Yersinia pestis. Our data demonstrate that it is possible to further augment the F1-V vaccine strategy in order to optimize and augment vaccine efficacy.Relatively recent advances in plague vaccinology have produced the recombinant fusion protein F1-V plague vaccine. This vaccine has been shown to readily protect mice from both bubonic and pneumonic plague. The protection afforded by this vaccine is solely based upon the immune response elicited by the F1 or V epitopes expressed on the F1-V fusion protein. Accordingly, questions remain surrounding its efficacy against infection with non-encapsulated (F1-negative) strains. In an attempt to further optimize the F1-V elicited immune response and address efficacy concerns, we examined the inclusion of multiple toll-like receptor agonists into vaccine regimens. We examined the resulting immune responses and also any protection afforded to mice that were exposed to aerosolized  Yersinia pestis, whose natural life cycle includes rodents and fleas  and has a key role in the translocation of the cytotoxic Yersinia outer proteins (Yops) into host cells, as well as stimulating the production of immunosuppressive cytokines  are compounds that enhance immunization to vaccines and experimental antigens for specific pathogen by a variety of mechanisms . The resY. pestis CO92 challenge and shifted the immune response towards a more protective Th1-like polarization , can augment the immune response to Alhydrogel-formulated vaccines . ODNs arrization .Y. pestis CO92 versus Y. pestis C12  aerosol challenge in mice vaccinated with rF1V. Secondly, we assessed a select number of TLR agonists as adjuvants with the rF1V subunit vaccine by monitoring their ability to enhance both the antibody and cellular immune responses, and the level of protection against Y. pestis C12 aerosol challenge. We predicted that the addition of a TLR agonist will augment antibody-mediated response against the V antigen, thereby enhancing protection against the F1-negative Y. pestis C12 strain.In this study, we compared the level of protection against Y. pestis CO92  in a total volume of 0.1 ml. PolyICLC (Hiltonol™) was provided by Oncovir, Inc., whereas Pam3CSK4, MPLA, CpG2006, Imiquimod and R848 were purchased from Invivogen and reconstituted per manufacturer’s recommendation. The number of mice, the amount of rF1-V, and TLR Agonists for each study are stated in the figure legends or tables. After 25 to 28 days post vaccination, depending on the study, blood was obtained by intracardiac or axillary vessel collection for serum isolation and antibody titer determination from deeply anesthetized mice. In addition, spleens were harvested for cytokine profiling from select studies. After 26-43 days, depending on the study, mice were challenged via s.c. or aerosol routes. The calculated colony-forming units (CFU) doses for each study are stated in the figure legends.Female, 6–8-week-old BALB/c and CD-1 mice were purchased from the Charles River Laboratories . Mice were vaccinated once or twice (three weeks apart), depending on the study, subcutaneously (s.c.) with rF1-V, in the presence or absence of TLR Agonists Y. pestis (pneumonic plague model) were prepared as previously described (50 doses indicated in the tables. Exposure of mice to aerosolized bacteria was accomplished as previously described (Y. pestis strain CO92 (encapsulated) or C12 (non-encapsulated) created by a three-jet collision nebulizer. Up to 48 mice can be exposed at one time to aerosolized Y. pestis. Samples were collected from the all-glass impinger (AGI) vessel and analyzed by performing CFU calculations to determine the inhaled dose of Y. pestis. The inhaled dose was calculated using Guyton’s formula: Respiratory volume per minute in ml = 2.10 x (weight in grams)0.75 0.75 , 44. Thevia the subcutaneous route (bubonic plague model). These mice were inoculated with 0.2 ml volumes of potassium phosphate buffer (Kphos) that contained CFU harvested directly from the tryptose agar blood base slants with no further culturing  in RPMI complete media for 48 hrs. The cells were centrifuged at 1200 xg for 10 min, the supernatant was collected, and then processed as per the IFN-γ Mouse ProcartaPlex Simplex Kit protocol (ThermoFisher). The concentration of the cytokines in the supernatant samples was calculated by plotting the expected concentrations of the standards against the MFI generated by each standard. A five-parameter logistic (5PL) standard curve was used for best curve fit model.Interferon gamma (IFN-γ) expression profiles were measured by the Luminex MagPix . Briefly, splenocytes were plated in 48 well plates  at 102 for 15 min. Splenocytes were resuspended in CTL-Medium with 1% L-Glutamine and seeded at 8x105 cells per well for rF1V stimulation. Plates were incubated for 24 hr at 37°C 9% CO2 and splenocytes were removed and plates were washed twice with PBS alone and then twice with PBS and 0.05 % Tween. 80 μL/well biotinylated detection anti-mouse IFN-γ monoclonal antibody was added. After 2 hr incubation at room temperature (RT), plates were washed three times with PBS and 0.05 % Tween. 80 μl of Strep-AP antibody solution was added to the wells, and the plates were incubated for 30 min at RT. Development reagents were added and incubated for 15 min at RT according to manufacturer recommendations. The colorimetric reaction was stopped by washing the plates three times with distilled water and air drying overnight. Spots were scanned and analyzed using an automated ELISPOT reader . The splenocyte response was assessed as spot forming cells (SFC), adjusted to 106 cells per well, which was automatically calculated by the ImmunoSpot® Software for each stimulation condition and the medium only control.Interferon gamma (IFN-γ) expression was measured by seeding purified splenocytes in the presence of rF1V. Briefly, 96-well plates were coated overnight at 4°C with 80 µl/well capture anti-mouse IFN-γ monoclonal antibody. Plates were washed one time with 1x PBS. rF1V (25 µg/ml) was re-suspended in CTL-Medium with 1% L-Glutamine and 100 µl was added to each well. The plates were incubated at 37°C, 9% COImmunoglobulin (Ig) class IgG titers  from vaccinated mice were determined by an ELISA performed in 96-well, Immulon 2 HB, round-bottom plates (ThermoFisher). rF1V , F1 , and V  were individually used as antigens diluted in 0.1 M carbonate buffer, pH 9.5, to a concentration of 2 µg/ml. Plates were covered and stored overnight at 4° C. The plates were washed five times with 1X wash buffer  with a Biotek ELx405ts plate washer , and incubated with blocking buffer  for 30 min at 37°C. Blocking buffer was removed by washing as stated above, then twofold serial dilutions of mouse sera were made with antibody assay diluent  in triplicate, and plates were incubated for 1 h at 37°C. Then the plates were washed as previously mentioned; diluted anti-IgG, -IgG1, or -IgG2a horseradish peroxidase conjugate  was added to each well and plates were incubated for 30 min at 37°C. After the plates were washed as previously stated, buffered hydrogen peroxide and 3,3′,5,5′-tetramethylbenzidine solution  was added to each well, and plates were incubated for 20 min at 37°C. The reaction was stopped with 2 N sulfuric acid, and the amount of bound antibody was determined colorimetrically by reading at 450 nm with a reference filter (570 nm) with a Biotek ELx808 plate reader . The results are reported as the reciprocal of the highest dilution giving a mean OD of at least 0.1 (which was at least twice the background) ± 1 SD.rF1V dilutions of between 5 and 100 µg protein in either 0 or 100 µg Alhydrogel were processed by Filter-Aided Sample Preparation (FASP) . Briefly6 ions and a maximum injection time of 200 ms. MS/MS spectra were acquired at a resolution of 60,000 (FWHM at m/z 400) with an AGC target value of 5x105 ions and a maximum injection time of 200 ms. The isolation width for MS/MS CID fragmentation was set to 2 Daltons. The normalized Collision energy was 35% with an activation time of 0.1 ms. The dynamic exclusion duration was 30 s.Sample digests were re-suspended in 40 μL of 0.1% formic acid in HPLC grade water (Buffer ‘A’). A Dionex 3000 RSLCnano system (Thermo Scientific) injected 5 μL of each digest onto a pre-column  using a flow rate of 10 µL/min. Peptides were then loaded onto an Easy-Spray analytical column (15 cm × 75 um) packed with PepMap C18, 3 um particle size, 100 A porosity particles . A 5–60% B gradient elution in 60 min was formed using mobile phase A and B (85% acetonitrile in 0.1% formic acid) at a flow rate of 300 nL/min. The column eluent was connected to an Easy-Spray source (Thermo Scientific) with an electrospray ionization voltage of 2.2 kV. An Orbitrap Elite mass spectrometer  with an ion transfer tube temperature of 300 °C and an S-lens setting of 55% was used to focus the peptides. A top 20 data dependent MS/MS method was used to select the 20 most abundant ions in a 400–1600 amu survey scan  with a full AGC target value of 10Y. pestis  and Human  subset of the SwissProt_2017_01_18 database Variable modifications used were Carbamyl (KMR), Methyl (DE), Acetyl (K), Deamidated (NQ), and Oxidation (M). Cysteine carbamidomethylation was specified as a constant modification. The peptide-level false discovery rate (FDR) was set at 0.1% using Posterior Error Probability validation. Only proteins having at least 2 Peptide Spectral Matches (PSM) were considered, with both unique and razor peptides used for spectral counts. Mass tolerances were 10 ppm for the MS1 scan and 0.6 Da for all MS/MS scans. Relative quantitation was performed using spectral peptide match counting of F1 or V peptides using the criteria above.Acquired MS/MS protein searches were performed with ProteomeDiscoverer 2.1 Sequest HT (Thermo Scientific) using a t-test on log transformed values and were summarized as Geo Mean and GSE. Fisher exact tests and log-rank tests were used to compare mouse survival curves and time to death post challenge, respectively. Median lethal doses (LD50) were estimated by probit regressions, with confidence limits by the delta method. Analysis was implemented in SAS version 9.4 .IFN-γ values were log-transformed for analysis and were summarized as geometric means (Geo Mean) and geometric standard error (GSE), and p-values indicate the main effect contrasts from a two-way ANOVA (treatment by assay date). ELISA antibody titers were compared by Welch’s Y. pestis was evaluated (50) aerosol values of Y. pestis CO92 are similar in BALB/c (6.80x104 CFU) and CD-1 mice (3.30x104 CFU) , 0.1 µg rF1V, 1.0 µg rF1V or 10 µg rF1V. All vaccine preparations including the PBS group contained 500 µg/dose of Alhydrogel.To facilitate subsequent studies on vaccine efficacy and future improvements to the formulation, the susceptibility of inbred BALB/c and outbred CD-1 mice to F1+ (CO92) strain of valuated , 48. The50 [2.02x106 CFU] and CD-1 mice received approximately 53 LD50 [1.75x106 CFU]). Mice were monitored for 21 days post challenge. All (10/10) BALB/c control group mice challenged with Y. pestis CO92 succumbed or were euthanized within 4 days of infection while all mice vaccinated with 0.1 µg rF1V died or were euthanized within 7 days post challenge (p=0.2 vs PBS for). Of the remaining rF1V vaccinated mice that were challenged with Y. pestis CO92, 1/10 (p=0.1 vs PBS) and 5/10 (p<0.001 vs PBS) mice survived the challenge that were vaccinated with 1.0 µg and 10 µg of rF1V, respectively (Y. pestis CO92 succumbed or were euthanized within 4 days of infection. Only 1/10 (p=0.1 vs PBS) CD-1 mice vaccinated with 0.1 