Significant titers of anti-P18I10 binding antibodies (p < 0.01%) and P18I10-specific IFN-γ secreting splenocytes (p = 0.0002) were detected in L1:P18I10 VLP-immunized mice in comparison with licensed Gardasil-9 HPV vaccine. Furthermore, we demonstrated that insertion of HIV-1 P18I10 peptide into HPV16 L1 capsid protein did not affect the induction in anti-L1 antibodies. All in all, we expected that the mammalian cell expression system and chromatographic purification methods could be time-saving, cost-effective, scalable platforms to engineer bivalent VLP-based vaccines against HPV and HIV-1Human papillomavirus (HPV) vaccines based on HPV L1 virus-like particles (VLPs) are already licensed but not accessible worldwide. About 38.0 million people were living with HIV in 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against both viruses are an urgent need. In this study, the HIV-1 P18I10 CTL peptide from the V3 loop of HIV-1 gp120 glycoprotein was inserted into the HPV16 L1 protein to construct chimeric HPV:HIV (L1:P18I10) VLPs. Instead of the traditional baculovirus expression vector/insect cell (BEVS/IC) system, we established an alternative mammalian 293F cell-based expression system using cost-effective polyethylenimine-mediated transfection for L1:P18I10 protein production. Compared with conventional ultracentrifugation, we optimized a novel chromatographic purification method which could significantly increase L1:P18I10 VLP recovery (~56%). Chimeric L1:P18I10 VLPs purified from both methods were capable of self-assembling to integral particles and shared similar biophysical and morphological properties. After BALB/c mice immunization with 293F cell-derived and chromatography-purified L1:P18I10 VLPs, almost the same titer of anti-L1 IgG ( Human immunodeficiency virus-1 (HIV-1), which causes acquired immunodeficiency syndrome (AIDS), was discovered in the early 1980s, and since then, it became a global pandemic , includiOver a hundred types of human papilloma virus (HPV) were already known and the HPV genotypes 16 and 18 are defined to be the cause for about 70% of women cervical cancer all over the world ,10,11. RIn our prior review papers, titled design concepts of VLP-based HIV-1 and HPV vaccines, we mentioned that papillomavirus VLPs, defined as non-enveloped VLPs, could play a functional role as delivery vehicles to present HIV-1 neutralizing antibody or CTL epitopes ,14. ThisThe recombinant HPV16 L1 proteins were successfully produced in insect cells ,25, yeasUltracentrifugal approaches, such as sucrose cushion (SC) or cesium chloride (CsCl) density gradients, were widely used to isolate the HPV6 L1 VLPs previously because the large VLP mass  was separated to most of contaminants ,33,34,35In this study, the HPV 16 L1 protein acted as the structural vaccine scaffold, and the P18I10 peptide was selected as HIV-1 immunogen and inserted into DE loop of HPV16 L1 protein. The immunodominant P18I10 CTL epitope comprising 10 amino acids (residues 311–320: RGPGRAFVTI) is derived from the third variable domain (V3) of the HIV-1 Env gp120 ,44. The The chimeric L1:P18I10 DNA coding sequence was cloned into pFastBac1 and pcDNA3.1 plasmid DNA expression vector, respectively, as shown in We used immunofluorescence staining to determine the expression of L1:P18I10 proteins and evaluate the polyethylenimine (PEI) or baculovirus-mediated transfection efficiency from day 0 to day 4 D,E. The Since frequency and intensity of L1 signals detected by immunofluorescence did not directly correlate with L1:P18I10 protein expression level in the host cells, we further quantified L1:P18I10 capsid proteins by Western blot analysis F,G. L1:P8 293F cells) was approximately ~39% lower than BEVS/IC system (137.87 µg per 1 × 108 Sf9 cells) in day 4 post-transfection. As shown in 8 293F cells) could be higher than BEVS/IC system (160 µg per 1 × 108 Sf9 cells) in day 4 post-transfection  containing approximately 2% of L1:P18I10 VLPs produced from 293F expression system was loaded into the size exclusion chromatography (SEC), heparin affinity chromatography (H-AC), and ion exchange chromatography (IEX) columns, respectively C–F. As sAlthough an application note of the general electric (GE) company indicated that disassembled HPV16 L1 monomers can bind to anion exchange chromatography media (AEC) at pH 8.5 , we obseBy following previous studies ,35,48, LBased on optimized chromatographic parameters obtained from the previous section, we designed a capture, intermediate purification and polishing (CiPP) chromatographic strategy to purify 293F cell-derived L1:P18I10 VLPs in flow-through mode using CEC, SEC, and H-AC C. Most oIn order to assess in vitro stability of purified L1:P18I10 VLPs, we performed non-reducing SDS-PAGE to evaluate disulfide cross-linking of L1:P18I10 capsid proteins A. It is In order to demonstrate that purified L1:P18I10 proteins from both methods are able to self-assemble to icosahedral particles, we further performed molecular mass assessment. As shown in Transmission electron microscopy (TEM) was used to examine the morphologic conformation of purified L1:P18I10 VLPs. As shown in The chromatography-purified L1:P18I10 proteins can also in vitro self-assemble into VLPs in a diameter of around 50–60 nm B. The moTo confirm whether HIV-1 P18I10 epitopes were expressed on chimeric HPV-16 L1 capsid proteins, we first extrapolated actual concentration of purified L1:P18I10 VLPs from a standard curve of commercial HPV16 L1 VLPs A. Equal p = 0.6409). Moreover, the L1:P18I10 VLP-induced IgG in mice sera was able to bind L1:P18I10 VLPs, but not HPV16 L1 VLPs (p < 0.01%). One group of mouse was immunized with only PBS buffer as a naïve group  to set up the cutpoint. Our immunogenicity data revealed that L1:P18I10 VLPs induced high anti-HPV16 L1, but lower anti-HIV-1 P18I10 antibodies (around 0.15 at MRD 1:50). After statistical analysis, the OD value for P18I10 induced IgG was significant from the naive PBS group. Therefore, we thought there is no pre-existing antibody against P18I10. Many other critical factors that could affect the immunogenicity of L1:P18I10 VLPs should be also considered, such as immunogen insertion site among different loops of VLPs, dose, prime-boost intervals, and administration route, etc. Overall, these results suggested that chimeric L1:P18I10 VLP-immunized mice produced almost the same level of anti-L1 IgG as Gardasil-9-immunized mice, and also elicited low anti-P18I10 binding antibodies.To evaluate whether chimeric L1:P18I10 VLPs induce HPV-16 L1 and HIV-1 P18I10-specific humoral and cell-mediated immune responses in BALB/c mice, the immunization schedule was designed as shown in  L1 VLPs C. After p = 0.0132) between L1:P18I10 VLP and Gardasil-9 immunization groups. The difference might be attributed to the unspecific adjuvanticity of L1:P18I10 VLPs according our modified formulation. After unpaired t test analysis, an extremely higher frequency of IFN-γ secreting splenocytes was observed in mice homologously immunized twice with chimeric L1:P18I10 VLPs, as compared to mice receiving HPV16 Gardasil-9 vaccines in response to P18I10 peptide-stimulated splenocytes (p = 0.0002)  E. These The development of an affordable, safe, and effective preventive vaccine against HPV and HIV is still an urgent need. The capacity of production and purification system to engineer preparative expression level, purity, and yield (recovery) of chimeric HPV:HIV (L1:P18I10) VLPs may facilitate the development of VLP-based vaccine against both viruses. In this study, (i) we demonstrated that the 293F expression system could be an alternative of BEVS/IC system to produce comparable expression level of L1:P18I10 VLPs for downstream purification; (ii) the chromatographic purification method could significantly increased L1:P18I10 VLP recovery (56%) approximately 6-fold higher than ultracentrifugal approaches (11%); (iii) both ultracentrifugation and chromatography-purified L1:P18I10 VLPs shared similar biophysical properties in vitro: stability, in vitro self-assembly and morphology. However, ultracentrifugation provided a milder purification condition for integral L1:P18I10 VLPs formation; (iv) both ultracentrifugation and chromatography-purified L1:P18I10 VLPs could be characterized by HPV16 L1 and HIV-1 V3 (P18I10) mAbs; (v) chromatography-purified L1:P18I10 VLPs were immunogenic after BALB/c mice immunization. We anticipated that this scalable chromatography-based purification procedures will reduce the time, cost, and labor involved in industrial-scale manufacturing of VLP-based vaccines. This work contributes towards developing an alternative platform for production and purification of a bivalent VLP-based vaccine against HPV and HIV-1, which is urgently needed in developing and developed countries.Mycobacterium bovis Bacillus Calmette-Guérin (rBCG) expressing HIVA immunogen and boosting with recombinant viral vector MVA.HIVA was safe and elicited HIV-1-specific T-cell immune responses in BALB/c mice [The P18I10 peptides derived from HIV-1 gp120 V3 loop are presented in HIV-infected cells by major histocompatibility complex (MHC-I) class I molecules . CD8+, cB/c mice ,65,66. TB/c