To the extent a distribution on our ordinary shares does not constitute an excess distribution to a Non-Electing U.S. Holder, such Non-Electing U.S. Holder generally will be required to include the amount of such distribution in gross income as a dividend to the extent of our current and/or accumulated earnings and profits (as determined for U.S. federal income tax purposes) that are not allocated to excess distributions. The tax consequences of such distributions are discussed above under “Taxation of U.S. Holders—Distributions.” Each U.S. Holder is encouraged to consult its own tax advisor with respect to the appropriate U.S. federal income tax treatment of any distribution on our ordinary shares.

For western blot, equal amounts of protein lysates were separated using 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis. The gels were blotted onto a nitrocellulose membrane and incubated with the primary antibodies of phosphorylated-eNOS (Ser1177) and total eNOS (Abcam, cambridge, UK). Binding of primary antibody was detected with a secondary anti-rabbit antibody and visualized by the enhanced chemiluminescence method. β-actin was used as a loading control.

When CR is compared with IF in regards to lower body fat % IF is also better then CR but only marginally, but IF does twice as well in regards to where it matters the most, the fat around the stomach.

Using chromatin immunoprecipitation (ChIP) assay, we determined the binding of HIF-1α to the miR-322 promoter with and without CoCl2 treatment or with ODDD-wt/-mut overexpression. As shown in Fig. 3g, binding of HIF-1α to the HRE site increased in A7r5 cells, but HIF-2α binding did not exhibit any change. We validated the system by using antibodies against IgG or RNA polymerase II (Pol II) and primers for a region lacking HRE site (data not shown). The ChIP assays indicated that HIF-1α directly binds to the HRE site on the miR-322 promoter in vitro. Altogether, the results above demonstrated that miR-322 is transcriptionally regulated by the hypoxia-responsive factor HIF-1α in hypoxia.

(a) Schematic diagram of cloned rat putative promoter region of miR-322, stretching from upstream 1000 bp. The position of the putative HRE matching the core sequence (A/G)CGTG is labeled in box, between two functional CACAG elements (P1k). A mutant promoter was constructed with the sequence as shown below (P1k-m). (b) Luciferase reporter assays of miR-322 promoter activity with both wild (pGL4-P1k) and mutant constructs (pGl4-P1k-m) in the presence or absence of CoCl2 (200μM) (left panel); Under these conditions, the protein levels of HIF-1α and HIF-2α were stabilized after CoCl2 treatment in the cells as determined by western blot analysis and normalized to β-actin levels (right panel). (c) miR-322 promoter activity assessed by luciferase reporter assay after CoCl2 treatment was diminished by HIF-1α knockdown. Cells were transduced with shRNA targeting HIF-1α (shHIF-1α) or HIF-2α (shHIF-2α) and miR-322 promoter activity was determined in the luciferase reporter assay after transfection of wild type and mutant constructs and CoCl2 treatment (left panel) as described under Methods; Control (sh-Con) or HIF-1α/-2α shRNA-transfected cells were harvested for protein analysis by western blot to determine knockdown specificity (right panel); *p < 0.05 compared with sh-Con. (d & e) A7r5 cells were transfected with recombinant adenoviruses expressing oxygen-dependent degradation domains (ODDD-wt) or mutated ODDD (ODDD-mut) under normoxic conditions. The influence on the miR-322 promoter reporter activity (d) and the endogenous expression levels (e) of miR-322 were determined by real-time PCR. All the bar plots represent means ± SD. *p < 0.05, **p < 0.01,compared with ODDD-mut. (f) Western blot for HIF-1α and HIF-2α in ODDD–transfected A7r5 cells after 24 hours. β-actin served as loading control. The full-length blots with these antibodies were presented in supplementary Figure S3. All gels have been run simultaneously under the same experimental conditions. (g) HIF-1α dynamic binding on the HRE site (−797 to −793) on the miR-322 promoter. ChIP assays were performed with indicated antibodies in the absence or presence of CoCl2 (left), or transfected with ODDD-wt or ODDD-mut (right) as described under Methods. Representative gel with input lanes showing products after PCR amplification and before immunoprecipitation.

To establish tumor in the flanks of C57BL/6 and TRPM2−/− mice, tumor cells were resuspended in 200 μl of PBS and injected subcutaneously. The tumor developed at the site of injection was measured and the mean tumor size was calculated [length (cm) × width (cm)]. To produce experimental lung metastasis, C57BL/6 mice received intravenous injections of B16F10 cells (1 × 105 cells/mouse). After 2 d, NK cells (2 × 106 cells/mouse) from Cd38+/+, Cd38−/−, or TRPM2−/− mice were injected intravenously into wild-type or TRPM2−/− mice. Mice were sacrificed 14 d later. Lungs were fixed with 4% paraformaldehyde and metastatic nodules on the surface of the lungs were counted.

Our management assessed the effectiveness of our internal control over financial reporting as of December 31, 2018. In making this assessment, our management used the criteria set forth by the Committee of Sponsoring Organizations of the Treadway Commission (COSO) in Internal Control-Integrated Framework (2013). Based on that assessment, our management concluded that as of December 31, 2018, our internal control over financial reporting was effective.

Endo Pharmaceuticals (NasdaqGS:ENDP ) Acquired Indevus Pharmaceuticals, March 2009. Endo Pharmaceuticals Inc is a specialty pharmaceutical company with a broad portfolio of branded and generic prescription products focused on providing relief from pain. Endo is dedicated to improving the lives of patients through identifying, developing, and marketing innovative and differentiated products in pain management and related therapeutic areas

        The containment of healthcare costs also has become a priority of federal, state and foreign governments and the prices of drugs have been a focus in this effort. Governments have shown significant interest in implementing cost-containment programs, including price controls, restrictions on reimbursement and requirements for substitution of generic products. Adoption of price controls and cost-containment measures, and adoption of more restrictive policies in jurisdictions with existing controls and measures, could further limit our net revenue and results. Coverage policies and third-party reimbursement rates may change at any time. Even if favorable coverage and reimbursement status is attained for one or more products for which a company or its collaborators receive regulatory approval, less favorable coverage policies and reimbursement rates may be implemented in the future.

        The consolidated financial statements together with the report of our independent registered public accounting firm required to be filed pursuant to this Item 8 are appended to this Annual Report on Form 10-K. An index of those consolidated financial statements is found in Item 15 of this Annual Report on Form 10-K.

For residents, or deemed residents, of the United States, the 2013 Plan provides grants, which are pursuant to Section 422 of the Internal Revenue Code of 1986, as amended, or the Code, as incentive stock options, or ISOs, and any other participants which do not qualify for ISOs, as non-statutory stock options, or NSOs, pursuant to the Code.

        Our competitors may succeed in developing, acquiring or licensing technologies and products that are more effective, have fewer side effects or more tolerable side effects or are more convenient or less costly than any product candidates that we are currently developing or that we may develop, which could render our product candidates obsolete and noncompetitive.