µg rF1V survived, but despite a higher challenge dose, all (p<0.0001 vs PBS) mice in the 1.0 µg and 10 µg rF1V groups survived for the duration of the study .Both BALB/c and CD-1 mice were exposed to aerosolized bacteria 26 days post vaccination  and 10 µg  rF1V dose which likely contributed to the higher level of protection against Y. pestis CO92 challenge . Furthermore, IgG1 titers were also higher in CD-1 versus BALB/c mice but IgG2a titers were lower. Statistical significance in antibody titers was reached for all groups within both strains of mice relative to PBS vaccinated group with the exceptions of antibody titers in CD-1 mice vaccinated with 0.1 µg rF1V dose (p=0.05-0.1) and in IgG2a titers in CD-1 mice vaccinated with 1.0 µg rF1V dose (p=0.1). Overall, the predominant antibody response was IgG1 biased in both strains of mice. Due to the high level of protection seen in CD-1 mice, the more susceptible BALB/c mouse model was chosen for further studies.On the day prior to challenge (day 25), 4 mice from each group were euthanized and assayed for antibody titers against rF1V. Antibody response (IgG) increased with increasing vaccine dose in both BALB/c and CD-1 mice. Relative to BALB/c mice, CD-1 mice had a significantly greater overall antibody response at 1.0 µg  strain, whereby the immune response generated by rF1V would be exclusively reliant on the V portion of the vaccine to protect against Y. pestis C12 challenge. We determined LD50 values for the C12 strain in both BALB/c mice and CD-1 mice by both subcutaneous (bubonic) and aerosol (pneumonic) routes  or Y. pestis C12 . Mice were monitored for 21 days post challenge. All (10/10) control group mice challenged with Y. pestis CO92 succumbed or were euthanized within 4 days of infection. Of the rF1V vaccinated mice that were challenged with Y. pestis CO92, 6/10 (p=0.0001 vs PBS) and 8/10 (p<0.0001 vs PBS) mice survived the challenge that were vaccinated with 10 µg and 20 µg of rF1V, respectively . Of the mice challenged with Y. pestis C12, all (10/10) control-infected animals succumbed to infection or were euthanized within 5 days of challenge, while 2/10 (p= 0.0208 vs PBS) and 4/10 (p=0.0019 vs PBS) mice survived the challenge that were vaccinated with 10 µg and 20 µg of rF1V, respectively . Based on survival analyses, the capsule negative (F1 deficient) Y. pestis C12 strain is more refractory to a single vaccine dose of rF1V than wild-type Y. pestis CO92 strain.We vaccinated BALB/c mice with a single subcutaneous dose of rF1V . All vaccine preparations including the PBS group contained 500 µg/dose of Y. pestis CO92 and Y. pestis C12. To this end, we vaccinated 4 groups of BALB/c mice with two subcutaneous doses of rF1V, 3 weeks apart. The groups were as follows: Control (PBS), 0.5 µg rF1V, 1.0 µg rF1V and 3.0 µg rF1V. All vaccine preparations, including the PBS group contained 500 µg/dose of Alhydrogel. The mice were challenged with aerosolized bacteria 29 days post boost vaccination with Y. pestis CO92  or Y. pestis C12 . Mice were monitored for 21 days post challenge. All (10/10) control group mice challenged with Y. pestis CO92 died or were euthanized within 4 days of infection. Of the rF1V vaccinated mice that were challenged with Y. pestis CO92, 10/10 (p<0.0001 vs PBS) mice in all groups survived the challenge that were vaccinated with 0.5 µg, 1.0 µg and 3.0 µg of rF1V . Of the mice challenged with Y. pestis C12, all (10/10) control-vaccinated animals succumbed to infection within 4 days of challenge, while 5/10 (p=0.0017 vs PBS) of the 0.5 µg rF1V vaccinated mice and 8/10 (p<0.0001 vs PBS) of the 1.0 µg and 3.0 µg of rF1V vaccinated mice survived the challenge . These results demonstrate that a double dose regimen of rF1V confers a greater level of protection against both Y. pestis CO92 and Y. pestis C12 relative to a single dose vaccine regiment. Although, protection is appreciably lower in mice challenged with the F1-negative (capsule deficient) Y. pestis C12 strain.Our next objective was to determine the level of antibody titers required to achieve 100% protection from an aerosol challenge with . Based on the comparisons between the survival analysis and antibody response, a total IgG antibody titer of 3.58x105, generated from 0.5 µg of rF1V, was sufficient to achieve 100% protection against CO92 challenge while only affording 50% protection against C12 challenge. An antibody titer of 1.27x106 resulted in 80% survival in mice challenged with Y. pestis C12, one can therefore speculate that at an even higher antibody titer could achieve 100% protection against the capsule negative strain. A stronger Th1 mediated immune response (IgG2a/IgG1 Ratio) was induced at higher rF1V doses, 0.0158 (3.0 µg rF1V) > 0.0012 (0.5 and 1.0 µg F1V) . Furthermore, two lower vaccine doses, ranging from 0.5 – 3.0 µg, of rF1V (3.58x105 - 1.27x106) administered during this study produced a significantly greater antibody response than one high dose of 10 µg (1.66x105) which was administered in the previous experiment.On the day prior to challenge, 4 mice from each group were euthanized and assayed for antibody titers against rF1V Y. pestis challenge by mainly inducing humoral immune response  and elicit an anti-Y. pestis Th1 immune response, we tested rF1V in combination with various Toll-like receptor agonists. Due to the higher level of protection  based on the saturation data presented above (We performed our immunomodulatory studies with the addition of low (5 µg) or high (25 µg) dose of TLR agonists; Pam3CSK4 (TLR2), Poly(IC)LC (TLR3), MPLA (TLR4), Imiquimod (TLR7), R848 (TLR7/8) or CpG2006 (TLR9) to our rF1V formulation with which we proceeded to vaccinate BALB/c mice. We vaccinated BALB/c mice with two subcutaneous vaccine doses, 3 weeks apart. The groups were as follows: Control (PBS), 25 µg TLR agonist alone, 0.5 µg rF1V, 0.5 µg rF1V + 5 µg TLR agonist, or 0.5 µg rF1V + 25 µg TLR agonist. All vaccine preparations including the PBS group contained a reduced amount of .There was enhanced IFN-γ production, in re-stimulated splenocytes from vaccinated mice, in the presence of either the higher  or more so, at the lower concentration  dose of CpG2006 compared to rF1V only vaccinated group (30.9 pg/mL). There was no significant change in IFN- γ production in the presence of MPLA or low dose of Imiquimod, while there was a significant inhibition in IFN-γ levels in the presence of all other TLR agonists . The highest IgG1 titers were produced in mice vaccinated with rF1V along with either, the low or the high CpG2006 doses, or with the high dose of Imiquimod. The IgG2a antibody titers increased in the presence of all TLR agonist although significance was not reached with the addition of lower dose of Pam3CSK4 or the lower or higher doses of R848. Although, the most pronounced increase in the IgG2a titer was only in the presence of CpG2006  relative to rF1V only (1.9x104) vaccinated group . Based on the IgG, IgG1 and IgG2a end point titer and the IgG2a/IgG1 ratio, the highest induction of a Th1 response was in the presence of CpG2006 with an increase of 13.5 and 30 fold in the presence of 5 µg and 25 µg CpG2006 relative to rF1V alone, respectively . Accordingly, CpG2006 was down selected for further vaccine formulations.Mice vaccinated with rF1V along with high dose (25 µg) of CpG2006, Imiquimod, R848, or with the low dose (5 µg) of Pam3CSK4 had demonstrably higher titers compared to mice vaccinated with rF1V alone 5) vaccinated mouse groups was in the presence of both the low and the high dose CpG2006     likely due to group size (N=8 in this iteration), the median time to death or euthanasia of the double dose proved to be statistically significant relative to both the single dose and control (PBS) vaccinated mice . Addition of CpG to the single dose of rF1V enhanced survival (0.1 µg rF1V + CpG [2/8 survived] vs 0.1 µg rF1V x1 [0/8 survived]) but this did not reach significance. Double dose of rF1V with CpG (0.1 µg rF1V + 5 µg CpG x 2) conferred the highest level of protection (6/8 survived) reaching significance relative to single dose of rF1V (p=0.007 vs 0.1 µg rF1V x 1) furthermore, the median time to death or euthanasia was also enhanced attaining significance against the single doses of rF1V +/- CpG .Lastly, in order to determine if the immune enhancement observed with the inclusion of CpG2006 translates to increased protection against vs 0.1 µg rF1V x 2), as determined by an ex vivo ELISpot assay , 0.1 µg rF1V +/- 5 µg CpG2006, and 0.5 µg rF1V +/- 5 µg CpG2006. The vaccinated mice were challenged with aerosolized bacteria 29 days post vaccination with Y. pestis C12  while statistical significance was reached in mice vaccinated with 0.5 µg rF1V . Addition of CpG to rF1V improved the survival of vaccinated mice post Y. pestis C12 exposure, with 8/10 (p=0.0007 vs PBS) of 0.1 µg rF1V + CpG vaccinated mice and 9/10 (p=0.0001 vs PBS) of 0.5 µg rF1V + CpG vaccinated mice surviving the challenge. Furthermore, mice vaccinated with 0.1 µg rF1V + CpG and 0.5 µg rF1V + CpG survived a high subcutaneous challenge dose  with Y. pestis C12 on day 29 post-vaccination resulting in 8/10 (p=0.0007 vs PBS) and 10/10 (p<0.0001 vs PBS) mice surviving, respectively . Once against, addition of CpG to rF1V improved the survival of vaccinated mice post Y. pestis C12 exposure even with a greater time between vaccination and bacterial challenge, with 7/10 (p=0.0031 vs PBS) of 0.1 µg rF1V + CpG vaccinated mice surviving, while mice vaccinated with 0.5 µg rF1V + CpG were fully protected against challenge . Therefore, addition of CpG to rF1V in the presence of alhydrogel enhances the level of protection conferred by rF1V vaccination.An additional set of vaccinated mice were challenged with aerosolized bacteria 43 days post vaccination with a lower dose of Yersinia pestis infection. However, unlike the historical rF1V or rV bench-scale preparations generated at the USAMRIID that demonstrated equivalent protection of mice challenged with Y. pestis CO92 or Y. pestis C12 the most recent cGMP rF1V formulation routinely protects less mice challenged with Y. pestis C12 compared to those challenged with Y. pestis CO92  post challenge, while the vaccine conferred protection in State 6-/- mice. The Stat 4-/- mice are not able to properly utilize IL-12 and IFN-y and are predisposed to a Th2-like immune response profile, while the Stat 6-/- mice are not able to use IL-4 and IL-13 and have reduced levels of IgG1 and hence have a biased Th1-like response . Of the six immunomodulators tested the TLR 9 agonist, CpG2006, produced the strongest IFN-γ response at both concentrations tested, although the response was greatest at the lower concertation . High levels of IFN-γ are indicative of Th1-like response stimulation. Furthermore, the induction of a stronger IFN-γ response in the presence of lower concentration of CpG2006 (5 µg) may be partially attributed to the oversaturation of Alhydrogel at the higher concentration of CpG2006 (25µg) resulting in inadvertent competition between CpG2006 and rF1V for the binding to Alhydrogel and hence reduction in rF1V interaction with Alhydrogel. Our preliminary results indicate that 100 µg/ml of Alhydrogel is saturated with CpG2006 in the range between 10 to 25 ug  that are based on varying structural characteristics and immunostimulatory effects . CpG-A pY. pestis strains.All TLR agonists tested, with the exception of the lower dose of Poly(IC)LC (TLR3) and R848 (TLR 7/8), to some degree promoted a stronger anti-F1V IgG antibody response in mice over that of mice vaccinated with rF1V alone. The anti-F1V IgG1 