mice . In our B/c mice . These f8 Sf9 cells in 80 mL Grace’s insect/TNM-FH medium was around 137.87 µg. Therefore, the overall yield per unit culture volume (mg/L) of our BEVS/IC-derived L1:P18I10 VLPs was calculated to be 1.72 mg/L. In our laboratory, the Sf9 cell density at 96 h of harvest could only reach approximately 1.2 to 1.5 × 106 cells/mL. This pattern match Merck’s application note indicating that the Sf9 cell density could expand from 1.0–1.2 × 106 cells/mL to 1.5–2 × 106 cells/mL through shake flask cultures to a bioreactor [6 cells/mL by using fed-batch bioreactors under tightly monitored culture conditions for large-scale manufacturing production [A comparison of the expression level of L1:P18I10 proteins between BEVS/IC and 293F expression systems is not always straightforward, since production is also affected by complexity of VLPs and different cell culture conditions . For insoreactor . However2 to 105 baculovirus particles during VLP purification [The BEVS/IC system was widely used in the pharmaceutical industry. Although both BEVS/IC and mammalian systems have post-translational modifications (PTM), the BEVS/IC system could only perform simpler glycosylation PTM, which is not in favor of enveloped VLP production . Anotherfication .8 293F cells in 30 mL FreeStyle 293 expression medium was 85.39 µg. Thus, the overall yield per unit culture volume of 293F-derived L1:P18I10 proteins was calculated to be 2.85 mg/L. Since mammalian cells tend to be lower VLP-producers [8 293F cells) was lower than BEVS/IC system (137.87 µg per 1 × 108 Sf9 cells). However, the 293F cells in shake flask suspension cultures can grow until a defined density of 3.0 to 3.6 × 106 cells/mL, compared to Sf9 cell density of 1.2 to 1.5 × 106 cells/mL. When we changed the measure of the L1:P18I10 protein expression level (yield) from weight per unit cell (µg/1 × 108 cells) to weight per unit culture volume (mg/L), the overall L1:P18I10 protein yield using 293F expression system (2.85 mg/L) was higher than BEVS/IC system (1.72 mg/L). Therefore, we could re-clarify that the 293F expression system is capable of reaching an overall level of L1:P18I10 protein expression that could be comparable with BEVS/IC system.According to pre-studies, the expression level of recombinant proteins produced by using PEI-mediated transfection in 293E cells is around 22–50 mg/L ,73,74. Oroducers , the oveAlthough 293F cells provide a baculovirus-free platform to generate VLPs, the mechanistic understanding about polyethylenimine (PEI)-mediated plasmid DNA delivery is still unclear. The branched PEI-25K was demonstrated to be efficient for transient transfection . HoweverRegarding to VLP purification methods from former studies, the recovery of HPV16 L1 VLPs using 40% or 45% sucrose cushion (SC) is 27% and 18.1%, respectively, and the purity is ranged between 2.2 and 5.4% . Our ultPichia pastoris yeast. After chromatographic and ultracentrifugal purification process, the L1:P18I10 VLPs were recovered with 96% purity and 9.23% overall recovery [The recovery of L1:P18I10 VLPs during downstream purification is critical, because it affects overall costs in bioprocessing . To obtarecovery . Accordirecovery . Anotherrecovery . Since wrecovery . We showrecovery . Heparinrecovery ,83. Our recovery , removalIn our present study, we established optimum production and purification methods to engineer chimeric HPV:HIV (L1:P18I10) VLPs. Although the BEVS/IC system was widely used in licensed HPV prophylactic vaccine  manufacturing, low expression level of L1 capsid proteins remained challenging for the production of certain HPV types  or as ye2. The 293F cells (Gibco), derived from human embryonic kidney (HEK) 293 cells, were cultured in FreeStyle 293 expression medium (Gibco) supplemented with 5 mL/L of penicillin-streptomycin (Gibco) and incubated in a 37 °C incubator containing a humidified atmosphere of 5% CO2 on an orbital shaker platform rotating at 125 rpm.The insect Spodoptera frugiperda 9 (Sf9) cells  were grown in Grace’s insect medium (Gicbo), supplemented with 10% fetal bovine serum (FBS) (Sigma) and 100 U/mL of penicillin-streptomycin (Gibco), and incubated in a 27 °C incubator without a humidified atmosphere and COE. coli. DH10Bac contains a parent bacmid with a lacZ-mini-attTn7 fusion. When the transposition was successful, the Sf9 cells were transfected with isolated DNA to produce first generation of recombinant baculovirus. The viral titer of amplified recombinant baculovirus was determined by plaque assay. A density of 1 × 106/mL of Sf9 cells (~90% confluent) was seeded in 75 cm2 flask  with 10 mL Grace’s insect/TNM-FH medium and infected with recombinant baculovirus at a multiplicity of infection (MOI) of 1 to generate the chimeric L1:P18I10 proteins. After 96 h post-infection, the Sf9 cell density can be about 1.2 to 1.5 × 106 cells/mL with at least 20% viability.The HIV-1 P18I10 CTL peptide (RGPGRAFVTI) was inserted into the DE loop of HPV16 L1 capsid protein. HPV16 L1 DE loop sequence encoding 130–136 amino acids was replaced with P18I10I10 peptide. The recombinant baculoviruses were produced according to the manufacturer’s instructions of Bac-to-Bac BEVS/IC system with pFastBac kit . In brief, the chimeric L1:P18I10 DNA coding sequence was cloned into a baculovirus donor plasmid (pFastBac1) and transformed into competent 6/mL and transiently transfected with L1:P18 I10 pDNAs using the branched polyethylenimine with a MW of 25 kDa (PEI-25K)  at an optimized ratio of DNA to PEI 1:3 (w/w) and DNA to culture medium 1:1 (w/v) according to manufacturer’s instructions [6 cells/mL with around 50% viability.The HIV-1 P18I10 CTL peptide (RGPGRAFVTI) was inserted into the DE loop of HPV16 L1 capsid protein. HPV16 L1 DE loop sequence encoding 130–136 amino acids was replaced with P18I10I10 peptide. The L1:P18I10 DNA coding sequence was modified with Kozak sequence, optimized with human codon, flanked by the restriction enzyme sites of HindIII and XbaI and cloned into pcDNA3.1(+) vector by using GeneArt gene synthesis services . The recombinant plasmid DNA (pDNA) was transformed into DH5α competent cells (Invitrogen) for amplification and extracted by using plasmid Maxi kits . The 293F cells were cultured with 30 mL FreeStyle 293 expression medium in a 125 mL Erlenmeyer flask (Corning) to a density of 1.0 × 10ructions . The 2935 Sf9 cells) from a total of 1.2 × 107 infected Sf9 cells in a 75 cm2 flask (10 mL culture medium/flask). Additionally, we collected 100 μL culture medium  from ~1.1 × 108 transfected 293F cells in a 125 mL Erlenmeyer flask (30 mL culture medium/flask). The cells were permeabilized on the glass slide with 100% cold acetone. Subsequently, the fixed cells were probed with anti-HPV16 L1 antibody CAMVIR-1  and captured with anti-mouse IgG-FITC . Immune-stained cell monolayers were thoroughly washed with PBS and covered with mounting medium with DAPI (Abcam). The immunofluorescence images were inspected under an inverted microscope at 40× magnification. Transfection efficiency was determined by the ratio of FITC (green)-positive cells to DAPI (blue)-stained cells.We collected 100 μL culture medium  infected Sf9 cells in eight 75 cm2 flasks (10 mL culture medium/flask) or 1.0 × 108 (~1.1 × 108) transfected 293F cells in a 125 mL Erlenmeyer flask (30 mL culture medium/flask) were collected by centrifugation at 1500 rpm for 5 min and washed twice with PBS. The pellet was re-suspended with cell lysis buffer formulated with 20 mM Tris (pH = 7.1), 20 mM NaCl, 1% Triton X-100, protease inhibitor (1:100) , and Benzonase (25 U/mL) (Millipore) and 2 mM MgCl2 and was then incubated at 4 °C for 24 h for complete lysis and DNA degradation. The crude cell lysates were centrifuged at 13,000 rpm for 10 min at 4 °C, and, subsequently, clarified by the 0.45 µm syringe filter (Millipore) to remove cell debris and clumps.A total of 1.0 × 10w/v) were prepared in PBS . The clarified Sf9 cell lysate adjusted in PBS was carefully layered on the top of two-step (20% and 70%) sucrose cushion in a thin wall ultracentrifugation tube . After ultracentrifugation at 40,000 rpm  for 4 h at 4 °C, the tube was placed on ice to avoid the interface layer being re-suspended. The practically purified and concentrated L1:P18I10 VLP sample was located by UV light and collected through a puncture using a 1 mL sterile syringe.The 20% and 70% sucrose cushions (SC) (g) for 16–24 h at 4 °C. After ultracentrifugation, the tube was placed on ice to avoid the layer being re-suspended. The CsCl gradient was fractionated from the top of the tube (400 μL per fraction). The signal of L1:P18I10 VLPs in each fraction was detected by dot blot, using anti-HPV16 L1 mAb CAMVIR-1.The SC-purified L1:P18I10 VLP sample was mixed with 40% CsCl solution in PBS and scattered with the sonicator . The L1:P18I10 VLP sample was ultracentrifuged at 40,000 rpm . Since we used HiLoad Pump P-50 (GE), pH and conductivity of flowthrough (FT), eluates or eluents were determined by pH papers (Sigma) and the EC/salinity meter. The clarified 293F cell lysate were adjusted to a volume of 5 mL with the starting buffer and loaded on to the column at a flow rate of 