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Royal jelly (RJ), a natural honeybee product, has a wide range of antibacterial activities. N-glycosylated major royal jelly protein 2 (N-MRJP2), purified from RJ, can inhibit the growth of Paenibacillus larvae (P. larvae, Gram-positive), a contagious etiological agent of the American foulbrood disease of honeybees. However, the inhibitory mechanism is largely unknown. Antibacterial assay and membrane proteome were conducted to investigate the inhibition capacity of RJ from different instar larvae and P. larvae treated by N-MRJP2, respectively. The similar antibacterial efficiency of RJ from different larval instar indicates that RJ is vital for the adaptive immune defense of small larvae. The killing of P. larvae by N-MRJP2 is achieved by disturbing the cell wall biosynthesis, increasing the permeability of cell membrane, hindering aerobic respiration, restraining cell division and inducing cell death. This demonstrates that RJ is critical for the passive immunity of immature larvae and N-MRJP2 can be used as natural antibiotic substance to resist P. larvae, even for other gram-positive bacteria. This constitutes solid evidence that RJ and N-MRJP2 have potentials as novel antibacterial agents.

Additional file 1: Fig. S1. Hydrodynamic diameters and PDI of CNSD after storage in 1�� PBS buffer (pH = 7.4) during 10 days (n = 3). Fig. S2. NIR images of CNS0, CNSC, CNSJ, and CNSD ([CuS] = 100 ��g mL-1) under the NIR-II laser irradiation (1064 nm, 1.0 W cm���2) for 5 min. Fig. S3. The time constant for heat transfer from the system is determined by applying the linear time data from the cooling period of (a) versus the negative natural logarithm of driving force temperature. Fig. S4. Release of CpG from CNSD with or without laser irradiation (1064-nm, 1 W cm���2) for 5 min (the concentrations of CuS and CpG for CNSD were 100 and 2.3 ��g/mL, respectively). Fig. S5. Schematic illustration of the synthesis of ICG-loaded CNS nanoparticles (CNS0@ICG, CNSC@ICG, CNSJ@ICG, CNSD@ICG) for tracing the nanoparticle trajectory. Fig. S6. UV-vis absorption spectrums of CNS0@ICG, CNSC@ICG, CNSJ@ICG, and CNSD@ICG. Fig. S7. Fluorescence intensity of 4T1 cells treated with PBS (control) or various ICG-loaded CNS nanoparticles ([ICG] = 20 ��g mL���1) for 24 h via flow cytometry. Fig. S8. Relative mean fluorescence intensity (MFI) of CRT in Panc02 cells after different treatments. Fig. S9. The NIR fluorescence imaging of xenograft Panc02tumor-bearing C57BL/6 living mice at 0, 8, 24, and 36 h after systemic administration of CNS@ICG through tail-vein administration (0.2 mL, [ICG] = 2 mg kg-1). The fluorescence images were collected with excitation at 710 nm and emission at 790 nm, and the tumors were marked by white circles. (b) The fluorescence intensity of tumor regions of mice at different post-injection times of (n = 3). Fig. S10. (a) NIR thermal photos of 4T1 tumor-bearing mice under laser irradiation at 24 h post-injection of PBS, CNS0, CNSC, CNSJ, and CNSD through tail-vein injection (0.2 mL, the concentration of CuS = 300 ��g/mL for CNS0, CNSC, CNSJ, and CNSD); (b) Temperature elevation curves of tumors in 4T1 tumor-bearing mice after administration of Control (PBS), CNS0, CNSC, CNSJ and CNSD under NIR-II laser illumination (1064 nm, 1W cm-2, 5 min). Fig. S11. Gating strategies for flow cytometry assay of matured CD80+CD86+ DCs in tumor-draining lymph nodes of Panc02 tumor-bearing C57BL/6 mice. Fig. S12. Western-blot analysis of JQ1-induced downregulation of PD-L1 in Panc02 cells (200 nM of JQ1 and 100 ng/ml of IFN-��). Fig. S13. Gating strategy for flow cytometry assay of CD4+ T cells and CD8+ T cells in distant tumors of Panc02 tumor-bearing C57BL/6 mice. Fig. S14. A volcano plot showing the up-regulated or insignificantly expressed or down-regulated genes when comparing the CNSD-treated group with the Control (PBS) group. Fig. S15. Body weights of Panc02 tumor-bearing C57BL/6 mice in different groups within 14 days of treatment (Control (PBS), CNS0, CNSC, CNSJ and CNSD, 0.2 mL, [CuS] = 300 ��g/mL, n = 5). Fig. S16. Body weights of 4T1 tumor-bearing Balb/c mice in different groups within 14 days of treatment (Control (PBS), CNS0, CNSC, CNSJ and CNSD, 0.2 mL, [CuS] = 300 ��g/mL, n = 5). Fig. S17. Representative histological H&E staining images of major organs (heart, liver, spleen, lung, and kidney) were collected from Panc02 tumor-bearing C57BL/6 mice in different treatment groups at the end of treatment (Control (PBS), CNS0, CNSC, CNSJ and CNSD, 0.2 mL, [CuS] = 300 ��g/mL). The scale bar represents 50 ��m. Fig. S18. Representative histological H&E staining photos of major organs (heart, liver, spleen, lung, and kidney) in healthy C57BL/6 mice before treatment (Control) and after tail-intravenous injection of CNSD (0.2 mL, [CuS] = 00 ��g/mL) for 15 (Day 15) and 30 days (Day 30). The scale bar represents 50 ��m. Fig. S19. The levels of (a) alanine aminotransferase (ALT), (b) aspartate aminotransferase (AST), (c) ��-glutamyl transpeptidase (GGT), (d) urea, (e) creatinine (CREA), (f) red blood cells (RBC), (g) hemoglobin (HGB), (h) mean corpuscular hemoglobin (MCH), (i) hemoglobin concentration (MCHC), (j) red cell distribution width (RDW-SD), (k) red cell volume distribution width (RDW-CV), (l) platelet (PLT), (m) plateletcrit (PCT), (n) mean platelet volume (MPV), and (o) platelet distribution width (PDW) in the blood of healthy C57BL/6 mice before treatment (D0) and after tail-intravenous administration of CNSD (0.2 mL, [CuS] = 300 ��g/mL) for 15 (D15), and 30 days (D30) (n = 3).

[Objectives] The purpose was to establish an induction system for friable callus of Hedera nepalensis var. sinensis with different parts. [Methods] By screening the most suitable explant and adjusting the hormone ratio of medium, friable calli of H. nepalensis var. sinensis were induced. [Results] The calli could be induced from leaves, petioles and stem segments, but the ideal explant was stem segments, with induction rate reaching 98%. The optimal medium for callus proliferation was MS + 0.5 mg/L KT + 1.0 mg/L 2,4-D + 30.0 g/L sucrose. After 3-4 generations of subculture on MS+0.5 mg/L BA+1.0 mg/L 2,4-D+30.0 g/L sucrose, favorable friable calli of H. nepalensis var. sinensis were obtained. [Conclusions] The friable calli induced in this experiment can lay a foundation for in-vitro regeneration and cellular secondary metabolite production of H. nepalensis var. sinensis.