Background: MG53 is a membrane repair gene whose role in wound healing has not been studied. Results: Topical administration of MG53 protein facilitates wound healing and reduces scar formation. Conclusion: This study establishes MG53 as facilitator of injury repair and inhibitor of myofibroblast differentiation during wound healing. Significance: MG53 has therapeutic benefits in treating wounds and fibrotic diseases.

Store-operated Ca2+ entry (SOCE) has recently been shown to be of physiological and pathological importance in the heart, particularly during cardiac hypertrophy. However, measuring changes in intracellular Ca2+ during SOCE is very difficult to study in adult primary cardiomyocytes. As a result there is a need for a stable and reliable in vitro model of SOCE which can be used to test cardiac drugs and investigate the role of SOCE in cardiac pathology. HL-1 cells are the only immortal cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining phenotypic characteristics of the adult cardiomyocyte. To date the role of SOCE has not yet been investigated in the HL-1 cardiac cell line. We report for the first time that these cells express stromal interaction molecule 1 (STIM1) and the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, which are essential components of the SOCE machinery. In addition, SOCE is tightly coupled to sarcoplasmic reticulum (SR)-Ca2+ release in HL-1 cells, and such response was not impaired in the presence of voltage dependent Ca2+ channels (L-type and T-type channels) or reverse mode Na+/ Ca2+ exchanger (NCX) inhibitors. We were able to abolish the SOCE response with known SOCE inhibitors (BTP-2 and SKF-96365) and by targeted knockdown of Orai1 with RNAi. In addition, knockdown of Orai1 resulted in lower baseline Ca2+ and an attenuated response to thapsigargin (TG) and caffeine, indicating that SOCE may play a role in Ca2+ homeostasis during unstressed conditions in cardiomyocytes. Currently, there is little knowledge about SOCE in cardiomyocytes, and the present results suggest that HL-1 cells will be of great utility in investigating the role of SOCE in the heart.

Background/Aims: Ischemic stroke is a leading cause of long-term disability. To date, there is no effective treatment for stroke. Previous studies have shown that Ginkgo biloba extract has protective effects against neurodegenerative disorders. In this present study, we sought to test the potential protective role of an active component of Ginkgo biloba extract, bilobalide, in a rat model of middle cerebral artery occlusion (MCAO). Methods: A rat model of MCAO was used to test the potential protective effects of Bilobalide B on stroke protection. TTC staining was performed to evaluate infarct size of the brains. Neurological deficit score was measured to reveal the effects of the treatments on animal behavior and cognition. Immunohistochemical staining and transmission electronic microscope analysis were performed to measure the cellular responses to drug treatment. Western blotting and ELISA were performed. The expression of Cleaved- Casepase 3, Beclin-1, p62 and LC3I/II were quantified, and the Phosphorylation of eNOS and Akt were evaluated. The ratio of Bcl-2/ Bax was determined to reveal the molecular pathways that are involved in the drug treatment. Results: We found that intraperitoneal delivery of various Bilobalide doses during ischemia can protect against brain injury, as evidenced by reduced infarct size and improved neurological scores after surgery. Histochemical analysis revealed that treatment with bilobalide can significantly reduce apoptosis, autophagy, and promote angiogeneis following ischemia/reperfusion injury to the brain. The performence of increased phosphorylation of eNOS and Akt suggested that bilobalide can activate Akt prosurvival and eNOS pathways to promote cell survival and angiogenesis, respectively. Conclusions: Our results suggested that bilobalide benefits stroke symptoms by reducing cell death pathways and promoting angiogenesis. As such, bilobalide may be a potential agent for improving self-repair after ischemic stroke.

Background: Preoperative diagnosis of pancreatic cystic lesions (PCLs) must be reliable as the current standard treatment, major or total pancreatectomy, dramatically affects quality of life. Additionally, early diagnosis of malignancy is essential to an improved prognosis. The diagnostic accuracy of fluid analysis using endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) has been demonstrated in pancreatic solid lesions. The utility of this technique in the diagnosis of PCLs is still unknown. Methods: A comprehensive search was performed in multiple databases. Studies differentiating benign and malignant PCLs via EUS-FNA were included in this meta-analysis. The quality of diagnostic accuracy studies (QUADAS) was adopted to evaluate the selected studies. Pooled sensitivity, specificity, likelihood ratio, diagnostic odds ratio, and summary receiver operating characteristic (sROC) curve analyses were conducted. Two main classification types of malignancy were characterized and analyzed. We also generated a subgroup analysis of available clinical factors. Publication bias was evaluated by Begg's and Egger's tests. Results: Sixteen studies containing 1024 subjects have been published. The pooled sensitivity for malignant cytology according to classification 1 was 0.51 (95% CI, 0.45-0.58), and pooled specificity was 0.94 (95% CI, 0.92-0.96). When the detected PCLs were identified as classification 2, suspicious malignancy or potential malignancy, sensitivity and specificity were similar, 0.52 (95% CI, 0.46-0.57) and 0.97 (95% CI, 0.95-0.98) respectively. Conclusion: This meta-analysis demonstrates that EUS-FNA is a reliable clinical tool for the diagnosis of PCLs. However, a more accurate algorithm is needed to reduce various biases and to improve the sensitivity of EUS-FNA in the detection of malignant PCLs.

