of CD68â /â osteoclasts revealed that, in the absence of CD68, osteoclasts demonstrate an accumulation of intracellular vesicle-like structures, and do not efficiently resorb bone. These findings demonstrate a role for CD68 in the function of osteoclasts, and future studies will determine the mechanistic nature of the defects seen in CD68â /â osteoclasts.
    • Human Platelet-Rich Plasma- and Extracellular Matrix-Derived Peptides Promote Impaired Cutaneous Wound Healing In Vivo

      Demidova-Rice, Tatiana N.; Wolf, Lindsey; Deckenback, Jeffry; Hamblin, Michael R.; Herman, Ira M.; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2012-02-23)
      Previous work in our laboratory has described several pro-angiogenic short peptides derived from endothelial extracellular matrices degraded by bacterial collagenase. Here we tested whether these peptides could stimulate wound healing in vivo. Our experiments demonstrated that a peptide created as combination of fragments of tenascin X and fibrillin 1 (comb1) applied into cranial dermal wounds created in mice treated with cyclophosphamide to impair wound healing, can improve the rate of wound closure. Furthermore, we identify and characterize a novel peptide (UN3) created and modified from two naturally-occurring peptides, which are present in human platelet-rich plasma. In vitro testing of UN3 demonstrates that it causes a 50% increase in endothelial proliferation, 250% increase in angiogenic response and a tripling of epithelial cell migration in response to injury.
    • Lack of Correlation between Outcomes of Membrane Repair Assay and Correction of Dystrophic Changes in Experimental Therapeutic Strategy in Dysferlinopathy

      Lostal, William; Bartoli, Marc; Roudaut, Carinne; Bourg, Nathalie; Krahn, Martin; Pryadkina, Marina; Borel, Perrine; Suel, Laurence; Roche, Joseph A.; Stockholm, Daniel; et al. (2012-05-29)
      Mutations in the dysferlin gene are the cause of Limb-girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. The dysferlin protein has been implicated in sarcolemmal resealing, leading to the idea that the pathophysiology of dysferlin deficiencies is due to a deficit in membrane repair. Here, we show using two different approaches that fullfiling membrane repair as asseyed by laser wounding assay is not sufficient for alleviating the dysferlin deficient pathology. First, we generated a transgenic mouse overexpressing myoferlin to test the hypothesis that myoferlin, which is homologous to dysferlin, can compensate for the absence of dysferlin. The myoferlin overexpressors show no skeletal muscle abnormalities, and crossing them with a dysferlin-deficient model rescues the membrane fusion defect present in dysferlin-deficient mice in vitro. However, myoferlin overexpression does not correct muscle histology in vivo. Second, we report that AAV-mediated transfer of a minidysferlin, previously shown to correct the membrane repair deficit in vitro, also fails to improve muscle histology. Furthermore, neither myoferlin nor the minidysferlin prevented myofiber degeneration following eccentric exercise. Our data suggest that the pathogenicity of dysferlin deficiency is not solely related to impairment in sarcolemmal repair and highlight the care needed in selecting assays to assess potential therapies for dysferlinopathies.
    • Palmitoleate Induces Hepatic Steatosis but Suppresses Liver Inflammatory Response in Mice

      Guo, Xin; Li, Honggui; Xu, Hang; Halim, Vera; Zhang, Weiyu; Wang, Huan; Ong, Kuok Teong; Woo, Shih-Lung; Walzem, Rosemary L.; Mashek, Douglas G.; et al. (2012-06-29)
      The interaction between fat deposition and inflammation during obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD). The present study examined the effects of palmitoleate, a monounsaturated fatty acid (16⠶1n7), on liver metabolic and inflammatory responses, and investigated the mechanisms by which palmitoleate increases hepatocyte fatty acid synthase (FAS) expression. Male wild-type C57BL/6J mice were supplemented with palmitoleate and subjected to the assays to analyze hepatic steatosis and liver inflammatory response. Additionally, mouse primary hepatocytes were treated with palmitoleate and used to analyze fat deposition, the inflammatory response, and sterol regulatory element-binding protein 1c (SREBP1c) activation. Compared with controls, palmitoleate supplementation increased the circulating levels of palmitoleate and improved systemic insulin sensitivity. Locally, hepatic fat deposition and SREBP1c and FAS expression were significantly increased in palmitoleate-supplemented mice. These pro-lipogenic events were accompanied by improvement of liver insulin signaling. In addition, palmitoleate supplementation reduced the numbers of macrophages/Kupffer cells in livers of the treated mice. Consistently, supplementation of palmitoleate decreased the phosphorylation of nuclear factor kappa B (NF-κB, p65) and the expression of proinflammatory cytokines. These results were recapitulated in primary mouse hepatocytes. In terms of regulating FAS expression, treatment of palmitoleate increased the transcription activity of SREBP1c and enhanced the binding of SREBP1c to FAS promoter. Palmitoleate also decreased the phosphorylation of NF-κB p65 and the expression of proinflammatory cytokines in cultured macrophages. Together, these results suggest that palmitoleate acts through dissociating liver inflammatory response from hepatic steatosis to play a unique role in NAFLD.
    • Rhabdomyosarcomas in Aging A/J Mice

      Sher, Roger B.; Cox, Gregory A.; Mills, Kevin D.; Sundberg, John P.; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2011-08-10)
      Rhabdomyosarcomas (RSCs) are skeletal muscle neoplasms found in humans and domestic mammals. The A/J inbred strain developed a high frequency (between 70â 80%) of adult pleomorphic type (APT) RSC at >20 months of age while BALB/cByJ also develop RSC but less frequently. These neoplasms invaded skeletal muscle surrounding either the axial or proximal appendicular skeleton and were characterized by pleomorphic cells with abundant eosinophilic cytoplasm, multiple nuclei, and cross striations. The diagnosis was confirmed by detection of alpha-sarcomeric actin and myogenin in the neoplastic cells using immunocytochemistry. The A/J strain, but not the related BALB/c substrains, is also characterised by a progressive muscular dystrophy homologous to limb-girdle muscular dystrophy type 2B. The association between the development of RSC in similar muscle groups to those most severely affected by the progressive muscular dystrophy suggested that these neoplasms developed from abnormal regeneration of the skeletal muscle exacerbated by the dysferlin mutation. Transcriptome analyses of RSCs revealed marked downregulation of genes in muscular development and function signaling networks. Non-synonymous coding SNPs were found in Myl1, Abra, Sgca, Ttn, and Kcnj12 suggesting these may be important in the pathogenesis of RSC. These studies suggest that A strains of mice can be useful models for dissecting the molecular genetic basis for development, progression, and ultimately for testing novel anticancer therapeutic agents dealing with rhabdomyosarcoma.