response was predominantly enhanced with the inclusion of almost all TLR agonists but the enhancement in titers was most consistent with the addition of CpG2006 at both concentrations tested. The most prominent difference that was observed in the presence of TLR agonist was in the increase of IgG2a titers in comparison to rF1V only vaccinated mice. Furthermore, addition of CpG2006 to rF1V significantly increased the IgG2a antibody titers. The increase of IgG2a isotype production further indicates the stimulation of Th1-like immune response. Adjuvanticity of CpG promoted a more Th1-like response based on the increase of the IgG2a/IgG1 ratio, which is a reflection of the Th1/Th2 immune polarization . Since rY. pestis C12 strain in rF1V vaccinated mice. With the inclusion of CpG2006 in the rF1V vaccine formulation, there was an enhancement in the level of protection against Y. pestis C12 challenge (Y. pestis C12 challenge, in the BALB/c mouse bubonic plague model (The ultimate goal of this study was to assess and improve the level of protection against challenge with aerosolized F1-negative Y. pestis antigens or potentially pairing the subunit rF1V with promising live attenuated vaccines. Additional effort should focus on elucidating the induction of Th17 response by the rF1V vaccine, specifically at the lung mucosal sites, since the production of IL-17 and IL-22 has been shown to correlate with vaccine-induced protection against aerosolized Y. pestis (Future work will be directed at refining rF1V immune response and enhancing protection of vaccinees may investigate combinations of adjuvants, such as a mixture of CpG with MPLA, adjusting the vaccine regiment to include additional boost vaccinations, supplementing the F1 and V proteins with additional protective . pestis –71. The . pestis –74.Y. pestis (Y. pestis when compared to vaccinated male mice. While the authors could not elucidate the reason for these differences, these differences must be explored later in more advanced studies. While we only examined female mice in this current study, male mice will be examined during advanced development of novel vaccine formulations such as those described here.Importantly, there are examples of differences in immune responses generated by vaccines associated with sex –77. Bowe. pestis . These aIn conclusion, the addition of CpG2006, enhanced both IFN-γ and IgG2a production in rF1V vaccinated mice. The observed dual humoral and CMI enhancement makes CpG2006 a candidate for inclusion with rF1V vaccination.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Research was conducted under an IACUC approved protocol in compliance with the Animal Welfare Act and other federal statutes and regulations related to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011.Conceptualization: CKC, SSB, KA, PLW; methodology: CKC, SSB, KA; formal analysis: CKC, SSB, KA, DPF; investigation: CKC, KA, JAB, SSB, MH, ZS, IV, NOR, CPK, JLS, RRA, JLD, LHC; resources: PLW, CKC; data curation: CKC, SSB; writing—original draft preparation: CKC and SSB; writing—review and editing: CKC, SSB, JAB, MDW, LHC, ; supervision: CKC; project administration: CKC; funding acquisition: PLW and CKC. All authors have read and agreed to the published version of the article.Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cutaneous melanoma is the deadliest type of skin cancer, although it accounts for a minority of all skin cancers. Oxidative stress is involved in all stages of melanomagenesis and cutaneous melanoma can sustain a much higher load of Reactive Oxygen Species (ROS) than normal tissues. Melanoma cells exploit specific antioxidant machinery to support redox homeostasis. The enzyme UBIA prenyltransferase domain-containing protein 1 (UBIAD1) is responsible for the biosynthesis of non-mitochondrial CoQ10 and plays an important role as antioxidant enzyme. Whether UBIAD1 is involved in melanoma progression has not been addressed, yet. Here, we provide evidence that UBIAD1 expression is associated with poor overall survival (OS) in human melanoma patients. Furthermore, UBIAD1 and CoQ10 levels are upregulated in melanoma cells with respect to melanocytes. We show that UBIAD1 and plasma membrane CoQ10 sustain melanoma cell survival and proliferation by preventing lipid peroxidation and cell death. Additionally, we show that the NAD(P)H Quinone Dehydrogenase 1 (NQO1), responsible for the 2-electron reduction of CoQ10 on plasma membranes, acts downstream of UBIAD1 to support melanoma survival. By showing that the CoQ10-producing enzyme UBIAD1 counteracts oxidative stress and lipid peroxidation events in cutaneous melanoma, this work may open to new therapeutic investigations based on UBIAD1/CoQ10 loss to cure melanoma. •UBIAD1 is associated with poor overall survival (OS) of melanoma patients and is highly expressed in melanoma cell lines.•Melanoma cell lines are dependent on UBIAD1 and plasma membrane CoQ10 for cell survival.•UBIAD1 and COQ10 protect melanoma cells from lipid peroxidation and apoptotic cell death.•NQO1 is required for UBIAD1/CoQ10-mediated melanoma lipid peroxidation protection and cell survival. MelanomIt has been recently suggested that the induction of lipid peroxidation (LP) could serve as a special target in melanomagenesis. Several protective mechanisms against lipid peroxidation in melanoma cells have been described by different studies . For exa2) mainly on the plasma membrane + and [M + NH4]+ adducts ions. Quantification of CoQ10 concentration was done relative to the internal standard by an external calibration curve. Results are presented as ng of CoQ10 per mg of proteins. The UHPLC/MS analysis was done on a Hybrid quadrupole-Orbitrap system  coupled to an UHPLC system  via a heated electrospray ionization source. Four μl of each sample was injected in the Accucore C18 100 × 2.1 (2.6 μm particle size) column. (Thermo Fischer Scientific) The UHPLC--/MS was operated at a flow rate of 250 μl/min with a gradient elution of 10 mM of ammonium formate in water (phase A) and 10 mM of ammonium formate in methanol/2-propanol 80:20 (phase B). Data were acquired in full MS/ddMS2mode with positive electrospray ionization mode. Similar procedure was used to analyze CoQ10 level in plasma membrane fraction. Ceramide 16:0 (Cer16:0) was used as internal control to normalize the CoQ10 amount among samples.Cells were seeded in 10 cm plates in DMEM supplemented medium and let to reach 80% of confluence. Cells were washed with ice-cold 1× PBS 3 times and scraped in 200 μl of 1× PBS on ice. Part of the suspension (20 μl) was used to measure protein concentration by BSA method and WB analysis to check UBIAD1 knockdown. The remaining volume (180 μl) was immediately frozen in dry ice and stored at −80°C. For the extraction of CoQ10 30 μg of protein were transferred to 300 μl of ice-cold extraction solution (ethanol/hexane 1:2) with 0.2 μM of CoQ9 used as internal standard followed by vortexing. Cells were then centrifuged at 13,200 g for 5 min at 4°C. The upper phase was transferred to a glass mass spectrometry vial. The extraction procedure was performed 2 times. The hexane was dried under a stream of N2.15trans-esterified and extracted with hexane. Hexane containing fatty acid methyl esters (FAME) was directly used for GC analysis on Agilent 5890 gas chromatograph (GC) equipped with a flame-ionization detector. FAMEs derived from TG were resolved with an Omegawax column  with injection of 1 μl running in on-column mode. The program for oven temperature was set up next: 60 °C for 3 min, increased 20°C/min to 205°C, then remained constant for 15 min. Temperature then increased 0.4°C/min up to 213°C, which was maintained for 10 min and finally increased to 240°C at 5.0 °C/min and held for 8 min. Peaks were determined in relation to a reference standard mixture .The following internal standards were used: trinonanoylglycerol and tripentadecanoylglycerol for triacylglycerols (TG), 5-α-cholestane for cholesteryl esters (CE), - cholestan-3-ol (epicoprostanol) for free cholesterol (CO). Lipids were extracted from 100 μl of sample using a chloroform-methanol solution in accordance with the Folch method (Folch J 1957). Extracted lipids were separated to classes by thin layer chromatography as previously reported . The TG 2. CE have been hydrolyzed by saponification and obtained free CO were extracted with hexane. In the end, both dried residues containing CO were derivatized in 50 μl of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Merck) for 30 min at RT. 1 μl of the resulting mixture was injected for GC-MS analysis. Measurements of sterols were recorded by gas chromatography-mass spectrometry (GC-MS) instrument Agilent 6890 GC coupled with Agilent 5973 inert MS on the single ion monitoring (SIM) mode. Splitless mode was set up for the samples injection at 270°C. Separation was done using an HP-5ms column . The oven temperature was set up initially at 200°C for 1 min, raised to 275°C at 10°C/min, then to 277°C at 0.1°C/min and finally increased to 290°C at 10 °C/min and was held for 3 min. Determination of peak was done by comparing the retention time and matching the height ratios of the characteristic ions. The internal standard method was used for quantification. 25 mm.CO and CE were eluted from the silica with 5 ml of mixture of chloroform-methanol 2:1. The solvent was collected and evaporated to dryness under a stream of N2.16Publicly available RNA expression levels and associated clinico-pathological and survival data of the skin cutaneous melanoma patients enrolled in the TCGA-SKCM cohort  were ret2.17t-test were applied. Specified number of biological replicates are defined in the legends. A p-value of less than 0.05 was considered significant. Statistical significance is reported as exact p-value or ns, when not significant.GraphPad Prism 8 was used for statistical analysis. For 2 experimental comparisons, 2-tailed unpaired Student’s t-test was used. For multiple comparisons, 1- and 2-way ANOVA and 1-sample 33.1UBIAD1, with the group with the higher expression demonstrating a worse prognosis as compared to the group with lower expression . The poor prognostic behavior of the UBIAD1high group persisted even in multivariable analysis  after correcting for standard prognostic factors such as tumor stage, age, and sex of the patient  cohort, which represents until now the largest collection of clinico-pathological information of SKCM patients with associated publicly available transcriptomics data A. We obs patient B, thus dNext, we investigated UBIAD1 expression across multiple human melanoma cell lines both at protein and mRNA levels. As control we analyzed normal human melanocytes (HEMa-LP and NHEM) and immortalized epidermal cells (HaCaT). We observed a significant expression of UBIAD1 protein in most of the melanoma cells tested, with BRAF-mutated cell lines (except for RPMI-7951) showing the highest amount of UBIAD1 . UBIAD1 UBIAD1 enzyme is responsible for the biosynthesis of the non-mitochondrial pool of CoQ10 and it plays an important role as an antioxidant enzyme . Using hUBIAD1 can be localized both in the ER and the Golgi apparatus as previously demonstrated ,30. SuchOverall, we conclude that UBIAD1 and CoQ10 are significantly expressed during melanoma progression as well as in melanoma cells carrying both BRAF and NRAS mutations. These results open the question whether UBIAD1 and CoQ10 might represent an important part of the redox systems which characterize melanoma progression and melanoma cells .3.2V600E-mutation, and the Mel Juso cell line, carrying the NRASQ61L-mutation. We then