1 mL/min. After washing with 10 mL of the starting buffer, the CEC-captured L1:P18I10 VLPs were one-step eluted with 5 mL of elution buffers  at a flow rate of 1 mL/min. The column was regenerated by washing with 5 mL of 2 M NaCl in 20 mM Tris-HCl buffer at pH 7.1 to remove remaining ionically bound proteins.The HiTrap Capto SP ImpRes column  was washed with 5 mL of ddH2O and equilibrated with 10 mL of the running buffer . The CEC-purified L1:P18I10 VLP sample was adjusted to a volume of around 20 mL with the running buffer and loaded on to the column at a flow rate of 0.6 mL/min. The first and second fractions (10 mL per fraction) containing the SEC-purified L1:P18I10 VLPs were collected through washing with the running buffer. Finally, a CIP procedure was performed again to clean the column.Before using HiTrap Capto Core 700 column , cleaning-in-place (CIP) was performed to remove the bound impurities. The column was washed with 5 mL of ddH2O and equilibrated with 10 mL of the binding buffer . The SEC-purified L1:P18I10 VLP sample (10 mL) was loaded on to the column at a flow rate of 0.5 mL/min. After washing with 10 mL of the binding buffer, the heparin-bound L1:P18I10 VLPs were one-step eluted with 5 mL of elution buffers  at a flow rate of 1 mL/min. The column was regenerated by washing with 5 mL of 2 M NaCl in 20 mM Tris-HCl buffer at pH 7.1 to remove remaining impurities. The eluates were subsequently diafiltrated 10-folds with Tris-HCl  by using 100 kDa Ultra-4 centrifugal filter devices .The HiTrap Heparin HP column  was washed with 5 mL of ddHThe L1:P18I10 VLPs purified by ultracentrifugal and chromatographic methods were mixed with 2× Laemmli sample buffer  in the presence or absence of 20 mM dithiothreitol (DTT) and reacted at room temperature (RT) for 15 min. Samples were separated by 8–16% TGX stain-free protein gels (BIO-RAD). Then, the gels were transfer to PVDF membranes. The membranes were probed with the anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:4000. After that, the membranes were incubated with anti-mouse IgG Peroxidase Conjugate  at a dilution of 1:4000. The signal was developed and visualized by chemoluminiscence using Western Blot ECL substrate kit (Bio-Rad). The blot images were acquired by using Odyssey Fc imaging system.L1:P18I10 VLPs purified from both methods were treated or un-treated with 20 mM DTT for 15 min and were then filtered out through 1000 kDa  or 100 kDa (Amicon) molecular weight cutoff (MWCO) ultrafiltration devices. The retentates were reconstituted to the original volume and collected from the filter device sample reservoir, while the filtrates were collected at the bottom of the centrifuge tube. The L1 signal was measured by using dot blot probed with anti-HPV16 L1 mAb and detected by anti-mouse IgG-peroxidase conjugate (Sigma-Aldrich). Images were acquired using Odyssey Fc Imaging System at a chemiluminescence channel.After charging the carbon-coated copper grids (Sigma-Aldrich) under ultraviolet light for 5 min, purified L1:P18I10 VLPs were absorbed on grids for 1 min and rinsed 3 times by miliQ water. The L1:P18I10 VLPs purified by ultracentrifugation in PBS  were negative-stained with 2% phosphotungstic acid (PTA) at pH 7.0 (Sigma-Aldrich) for 1 min. The L1:P18I10 VLPs purified from chromatography in Tris-HCl  were negative-stained with 2% uranyl acetate at pH 4.5 (Sigma-Aldrich) for 1 min. Excess staining agents were removed by Whatman qualitative filter paper (Sigma-Aldrich). Grids were placed in a dehumidifier chamber at least 2 h before observation. Images were acquired using a transmission electron microscopy  at magnification SA270K (50 nm), SA59000 (200 nm) and SA529500 (400 nm), respectively.The band intensity of L1 from the Western blot was quantified by densitometric assay using Image Studio Lite version 5.x software. The purified L1:P18I10 VLPs were quantified by indirect ELISA. The band intensity of total host cellular proteins (HCPs) from Coomassie-stained SDS-PAGE was quantified by densitometric assay using Image Studio Lite 5.x software. The HCPs were also quantified by BCA protein assay kit (Thermo Fisher). 2 mg/mL of bovine serum blbumin Standard (Thermo Fisher) were used to construct a standard curve plotting concentration versus absorbance. The total protein from each purification step was extrapolated from this standard curve to determine the actual amount of HCPs by using a NanoDrop 2000 spectrophotometer.Equal amounts (200 ng) of HPV16 L1 protein (Abcam) and L1:P18I10 VLPs purified from both methods were mixed with 2× Laemmli sample buffer containing 5% 2-ME and boiled at 95 °C for 5 min. Samples were separated by 8–16% TGX Stain-free protein gels and were then transferred to a PVDF membrane (Millipore) using a Semi-Dry transfer device (Bio-Rad). The membrane was blocked with 5% skim milk in TBST. Then, the membranes were probed with the anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:4000 and anti-HIV1-V3 loop mAb  at a dilution of 1:500, respectively. After that, the membranes were incubated with anti-mouse IgG Peroxidase Conjugate (Sigma-Aldrich) at a dilution of 1:4000. The signal was developed and visualized by chemoluminiscence using Western Blot ECL substrate kit (Bio-Rad). The blot images were acquired by using Odyssey Fc imaging system.n = 4 and female n = 4 per group). In the group A, BALB/c mice were immunized intramuscularly (i.m.) with 10 μg of L1:P18I10 VLPs, respectively by following a homologous prime-boost regime. In the group B, positive control mice were offered Gardasil-9 prime followed by Gardasil-9 boost intramuscularly with 10 μg of HPV16 L1 VLPs. The prime-boost interval was 2 weeks. Mice were sacrificed on day 28. Blood samples were collected from the heart of mice. Sera were recovered by centrifugation and stored at −20 °C for ELISA assay. Murine spleens were removed and pressed individually through a cell strainer  with a 5 mL syringe rubber plunger. Following the removal of red blood cells with ACK lysing buffer (Lonza), splenocytes were washed and resuspended in lymphocyte medium R10 , penicillin-streptomycin, 20 mM HEPES and 15 mM 2-ME) at a concentration of 2 × 107 cells/mL.Chromatography-purified L1:P18I10 VLPs were emulsified with an equal volume of (225 μg per each 0.5 mL dose) aluminum hydroxyphosphate sulfate (Thermo Fisher), to ensure a similar formulation to the licensed Gardasil-9 HPV vaccine . All mou2SO4. The OD of each well was measured at a wavelength of 450 nm by using ELx800 absorbance microplate reader.To measure the VLP-induced antibodies in the sera of BALB/c mice, the microtiter plates were coated with 50 µL of 2 µg/mL HPV16 L1 VLPs and HIV-1 P18I10 peptide , respectively, with 50 mM carbonate–bicarbonate buffer (pH = 9.6). The plates were incubated at 4 °C overnight. Plates were blocked with the blocking buffer (5% skim milk in TBST) at 37 °C for at least 2 h. At the same time, sera collected from group A and B immunized mice were two-fold serially diluted with 5% skim milk in TBST from a ratio of 1:50 to 1:800. After washing twice with TBST, the plates were added with the diluted sera and incubated at 37 °C for 2 h. After washing 3 times with TBST, the plates were added with recombinant protein G HRP conjugate at a dilution of 1:4000 in blocking buffer and incubated at 37 °C for 1 h. TMB was used to develop the ELISA signal and stopped with 50 µL of 2 M H5 fresh splenocytes were added to each well. Subsequently, the cells from group A and B were stimulated with 2 μg/mL of HPV16 L1 VLPs and HIV-1 P18I10 peptides, respectively, and the plates were incubated at 37 °C with 5% CO2 for 24 h. After emptying the cells by washing 5 times with PBS, the plates were added with biotinylated anti-IFN-γ detection mAb diluted to a concentration of 1 μg/mL in PBS containing 0.5% FCS and incubated for 2 h at RT. After washing 5 times with PBS, the plates were added with diluted Streptavidin-ALP (1:1000) in PBS-0.5% FCS and incubated for 1 h at RT. After the final wash, the alkaline phosphatase conjugate substrate (BIO-RAD) was added to the plate until distinct spots emerge. Color development was stopped by washing extensively in tap water, and the count spots were inspected using an ELISpot reader .The ELISpot assay was performed according to the manufacturer’s instructions . The PVDF plates  pre-treated with 70% EtOH were coated with anti-mouse IFN-γ capture mAb (15 μg/mL) in PBS and incubated at 4 °C overnight. After removing excess antibody by washing 5 times with PBS, a total of 2.5 × 10t test to determine the statistical significance between two groups.Statistical analysis was performed using Prism 6 GraphPad software . The line graph of ELISAs were analyzed by simple linear regression test to compare the slope of the two lines together and to confirm two data sets were significant different. ELISpot data were tested by unpaired Six to eight-week-old BALB/c mice were purchased from Envigo  and approved by local authorities  and the Universitat Autònoma de Barcelona Ethics Commitee. The animal welfare legislation strictly conformed to the Generalitat de Catalunya. All experimental works were approved by the local Research Ethics Committee .


