Actinomycin

Since its inception in 2015, MIBiG has provided an online submission form for adding new entries. To submit a new entry, a user starts by requesting a MIBiG accession number. This is done through submitting the product name(s) and the sequence information of the BGC, preferably in the form of a set of coordinates corresponding to the BGC’s position within an NCBI Genbank accession. After the request is approved by MIBiG staff, the workflow subsequently provides an extended entry form where users can input more detailed information. This crowdsourcing, open-for-all approach has garnered 140 new entries since 2015, with contributions coming from various experts all over the world.
Because not all newly characterized BGCs are submitted to the database, we actively complemented this crowdsourcing approach by periodically organizing in-house ‘Annotathons’, where multiple scientists sat together for an entire day to work on MIBiG curation (Supplementary Table S1). This has yielded 702 new entries, and annotation quality improvements for over 600 BGCs.
More recently, we have introduced an additional MIBiG curation process into the classroom environment with the help of a comprehensive and very specific set of guidelines for the students (10 (link),11 (link)). By giving one task to multiple students to work on independently, and later on having an expert (the teacher) to combine and validate the results, we have generated an additional 10 high quality BGC entries, for actinomycin, carbapanem, daptomycin, ebelactone, lipstatin, nocardicin A, obaflourin, oxazolomycin, salinosporamide and tabtoxin. Scaling up this process in the future may allow the annotations of many more important entries, which have remained incomplete, because, e.g. the scientists who have worked on the pathway are no longer active in the field.

Isolation and separation of the ovary and subsequent flow cytometry were performed as previously described³⁰ . Hamster anti-mouse CD11c monoclonal conjugated with PE (Cat# 553802), and 7-amino-actinomycin D [7AAD] (Cat# 559925) were obtained from BD Biosciences (Tokyo, Japan). The rat monoclonal antibody for anti-mouse CD206 conjugated with alexa fluor 647 (MCA2235A647) and the rat IgG2a conjugated with alexa flour 647 isotype antibody (Cat# 1212A647) were obtained from AbD Serotec Co. (Oxford, UK). In ovarian cells, after exclusion of dead cells by gating with 7-amino-actinomycin D, live cells were used for further analysis. M1 or M2 MΦs were identified as CD45+/F4/80+/CD11c+/CD206− or CD45+/F4/80+/CD206+/CD11c− cells, respectively (Fig. 1a). DCs were identified as CD45+/F4/80−/CD11c+ cells (Fig. 1a). These experiments were performed with a FACS Diva Version 6.1.2 automated cells analyzer (Becton Dickinson FACS Canto II). Data analyses were performed using Flow Jo software. Unstained specimen and isotype negative control were used for all relevant samples to justify gating strategy. Fluorescence minus one (FMO) control was used wherever needed.Figure 1

M1 and M2 macrophages (MΦs) in wild type (WT) mice ovary. (a) Representative flow cytometry analysis of M1 and M2 MΦs in the WT mice ovary. M1-like MΦ was defined as CD45+/F4/80+/CD11c+/CD206− cells, and M2-like MΦ was defined as CD45+/F4/80+/CD206+/CD11c− cells and Dendritic cells (DCs) was defined as F4/80− CD11c+ cells. (b) The proportion of CD11c+F4/80+ cells, M1-like MΦs (left panel) and CD206+ F4/80 cells, M2-like MΦs (middle panel) and F4/80− CD11c+ cells, DCs (right panel) in ovary. The proportion of M1-like MΦs significantly increased following follicular induction with PMSG 48 h, while that of M2-like MΦs and DCs was not increased. The data are shown as the means ± standard error of the mean (SEM). A P-value of < 0.05 was considered statistically significant by Mann-Whitney U test. N.S; not significant compared to WT mice. n; the number of mice.