knocked-down UBIAD1 (UBIAD1KD) in these cell lines and performed a growth curve assay to determine whether UBIAD1 loss could affect melanoma proliferation. UBIAD1KD resulted in a significant reduction of cell viability in these cell lines with strongest effects on SkMel28 and A375 cell lines  protein, S6 signaling and AMPK in SkMel28, while UBIAD1KD affect significantly only AKT and mTOR/s6 signaling in A375. Compared to control cells, UBIAD1KD decreased the protein level of AKT and ERK1/2 in SkMel28 and Mel Juso, but not in A375. Hence, we observed different responses in all three cell lines. This experimental evidence raises the question of the involvement of different signaling pathways in each cell line and remains for now inconclusive.To further understand the effect of UBIAD1tabolism  [[32], [KD cells  from melanoma cells and measured CoQ10 levels  , supporting the functional role of CoQ10 in UBIAD1-dependent melanoma survival.To confirm that the loss of UBIAD1-dependent CoQ10 synthesis is responsible for cell viability defects, we performed a rescue-viability assay by treating UBIAD1ed CoQ10 . Treatme[KD Low) F. To fur[KD Low) G. We fouAltogether, these data demonstrate that UBIAD1 is required to sustain survival and that it exerts this effect through the synthesis of CoQ10 in melanoma cells.3.3KD  cell death staining. Analysis of AnnexinV/PI staining by flow cytometry allows to distinguish between “early apoptotic cells”, which are AnnexinV-positive and PI-negative (AnnexinV+/PI−) and “late apoptotic cells”, which are instead AnnexinV+/PI+. Compared to control cells, UBIAD1KD led to a significant increase of AnnexinV+/PI− signal in A375, but not in SkMel28  cell death.These results suggest that by lowering the amount of plasma membrane CoQ10, UBIAD13.42 or ubiquinol) [2 and functions as a radical-trapping antioxidant suppressing the propagation of lipid peroxides in melanoma cells. For this purpose, we evaluated FSP1 and NQO1 protein and mRNA levels in a panel of melanoma cell lines H:ubiquinone oxidoreductase activity and functions as a component of the plasma membrane redox system generating the reduced forms of CoQ10 (CoQ10Hiquinol) . It has iquinol) ,43. FSP1iquinol) . We thusll lines A–C. FSP1ll lines . UBIAD1 ll lines . InteresKD. NQO1 loss promotes increase of ROS levels similarly to UBIAD1KD  (KD and NQO1KD (KD Low) or NQO1 (NQO1KD Low) had less extreme effects on cell survival than those caused by a stronger KD (UBIAD1KD or NQO1KD). However, the concomitant mild silencing of both UBIAD1 and NQO1 enzymes had a dramatic effect on cell survival (UBIAD1KD Low + NQO1KD Low), suggesting a synergistic effect of the two enzymes in protecting melanoma cells from cell death.To further explore the role of NQO1 in melanoma cells-survival, we knock-downed NQO1 in SkMel28 and A375 melanoma cells and performed cell proliferation assays. NQO1ll lines F. Also iferation . To demo1KD Low)  to avoidd NQO1KD . Next, wd NQO1KD G. We fouKD or NQO1KD in melanoma cells  ,55.LA and MMS conceived the concept of the study. MMS provided supervision and wrote the manuscript. MMS, LA, GT and MR were involved in the experimental design and writing. MS, PC performed the metabolomics analyses. SP, FAT performed bioinformatic analyses on TCGA dataset. LA, GT carried out biological activity assays. LA, GT and MR performed cell culture experiments. LA, GT, MR, MS, PC and MMS interpreted the data. All authors agreed on the final version of the manuscript. All authors commented on the manuscript.The authors declare no competing interests.


The B2T@DMSNs formulation contained the peptide vaccine and the DMSNs without further need of other compounds neither adjuvants nor emulsions. We measured in vitro a sustained release up to 930 h. B2T@DMSNs-57 and B2T@DMSNs-156 released 23.7% (135 µg) and 22.8% (132 µg) of the total B2T. The formation of a corona of serum proteins around the DMSNs increased the B2T release up to 61% (348 µg/mg) and 80% (464 µg/mg) for B2T@DMSNs-57 and B2T@DMSNs-156. In vitro results point out to a longer sustained release, assisted by the formation of a protein corona around DMSNs, compared to the reference formulation . We further confirmed in vivo immunogenicity of B2T@DMSNs in a particle size-dependent manner. Since B2T@DMSNs elicited specific immune responses in mice with high IgG production like the reference B2T@Montanide™, self-adjuvant properties of the DMSNs could be ascribed. Our results display DMSNs as efficacious nanocarriers for peptide-based vaccine administration.Mesoporous silica nanoparticles have drawn increasing attention as promising candidates in vaccine delivery. Previous studies evaluating silica-based vaccine delivery systems concentrated largely on macromolecular antigens, such as inactivated whole viruses. In this study, we synthesized dendritic mesoporous silica nanoparticles (DMSNs), and we evaluated their effectiveness as delivery platforms for peptide-based subunit vaccines. We encapsulated and tested  Adjuvants of different kinds, such as aluminum hydroxide, mineral salts, water-oil emulsions, or liposome-based formulations have been developed to enhance efficacy  whole virus-based vaccines –3. They efficacy . Althougefficacy , 9.in vivo  in swine  (2T is currently administered emulsified with Montanide™ ISA 50V2 W/O (water in oil) . This formulation has some drawbacks. For instance, there are several studies reporting unacceptable local reactions toward the Montanide adjuvant  and have loaded them with B2T, naming the resulting nanoformulation B2T@DMSNs. Both sizes exhibited high B2T loading capacities (570 µg/mg for DMSNs-57 and 580 µg/mg for DMSNs-156) and an in vitro sustained B2T release profile over 930 h. Furthermore, RAW 264.7 macrophage cells efficiently internalized the fluorescent version of both nanoformulations in a size-dependent manner. Finally, we have confirmed a specific immune response with high IgG production upon vaccination of outbred Swiss mice (Swiss ICR-CD1) with two doses of B2T@DMSNs, obtaining similar antibody titers than those elicited by the previous gold standard B2T@Montanide™.In this study, we extend the use of DMSNs to delivery platforms for peptide-based vaccines and evaluate their in swine , 31. Preduction) . B2T is adjuvant . Moreoveadjuvant . To overFor a detailed description of the procedures and more results, we refer the readers to the The DMSNs with a diameter of 156 nm (designated as DMSNs-156) were synthesized using a modified version of a previously reported method . BrieflyThe structure of both DMSNs types was imaged with a transmission electron microscope  at an acceleration voltage of 80 kV. TEM specimens were prepared by evaporating one drop of ethanolic nanoparticle solution on Ted Pella Formvar carbon-coated copper grids. The z-potential and hydrodynamic diameter of the samples was determined in a Malvern Zetasizer ZS instrument at 25°C. Samples were dispersed in water and transferred into disposable polystyrene cuvette. The given values are the average of triplicate readings.§SI-1.1) for complementary information.See de 2T immunogen was produced as described earlier  and then properly dispersed by sonication for 5 min. The resulting mixture was gently shaken at 200 rpm for 5 h at RT. Afterward, the products were separated by centrifugation at 12,000 rpm for 10 min and washed twice with PBS. B2T encapsulation efficiency (EE%) was defined as follows:We followed the same methodology to load B2T was determined by subtracting the free B2T in the supernatant from the total amount, and the amount of free B2T in the supernatant was calculated based on the B2T calibration curve obtained in DPBS  on the loading efficiencies, and section §SI-2.1 for the loading of fluoro-B2T into DMSNs.where the amount of loaded B2T stock solution  was prepared by dissolving 2 mg lyophilized B2T powder in 2 ml DPBS. From this stock solution serial dilutions in DPBS  were prepared and measured on a Biochrom™ Ultrospec 2100 Pro UV/Vis spectrophotometer using a quartz cuvette with a 1-cm path length (with DPBS as blank). The calibration curve was constructed by plotting the absorbance at 225 nm against the corresponding B2T concentrations. See section §SI-1.3 for complementary information.A B2T and 1.0 ml DPBS (pH 7.4). Samples were gently shaken at 37°C and, at predetermined time points, the suspension was centrifuged at 12,000 rpm for 10 min. We took the supernatant and measure the absorbance (225 nm) of B2T released. The procedure was repeated for each time point and for both DMSNs. Fresh DPBS  was added to redisperse the pellet. All release measurements were performed in duplicate.Release experiments were carried out in 1.5 ml Eppendorf tubes containing 1.0 mg DMSNs loaded with Bl-glutamine, 10% heat-inactivated FBS, and 1% penicillin and streptomycin) were seeded in Ibidi μ-slide 8 well at a density of 5.0 × 104 cells/well. Cells were incubated at 37°C in an atmosphere of 5% CO2 for 24 h. Then, 30 μg/ml of fluoro-B2T@DMSNs was added to the cells. Following 0.5, 1, 2, 4, 8, and 16 h incubation, cells were washed three times with PBS, and fresh growth medium containing CellMask deep red plasma membrane was added and incubated for 8 min. After three washes with PBS, fresh PBS was added, and cells were imaged by confocal laser scanning microscopy (CLSM). See section §SI-2.2 for complementary information.RAW 264.7 cells in RPMI 1640 medium  at a density of 1.0 × 106 cells/well. Cells were incubated at 37°C in an atmosphere of 5% CO2 for 24 h. Then, 30 μg/ml fluoro-B2T@DMSNs was added. Following 0.5, 1, 2, 4, 8, and 16 h incubation, cells were washed three times with PBS, new PBS was added, and cells were carefully detached from the plates with a Falcon cell scraper. The collected cells were transferred to tubes, were placed in ice, and the nuclear dye, DAPI was added to a final concentration of 1.0 µg/ml, and incubated for 2 min. The labelled cells were then measured by flow cytometry (FC) in a BD LSRFortessa X-50 flow cytometer. The mean fluorescence intensity (MFI) and percentage of cells with a positive fluorescent signal compared to the control (untreated cells) were determined on 5,000 gated single-living cells. FACS data were processed by the method described in section §SI-2.3.RAW 264.7 cells were seeded in six-well plates in RPMI 1640 medium , in agreement with EU  and domestic  regulations. The protocol to produce antibodies was in accordance with institutional guidelines under a license from the local government (DAAM 7463) and was approved by the Institutional Animal Care and Use Committee at the CID-CSIC.All formulations were prepared on the day of injection. Mice were randomized into groups and inoculated by two subcutaneous injections over the interscapular area at day 0 and day 21. All mice were euthanized at day 40 by carbon dioxide inhalation. Animals were monitored three times per week for health during the study.2T@DMSNs in mice, two trials were performed (section §SI-3). In the first one, mice were divided into three groups as shown in 2T (B2T@Montanide™), following earlier studies (2T loaded in DMSNs-156 (B2T@DMSNs-156) in 200 µl DPBS, and the third group was treated with the same amount of DMSNs alone (163 µg DMSNs-156) in 200 µl DPBS. All groups were boosted at day 21. Blood samples were collected before vaccination (day 0) and at days 14, 20 (pre-boost), and 40 . In the second trial, aimed at assessing the impact of DMSNs size on mice immunization, mice were divided into three groups as shown in 2T@Montanide™; three mice). The second (five mice) group was treated with the formulation B2T@DMSNs-57 and the third (five mice) group with B2T@DMSNs-156. All mice were treated with the same dose of peptide vaccine (100 µg B2T). Blood sample collection was extended until day 80, to study the long-term immune effect of B2T@DMSNs.To assess immunogenicity of B studies ; the sec® plates were coated with 50 µl B2T (15.4 µg/ml) in bicarbonate/carbonate coating buffer  and incubated at 4°C overnight. After washing three times with DPBS, 50 µl of diluted serums  were incubated for 1 h at 37°C, followed by four DPBS washes. Pre-immune sera from mice were used as negative controls. Next, 50 µl of a 1:4,000 dilution of HRP-labeled rabbit anti-mouse IgG were added and incubated for 1 h at 37°C followed by five washings with DPBS. Then, 100 µl of TMB substrate solution was added for 20 min at RT in the dark. Finally, the reaction was stopped by adding 100 µl of 1 M H2SO4. The optical density (OD) of the samples was measured in an ELISA reader  at 450 nm. Titers in a log10 scale were expressed as the reciprocal of the last dilution giving the absorbance recorded in the control wells (serum at day 0) plus 2 SD. See section §SI-3.2 for complementary information on the individual response of each mice to the treatment.Specific antibodies were detected by enzyme-linked immunosorbent assay (ELISA). 