The aim of the study was to investigate clinical significance of soluble PD-L1 (sPD-L1) serum level in head and neck cancer and to evaluate its role as a possible prognostic and predictive biomarker.A prospective analysis of sPD-L1 levels in 60 patients diagnosed and treated due to malignant and non-malignant lesions in the region of head and neck was performed in peripheral blood by an ELISA test.p =  0.006) depending on the histopathological advancement of the lesions, 0.704 ± 0.349 and 0.512 ± 0.177 respectively in the malignant and benign group. The separate analysis of laryngeal lesions confirmed statistical difference in sPD-L1 (p =  0.002) for the malignant lesions (0.741 ± 0.353) compared with the benign (0.489 ± 0.175). The sPD-L1 level of 0.765 ng/mL or higher, revealed 35% sensitivity and 95.5% specificity for the diagnosis of head and neck malignant lesions . The 1-year DFS was 83.3% in the group of patients with low sPD-L1 levels (< 0.765 ng/mL) and 53.8% in patients with high sPD-L1 (≥0.765 ng/mL). The 2-year OS were 68% and 69.2% respectively in both groups. The log-rank test confirmed statistically significant prognostic value of sPD-L1 level for 1-year DFS .The range of sPD-L1 in the study group was 0.16–1.63 ng/mL, mean 0.64 ± 0.32. There were no differences in the mean sPD-L1 regarding patients’ age, sex, and the localization of the lesion. Statistically significant difference was revealed in the average sPD-L1 level (sPD-L1 is a promising prognostic and early recurrence predictive biomarker for head and neck cancers, most significantly for laryngeal lesions.3. Head and Neck Cancers (HNCs) include malignancies of oral cavity, oropharynx, larynx, hypopharynx, salivary glands, nasopharynx and paranasal sinuses. HNCs are one of the most common malignancies worldwide, accounting for 5.3% of all cancers with 890,000 incidence, and 5.3% of cancer mortality with 507,000 deaths reported annually.Programmed Death protein-1 (PD-1) is an inhibitory receptor expressed on various immune response cells, including activated T-cells, B-cells, natural killer T-cells, activated monocytes and dendritic cells.Wide range of malignant tumors are known to express PD-L1 to successively escape immune response. The main form of PD-L1 is membrane-bound PD-L1 (mPD-L1), present on the cell surface. Its soluble form (sPD-L1) can be detected circulating in the blood flow of patients with solid tumors, autoimmune diseases and some other chronic conditions.One of the causes for current attention to PD-1/PD-L1 signaling pathway are monoclonal antibodies, that target it with remarkable clinical outcomes. Pembrolizumab and Nivolumab registered in the treatment of HNCs are antibodies directed at PD-1.11In recent years a growing number of studies focused on sPD-L1 is evaluating its role as a potential biomarker. Detection of this protein is convenient, fast, repeatable and objective. So far few meta-analyses have proven that high sPD-L1 level is correlated with poor survival outcomes in numerous solid tumors.17In this study we investigate the pretherapeutic levels of sPD-L1 in serum of patients with different sites of HNCs, dysplastic lesions and benign tumors. The data is analyzed and correlated with clinical features, histopathological staging and treatment outcomes.The research was approved by the local Ethics Committee, and written consent was obtained from each patient. All procedures were conducted in accordance with the ethical standards of the Declaration of Helsinki.The prospective study included 60 patients hospitalized and treated in the Department of Otorhinolaryngology, Head and Neck Surgery between June and December 2018 due to malignant and non-malignant tumors in the region of head and neck. The inclusion criteria were age above 18 years and primary tumor diagnosis. Patients with the history of another malignancy, acute inflammatory condition, chronic inflammatory disease, systemic immunotherapy were excluded. Each participant enrolled was subjected to a surgical treatment of the lesion and was observed for minimum of two years.The final diagnosis and staging were based on histopathological examination, conducted according to World Health Organization (WHO) classification for each type of malignancy. Laryngeal lesions were staged into adequate categories: non-dysplastic, Low-Grade Dysplasia (LGD), High-Grade Dysplasia (HGD) and invasive cancer. The study population included 17 female and 43 male participants; the mean age was 63.47 years . The site of a lesion was in 46 cases larynx, in 9 cases oral cavity, in 3 cases salivary gland, and in 2 cases neck . For statistical analysis the patients were divided into two groups based on a histopathological advancement. The malignant lesions group included invasive cancers (n = 34) and HGD (n = 6), and the benign lesions group consisted of non-dysplastic lesions (n = 18) and LGD (n = 2). All malignant lesions were squamous cell carcinomas Neither of the malignancy has positive expression of human papilloma virus. The non-dysplastic group clinically presented with laryngeal leukoplakia and hypertrophic mucosal changes, adenolymphoma and pleomorphic adenoma of salivary gland and two lateral neck cysts. The demography and clinical characteristics of the study population are summarized in The blood samples were obtained from peripheral vein with an interval of 0–2 days before the surgical treatment. The samples were centrifuged at 4 °C to retrieve serum and further stored at −80 °C in the biological resource repository at the Department of Laboratory Diagnostics. The national standards and protocols for collecting, processing, and storing human tissues were maintained. The serum samples were thawed shortly before determination of human Programed Death Ligand-1 (PDL-1) expression by enzyme-linked immunosorbent assay (PDL-1/B7-H1/CD274 Sunred Bio ELISA kit). The ELISA detection was performed according to manufacturer’s instruction. Individual serum concentration was reported in ng/mL.p-value lower than 0.05 was considered statistically significant for all analyses. For each statistical test, the assumptions for the tests were checked, and depending on the results, the parametric independent two-sample t-tests was used. For the graphical presentation of the results, box plots were created showing the mean, Standard Error (SE) and Standard Deviation (SD) values. Using the ROC curve analysis, the level of sPD-L1 with the highest sensitivity and specificity for differentiation between malignant and benign lesions was determined. The next part of the analysis was conducted only for malignant lesions (HGD + invasive cancer). The Kaplan-Meier curves for DFS and OS were determined depending on the critical level of sPD-L1. Log-rank test was calculated for all groups. The Cox proportional hazards model was used to quantify risk factors for recurrence and death.Parameters were evaluated using IBM SPSS Statistics 26.0 and Statistica 13.3. A t-test, statistical difference was revealed in the average sPD-L1 level (p = 0.006) depending on the histopathological staging of the lesions. The separate analysis of patients with laryngeal site also confirmed a statistical difference in the serum levels of sPD-L1 (p = 0.002) for the malignant group (0.741 ± 0.353) compared with the benign origin group (0.489 ± 0.175). The analysis of correlations between sPD-L1 levels and clinical staging of malignant lesions did not reveal significant differences, neither for early and advanced T-stages , nor for the nodal advancement (N0 vs. N1‒N3 stage). There were no significant differences in the mean serum level of sPD-L1 in regard to histopathological tumor grading, however the data distribution was in favor of G2 (n = 20) and the grading was not provided for five patients. The comparison of sPD-L1 level with smoking status and with the history of gastroesophageal reflux disease did not reveal significant differences . The 54.8% sensitivity and 93.3% specificity for identification of malignant laryngeal lesion were established for the serum sPD-L1 concentration of 0.67 ng/mL ; ; .Figure 2Disease Free Survival (DSF) and Overall Survival (OS) analysis with the Kaplan-Meier curves and the Cox proportional hazards models were performed to determine a prognostic utility of sPD-L1 in head and neck malignant lesions. The cut-off value of sPD-L1 was 0.765 ng/mL, as this level of biomarker had the highest predictive and prognostic value. The follow-up time for the study group was 24 months .Figure 3p-value = 0.035).The 1-year DFS was 83.3% in the group of patients with low sPD-L1 level (<0.765 ng/mL) and 53.8% in patients with high sPD-L1 level (≥0.765 ng/mL). The 2-year DFS in both groups were 64% and 46.2%, respectively. For the whole study population 1-year DFS amounted 73%, and 2-year DFS ‒ 57.9%. The 1-year OS valued 88% among patients with low sPD-L1 level and 69.2% among patients with high sPD-L1 level. The 2-year OS were 68% and 69.2% in both groups, respectively. The OS for study population were 81.6% for one year and 68.4% for two years. The log-rank test was calculated for all 1- and 2-years DFS and OS, but only in case of 1-year DFS the difference was statistically significant  and probability of death on average 6.68 times . The remaining variables did not have a statistically significant effect on the endpoints, although it should be noted that the sPD-L1 value was at the borderline of the significance level  for DFS  was considered statistically significant and compared to the regional stage (N-), it increased the probability of recurrence on average 3.93 times  delivered very promising results. The level of sPD-L1 was significantly related to grade, clinical stage and lymph node advancement.Clinical importance of proven and reliable prognostic biomarkers in oncological setting cannot be overestimated. They enable individualized, reasonable intensification or de-escalation of the therapy and the most appropriate follow-up. So far, a few meta-analyses have proven that high sPD-L1 level is correlated with poor survival outcomes in solid tumors,Although our study reveals some encouraging results, it is not free of limitations. The study group was fairly small. Moreover, the distribution of cancer localization within the head and neck region was not sufficient, however the reason for that was the consecutive inclusion into the study group. The representation of patients with LGD was deficient and therefore it was not possible to assess the predictive role in regard to the histopathological staging. Another limitation of the study is the lack of comparison of serum sPD-L1 level to tissue expression of PD-L1. This analysis could be conducted retrospectively, after securing grant for further investigations. The bias regarding the prognostic role of PD-L1 may be involved with the type of curative therapy, what in the future will require analysis of large and comparable in advancement cohorts.Presented results encourage further cohort-based and multicenter studies regarding predictive and prognostic role of serum sPD-L1 in head and neck cancers. Future research concerning comparison of sPD-L1 level with a tissue expression in this group is required for more comprehensive understanding and individualized treatment options.sPD-L1 is a promising predictive and prognostic biomarker for head and neck cancers, particularly for laryngeal lesions. Further research concerning this protein is required to fully understand its significance in HNCs.Conceptualization: AR, MMM, MŻ; methodology AR, MMM, MŻ; validation, AR, MMM, MŻ, IK; investigation MMM, AR, MŻ, IK; data curation MMM, MŻ, AR; writing-original draft preparation, MMM; writing-review and editing, AR, MŻ; visualization AR, MMM, MŻ; supervision, KN, UD; project administration AR, MMM, MŻ. All authors have read and agreed to the published version of the manuscript.The authors declare no conflicts of interest.


