PNH.    The principal competitors for our program in PNH are eculizumab and ravulizumab-cwvz, both of which are monoclonal antibody C5 inhibitors marketed by Alexion Pharmaceuticals. In addition, we are aware that there are a number of other companies that are actively developing product candidates for the treatment of PNH. These include product candidates directed at C5 inhibition such as Coversin, a small protein inhibitor of C5 in clinical development by Akari Therapeutics; ABP 959, a biosimilar product candidate in clinical development by Amgen; tesidolumab, a monoclonal antibody inhibitor of C5 in clinical development by MorphoSys AG and Novartis Pharmaceuticals; pozelimab, a monoclonal antibody inhibitor of C5 in clinical development by Regeneron Pharmaceuticals; and RG6107, a monoclonal antibody inhibitor of C5 in clinical development by F. Hoffmann La Roche. In addition, competitors include product candidates directed at C3 inhibition, such as APL 2, currently in clinical development by Apellis Pharmaceuticals, and AMY-101, currently in clinical development by Amyndas Pharmaceuticals. Lastly, we face competition from orally available small molecule inhibitors of Factor D, such as ACH 4471, in clinical development by Achillion Pharmaceuticals, and LNP023, a Factor B inhibitor in clinical development by Novartis.

(a)      Designed such disclosure controls and procedures, or caused such disclosure controls and procedures to be designed under our supervision, to ensure that material information relating to the Company, including its consolidated subsidiaries, is made known to us by others within those entities, particularly during the period in which this report is being prepared;

Pentobarbital sodium was purchased from Genview (St. Galveston, TX, USA). Isoflurane was from Jiupai Pharmaceutical Co. Ltd. of HeBei (Jiu-pai, Shijiazhuang, China). HE was from Zhongshan Goldenbridge biotechnology Co. Ltd (Beijing, China). Wheat germ agglutinin (WGA) and rhodamine phalloidine were purchased from Invitrogen (Carlsbad, CA, USA). Tris, urea, 3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPS), DL-Dithiothreitol (DTT), sodium dodecyl sulfate (SDS) and iodoacetamide were purchased from AMRESCO (Solon, OH, USA). Glycerol and paraformaldehyde were from Beijing Chemical Works (Beijing, China). Thiourea and PhastGel Blue R were purchased from GE Healthcare (Piscataway, NJ, USA). Trypsin solution was purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies against reduced glyceraldehyde-phosphate dehydrogenase (GAPDH), ALDOA, ENOβ, ECH1, HSP70, HIF1, CPT1A, PFK2, HSPB1 and RhoGDI1 were from Abcam (Cambridge, USA), the antibodies against ENOα, SODM and PDH were from Santa Cruz Biotechnology (Santa Cruz, Calif, USA). ELISA kits for ATP, ADP, AMP, and MDA were purchased from Beijing Huanya Biomedicine Technology Co. Ltd (Beijing, China).

        All financial statement schedules have been omitted because they are not applicable, not required or the information required is shown in the financial statements or the notes thereto.