As a de-ubiquitin enzyme, ubiquitin C-terminal hydrolase (UCH)-L1 has been shown to be overexpressed in several human cancers. However, the function of UCH-L1 in invasion of breast cancers is still unclear. Here we report that the expression of UCH-

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy. Video LinkThe video component of this article can be found at https://www.jove.com/video/2717/ Protocol Treadmill Running for Revealing the Extent of Muscle Injury in Mouse Models1. Establish the angle of the treadmill surface for use during the running protocol. Generally, a flat level or an angle between 7°and 15°degrees downhill or uphill is used. Some treadmills have in integral apparatus to adjust the incline while others require the treadmill to be elevated through other means. 2. Place a tray or a blue lab pad underneath the treadmill before putting the animals in the treadmill to collect any waste from the animals during the running protocol. 3. Before running mice should be acclimated to the environment of the treadmill. This involves placing the animals in the treadmill for 15 minutes with the electric grid off and the belt drive motor on but the belt not moving (i.e. with the speed set at 0 m/s). 4. Turn on the motivational electric grid. The intensity and frequency of the pulses used can usually be controlled on treadmills but varies from one manufacturer to another. Generally, maximum intensity is not required to motivate mice to run while a high frequency (at least once every two seconds) will improve compliance. 5. Activate the treadmill belt to begin the running protocol. An initial speed of approximately 5 meters/min should be used for a warm up period for the mice. The speed of the treadmill can be accelerated slowly, usually by adding 1-2 meters/min for each minute following the start of the treadmill. 6. Compliance of animals in most protocols can be improved by conducting a series of short runs (5 minutes) at warm up speed for 3 to 10 days before the experimental running begins. 7. An acute exhaustion exercise will usually involve increasing the speed of the treadmill with time until a maximum speed (generally < 30 m/ m) is achieved and maintained until the mice show signs of exhaustion. Criteria for judging exhaustion vary but generally include a mouse spending more than half of the time on the electric grid or 5 consecutive seconds on the electric grid without returning to the treadmill surface.After an individual mouse is exhausted it can be removed...

As a key component of caveolae structure on the plasma membrane, accumulated evidence has suggested that Polymerase I and Transcript Release Factor (PTRF) plays a pivotal role in suppressing the progression of human malignances. However, the function of PTRF in the development of colorectal cancers is still unclear. Here we report that the expression of PTRF is significantly reduced in tumor tissues derived from human patients with colorectal cancers, and that the downregulation of PTRF correlates to the advanced stage of the disease. In addition, we found that the expression of PTRF negatively regulates the tumorigenic activities of colorectal cell lines (Colo320, HT29 and CaCo2). Furthermore, ectopic PTRF expression caused significant suppression of cellular proliferation, and anchorage-independent colony growth of Colo320 cells, which have the lowest expression level of PTRF in the three studied cell lines. Meanwhile, shRNA mediated knockdown of PTRF in CaCo2 cells significantly promoted cellular proliferation and anchorage-independent colony growth. In addition, in vivo assays further revealed that tumor growth was significantly inhibited in xenografts with ectopic PTRF expression as compared to untreated Colo320 cells, but was markedly enhanced in PTRF knockdown CaCo2 cells. Biochemical studies revealed that overexpression of PTRF led to the suppression of the AKT/mTOR pathway, as evidenced by reduced phosphorylation of AKT, mTOR, and downstream MMP-9. Thus, these findings, for the first time, demonstrated that PTRF inhibits the tumorigenesis of colorectal cancers and that it might serve as a potential therapeutic target for human colon cancer patients.

Ischemic stroke is a leading cause of long-term disability. Although previous studies have identified some promising neuroprotective agents, such as cinnamophilin, EGCG, and oxyresveratrol, their protective effects are still very limited. Thus, identification of novel neuroprotective agents is highly desired for treatment of this disease. Previous studies have shown that Ginkgo biloba extract has protective effects against neurodegenerative disorders such as cerebral insufficiency, Alzheimer's disease, and dementia. In this present study, we sought to test the potential protective role of an active component of Ginkgo biloba extract, Bilobalide, in a rat model of middle cerebral artery occlusion (MCAO). We found that intraperitoneal (IP) delivery of various Bilobalide doses during ischemia can protect against brain injury, as evidenced by reduced infarct size and improved neurological scores after surgery. Histochemical analysis revealed that treatment with Bilobalide can significantly reduce apoptosis, autophagy, and promote angiogeneis following ischemia/reperfusion injury to the brain. In order to determine the molecular mechanisms underlying Bilobalide mediated neuroprotective effects, biochemical assays were performed, and the results suggested that Bilobalide can activate Akt prosurvival and eNOS pathways to promote cell survival and angiogenesis, respectively. To confirm these biochemical observations, we cotreated animals with a combination of Bilobalide and inhibitors of Akt and/or eNOS pathways. We found that treatment with inhibitors can partially abolish the beneficial effects of Bilobalide, indicating that Bilobalide indeed provides protection from neuronal injury through activation of these signaling pathways. Taken together, our results suggested that Bilobalide benefits stroke symptoms by reducing cell death pathways (apoptosis and autophagy) and promoting angiogenesis. As such, Bilobalide may be a potential agent for improving self-repair after ischemic stroke.