Allium sativum). The nanocomposite obtained is the result of a series of experiments combining the graphene oxide (GrOx) with two members of the Amaryllidaceae family; garlic and onion (Allium cebae), which contain different sulfur materials. The characterization arrays confirmed the successful production of silver crystal, graphene oxidation and the blending of both components. The role of the chitosan as a binder between graphene and silver nanocrystals is proved. Moreover, the study discusses garlic as an optimal source that permits the synthesis of silver nanocrystals (AgNCs) (⁓ 2 to 10 nm) with better thermal and crystallinity properties. It was also confirmed the successful production of the ChAgG nanocomposite. Escherichia coli and Staphylococcus aureus were used to demonstrate the antibacterial bioactivity and L-929 fibroblast cells were utilized to visualize their biocompatibility. The proposed ChAgG nanomaterial will be useful for functionalizing specific fiber network that represents current challenging research in the fabrication of bioactive wound dressings.An adequate wound dressing reduces time of healing, provides cost-effective care, thereby improving patients’ quality life. An antimicrobial bioactivity is always desired, for that reason, the objective of this work is to design an antimicrobial nanocomposite of chitosan/silver nanocrystals/graphene oxide (ChAgG). ChAgG nanostructured composite material is composed of chitosan from corn (Ch), and silver nanocrystals from garlic ( Based on World Health Organization statistics, almost 300,000 people worldwide die from burns each year. In severe burns, the affected cells and blood vessels are often injured and the blood supply to the wound is disturbed. Many factors such as oxygenation, infection, aging, hormones, and nutrition can potentially influence burn progression and disrupt repair with an unbalanced release of various growth factors and cytokines. Different treatment approaches such as dressings and skin substitutes have been applied to aid wound healing. A thorough understanding of the effective factors of burns can improve wound healing outcomes2.The main purpose of wound dressing is to generate a temporary protective physical barrier, absorb wound drainage, and provide the required moisture to optimize re-epithelialization. The choice of dressing depends on the anatomical and pathophysiological characteristics of the wound. Contemporary wound dressings provide additional benefits, such as antimicrobial properties and pain relief3.Despite all, the main issue to solve is bacterial infections in chronic wounds. Electrospun nanofibers offer a promising solution to the management of wound healing, and numerous options are available to load antibacterial compounds onto the nanofiber webs4.The development of new and/or improved antibacterial compound formulations with alternative mechanisms of action is taken into consideration as an important topic to include them in a wound dressing design for overcoming antimicrobial resistance5 demonstrated a strong antimicrobial capacity of a chitosan/silver nanoparticle/graphene oxide nanocomposite using both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The authors discussed that combining chitosan with graphene oxide promotes interesting properties for its proposal in a wide range of applications, especially in the biomedical field.In 2021, Su et al.6. On the other hand, different carbon nanostructures have been proposed to be used in biomedical applications, mainly graphene10. Currently, different reviews in the literature have reflected these studies, mainly in films of chitosan compounds with oxidized graphene11.Silver nanostructures such as nanocrystals, nanofibers, or nanocrystals possess antimicrobial activity which is useful for its loading in drug delivery systems such as wound dressings and topical gels to treat local wounds12. Chitosan is biodegradable, non-toxic, and promotes wound healing13. Silver on nanometer scales provides antimicrobial properties and reduces inflammation14, and graphene presents antibacterial properties on its own or mixed with different substances. Besides, it has excellent mechanical properties, conductivity, and biocompatibility7.The blend of chitosan, silver nanocrystals, and graphene has been previously investigated demonstrating a great capacity for biomedical applicationsEscherichia coli and Staphylococcus aureus was performed. Finally, a cytotoxicity biocompatibility test using L-929 fibroblast cells were used.Hence, this work aims to prepare and characterize a blend nanocomposite of chitosan, AgNCs, and graphene (ChAgG) and validate its potential biological characteristics to functionalize electrospun fibers for wound dressings. A physicochemical characterization is presented through simultaneous Thermal Analyzer DSC/TGA (SDT), X-ray diffraction (XRD), scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) demonstrating the nanocomposite conjugation. To demonstrate the potential use for its incorporation in an electrospun wound dressing, an antibacterial test using Ch) (Merck). Graphene powders (Gr) (LP Bond Research and Development of the Third Millennium S.A. of C.V), and AgNO3 (OMNICHEM) were used as received. Acetic acid at 1% v/v and H2O2 at 30% v/v were used as solvents (Merck). For the synthesis of the nanocrystals (AgNCs), AgNO3 from the commercial company OMNICHEM was used. NH4OH, distilled water, Allium sativum extract (garlic), and Allium cebae (onion) were obtained in the central market of the City of Acatlán de Osorio, México.The antimicrobial nanocomposite ChAgG was prepared using chitosan (Gr) were shredded in a food processor at 600 W for 30 s, they were dispersed at a concentration of 2.41% w/v in hydrogen peroxide (H2O2) at 30% v/v for a period of 30 s. Subsequently, this mixture was placed in a sonic bath at a frequency of 1 Hz for 25 min, until the mixture was homogeneous. The mixture was then placed in a conventional microwave oven for 30 s. After that, the container was removed and placed again in a sonic bath at 1 Hz for 60 s. These last steps were repeated 10 times. Completing these cycles of reactions by microwaves and sonic baths, the temperature increases, and a boiling graphene solution is observed, because of that, it was allowed to stand for 3–4 h cooling to an ambient temperature. To neutralize the H2O2, isopropanol was added in a 1:1 v/v ratio with respect of the H2O2. Finally, a filtering system which includes a Kitasato, Büchner funnel, and a 0.20 µm diameter nitrocellulose membrane was set up to bring the solid residues detained in the membrane, the filtered samples were dried in a conventional oven (FELISA brand) (± 5 °C) at 100 °C obtaining dry graphene. It was stored and labeled in an amber-colored vial until its use.Once the graphene powders (CH3–COOH). To obtain a homogeneous solution, the solution was blended in a food processor at 600 Watts for 30 s and then left to stand at room temperature until there are no more bubbles on the surface.A 2% w/v chitosan solution (Ch), from Merck, was prepared using 1% v/v acetic acid (Allium sativum) (A1–A5) and onion extracts (Allium cebae) (C1–C5), taking into account the base experience by some authors of this research group in the patent previously reported15.In order to obtain a higher yield in the synthesis of silver nanocrystals in our ChAgG nanocomposite, in this research, a series of experiments was performed to find the one with the highest yield, that is, choosing between garlic extracts (H2O) or in ammonium hydroxide (NH4OH), using silver nitrate (AgNO3) in crystal (solid) or in solution (liquid), maintaining in both cases a concentration of 0.98 mmol. To avoid confusing the reader, the TGA and XRD characterization of the samples with the highest concentration of silver nanocrystals between garlic and onion extracts, as well as the HRTEM of the silver nanocrystals used in the ChAgG nanocomposite are presented, where the A5 sample was selected for the synthesis of the ChAgG nanocomposite.Therefore, an experimental design was created where different variables were combined, such as the use of the plant extracts in water -S-methyl-l-cysteine sulfoxide, and γ-l-glutamyl-S-allyl-l-cysteine from garlic were extracted16 as well as thiopropanal sulfoxide, dipropenyl disulfides and propenyl propyl disulfides were obtained from onion17. At the end of this period, the organic solid residues were separated through a cloth mesh by vacuum filtration, and a reflux unit was used  to bring this garlic solution to a temperature of 75 °C. Once this temperature was reached, 1 mmol of AgNO3 was added for 30 min. A color change from turquoise blue to a brick-brown color is observed in the solution, indicating the formation of silver nanocrystals18. After 30 min of reaction at reflux conditions, the oxidized graphene was added in a 2:1 m/m ratio to the AgNCs’s solution, at the same temperature of 75 °C for another 30 min. After this time, the chitosan solution was added at 2% w/v, with an initial concentration of 0.6% v/v to the entire solution, maintaining the temperature reaction at 75 °C for the last 30 min. Finally, a filtering system with Kitasato, Büchner funnel, and 0.20 µm diameter nitrocellulose membrane was assembled and used to obtain the corresponding solids, thus comprising the formation of the graphene nanocomposite, silver nanocrystals, and chitosan (ChAgG compound).Based on the patent application for the synthesis of AgNCsSDT was carried out with a high-resolution TA Instruments 2950 thermogravimetric analyzer. Samples were evaluated in a temperature range from room temperature to 500 °C at a heating rate of 10 °C/ min in air. To perform this analysis of each of the samples, only 2 mL of each sample was taken in liquid, evaporation was not performed because it was possible to obtain results in the liquid state. The liquid samples were stored in a refrigerator (− 4 °C) before being analyzed.Ag crystal structure was confirmed by powder X-ray diffraction (XRD) in a Bruker AXS Model D-B Advance. At room temperature from the range of 5° to 70° for A1 and A5 samples, start: 2.000°–end: 70.000° for ChAgG sample—Step: 0.020°–Step time: 1.2 s–Time Started: 8 s–Cu radiation. The nanocrystals that were infiltrated in the garlic, were subjected to a temperature of 95 °C for 8 days. in an oven (± 5 °C) to perform the evaporation of the solvents and water, and later perform this analysis because of the detachment of the nanocrystals in each of the crystallizers. Any problem was identified at the laboratory temperature and humidity conditions. For the nanocrystals embedded in the onion, the temperature at which they were initially subjected was the same used for garlic, but the detachment of the nanocrystals was not achieved from the crystallizer; so, each of the samples of the onion was subjected to a temperature of 250 °C for 5 days. to achieve detachment of the onion and garlic of the nanocrystals and with this, the unit analysis of the nanocrystals can be performed to determine the crystal structure and morphology.The purity of the reagent and the Allium sativum) was performed by Scanning Electron Microscopy (SEM), on a Leica Stereoscan 440, and High-Resolution Transmission Electron Microscopy (HRTEM) on JEOL equipment