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Melanoma is the most dangerous type of skin cancer because of its high invasion and metastasis ability. Bromodomain-containing protein 4 (BRD4), an acetylation-recognizing reader, mediates the proliferation, metastasis, and invasion of melanoma, and is thus a potential therapeutic target. Mounting evidence suggests that inhibition of single bromodomain of BRD4 would improve specificity and reduce cytotoxicity to non-tumor tissues or cells. In this study, a hierarchical virtual screening campaign was performed against BRD4 BD2 from a chemical database including over 90,000 natural/natural-like compounds. Using various biochemical assays, the 7-methoxycoumarin derivative N13 was identified as a potent inhibitor of BRD4 BD2. Compared with the well-known BRD4 inhibitor JQ1, N13 exhibited higher potency against BRD4 BD2 and much higher specificity for BRD4 BD2 over BRD4 BD1. Additionally, N13 inhibited the proliferation of two kinds of BRD4-overexpressing melanoma cell lines. Mechanistically, N13 impaired the protein-protein interaction (PPI) between BRD4 BD2 and its acetylated ligand proteins (Twist1 K73/K76Ac and FOXO3a K242/245Ac), leading to reducing levels of Wnt5A and CDK6 expression, inducing cell senescence of melanoma cancer cells, and ultimately weakening the adhesion, metastasis, and invasion ability of melanoma cancer cells. To our knowledge, N13 is the first 7-methoxybicoumarin-based BRD4 BD2 inhibitor described to date and may function as a new scaffold for developing more specific and potent therapeutic agents against BRD4 BD2. In addition, our research highlights the druggability of BRD4 BD2 as a target for BRD4-overexpressing melanoma and provides a potential mechanism for the anti-melanoma activity of BRD4 BD2 inhibitor., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ethylene response factor (ERF) functions as an important plant-specific transcription factor in regulating biotic and abiotic stress response through interaction with various stress pathways. We previously obtained three ERF members, VpERF1, VpERF2, and VpERF3 from a highly powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata cDNA full-length library. To explore their functions associated with plant disease resistance or biotic stress, we report here to characterize three ERF members from this library. PM-inoculation analysis on three different resistant grapevine genotypes revealed that three VpERFs displayed significant responses, but a different expression pattern. Over-expression of VpERF1, VpERF2, and VpERF3 in transgenic tobacco plants demonstrated that VpERF2 and VpERF3 enhanced resistance to both bacterial pathogen Ralstonia solanacearum and fungal pathogen Phytophtora parasitica var. nicotianae Tucker. Importantly, VpERF1-overexpressing transgenic Arabidopsis plants increased susceptibility toward these pathogens. Investigation on drought, cold, and heat treatments suggested, VpERF2 was distinctly induced, whereas VpERF3 displayed a very weak response and VpERF1 was distinctly induced by drought and heat. Concurrently, VpERF3 was significantly induced by salicylic acid (SA), methyl jasmonate (MeJA), and ET. Our results showed that the three VpERFs from Chinese wild V. pseudoreticulata play different roles in either preventing disease progression via regulating the expression of relevant defense genes, or directly involving abiotic stress responsive pathways., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

DC-SIGN, a C-type lection expressed on dendritic cells, enhances HIV-1 infection in cis and in trans. HIV-1 circulating recombinant form (CRF) 07_BC viruses have been the predominant strain found among injection drug users in southern China and Taiwan. The goal of this study was to map the DC-SIGN-interactive domain on the gp120 of CRF07_BC. Pseudotyped viruses containing single (N233Q, N275Q, N330Q, N351Q, N355Q, N381Q, and N387Q), double (N233Q + N275Q, N233Q + N351Q, N275Q + N351Q), or triple (N233Q + N275Q + N351Q) N-glycan mutant gp120 were generated. Capture assays showed that the DC-SIGN-binding capacity of pseudoviruses with N275Q or N351Q decreased significantly. Rabbit antisera against synthetic peptides covering the N275 (R72 antiserum) or N351 (R77 antiserum) region blocked the interaction between wild-type gp120 and DC-SIGN in the capture assay. Furthermore, pseudotype viruses containing gp120 from five different CRF07_BC isolates were generated and R72 and R77 antisera blocked their interactions with DC-SIGN (80% for R72 and 40% for R77, respectively) in the capture assays. In conclusion, the N275 and N351 glycan sites on the CRF07_BC gp120 play an important role in mediating the interaction between gp120 and DC-SIGN. This information is valuable for developing both therapeutic and preventive agents for HIV-1 infection.

Chemical modification of glycerinated stalks of Vorticella with TNM is used to investigate the role of tyrosine residues in the Ca(2+)-induced contraction of the spasmoneme. Tetranitromethane (TNM) is often employed as a specific reagent for the nitration of tyrosine residues in a protein at neutral and slightly alkaline pHs although TNM can also oxidize cysteine residues in the acidic and neutral pH range. Prior incubation with Ca(2+) of stalks to be treated with TNM can protect the spasmoneme from irreversible denaturation. On the other hand, TNM treatment in the absence of free Ca(2+) causes an irreversible denaturation of the spasmoneme. It was revealed by us that an isolated Ca(2+)-binding protein called spasmin could not bind with Ca(2+) after TNM treatment, even if the treatment was performed in the presence of Ca(2+). In an additional experiment, we confirmed that the chemical modification of cysteine residues in the spasmoneme with N-7-dimethyl-amino-4methyl- coumarinyl- maleimide (DACM) has no effect on the contractibility. These results suggest that tyrosine residues in spasmin are essential for spasmoneme contraction and are protected from TNM in the presence of Ca(2+) when spasmin binds with its receptor protein in the spasmoneme.