96-well Costar2T@DMSNs-immunized groups in B2T-antibody titers were analyzed by one-way ANOVA, followed by Tukey’s post-hoc comparisons tests. Values are cited in the text as means ± SD. All p values are two-sided, and p values < 0.05 were considered significant. Statistical analyses were conducted using GraphPad Prism Software 5.0 .Differences among B§SI-1.1, TEM measurements showed that both types of DMSNs have an inorganic core diameter of 57 ± 9 nm (DMSNs-57) and 156 ± 10 nm (DMSNs-156) §SI-1.1, . The z-p2T peptide vaccine . The B2T amount loaded into both types of DMSNs was quantified based on its absorbance at 225 nm  reached 76% and 77%, respectively. Regardless the differences in DMSNs sizes, we measured similar loading efficiencies. We attribute this to their close z-potential values and to the equivalent hydrogen bonds and polar interactions with the peptide (2T loading capacities obtained are probably related to the strong electrostatic interaction between the anionic DMSNs and the positively charged B2T (pI 10.88) in DPBS (pH 7.4) and to the DMSNs central-radial pore structures with large surface areas  (vs. linear)  regardless of DMSNs charge. Up to 5× ionic strength, DMSNs-156 were more efficiently loading the peptide. Note that within this work the ionic strength was set at 1×. Furthermore, we observed that our synthesized, negatively charged DMSNs were significantly more effective in loading the B2T peptide than their positively charged counterparts (We performed the loading under different conditions (section §SI-1.4) , 39 to e linear) SI-5. Re2T release kinetics from the DMSNs. To this end B2T@DMSNs were dispersed in a saline buffer (1× DPBS). At given time points, we collected the supernatants after centrifugation, we measured their absorbance at 225 nm and with help of the calibration curve (2T released from the DMSNs. 2T up to 1000 h (41 days). After 700 h, the release curve reached a plateau. Both B2T@DMSNs-57 and B2T@DMSNs-156 showed similar release kinetics. The B2T amount released in B2T@DMSNs-57 and B2T@DMSNs-156 corresponds to 23.7% (135 µg) and 22.8% (132 µg) of the total amount loaded.Next, we investigated the Bin vivo administration. We therefore wanted to elucidate the impact of the protein corona on the B2T release kinetics , allowed the DMSNs to build their protein corona and measured the B2T release (section §SI-1.5) following the procedure described before. We took advantage of the distinct absorption peaks for B2T at 225 nm (section §SI-1.3) and for albumin at 280 nm  and 80% (464 µg/mg) of the total B2T loaded within B2T@DMSNs-57 and B2T@DMSNs-156, respectively. It seems evident, that the presence of BSA significantly enhances B2T release. We ascribed this effect to a competitive interaction towards the DMSNs in favor of BSA resulting in B2T displacement and release  in the supernatants. Afterwards, the BSA level in both formations kept fluctuating around 250 µg/ml  which points out to an absence of protein corona around the DMSNs. Although longer experiments would be required to draw a conclusion, these results may indicate a long-term sustained release promoted by the DMSNs. At any rate, they confirm vaccine release from the DMSNs in physiological complex media as the one in the cell.After the formation of the protein corona, B release , 46. To in vitro cellular internalization of our nanoformulations, using 1 mg DMSNs-57 and 1 mg DMSNs-156 loaded with 200 µg B2T labeled with a dye  (see section §SI-2 and 2T@DMSNs occurred in a size-dependent manner (2T@DMSNs-57 (57 nm size) and after 8 h for the fluoro-B2T@DMSNs-156 (156 nm size) . In both trials, we injected subcutaneously samples containing the same amount of B2T antigen (100 µg) at day 0 and boosted with the same dose at day 21. We performed an ELISA to detect specific anti-B2T antibodies in sera collected following the schedule shown in §SI-2). In the first trial (2T@Montanide™ (positive control), B2T@DMSNs-156, and bare DMSNs-156 (negative control). Results in 2T@DMSNs treatment elicits a consistent response with all treated mice, presenting an increase in anti-B2T IgG production values after the boost (day 40). Although the anti-B2T IgG level from B2T@DMSNs-156 is slightly lower than B2T@Montanide, these results confirm that B2T@DMSNs-156 successfully stimulates anti-B2T–specific immune response in mice. On the contrary, as expected, no enhancement of the immune response was found in mice treated with bare DMSNs-156.We next validated Bst trial Figure 42T@DMSNs and considering their long-time sustained release profile obtained in vitro  in the internalization of the B2T@DMSNs by macrophage cells in a size dependent manner. Finally, the effectivity of B2T@DMSNs as nanovaccine was validated in vivo by comparing the inmunogenic response to that of the positive control B2T@Montanide™. Mice vaccination trials showed that both DMSNs formulations increased specific B2T antibody titers in a similar manner. However, results revealed a trend toward higher antibody titers in the animal group immunized with DMSNs of smaller particle size (57 nm) in agreement with previous literature  as well as by Generalitat de Catalunya (grant 2009SGR492 to DA).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Streptomyces sp. AE170020, was isolated and selected for the purification of nematicidal substances based on its high nematicidal activity. Two highly active components, aureothin and alloaureothin, were identified, and their chemical structures were determined using spectroscopic analysis. Both compounds suppressed the growth, reproduction, and behavior of Bursaphelenchus xylophilus. In in vivo experiments, the extracts of strain Streptomyces sp. AE170020 effectively suppressed the development of pine wilt disease in 4-year-old plants of Pinus densiflora. The potency of secondary metabolites isolated from endophytic strains suggests applications in controlling Bursaphelenchus xylophilus and opens an avenue for further research on exploring bioactive substances against the pine wood nematode.Endophytic bacteria, a rich source of bioactive secondary metabolites, are ideal candidates for environmentally benign agents. In this study, an endophytic strain,  Bursaphelenchus xylophilus. B. xylophilus is a quarantined plant parasitic nematode of the Aphelenchoidoidea superfamily and belongs to clade 10 tylenchida1. Native to North America, it was first introduced to Japan at the beginning of the twentieth century2. Since then, PWN has spread further to China, South Korea, Portugal, and thereafter to Europe4. When pine trees are infected with pathogenic PWN, some dynamic host responses are induced by the nematode infection, such as the generation of superoxide anions, the development of vacuoles in ray parenchyma cells to capacity, and a dramatic increase in lipid peroxide levels, resulting in host mortality5. PWD, a disease that has reportedly caused significant damage to forestry, ecologies of the affected countries, and local economies, is becoming a matter of concern in several continents.Pine trees are one of the most important tree species in global ecosystems. They are not only a predominant component of natural reserves, parks, and urban ornamental landscapes but are also a valuable source of high-value timber carpentry items. The most serious threat to pine forests worldwide is pine wilt disease (PWD) caused by the pine wood nematode (PWN), 6. Indeed, the use of beneficial bacteria and bacterial nematicides has spawned intense interest in the development of safer alternatives for PWN management8. However, many problems remain regarding the bacterial control of nematodes, including the relative scarcity of nematicidal bacteria. Only a few bacteria have been reported to possess activity against nematodes and to show potential for the biological control of nematodes11. Identifying novel nematicidal bacteria has become urgent for the bacterial control of nematodes and is of vital practical and economic significance.The current means of controlling PWNs rely on chemical nematicides, which are expensive, inefficient and have adverse impacts on the environment and human health14. Several reports have investigated bacterial endophytes as possible agents against PWNs15. These bacteria kill nematodes by different mechanisms, including by mobilizing plant defenses and producing an array of secondary metabolites and enzymes.Endophytic bacteria that reside in the tissues of living plants and establish a symbiotic relationship with the host are relatively unstudied and potent sources of novel natural products for application in medicine, industry, and agriculturePinus spp. has been investigated using several methods, such as cultivation-based methodologies, biochemical characterization of the isolates by BIOLOG phenotypic assay16, and molecular approaches19. Strains belonging to the genera Bacillus, Paenibacillus, and Pseudomonas were isolated from several pine tree species, and some of these bacteria were found to produce siderophores17 or have the ability to fix nitrogen19. However, bacteria associated with Pinus spp. have rarely been explored with regard to their nematicidal activity against B. xylophilus20. Therefore, the objectives of this study are (i) to isolate the nematicidal substances from Streptomyces sp. AE170020, an endophytic bacteria isolated from root tissues of pine tree; (ii) to investigate the effects of active substances on B. xylophilus; and (iii) to evaluate the disease control efficacy of the strain as a biocontrol agent in vivo.The community of endophytic bacteria in 1H nuclear magnetic resonance (NMR) and 13C NMR spectra were obtained on a Bruker Biospin Avance 400 spectrometer with tetramethylsilane (TMS) as the internal standard. Electrospray ionization–mass spectrometry (ESI–MS) was performed on an Agilent 6430 LC/MS/MS and a 1100 LC/MS spectrometer. The high-performance liquid chromatography (HPLC) system comprised a Hitachi Model L-2130 pump, L-2400 UV detector, and YMC J’sphere ODS H-80 column .The 3), ethyl acetate (EtOAc), n-buthanol (n-BuOH), and acetone were purchased from Samchun Pure Chemical Co., Ltd., Korea. Dimethyl sulfoxide , trifluoroacetic acid , proteinase K, abamectin, propylene glycol monomethyl ether (PGME), propylene glycol (PG), thiobarbituric acid, trichloroacetic