Additional file 4: of Blockade of TGF-β/Smad signaling by the small compound HPH-15 ameliorates experimental skin fibrosis

Figure S4. The inverse proportional change of Ly6Chi macrophages and CD206+ M2 macrophages from the inflammation stage (day 7) to the fibrotic stage (day 21). The single-cell suspension obtained from the back skin of bleomycin-injected C57BL/6 mice on day 7 and day 21 was stained with the mAbs against CD45, CD11b, Ly6C, and CD206. Stained samples were analyzed using the FACSCanto II system. Data were analyzed using FlowJo software version 7. All values represent mean ± SEM. n = 3 at each time point. ** p ≤ 0.01. (PDF 49 kb

Additional file 2: of Blockade of TGF-β/Smad signaling by the small compound HPH-15 ameliorates experimental skin fibrosis

Figure S2. HPH-15 treatment did not affect the growing of the mice. The body weight changes of mice treated with vehicle or HPH-15 were assessed every 3 days. All values represent mean Âą SEM; n = 5 in each group. (PDF 6 kb

Additional file 1: of Blockade of TGF-β/Smad signaling by the small compound HPH-15 ameliorates experimental skin fibrosis

Figure S1. The molecular structure of HPH-15. (PDF 7 kb

Additional file 3: of Blockade of TGF-β/Smad signaling by the small compound HPH-15 ameliorates experimental skin fibrosis

Figure S3. Every-other-day injection of bleomycin for 2 weeks induced significant skin fibrosis. Back skin of NaCl- or bleomycin-injected mice was harvested on day 14 and stained with H&E. Scale bar = 100 Îźm. n = 3 in each group. (PDF 80 kb

Difference in the Inhibitory Effect of Thiol Compounds and Demetallation Rates from the Zn(II) Active Site of Metallo-β-lactamases (IMP‑1 and IMP-6) Associated with a Single Amino Acid Substitution

Gram-negative bacteria producing metallo-β-lactamases (MBLs) have become a considerable threat to public health. MBLs including the IMP, VIM, and NDM types are Zn(II) enzymes that hydrolyze the β-lactam ring present in a broad range of antibiotics, such as N-benzylpenicillin, meropenem, and imipenem. Among IMPs, IMP-1 and IMP-6 differ in a single amino acid substitution at position 262, where serine in IMP-1 is replaced by glycine in IMP-6, conferring a change in substrate specificity. To investigate how this mutation influences enzyme function, we examined lactamase inhibition by thiol compounds. Ethyl 3-mercaptopropionate acted as a competitive inhibitor of IMP-1, but a noncompetitive inhibitor of IMP-6. A comparison of the crystal structures previously reported for IMP-1 (PDB code: 5EV6) and IMP-6 (PDB code: 6LVJ) revealed a hydrogen bond between the side chain of Ser262 and Cys221 in IMP-1 but the absence of hydrogen bond in IMP-6, which affects the Zn2 coordination sphere in its active site. We investigated the demetallation rates of IMP-1 and IMP-6 in the presence of chelating agent ethylenediaminetetraacetic acid (EDTA) and found that the demetallation reactions had fast and slow phases with a first-order rate constant (kfast = 1.76 h–1, kslow = 0.108 h–1 for IMP-1, and kfast = 14.0 h–1 and kslow = 1.66 h–1 for IMP-6). The difference in the flexibility of the Zn2 coordination sphere between IMP-1 and IMP-6 may influence the demetallation rate, the catalytic efficiency against β-lactam antibiotics, and the inhibitory effect of thiol compounds