To obtain single-cell suspension from dorsal skin, samples were removed from the subcutaneous fat and mucosal tissue with the scalpel and spread into a Petri dish. The samples were then incubated in 2.4 U/mL dispase II overnight at 4 °C and then immersed in DMEM containing 50% (v:v) FBS to inactivate the dispase II. Gently scrape off the epidermal layer and add 5 mL of 0.25% EDTA-free trypsin (Thermo Fisher, 15050057) to the tube to obtain a single cell suspension, after digestion at 37 °C for 20 min. Finally, neutralized with 5 mL DMEM medium (GbicoTM, LS11995065). Single-cell suspension of epidermis cells was made followed by mechanical dissociation with a gentle MACS dissociator (Miltenyi Biotech, Bergisch Gladbach, Germany), and filtered sequentially through 70 μm cell strainers (BD Bioscience, 352350), and cells were washed once with PBS.
To obtain single-cell suspension from lymph nodes, samples were ground in 40 μm cell strainers (BD Bioscience, 352340) with 5 mL PBS solution, and then filtered with 70 μm cell strainers (BD Bioscience, 352350). Cells were washed once with PBS.
For surface staining, the cells were stained with appropriate antibodies against surface antigens in PBS on ice for 30 min. The cellular viability was assessed by staining with 7-amino actinomycin D (7-AAD) (BioLegend, 420404; 0.5 μg/mL) to exclude dead cells. For the analysis of IL-4、IFN-γ and IL-17A production, in vitro re-stimulation, and intracellular staining, single-cell suspensions were incubated for 4 h at 37 °C with PMA (Sigma-Aldrich, p1585; 200 ng/mL), brefeldin A (BioLegend, 420601; 5 μg/mL), and ionomycin (Abcam, ab120116; 1 μg/mL). The cells were then washed and stained with the fixable viability stain 620 (FVS 620; BD-Biosciences, 564996) for 10 min. After performing surface staining as described above, cells were fixed with 4% paraformaldehyde and permeabilized with PBS supplemented with 0.1% Triton X-100. Intracellular staining with fluorescent-labeled antibodies was per- formed for 30 min in PBS. For flow cytometric analysis, the cells were washed and resuspended in PBS. Flow cytometry was per- formed using the NovoCyte flow cytometer and ACEA NovoExpressTM software (ACEA Biosciences, San Diego, CA, USA) and BD LSRFortessaTM and Flow Jo™ software (BD Biosciences, USA). The single-cell suspensions were stained with the following antibodies: CD3-APC-CY7 (100222), CD4-PerCP/Cy5.5 (100434), CD8-PE-CY7 (100722), IFN-γ-FITC (505806), IL-4-APC (562045), IL-17A- PE (506903), CD11b -FITC (11-0112-82), Ly6G-PE-cy7 (108416), Ly6C-APC/Cy7 (1208026), Siglec-F-Bv421 (E50-2440), CD11c-APC/Cy7 (117324), MHCII-FITC(I-A-I-E, 107606). Antibodies were purchased from eBioscience and BioLegend and used at 1:100 dilution.

The cells were digested into a single-cell suspension. Annexin V-phycoerythrin/7-amino-actinomycin D (PE/7AAD; KeyGEN BioTECH, Nanjing, China) was added according to the manufacturer’s instructions. The cells were incubated at room temperature for 15 min in the dark. They were then analyzed by flow cytometry (BD FACSCalibur; BD Biosciences, NJ, USA).

TR is structurally identical to the known anti-cancer drug; actinomycin, except in the valine motif and additional oxygen (Fig 1). Synthesis of TR analogs was carried out by following the methods as described elsewhere for the synthesis of actinomycin drug analogs [9 (link)–12 (link)]. Analogs of the parent compound were synthesized by deactivation of NH₂ group by N-substitution via chemical modification and deactivation of the keto group by chemical reduction methods.
Analogs of TR namely, TR-NC6, Tr-SUND, TRR, TR-RB, FN-Me, and C-tertbutyl were synthesized. TR-NC6 was synthesized by N-Alkylation of TR using 4-Hydroxy (hexyl)-Coumarin. Tr-SUND was derived by N-Glycosylation of TR using Acetobromo-α-D-Glucose. TRR was prepared by sodium borohydride (NaBH₄) mediated reduction of TR’s keto group. Benzoylation of the TR OH group using 4-Methoxy-Benzoyl Chloride resulted in the TR-RB. N-Alkylation of TR using Iodomethane was done to synthesize FN-Me and Bromination-alkylation of TR using 3,5-Ditert-buty-4-hydroxybenzoic acid methyl ester was used to prepare C-terbutyl-TR (S14 Fig in S1 File).