acid, and Na2CO3 were obtained from Sigma–Aldrich Chemie GmbH, Steinheim, Germany. MeOH and water for HPLC analysis were purchased from Duksan Pure Chemicals Co., Ltd., Korea. Folin-Ciocalteu’s phenol reagent was obtained from Merck KGaA, Darmstadt, Germany. Tris buffer and gallic acid were purchased from Bioneer Corp and MP Biomedicals, Korea, respectively. Alloaureothin and aureothin used in the experiment were separated by prep TLC, and then used as a compound with a purity of 90% or more by HPLC.Highly pure, chemical-grade n-Hexane, chloroform . After 6 weeks of inoculation, the isolate was selected and purified. Genomic DNA was extracted, and the 16S rRNA gene was amplified using polymerase reaction. The resulting 16S rRNA gene sequence was compared with the curated sequences from EzBioCloud (http://www.ezbiocloud.net/) and a phylogenetic tree was built with the neighbor-joining method.Strain AE170020 was isolated from root tissues of pine tree samples collected from Jinju , Korea. Briefly, plant samples were washed with an ultrasonic step  to remove the surface soils and epiphytes. The washed plants were put into sterilized Petri dishes with aseptic filter paper to remove the surface water. After drying, plants were subjected to a seven-step surface sterilization procedure: immersion in 0.1% Tween 20 for 1 min, followed by a 6-min wash in 5% sodium hypochlorite, a 10-min wash in 2.5% sterilized sodium thiosulfate, three washes in sterile water, a 4-min wash in 70% ethanol, five washes in sterile distilled water, and a final rinse in 5% sodium bicarbonate for 10 min. Samples were then put into 2 ml sterile tubes and grinded using liquid nitrogen and then put onto TWYE media  for 36 h. Two percent of the seed was transferred to a 500 mL flask containing 50 mL of GSS broth and incubated on a rotary shaker at 150 rpm at 28 °C for 1–10 days. The culture of strain AE170020 was then centrifuged at 13,000 rpm for 10 min to separate the broth and cells. The resultant supernatant was aseptically transferred to sterile tubes and used for a nematicidal bioassay, as described by Gao et al., with some modification22. Briefly, 50 μL of the supernatant and bacteria-free J2s of B. xylophilus (n = 30–40) specimens were added to each well of a 96-well tissue culture plate. Later, 100 μg mL−1 streptomycin was also added to each well to inhibit bacterial contamination, and GSS broth was used as a control.The strain was inoculated into the GSS broth . After incubation at 28 °C for 10 days, the morphological characteristics of the strain AE170020 strain were observed under an optical microscope (Nikon Labophot-2).To investigate morphological characteristics, strain AE170020 strain was streaked on a Bennett's agar medium. The strain was cultured at 28 °C for 14 days color of the mycelium of the metal-bearing strain were observed using a scanning electron microscope . The spore morphology was photographed as follows. Spore and spore form, size of strain AE170020 were determined on M3 agar media  with a gradient solvent system of aqueous 85% MeOH, containing 0.04% TFA, as a mobile phase was used to check the purity of the compounds. The peaks of the two active compounds were confirmed at 22 min. They were named 1 and 2, respectively  = dead juveniles/total juveniles × 100.The treated plates were stored in the dark at 25 °C, and the mortality of PWNs was recorded after 24-h treatment by visualization under a light microscope . The LC23. To assess the impact of alloaureothin and aureothin on B. xylophilus mortality, thrashing of J3s nematode was assayed. Thrashing rate was scored for 1 min at the following time points: 2, 6, and 24 h in the presence of two compounds at the indicated concentrations . Control assays were conducted in the presence of DMSO or abamectin, and all assays were conducted at 22 °C. Two trials were performed for this experiment, and six numbers of surviving nematode in each treatment were used to test the thrashing behavior.In liquids, a single movement of the stylet knob forwards and then backwards to its original position was called “thrashing”23 with some modification. Briefly, adult nematodes (~ 500) were initially treated with alloaureothin and aureothin at concentrations of 0.5 and 1.0 μg mL−1, and incubated for 24 h at 22 °C. After 24 h, 10 female and male nematodes were randomly selected and placed on a PDA plate fully covered with B. cinerea and allowed to grow. When the B. cinerea has been completely consumed by nematodes in control plates, nematodes were extracted from plates using distilled water. The recovered nematodes were then serially diluted and numbers of nematodes in 100 μL suspensions were counted. The reproduction rates  were calculated. The experiment was conducted twice, with three replicates per treatment.A population inhibition assay was conducted according to the method described by Cheng et al.23. Egg suspensions were exposed to alloaureothin and aureothin at final concentrations of 20, 15, 10, 5, 2, 1, 0.5, and 0.1 μg mL−1. Two microliters of DMSO and abamectin at the same concentration were served as negative and positive, respectively. Plates were incubated at 22 °C and nematodes of the J2s stage were scored after 48 h. The hatching rate was calculated according to the following formula: Hatching rate  = [juveniles/(eggs + juveniles) × 100]. Each treatment was repeated in three wells, and the experiment was repeated three times.Nematode eggs were obtained as above and used for the embryonic lethality tests. Experiments were conducted using established procedures−1) were used in the egg deposition assay according to an established protocol23.B. xylophilus (about 100 numbers) were treated with compound for 24 h in the 96-well plates. Approximately 10 male and 10 female nematodes were then selected randomly and transferred to Petri dishes and allowed to lay eggs for 24 h. The eggs laid by a single female were then recorded and the nematodes inside the female body were also imaged. The experiments were performed two times with six repetitions.One-day-old female nematodes in the presence of alloaureothin, aureothin or abamectin  and Pinus densiflora were transferred to a greenhouse for at least 1 month prior to the pot test and watered every other day and fertilized if necessary. The average temperature of the greenhouse was 25 ± 5 °C. Acetone extracts of strain AE170020 were prepared as described above and dissolved using DPPT solvent . The solution of abamectin in DPPT solvent served as a positive control. Holes were made in the trunk of pine trees  by using an electric drill about 5 cm above the ground level. Then, 200 μL of strain extract was injected into holes at different concentrations, and holes were immediately covered with parafilm. The final concentration of the acetone extract was 7.2 mg, 3.6 mg, and 1.8 mg per tree and abamectin was 3.6 mg per tree. Control plants were injected with the DPPT solution. Inoculations of B. xylophilus were performed 1 week after the injection of chemicals in the trunk. B. xylophilus were reared on B. cinerea and extracted; they were then washed with distilled water at least five times to remove any fungal hyphae present. Final nematode density in the suspension was adjusted to about 20,000 nematodes per mL. A hole in the trunk of each pine tree was made approximately 20 cm above the soil. A 0.1 mL aliquot of the nematode suspension (containing about 2000 nematodes: a mixture of adults and juveniles) was pipetted into the holes and the holes were covered with parafilm. Control plants were also wounded but distilled water was used instead of the PWN solution. Pine trees were watered three times a week. Three runs with five replicates for each treatment were conducted. External symptoms of pine trees were visually assessed at 15, 30, 45, and 60 days after the inoculation and results were recorded at 60 days.Injection of active substances into pine trees was performed to evaluate the control efficacy in vivo, as previously describedSixty days after inoculation, three pine trees from each treatment in one trial were randomly selected and collected. Plants were cut immediately above the ground. Needles and the twigs were removed, and the stems were chopped into small pieces. Stems were immediately weighed and used for nematode quantification. The remaining stems and leaves were stored at − 80 °C for chemical analysis.25. Five grams of the stem pieces were weighed and transferred into gauze equipped in the upper part of a 50 mL falcon tube. The plant tissues were then immersed in water for 24 h, and after that the gauze and plants were gently removed from tubes and allowed to precipitate for 12 h. Nematodes were mostly located at the bottom of the falcon tube after the precipitation and the upper water was removed to make a final volume of 10 mL. Then, 100 μL of the extracted nematodes was transferred into 96-well tissue plates, and live nematodes were counted under a light microscope. Nematode density was expressed as the number of nematodes in the stem per gram of wood. The data of each treatment were generated from three trees and obtained in triplicate.The nematode population was measured using established procedures25. Briefly, needles were homogenized with liquid nitrogen, and samples (about 0.1 g) were extracted with 10 mL of cold acetone/Tris buffer . The samples were incubated at 4 °C for 72 h, and the tubes were centrifuged at 13,000 rpm for 5 min. The absorbances were then recorded at 537, 647, and 663 nm in a UV/visible spectrophotometer . The amount of chlorophyll was calculated as follows: total chlorophyll concentration in the extract (mmol mL−1) = (0.001373A663 – 0.000897A537 – 0.003046A647) + (0.02405A647 – 0.004305A537 – 0.005507A663).Chemical analyses were performed on leaf tissues  and stem tissues (water content). Leaves collected as previously described were used for chlorophyll quantification. The extraction and determination of the chlorophyll content were performed as per the method described by26. Needles were ground with liquid nitrogen; about 100 mg of leaves was extracted with 95% methanol and sonicated for 15 min. Samples were then gently shook and allowed to react overnight in the dark at room temperature. Later, 200 μL of 10% (vol/vol) Folin–Ciocalteu’s phenol reagent was added to 100 μL of the methanolic extract. The solution was mixed thoroughly and allowed to stand for 5 min, after which 800 μL of 700 mM Na2CO3 was added, and the tubes were incubated at ambient room temperature for 2 h. Next, 150 μL of each sample solution was loaded on a 96-well microplate and the absorbance was measured at 760 nm with a spectrophotometric microplate reader (VERSAmax™). The total phenolic concentration was calculated from the gallic acid calibration curve. Data were expressed as gallic acid equivalents (GA)/g of extracts averaged from each treatment.The concentration of total soluble phenolics was determined using the protocol described by Ainsworth et al.25. After homogenization with liquid nitrogen, about 0.1 g of leaf sample was extracted with 10 mL 0.5% thiobarbituric acid in 20% trichloroacetic acid. The sample was then vortexed thoroughly and incubated at 100 °C for 30 min. Next, reaction solutions were immediately transferred to ice for 5 min and then centrifuged at 4,000 rpm for 15 min. The absorbance was monitored at 450, 532, and 600 nm in a UV/visible spectrophotometer. Calculation of MDA was based on the following formula: MDA concentration (μmol L−1) = 6.45(A532 – A600) – 0.56A450.To gain insights about plant oxidative stress and cell damage, lipid