model JEM-1200EX. The analysis by SEM required that samples of garlic (Allium sativum) and onion (Allium cebae) were subjected to a solvent evaporation process in an oven at a temperature of 95 °C, for 5 days and then the nanocrystals were removed from the containers . For this analysis HRTEM, the garlic (Allium sativum) and onion (Allium cebae) samples were subjected to the same treatments as in the previous analyzes, but for better evaporation of the solvents, the temperature was adjusted at 400 °C for 3 h. To assemble the samples, a solution with nanocrystals in methanol was made by 10 min shaking in an ultrasound bath. A count of the size of 50 nanocrystals presents in the SEM figure field (50 nm) and 30 nanograins conglomerates in the HRTEM figure (1 µm) were measured, and an average particle <  = change to grain diameters and standard deviations were calculated using the Image J® software.A verification of the nanocrystal morphology on garlic .The XPS apparatus also possesses a μ-FOCUS 500 X-ray monochromator . Binding energies were referenced to the adventitious C1s, O1s, and Ag peaks. Background subtraction was performed using a Shirley baseline, while mixed Gaussian/Lorentzian functions were used to fit core-level spectra.19 and carried out by inoculating the pathogenic strains Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923), previously incubated for 24 h at 35 °C in Mueller–Hinton medium (MHB at 23 g/L) in a 96 microwell plate, where the inoculated medium was placed, adding the sample at a concentration of 10 mg/mL in 0.1% DMSO solvent.ChAgG sample powder was dissolved in 0.1% dimethyl sulfoxide (DMSO) solvent at a concentration of 10 mg/mL. The antimicrobial activity test was performed using the serial dilution methodControls (gentamicin as a positive control and inoculated MHB culture medium as negative control), were handled in parallel to verify the inhibition and normal growth of the pathogens. After the inoculation was completed, the absorbance of each well was read at 620 nm using a microplate spectrophotometer (Thermo Scientific Multiskan). Subsequently, the microplates were incubated at 35° for 24, 48, and 72 h, in order to observe the inhibitory effect on the strains of the samples.A volume of 96 μL of MHB medium was inoculated with the pathogen strain to be tested and deposited in row A of the microplate, to which 4 μL of the sample (at a concentration of 10 mg/mL in 0.1% DMSO solvent) was added and homogenized. From rows B to H, a volume of 50 μL of inoculated MHB medium was deposited. A volume of 50 μL of the homogenate from row A was taken and transferred to row B by homogenizing and completing a total volume of 100 μL, of which a volume of 50 μL was taken, repeating this procedure consecutively until reaching the wells of row H, from which the remaining 50 μL were discarded. Eight dilutions of the sample of interest were obtained from the dilution process , and incubated at 35° for 24, 48, and 72 h, after which the absorbance of the microwell plate was measured at 492 nm, to calculate the inhibitory effect for each of the established concentrations. An inhibition curve was constructed at 24, 48, and 72 h of exposure, separately calculating dose response at these times, which would establish the minimum inhibitory concentration (MIC) for each sample. All experiments were done in triplicate. Statistical analysis (ANOVA P > 0.05) was performed for all groups.mus musculus tissue, were used for culturing. The cells were cultivated in DMEM, supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin–streptomycin, at 37 °C in 5% CO2, until they reached the desired confluency (80%). ChAgG samples were prepared using the eight dilutions  prepared for the antibacterial test. Then, 10, 000 cells were added per well of the L-929 fibroblasts exposing for 24 h to the ChAgG samples under the same conditions of cell incubation. Where diluted phenol 10 mg/mL was used as the positive control and the negative control was the cell suspension without any treatment.The prepared ChAgG sample (10 mg/mL 0.1% DMSO solvent), was also used for the MTT cell viability test. ATCC CCL-1, fibroblast cell line NCTC Clone 929  from After time passed, 10 µL MTT solutions to the 100 µL of the medium in each well were added. The plate was mixed by tapping gently on the side of the tray. Then, it was incubated at 37 °C for 4 h. Subsequently, 200 µL DMSO directly into the medium in each well was added and pipetted up and down several times to dissolve the formazan salt. Finally, the measure of the absorbance signal was made on a spectrophotometer at 570/630 nm. Experiments were done in triplicate. Average absorbance and standard deviation were calculated. Statistical analysis (ANOVA P > 0.05) was performed for all groups.20, TGA also provides important information about the thermal stability and kinetic information of the compounds adding evidence of the incorporation of the silver nanocrystals (AgNCs) to the sample21. The obtention of silver nanostructures can be seen by thermal analysis such as TGA, on account of the high thermal resistance of silver nanocrystals. Despite changes in morphology and size that can be observed with the increment of temperature, silver nanocrystals remain unaltered at high temperatures. When silver nanocrystals are synthesized using a biological source, it is easy to detect because organic matter degrades around 100 °C22.Despite there are several characterization techniques for the determination of the presence of nanometric crystals such as Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD), Atomic Force Microscopy (AFM), Dynamic Laser Scattering (DLS), Small-Angle X-ray Scattering (SAXS), amongst othersNH4OH24 (38–58 °C) and AgNO3 where its operating temperature shift (212–444 °C) due to the presence of all materials. In Figure-It can be seen at the Supplementary Information Figure- of TGA tAgNO3 reagent present at ~ 250 °C, this observed residual mass represented the presence of silver nanocrystals only seen in the garlic source. Hence, it was decided to continue the further synthesis of the silver nanocrystals from the garlic sourceand discard the onion source due to the total discharge of AgNO3, significant residual mass that can compete with the alternative source (garlic).According to what it is seen in Figure-Oscillatoria sp., extract, where they observed a residual mass of 23.46% that was not degraded even at higher temperatures (884–94 °C), endorsing that SDT can be used to demonstrate the presence of silver structures25.Adebayo-Tayo et al. also use the thermogravimetric analysis to monitor the bioproduction of silver nanocrystals using As described in the methodology section, the crystallinity of the silver nanocrystals synthesized from garlic and onion, were compared using the X-Ray Diffraction (XRD) technique. AgNCs samples obtained from garlic presented a higher crystallinity and number of nanocrystals, being A1 and A5 samples the ones with the best results  and Ag (200)27.The graph shows signs which are assigned to A1 and A5 experiments in which more nanocrystals were obtained. In the garlic samples, the presence in the diffraction peaks at 2θ = 38.2° and 44.2° mainly represent crystal planes of Ag, corresponding to (111) and (200)AgNO3 and different solvents was carried out using the X-ray diffraction (XRD) technique. The graph shows signs which are assigned to A1 and A5 experiments in which more nanocrystals were obtained. In the garlic samples, the presence of AgCl as chlorargyrite (PDF 00-001-1238) was observed, presenting a crystalline cubic structure  planes: (120), (102), (022) and (200). Additionally, in red lines Carbon identifying through PDF 00-003-0401, exhibited in d values 3.4000, 2.0600 and 1.6800. As expected, AgNCs are not observed, since they are less than 3%. As it is known to all28 the DRX can detect the materials present in the range of 1–3%, depending on the material analyzed. Continuing with the analysis of the diffractogram as expected, the amorphous part that comprises the nanostructured, organic material and sample holder is observed like a very wide reflection.ChAgG observed in Fig. In another perspective in the diffractogram of the nanocomposite ChAgG, in Fig.  ± 168.2 nm . Samples without graphene oxide showed that the morphology of the AgNCs appears to be mostly spherical, and that they have smaller particle sizes (mean particle size ~ 27.5 nm), and are better dispersed than a group of samples with the presence of graphene oxide where the AgNCs appear to be more agglomerated (raspberry-like) and have a larger particle size (mean particle size ~ 73.69 nm).Su et al.2− in the sensor induced the aggregation of the functionalized AgNCs by selective diazotization coupling reaction in the nanoparticle system30.In another study, silver nanocrystals (AgNCs) using 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid monosodium salt (AHNDMS) and functionalized with p-Aminobenzoic acid (PABA) were prepared, as a sensitive and selective colorimetric sensor for nitrite ions. In that study, SEM results were described, the resulted AgNCs have spherical shape, but, when nitrite ion was introduced with certain concentration to the functionalized AgNCs, the spherical AgNCs changed to an aggregated one, indicating that the presence of the NONigella sativa leaf extract was compared with other chemical synthesis methods for AgNCs. The research group examined their samples trough SEM and particle size analysis that revealed that most of green synthesized AgNCs were spherical in shape with the average size ~ 15 nm and are often agglomerated into small aggregates, while comparing with chemically-synthesized nanocrystals predominately were spherical in shape with the average size 30 nm and were often agglomerated into large aggregates (more than ~ 500 nm)31.Moreover, in a study which a green synthesized AgNCs from E. coli) excellent bioactivity was demonstrated contrary to the S. aureus strain. It is well known that the antimicrobial properties of silver nanocrystals are related to size, whereas lower sizes have better antimicrobial properties32. Despite that, less cytotoxicity in our samples was demonstrated compared with the above studies at similar nanocomposite concentrations.In our study, nanocomposite sizes were bigger than in the previously mentioned studies, and this can be reflected in the antimicrobial properties observed, compared with the cited references, despite those studies which evaluated the antimicrobial bioactivity, different methods than ours were performed, reporting excellent antimicrobial properties. In our study, we do not have excellent results in both bacteria, but for Gram-negative bacteria , the sample used on the ChAgG nanocomposite. The presented micrograph shows the cubic morphology of the obtained nanocrystals in form of crystals, which have diameters in the order of ~ 2 to 10 nm. The calculated nanoparticle average diameter and standard deviation after the measuring of 50 crystals in the field were 5.6 ± 1.5 nm  in face center cubic (fcc), hexagonal close-packed (hcp) and body center cubic (bcc) structures and random compact packaging (rcp). This is due to the collective property, which is due to dipolar interactions or to the order itself (intrinsic), according to what