For proteome analysis, two-dimensional polyacrylamide gel electrophoresis was performed. Briefly, the heart tissue (n = 3) was homogenized in a homogenization buffer containing 30 mM Tris, 7 M urea, 2 M thiourea, 4% CHAPS, and 40 mM DTT. The lysates obtained were centrifuged at 15, 294 g for 30 minutes at 4 °C; the supernatant was collected and stored as aliquots at −80 °C until use. The samples were pre-treated with 2D Clean-Up Kit (GE Healthcare, Piscataway, NJ, USA) to remove the interfering components. The protein concentration of each sample was determined using the Bradford method. A total of 1 mg of protein was loaded onto a 24-cm linear IPG strip (pH 3–10, Amersham Biosciences, Piscataway, NJ, USA) for first-dimension isoelectric focusing. The strips were placed into a Ettan IPGphor 3 (GE Healthcare, Piscataway, NJ, USA) and rehydrated at 50 V for 12 hours, and the proteins were separated based on their protein isoelectric point with a protocol as follows: 250 V with linear climb for 30 minutes, 500 V with linear climb for 30 minutes, 1000 V with linear climb for 60 minutes, 4000 V with rapid climb for 60 minutes, 8000 V with linear climb for 5 hours, and 8000 V with rapid climb until a level of 60000 V-h was reached. After first-dimension isoelectric focusing, the IPG strips were equilibrated for 15 minutes in a buffer containing 50 mM Tris-HCl, pH 8.8, 30% glycerol, 7 M urea, 2% SDS, and 1% DTT, then further treated in a similar buffer containing 4% iodoacetamide instead of DTT for 15 minutes and directly applied onto 12% homogeneous SDS-PAGE gels for 6 hours electrophoresis using a Ettan DALT six system (GE Healthcare, Piscataway, NJ, USA). The gels were stained by PhastGel Blue R according to the manufacturer’s instructions. The stained gels were scanned and then analyzed using Image Master 2D platinum 7 software (GE Healthcare, Piscataway, NJ, USA). The intensity of each spot was quantified by calculating the spot volume after the gel image had been normalized. Each sample was processed in triplicate to ensure reproducibility, and the Student’s t-test was used to evaluate the average change in spot abundance corresponding to each target spot across the gels. Spots with more than a 2-fold change (either increasing or decreasing) in density were considered to be differentially expressed (paired t-test, p ≤ 0.05) and were selected for further identification via mass spectrometry analysis9. Briefly, differently expressed spots were manually excised from the gels, destained and dehydrated in acetonitrile. Subsequently, they were subjected to rehydration with a trypsin solution and in-gel digestion overnight at 37 °C. For each spot, 0.75 μL of digested peptides were spotted onto a MALDI-TOF target (GE Healthcare, Piscataway, NJ, USA) and allowed to dry. Then, 0.75 μL of matrix solution (saturated solution of α-cyano-4-hydroxycinnamic acid in 50% v/v acetonitrile and 0.5% v/v trifluoroacetic acid) was applied to the dried sample, and tryptic peptide masses were acquired on an Ettan MALDI-ToF Pro mass spectrometer (GE Healthcare, Piscataway, NJ, USA). Mass spectra were acquired in reflectron mode with an accelerating voltage of 20 kV using the Ettan MALDI Evaluation software (GE Healthcare, Piscataway, NJ, USA). Peptide masses were internally calibrated using the 842.509 and 2211.105 m/z trypsin autolysis peptides. Databases were searched for peptide identification using MASCOT Distiller 2.1 (Matrix Science, http://www.matrixscience.com).

Wang, Y. et al. Development of fluorescence imaging-based assay for screening cardioprotective compounds from medicinal plants. Anal. Chim. Acta 702, 87–94, 10.1016/j.aca.2011.06.020 (2011).

Raising additional capital may cause dilution to our stockholders, restrict our operations or require us to relinquish rights to our technologies or product candidates.

        5.     The registrant’s other certifying officer and I have disclosed, based on our most recent evaluation of internal control over financial reporting, to the registrant’s auditors and the audit committee of the registrant’s board of directors (or persons performing the equivalent functions):

Zhejiang Huahai’s improved process replaced tributyltin azide with the more toxic compound, sodium azide, says André. As a result, the yield of tetrazole formation was much better. However, sodium nitrite was used to destroy the excess sodium azide that remained after the synthesis step. Sodium nitrite is often used as a decontaminating agent of sodium azide in acidic conditions, André says. However, under these conditions, it forms nitrous acid, which could react with the residue of dimethylamine in dimethylformamide, the solvent that is used in the tetrazole-forming reaction, to generate NDMA, says André. 

The Pharma Services business of Thermo Fisher Scientific will invest $150 Million at three facilities.

In addition to approval in the United States, we currently intend to seek regulatory approval of Aramchol in the EU. As such, a summary of the EU regulatory processes follows below.

I appreciate your input. Thank you for the information about the optimal IGF -1 levels. Very helpful to know.