peroxidation was measured. The level of lipid peroxidation in tissues was determined in terms of the malondialdehyde (MDA) content by the method of Nunes et al.Specific water content was used as a proxy for water stress, which is usually associated with PWD. For water content determination, each wood sample was oven dried for 72 h, and the relative water content was evaluated through the following formula: relative water content (%) = (fresh weight – dry weight)/fresh weight × 100. All the chemical data were obtained from six trees and three samples per tree.50 values were determined via a probit analysis. Values are expressed as mean ± standard deviation (SD) unless indicated otherwise. The data were compared using analysis of variance (ANOVA) followed by a post-hoc test, as appropriate. Significant differences were determined according to thresholds of *p < 0.05; **p < 0.01, and ***p < 0.001. All charts and figures generated in this study were constructed using GraphPad Prism version 8.0.2.Data analyses were performed using SPSS 18.0 software (SPSS Inc.), and the LCThe morphological characteristics of the selected active strains AE170020 was analyzed. Morphological characteristics were observed by culturing three strains in M3 agar media, a basic medium for actinomycetes. The AE170020 stain formed circular colonies with white/Gray aerial mycelium and yellow substrate mycelium  sequence similarity with Streptomyces stramineus  with accession number "KCTC14792BP".The identification of 16S rRNA gene sequence using EzTaxon server revealed that strain AE170020 belongs to the genus B. xylophilus, with mortality ranging from 62.8 ± 2.5% to 94.2 ± 0.8%. The highest mortality was observed when B. xylophilus was exposure to 5-days and 6-days fermentation broths. The broth of GSS medium led to a modest mortality rate of 6.7 ± 0.6%.As shown in Fig. The nematicidal substances of strain AE170020 were also analyzed for their stability at different pH values. It was found that the supernatant of strain AE170020 exhibited high nematicidal activity within a pH range of 4.0–11.0. However, the nematicidal activity slightly decreased at pH 4.0 and 5.0, and no mortality was observed in strong acidic environments (pH 1.0–3.0) . 2 was obtained as a yellow Prism and exhibited the same UV spectrum as 1 . Its physico-chemical properties are summarized in Table 22H23NO6 from HR-ESI–MS data (m/z 398.1628). The purified 1 and 2 were a yellow powder; ESI–MS [M+H]+ m/z 398.0   could be identified and assigned to complete the 1H-13C NMR chart (Table H 5.23) and C-6 (δC 75.0), H-7  and C-7 (δC 38.5), H-9  and C-9 (δC 71.3), H-10 (δH 6.40) and C-10 (δC 120.9), H-12 (δH 6.46) and C-12 (δC 128.0), H-14 (δH 7.49) and C-14 (δC 130.8), H-15 (δH 8.15) and C-15 (δC 124.6), H-17 (δH 8.15) and C-17 (δC 124.6) and H-18 (δH 7.49) and C-18 (δC 130.8), 1-O-Me (δH 3.97) and 1-O-Me (δC 56.5), 2-Me (δH 1.81) and 2-Me (δC 7.1), 4-Me (δH 1.97)와 4-Me (δC 9.4), 11-Me (δH 2.07) and 11-Me (δC 24.3) was confirmed  could be identified and assigned to complete the 1H-13C NMR chart (Table H 5.15) and C-6 (δC 73.3), H-7  and C-7 (δC 38.3), H-9  and C-9 (δC 70.1), H-10 (δH 6.21) and C-10 (δC 126.0), H-12 (δH 6.38) and C-12 (δC 128.4), H-14 (δH 7.40) and C-14 (δC 129.6), H-15 (δH 8.21) and C-15 (δC 123.6), H-17 (δH 8.21) and C-17 (δC 123.6) and H-18 (δH 7.40) and C-18 (δC 129.6), 1-O-Me (δH 3.96) and 1-O-Me (δC 55.3), 2-Me (δH 1.86) and 2-Me (δC 6.9), 4-Me (δH 2.04) and 4-Me (δC 9.5), 11-Me (δH 2.06) and 11-Me (δC 17.7) was confirmed  and non-treated control (55 ± 3) after 24 h , aureothin (8 ± 3), and abamectin (9 ± 3) at a concentration of 1 μg mL4 h Fig. D. Upon eent Fig. E.B. cinerea were almost completely consumed by nematodes after 7 days in the non-treated plates and the DMSO-treated group exhibited a similar clearance zone to the non-treated plates  . These results indicate that these compounds prevented, or at least slowed, the beginning and progression of PWD. Although the two compounds isolated from different Streptomyces sp. than used herein have already been described and their biological activities have been studied, to our best knowledge, this is the first report exploring the nematicidal activity of alloaureothin and applying strain Streptomyces sp. AE170020 to the biocontrol of PWD under pot conditions.The trunk injection method involves injection of pesticides directly into the tree trunk; the liquids are then transported through the plant’s conductive tissues to the site of action. This method has been successfully used in biocontrol of PWD36. Phenolic substances have been considered responsible for the browning of injured or pathogen-infected plant tissues, and PWD may be a result of the production of such compounds37. In the current study, acetone extract of strain Streptomyces sp. AE170020 prevented the reductions of water content, total chlorophyll, and total polyphenolics. Lipid peroxidation is an indicator of membrane cellular damage and usually occurs in plants after PWN inoculation as a result of xylem parenchyma cell necrosis and partial destruction of the cortex and phloem38. The increasing concentration of malondiadehyde in the nematode-control group suggested a high degree of lipid oxidative damage caused by PWN, and the active substances successfully protected pine trees from this damage.In addition to the external symptoms, the examination of other characteristics of pine trees after treatments allowed a better assessment of the physiological impact of nematode infection and compound injection. Specific water content was used as a proxy for water stress, which is usually associated with PWD. A decrease in chlorophyll content was previously described in PWN-inoculated pine seedlings and is regarded as a symptom of the advanced stage of the PWD, which is induced by water deficiency in leavesStreptomyces sp. AE170020, and its related compounds for defense against B. xylophilus in vitro and in vivo under greenhouse conditions. These results support the development of endophytic microorganisms as alternatives for the management of PWD. Further investigation is needed to understand the molecular mechanisms responsible for the nematicidal activity. In addition, scale-up, modification for this strain, safety evaluation, and risk assessment should be further developed to control B. xylophilus in actual agricultural applications.Therefore, this study explored an endophytic strain, Supplementary Figures.


Kb) values of the order of 0.05 ± 0.2 μM, 0.12 ± 0.1 μM and 0.05 ± 0.3 μM for complexes in K+ alone or 40wt% PEG 200 or 20wt% PEG 8000 respectively. QW10 showed preferential cytotoxicity with IC50 values of 11.10 μM and 6.44 μM after 72 and 96 hours' incubation on Human Breast Carcinoma MDA-MB 231 cells and was found to be non-toxic with Human Embryonic Kidney (HEK-1) cells. Interestingly, we observed reduction of c-Myc gene expression by 2.5 fold due to QW10 binding and stabilizing c-MYC G4. Our study for the first time provides an expanded overview of significant structural change in human c-Myc promoter G-quadruplex upon peptide binding in potassium.We selected the G-quadruplex motif located in the nuclease-hypersensitive elements (NHE) III1 region of the c-Myc promoter and for the first time performed its interaction studies with a designed peptide (QW10). Our CD results showed that the peptide bound to the c-Myc G-quadruplex and induced a significant blue shift in the positive peak of 20 nm in KCl alone or with 40wt% PEG200 or 20wt% PEG8000 in comparison to NaCl. Our Native Gel results confirmed that peptide binding destabilized the duplex and stabilized the unimolecular G-quadruplex and not binding to i-motif. UV thermal results confirmed destabilization of bimolecular structure and stabilization of unimolecular G-quadruplex. QW10 showed preferential binding towards c-MYC promoter G4 with binding constant ( We selected the G-quadruplex motif located in the nuclease-hypersensitive elements (NHE) III1 region of the c-Myc promoter and for the first time performed its interaction studies with a designed peptide (QW10). The abnormal over-expression of c-Myc has been reported in human cancers such as breast, colon, and cervix carcinomas, as well as small-cell lung cancer, glioblastomas, osteosarcomas, and myeloid leukemia.11 The promoter region located 142–115 bp upstream from the P1 promoter of the c-Myc gene is composed of seven (NHEs), of which, NHE III1 controls 80–90% transcription of c-Myc gene and is responsible for mediating multiple pathways important in tumor cell survival. The (NHE) III1 region of the c-Myc promoter can fold up into the G-quadruplex structure,12,13 and compounds that stabilize the G-quadruplex can repress c-Myc gene expression.13–16 This could be due to the polymerase pausing at the site of G4 formation or resulted in altered protein expression and also can activate DNA damage responses, thereby potentiating their anticancer effects. Therefore, small molecule that binds and stabilizes G4 DNAs leading to be considered as an alternative and a promising strategy to effectively inhibit the growth of cancer cells and overcome the problems of drug resistance.17 Over the past decades, several derivatives of quindolines have shown effective to stabilize the c-Myc G-quadruplex and down-regulate the expression of c-Myc in cancer cells.19,20 In recent years, a number of metal complexes like ruthenium(ii) have been reported to effectively bind to, induce and stabilize c-Myc G-quadruplex DNA.21,22 Zhang et al. have been synthesized and characterized the complex [Ru(phen)2(p-tFMPIP)](ClO4)2 {p-tFMPIP = 2-(4-(trifluoromethyphenyl)-1H-imidazophenanthroline)} to stabilize the c-Myc G-quadruplex DNA and showed the growth inhibition in MDA-MB-231 cells through apoptosis pathway.18 Li et al. reported a series of aryl alkyne modified complex [Ru(phen)2(p-tFMPIP)](ClO4)2 {p-tFMPIP = 2-(4-(trifluoromethyphenyl)-1H-imidazophenanthroline)} as a groove binder of c-Myc G-quadruplex DNA which can also act as a potential luminescent switch-on probe through selectively recognizing and promoting self-assembly of c-Myc G-quadruplex DNA.21 Wu et al. demonstrated that a series of arene ruthenium(ii) complexes [(η6-C6H6)Ru(p-XPIP)Cl]Cl  {PIP = 2-phenylimidazolephenanthroline)} which were binding and stabilizing c-Myc G-quadruplex DNA and exhibit excellent inhibitory activity against MDA-MB-231 breast cancer cells.23 Recently, bioactive compound L755507 was reported as novel inhibitor blocks c-Myc–MAX heterodimerization and induces apoptosis in cancer cells.24 Despite the progress in the discovery of new G-quadruplex-interactive ligands, important questions remain regarding the ligand-binding specificity in the c-Myc G-quadruplex. To date, no small molecule inhibitors that directly target the c-Myc interaction have progressed to clinical trials except few report.25 This is due to less target selectivity, rapid metabolism and low potency.G-quadruplexes are one of the higher-order DNA structures which have been identified for their functional importance in the regions of the genome like human telomeres and oncogene promoters. In the human genome, about 376 000 putative motifs are present, which tend to form a G-quadruplex structure.in vitro studies were also employed to understand the cytotoxic effects of QW10 and its subcellular localization showing its potential to down-regulate c-Myc expression in human breast carcinoma cells and found to be non-toxic to human embryonic kidney cells. Hence, the present binding studies for the first time reveal important hints on the relationship between the structure and