is reported in the literatureIn this study, the spectroscopic characterization  gives evidence of the covalent and ionic interaction of the AgNCs with the oxygen atoms of the oxidized graphene and chitosan discussed in the mentioned sections, these AgNCs are shown in the HRTEM Fig. A.35 prepared through a rapid green synthesis AgNCs using curcumin. In that study, HRTEM was used with the unique purpose in calculating the size of the crystalline nanocrystals, recorded as 12.6 ± 3.8 nm (doubled higher size than our study), which was confirmed by HRTEM, while the face-centered cubic (fcc) crystallographic structure was confirmed by powder x-ray diffraction (PXRD) and selected area (electron) diffraction SAED35.Khan et al.36 discussed the green synthesis of AgNCs using plant sources such as Piper longum, Manilkara zapota, Prosopis juliflora, and Codium capitatum extracts, amongst others. The applications of these AgNCs are intended to be used in biomedical applications such as cancer treatment. As same as our study and Khan’s group35, the HRTEM and SEM were used to observe the morphology and calculate the average sizes of AgNCs (1–100 nm), and crystallographic structure characteristics were analyzed by the XRD36.In another study, Chung et al.36.Comparing the obtained results with the reported literature which synthesized AgNCs from plants, smaller particle sizes but with agglomerated cubic structures were obtained. The resulted nanostructured material presented excellent bioactive propertieshkl values of 0.235 nm and 0.2439 nm for a set of crystal planes at different sites on the AgNCs (8.18 nm of diameter) surface. HRTEM micrographs exhibited well-defined lattice fringes of d111Ag plane . The d-spacing values of the derived diffraction planes from d111Ag = 0.2428 nm and d200Ag = 0.2126 nm, are in good agreement with XRD diffraction pattern of AgNCs39.The XRD pattern of AgNCs presented earlier in Figure-−1, attributed to the G band and the disorder-induced D band, respectively, analogous to those of graphite. On the other hand, the Raman characteristics of graphene-based materials depend not only on the phonon properties but also on the corresponding electronic properties, for example, the linear band structure in single-layer graphene (1LG) and the structures of the different bands. In 1LG, the origin of the harmonic and combining modes, like the 2D modes, define a triple resonance Raman scattering (TRRS) process, which is related to the linear dispersion of its electronic bands. In other words, by keeping this 2D band in mind, we have multilayer graphene, and it appears around ∼ 2700 cm−140. The G band (1580 cm−1) corresponds to vibrations in the graphite plane with the phonon modes in the E2g symmetry  and transverse optics (TO) in the plane). Therefore, the maximum position of the G-mode is sensitive to external perturbations, such as defects, doping, strain, and temperature, and is therefore widely used to probe the responses of graphene-based materials and related devices to external perturbations. In the high-frequency region, due to the weak interlayer coupling in Multilayer Graphene (MLG), the maximum position of the G mode, Pos (G), in the intrinsic N-layer graphene (NLG) is almost 1582 cm−1 and it is not sensitive as such to the number of layers of graphene (N). However, in graphene structures with less than ten layers, they show parameters that depend significantly on the number of layers they have in graphene (N). The D band (1362 cm−1) is generally associated with the respiratory vibrations of the sp2 rings. Therefore, this band is associated with the different disorders that the different NLGs have, which exhibit similar spectral characteristics to the 2D mode. The corresponding fundamental modes of the 2D and D peaks require a surface defect for their activation in Raman scattering. Thus, the absence of the 2D band would give us evidence of the existence of a graphene monolayer of high quality (1LG)41.The strongest and most representative bands in the Raman study of carbon structures are at 1580 and 1362 cm42. On the other hand, to identify the characteristic bands of chitosan and silver nanocrystals, these are indicated based on what is cited in the literature, which indicates that chitosan can be found at 2885 cm−1 and 1654 cm−143, mainly the amine group of chitosan at 1593 cm−144. While AgNCs are concerned, as previously reported, where these nanostructures have decorated chitosan in aerogel, they showed an important interaction in the range of 1000–1800 cm−145, precisely where the ChAgG spectrum widens enormously, overlapping between them, the signals are interacting, corresponding to the D and G bands of graphene, chitosan and silver nanocrystals. This broadening has also been documented when silver nanocrystals interact with graphene bilayers46.According to the results obtained and the tabulated values Fig.  of D, 47.According to the symmetrical shape of the signal identified as 2D, it can be deduced that there is a graphene monolayer, with respect to the 2D/G signal ratio. It can also be concluded that graphene does not have important structural defects until it interacts with AgNCs. However, the increase in the D/G ratio makes it possible to identify surface defects in the oxidation and, at the same time, a disturbance in its lamellar structure when interacting with the silver nanocrystals and chitosan, which when the graphene is oxidized, these oxides are at the extremes of the sheets and that lead to the grafting between the silver nanocrystals and the graphene sheets. Chitosan plays as a binder for the stability of these two nanostructuresC1s) is at 295–281 eV, for oxygen (O1s) is at 540–528 eV and for silver is at 364–376 eV. The three band ranges were evaluated for the nanocomposite ChAgG and, the reference (Gr and GrOx) only C1s and O1s. The deconvolution of the C1s and O1s peaks was based on the common data of different reports on XPS51. For Gr and GrOx , at 287.6 eV for carbonyl (C=O), at 288.6 eV for carboxyl groups (–COOH) of oxidized graphene, at 290 eV for carbonate (C–O*–C=O) and, at 290.9 eV for pi–pi* (π–π*) electronic transitions characteristics in graphene sheets. O1s peaks at 531.2, 532.1, 533.4, 534.3, and 535.5 eV for the molecules of carboxyl (–COOH), carbonyl (O=C), hydroxyl (O–C), carbonate (C–O*–C=O) and water (H2O), respectively.XPS was used to identify the presence of AgNCs in the ChAgG nanocomposite with respect to carbon and oxygen. The XPS spectra of graphene (Gr) and oxidized graphene (GrOx), were recorded as a reference. The range of binding energy for carbon , covalent bond (Ag–O), hydroxyl (O–C), and carbonate (C–O*–C=O), respectively. Finally, there are the two bands that are fingerprints, about the presence of the AgNCs53, this refers to the binding energies of Ag3d5/2 and Ag3d3/2 were 367 eV and 373 eV, respectively.The nanocomposite ChAgG peaks Fig. B are pre51. On the other hand, we can also notice that the hybridization of the defects (sp3) rises with respect to the references, as well as the –OH and carboxyl54. The increase of the π–π* electronic transitions (at 290.9 eV) in oxidized graphene compared to only graphene, suggests that there is exfoliation of the graphene sheets due to peroxide55.Analyzing the percentage arrangement of the peaks described in C1s of the three samples , Supplementary Information Figure-53, It is observed that 50% belongs to an ionic interaction between oxygen-silver, while 21% of this band corresponds to a covalent interaction between these atoms, deducing that the nanocrystals are linked by these two means to oxidized graphene and to molecules of chitosan.To describe the O1s peak of the ChAgG nanocomposite, Supplementary Information Figure-In this study, the Raman and XPS of the chitosan and AgNCs were not performed, since with respect to the Raman assay, only the D/G, and 2D/G bands of the graphene were studied, which are considered fingerprints to demonstrate the grafting on the surface of our nanocomposite. Neither, the XPS of the AgNCs nor chitosan were necessary, because the objective of the XPS is to obtain the presence of silver and its interaction with the oxygen molecules, either from oxidized graphene or of chitosan, for which there is evidence and it has been reported.In this case, it can be said with certainty that the presence of the AgNCs is interacting with the oxygen atoms of the oxidized graphene and chitosan, which together with the other structural characterizations , we have a nanocomposite of graphene, chitosan and silver nanocrystals, called ChAgG.Escherichia coli (ATCC 25922) and Gram-positive Staphylococcus aureus (ATCC 25923) bacteria were selected to evaluate the antibacterial activity of ChAgG samples. In general, the bioactivity of the ChAgG sample differs from the two bacteria tested representing each of their differences in cell wall composition. Nevertheless, the results showed that the ChAgG sample decrease the bacterial population of both bacteria using the highest concentration (400 µg/mL). Still, ChAgG bioactivity was more effective in Escherichia coli (ATCC 25922) than its counterpart Staphylococcus aureus (ATCC 25923).Gram-negative Staphylococcus aureus (ATCC 25923) exposed to ChAgG samples no important reduction (ANOVA P > 0.05) of cell growth was observed, a greater decrease was seen with concentrations of 400 and 200 µg/mL, where approximately a ~ 20% of cell growth reduction was showed. The highest bioactivity observed was with a concentration of 200 µg/mL at 48 h, and 12.5 µg/mL at 72 h, with ~ 75% of cell growth reported. At the lowest concentrations such as 6.25 and 3.125 µg/mL, no alteration in cell proliferation was detected at any time. These results allow us to conclude that, in general, the ChAgG samples were not effective against Staphylococcus aureus (ATCC 25923).In the case of the Escherichia coli (ATCC 25922) showed a more notable antibacterial effect than in the study with Staphylococcus aureus (ATCC 25923), whereas with just 3.125 µg/mL of the ChAgG we observed a pretty interesting bacterial bioactivity reducing bacterial population about ~ 52, 54 and 62% at 24, 48 and 72 h, respectively. The highest bioactivity of ChAgG was detected using 200 µg/mL, at 48 and 72 h, where the % of cell growth was 27 ± 0.5 and 28 ± 1% respectively. Despite good results, our ChAgG samples decrease importantly (ANOVA P > 0.05) the % growth of E. coli (ATCC 25922) but not as high as the gentamicin which was used as a positive control, which reduce the ability of cell proliferation (~ 22%), but optimization of our formulation still can be made increasing its bioactivity.In contrast, Table All data are expressed in % of growth calculated using the growth of negative control (cell growth without any stimuli). Experiments were done in triplicate.Staphylococcus aureus is associated with skin and soft tissue infections in ambulatory care56, while Escherichia coli can be isolated from surgical and traumatic wounds, foot ulcers, and decubitus3. Likewise, each of them are widely studied bacterial models, so they can be used for comparative purposes and likewise as representatives in their cell walls, being E. coli Gram-negative and S. aureus Gram-positive.The selected bacterial strains in this study can cause serious skin damage, for