the selective binding of the peptide as next-generation G-4 targeting natural ligands for potential therapeutic treatment of cancer.We addressed this issue by using a designed peptide, QW10 (QQWQQQQWQQ), which may be possible to bind and stabilize c-Myc promoter G4 with high selectivity in cation specific manner. In the peptide, glutamine (Q) residues possibly may bind to the hydrogen bonding donor and acceptor sites with guanine bases followed by the intercalation of tryptophan residues. Based on the molecular design, it is possible to consider that binding modes of QW10 as hydrogen bonding and stacking interaction. In this study, we have used 22-mer studied c-Myc promoter G-quadruplex sequence and performed its interaction with designed peptide QW10. Biophysical studies such as circular dichroism, fluorescence spectroscopy, UV-thermal melting and biochemical studies Native Gel Electrophoresis were employed to investigate the binding mode of QW10 with c-Myc promoter G-quadruplex structure. Our results clearly showed that the peptide binds with c-Myc promoter G4 in cation specific manner and showed structural distinctiveness, binding with Hoogsteen bonded G-quadruplex, destabilizing the Watson–Crick hydrogen bonded DNA duplex and not binding with i-motif structure. This unique design of peptide makes the peptide structure specific and cation selective G4 targeting ligand which may open the new ways for selecting the peptide as a unique drug molecule in future for cancer patients. Furthermore, 22.126,27PAGE purified grade DNA oligonucleotide and HPLC purified peptide [QQWQQQQWQQ] was purchased from Helix Biosciences. The concentration of the peptide was determined by measuring the absorbance of Trp at the C-terminal at 280 nm at 25 °C. Single-strand concentrations of DNA oligonucleotides were determined by measuring the absorbance at 260 nm at a high temperature using a Shimadzu 1800 Spectrophotometer  connected to a thermoprogrammer. Single-strand extinction coefficients were calculated from mononucleotide and dinucleotide data using the nearest-neighbour approximation.2.2−1. Before measurement, the samples were heated to 95 °C in water bath and slowly cooled till water attains room temperature and incubated at 4 °C overnight to avoid any non-equilibrium structures. Average scans of the DNA samples were subtracted from the buffer scan and data was normalized as a function of DNA strand concentration and path length of the cuvette.CD spectra were carried out on JASCO-715 spectropolarimeter using a quartz cuvette of 1 cm path length. All the spectra were recorded in the range of 200–350 nm wavelengths at a scanning rate of 100 nm min2.3Tm values for 4 μM DNA structures were obtained from the UV melting curves as described previously (ref). The heating rates were 0.5 °C min−1. Before measurement, the samples were heated to 95 °C in water bath and slowly cooled till water attains room temperature and incubated at 4 °C overnight to avoid any non-equilibrium structures. Experiment has been repeated in triplicates to reproduce the data.UV absorbance of different samples were recorded with a Shimadzu 1800 spectrophotometer  equipped with a temperature controller. Melting curves of DNA structures were obtained by measuring the UV absorbance at 260 nm or 295 nm in buffer pH 7.0 containing 0.5 mM EDTA, 100 mM NaCl, or 100 mM KCl, with and without 40 wt% PEG 400 or 20 wt% PEG 8000 in the presence or absence of QW10 peptide at DNA : peptide ratio (1 : 0), (1 : 1), (1 : 2), (1 : 5) and (1 : 10). The 2.4For doing native gel experiment, 15% (w/v) polyacrylamide gel was used. Here in PAGE experiment, samples were prepared in 30 mM sodium cacodylate buffer (pH 7.4) containing 0.5 mM EDTA, 100 mM NaCl or 100 mM KCl, with and without 40 wt% PEG 200. The samples were heated to 95 °C in water bath and slowly cooled till water attains room temperature and incubated at 4 °C overnight. The running buffer TBE (pH 7.4) also contains the same concentration of salt and EDTA in gel as contained in oligonucleotide sample. Experiment was performed in cold room at constant 50 V. A 1 : 1 mixture of glycerol and orange-G was used for tracking the movement of DNA oligonucleotides in the gel. Finally, gel was stained using silver staining and imaged using Gel-Doc .2.5+ alone or with 40 wt% PEG 200 or 20 wt% PEG 8000.Fluorescence experiments were performed by utilizing a JASCO FP 8300 spectrofluorometer . Experiments were carried out at 25 °C in 1 cm path-length quartz cuvette. Peptide 4 μM was taken in 30 mM buffer of pH 7.0 containing 100 mM NaCl or 100 mM KCl, 0.5 mM EDTA with and without 40 wt% PEG 200 titrated with equimolar concentration of c-Myc G-quadruplex. The temperature of the cell holder was regulated by a JASCO ETC-273T temperature controller. Samples were prepared by same procedure. Excitation and emission slit width were 5 nm each and the samples were excited at 275 nm and the emission was recorded in a range of 300 nm to 500 nm. Experiment has been repeated in triplicates to reproduce the data. The fluorescence intensity of QW10 was plotted at 342 nm, 347 nm and 343 nm against DNA concentration in the presence of K2.62.6.12, humidified incubator at 37 °C. QW10 peptide-40 μM was dissolved in RPMI medium completely. The cells were maintained at 37 °C in a 5% CO2 humidified incubator using standard cell culture procedures. After 24 hours, the growth medium was removed and freshly prepared QW10 peptide stock was eleven times serially diluted by two-fold dilution  and each concentration of 100 μL was added in triplicates to the respective wells and incubated at standard conditions. Non-treated control cells were also maintained in the same conditions to compare the growth inhibition. The content in all respective wells including tests and control was decanted after 72 and 96 hours of treatment and 20 μL of reconstituted MTT (Sigma) was added. After 2 hours of dark incubation in a 5% CO2 humidified incubator, the supernatant was removed and 100 μL of MTT solubilisation solution was added and kept in a shaking incubator at 37 °C to solubilize formazan crystals. The absorbance was recorded at 570 nm using a microplate reader. The IC50 value was analyzed using GraphPad prism 8.MDA-MB-231  cells and human embryonic kidney (HEK-1) cells were procured from National Centre for Cell Sciences (NCCS), Pune, India, and grown in Roswell Park Memorial Institute Medium, RPMI . Epithelial Human breast cancer cell line MDA-MB-231 and HEK-1 were cell culture grown in 10% FBS (Gibco) and 1% HEPES (Gibco) supplemented RPMI medium (Sigma) with 1% penicillin/streptoMycin (Gibco) in 5% CO2.7We applied this qPCR to analyse the response of MDA-MB-231 cells towards QW10 peptides. QW10 peptide (100 μM) was dissolved in double distilled water to prepare its aqueous solution. MDA-MB-231 cells were grown at an exponential growth phase and subjected to QW10 treatment at two different concentrations (5 μM and 10 μM) for 24 hours. Non-treated cells were maintained at the same conditions to compare the gene expression profile. For RNA isolation, the sample content was removed and the cells were washed with phosphate buffer saline  followed by triazole washing. We have added chloroform and centrifuged at 12 000 rcf at 4 °C for 15 min. The supernatant was aliquoted and added isopropanol and centrifuged at 12 000 rcf at 4 °C for 15 min. Supernatant was discarded, 70% ethanol was added and centrifuged at 7500 rcf at 4 °C for 5 min. Ethanol was removed and nuclease-free water was added to resuspend the RNA. The RNA concentration was quantified with Nanodrop (Multiskan) and RNA integrity was evaluated through 1.3% agarose gel electrophoresis. For cDNA isolation, for each sample, a master mix  was prepared. To prepare cDNA, each PCR well contains 1000 ng RNA, master mix, and nuclease-free water. A short spin was used followed by a PCR run for 1 hour 10 minutes.−ΔΔCT method. All experiments were conducted in triplicates.qPCR was performed with applied biosystems step one plus real-time PCR system using a final volume reaction of 10 μL containing forward and reverse primer, 1× SYBR Green master mix, and cDNA. The following thermal profile was applied: 1 cycle at 95 °C for 10 min, 40 cycles at 95 °C for 10 s, 64 °C for 30 s and 72 °C for 15 s. Melting curve analysis was performed at ramping from 60 °C to 90 °C and rising by 0.5 °C every 2 s. Gene expression variations were analyzed in terms of fold induction concerning the untreated control cells by 22.7.1QW10 (QQWQQQQWQQ) is a designed peptide which is glutamine rich containing intermittent tryptophan residues. This peptide is designed to make it structure selective as glutamine contains carbonyl group and amino group which can bind to the hydrogen bonding sites of G base, hence the idea is to allow the binding of glutamine with each G so when all four Gs within G quartet plane will get saturated due to glutamine binding then tryptophan may intercalate within G-quartet planes. Hence, this arrangement of the recognition of glutamine with G base by hydrogen bonding makes the peptide structure selective and intercalation of tryptophan residues may further affect the geometry of the structure. We have systematically shown the arrangement of G-quadruplex forming motifs located in the NHE1 site in the promoter region of c-Myc gene . Fig. 1b2.7.2+ or 100 mM K+ in presence and absence of peptide  upon peptide binding. The structure of each DNA strand was recorded in the presence of 30 mM sodium cacodylate buffer pH (7.0) and 100 mM Na peptide . CD spectructure . Qualitaruplex27 . On titr2.7.3+ under cell-mimicking molecular crowding conditions using 40 wt% PEG 200  was evaluated by a curve fitting procedure as described previously.27 The melting curves with a single transition were obtained in all conditions. The Tm of the c-Myc promoter G4 was decreased initially from 63.1 °C, 61.1 °C, 51.1 °C, with the DNA peptide ratio of  and increased further to 57.1 °C, 58.1 °C at (1 : 5 and 1 : 10) respectively. These results indicated that peptide did not bind significantly with c-Myc promoter G4 structure in the presence of Na+. These results are consistent with our CD results showing the signatures of parallel G-quadruplex in control with slight decrease in CD intensity on increasing the peptide concentration in the presence of Na+ as shown above  at 48.0 °C is due to the melting of biomolecular structure while the upper transition (Tm2) at 88.1 °C is due to the melting of unimolecular G-quadruplex structure. Next, melting profiles were recorded with DNA: peptide ratio as . The Tm of lower transition (Tm1) decreased gradually from 48.1 °C, 48.0 °C, 42.0 °C, 39.1 °C and 37.1 °C while the Tm of upper transition (Tm2) was increased from 87.1 °C, 81.1 °C, 88.1 °C, 88.7 °C and 90.1 °C respectively  was 91.6 °C and 91.8 °C in the presence of 100 mM K+ and 40 wt% PEG 200 or 20 wt% PEG 8000  was evaluated as 0.05 ± 0.2 μM (+ and 40 wt% PEG 200 (+ and 40 wt% PEG 200 (Kb) values calculated were 0.12 ± 0.1 μM and 0.05 ± 0.3 μM with K+ and 40 wt% PEG 200  purine strand and (5′-TAACCCTAACCCTAACCCTAACCC-3′) by mixing purine and pyrimidine rich strand in 1 : 1 ratio at neutral pH Fig. S3a and i-mo Fig. S3b and 17.7 Fig. S3b respecti2.7.750). The results showed that QW10 significantly inhibited the proliferation (affecting cell viability) of human adenocarcinoma cells in a dose-dependent manner with IC50 values of 11.10 μM and 6.44 μM after 72 ”, Govt of India for research funding to this project (SAN No. 102/IFD/SAN/864/2018-2019).The authors declare no competing financial interest.RA-012-D2RA00535B-s001