example, Escherichia coli (ATCC 25922), however, against S. aureus (ATCC 25923) was not as effective, a factor to consider is the multiple drugs resistance phenotype of the S. aureus bacteria (ATCC 25923). That makes it one of the most difficult pathogenic bacteria to treat57, so one of the reasons to which can be attributed these results may be the low percentage of the antibacterial compound used. Hence, Ch-functionalized compound show higher efficacy for E. coli (ATCC 25922) than for S. aureus (ATCC 25923) due to the difference in their cell walls in a study published by Dutta et al., in which it has also been shown that the antibacterial activity of chitosan incorporated, either with silver ions (Ag) or nanocrystals, is greater than the activity of each component individually58.The results showed that ChAgG compound has better behavior against E. coli (ATCC 25922). Similar results were reported by Gu et al., where it was evaluated the antibacterial properties of graphene supplemented with chitosan, confirming that the inhibition effect was stronger and dependent on the concentration of said components59.It has also been shown that the potential of G-based bacterial surfaces can be enhanced by using silver, zinc, and iron nanocrystals. Our ChAgG sample, as already mentioned, showed greater efficacy against 60. In this case, it was carried out with mouse fibroblasts L-929 where after 24 h of incubation it was observed that there was no difference between cell proliferation when compared to control .The MTT assay was performed to measure the metabolic activity of cells and estimated through the ‘color-change’ phenomenon from yellow-colored tetrazolium salt, MTT 3--2,5-diphenyltetrazolium bromide to purple-colored formazanThe proliferation of the L-929 cultures showed that the ChAgG compound decreased by ~ 21%. A slight pattern can be observed where between higher the concentration of the ChAgG compound higher the alteration of cell growth (%). Nevertheless, at 100 µg/mL was observed the highest cytotoxicity with a ~ 16% of cell growth reduction, and at 3.125 µg/mL a slight increment of 2% was observed. Almost no alteration (ANOVA P > 0.05) in cell growth was observed between concentrations of 50–3.125 µg/mL. Despite that, all these positive or negative alterations do not represent a major change in cell behavior.When performing an analysis of variance (ANOVA P > 0.05) comparing the unexposed samples with those exposed for 24 h, it was observed that there was no significant difference in the normal growth of the L929 fibroblasts. Although, in the analysis performed comparing the several concentrations between them, they did not show a significant difference to decide which of the samples is better, however, by showing that there was no significant alteration in the normal growth of L929 fibroblasts after 24 h, we assume that the ChAgG compound is not toxic to the human body, but this biocompatibility will depend of concentration, where at highest concentration cytotoxicity can be increased Fig. .Figure 661.L929 cells were used in the present study because they can be easily cultured in a reproducible manner, and also this cell line is widely used for preliminary cytotoxicity evaluation for a wide range of biomaterials because of its easy proliferation and adherence on most of the biomaterial surface62. Moreover, Toullec et al. demonstrated that the presence of pure chitosan electrospun scaffolds does not provoke any alteration on L929 cell proliferation63.About the reported cytotoxicity of chitosan, silver nanocrystal, and graphene in the presence of L929 cells. Gao et al. discussed the good biocompatibility and improved cellular attachment of L929 fibroblast cells64 evaluated the cytotoxicity of pristine graphene monolayer using the murine fibroblast L929 cell line. In that study, graphene demonstrated no cytotoxicity in L929 fibroblast and increased cell adhesion and proliferation within 24 h of culture. The authors discussed the potential capacity of graphene for the recovery of damaged subcutaneous connective tissue after injury64.In the case of the cytotoxicity of graphene, Lasocka et al.65. Hence ChAgG sample’s biocompatibility will depend on the concentration of AgNCs present in the final formulation.Finally, AgNCs cytotoxicity on L929 cells is concentration-depended, hence, a higher concentration of AgNCs presence lower cell proliferation, this phenomenon is observed in our ChAgG samples as well as the study of Składanowski et al., where the patterns of the same results are observed between both studiesThe objective of this study is to report a sequential set of characterization techniques that helps in the decision making of the experimental steps for the antimicrobial ChAgG nanocomposite synthesis. From the selection of a natural source for AgNCs production to the demonstration of chemical interaction between chitosan, graphene oxide, and the resulting AgNCs. Table From an initial screening of 10 samples, 5 using garlic as a source (A1–A5) and 5 using onion (C1–C5), were tested and the residual mass percentage (Ag presence) was calculated selecting two samples from the garlic source. Secondly, the cubic structure of the resulting AgNCs was characterized with XRD and general morphologies, and the size of the AgNCs nanocrystals and agglomerated were tested with HRTEM, and SEM, respectively. Then, Raman and XPS demonstrated the chemical interaction between chitosan, graphene oxide, and AgNC. Finally, bioactivity and biocompatibility were successfully demonstrated.66.Traditional wound dressings e.g., bandages and gauzes, although highly absorbent and effective for drying mild, exuding wounds, require a regular application, which therefore can cause pain upon dressing change. In addition, they have poor adhesion properties and cannot provide enough drainage for the wound. In this regard, the normalization of the healing process in chronic wounds is an extremely urgent task of public health and requires the creation and implementation of affordable dressings for patients with chronic wounds67.Despite all, the main issue to solve is bacterial infections in chronic wounds. Electrospun nanofibers offer a promising solution to the management of wound healing, and numerous options are available to load antibacterial compounds onto the nanofiber webs2O3 fibrous scaffolds are non-toxic, flexible, with antimicrobial activity , and can absorb and retain water. These components were effectively to be used as wound dressing generating effective wound healing. The biocompatibility was evaluated in HFF-1 fibroblast after 24 h of exposure69.In our previous work, it was demonstrated that PCL/PVP 85:15 and PCL/PVP loaded with Ag-Si/Al12. Chitosan is biodegradable, non-toxic, and promotes wound healing13, silver nanocrystals (AgNCs) give antimicrobial properties and reduce inflammation14, and graphene also confers self-antibacterial property and antibacterial properties in combination with different substances, excellent mechanical properties, conductivity, and biocompatibility7.The blend of chitosan, silver nanocrystals, and graphene has been previously investigated demonstrating a great capacity for biomedical applicationsHence, our research group will prepare and characterize PCL/PVP-ChAgG fibers and test their morphological, physicochemical, and mechanical properties of the fibers to create potential wound dressing for further biological studies.Escherichia coli rather than Staphylococcus aureus and the final composite does not provoke important cytotoxicity to L-929 fibroblast cells validating its biocompatibility in vitro. For future work, the ChAgG nanocomposite will be used to functionalize electrospun nanofibers to create bioactive antimicrobial wound dressings.Bioactive wound dressings to treat superficial skin or burn wounds that generate high exudation are desired. One of the important issues in these treatments is microbial contamination and dressing attachment to the affected zones, complicating skin healing and recovery. Our work proposed an antimicrobial nanocomposite (ChAgG) that includes chitosan, silver nanocrystal, and graphene. These components will give the final wound dressing a potential bioactive characteristic. The characterization arrays which include TGA, XPS, Raman, HRTEM, and TEM demonstrated the successful production of silver crystal graphene oxidation and the blending of the components. TGA was useful to decide whether silver nanocrystals will be prepared from garlic. X-ray diffraction demonstrated that A5’s sample has a higher crystallinity with respect to A1’s sample, therefore, being A5 the ideal preparation for the synthesis of the ChAgG nanocomposite. HRTEM and TEM demonstrated the silver nanocrystal formation with cubic structures with sizes of 2–10 nm, Moreover, the Raman study provides evidence that chitosan plays a binder role between graphene and silver nanocrystals. XPS demonstrated the successful preparation of the whole ChAgG nanocomposite. Finally, it was demonstrated that the ChAgG composite possesses better antimicrobial bioactivity against Supplementary Information.


TAR DNA-binding protein 43 (TDP-43) is a versatile RNA/DNA-binding protein with multifaceted processes. While TDP-43 has been extensively studied in the context of degenerative diseases, recent evidence has also highlighted its crucial involvement in diverse life processes beyond neurodegeneration. Here, we mainly reviewed the function of TDP-43 in non-neurodegenerative physiological and pathological processes, including spermatogenesis, embryonic development, mammary gland development, tumor formation, and viral infection, highlighting its importance as a key regulatory factor for the maintenance of normal functions throughout life. TDP-43 exhibits diverse and sometimes opposite functionality across different cell types through various mechanisms, and its roles can shift at distinct stages within the same biological system. Consequently, TDP-43 operates in both a context-dependent and a stage-specific manner in response to a variety of internal and external stimuli.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01336-5. TARDBP gene, TDP-43 is highly conserved across various species . Additionally, TDP-43 exhibits a dual role in the complex regulation of cancer-related miRNAs, acting as a promoter of cancer progression through the regulation of miR-423-3p, while also exerting inhibitory effects on cancer progression through the regulation of miR-500a-3p [TDP-43 also affects diverse functions of downstream genes. In HeLa cells, TDP-43 inhibits CDK6 by recruiting UG-rich nscripts , whereas-500a-3p .These studies underscore the heterogeneity of cancer and highlight the intricate regulatory role of TDP-43 in cancer progression. Although the mechanisms by which TDP-43 regulates cancer are complex and not yet fully understood, they likely depend on the specific cellular context and cancer type.Accumulating evidence suggests the existence of an inverse comorbidity phenomenon between oncological and neurodegenerative conditions. In 2014, Ibanez et al. conducted a transcriptomic meta-analysis of several neurodegenerative diseases and three cancers . They discovered a significant overlap between up-regulated genes in neurodegenerative diseases and down-regulated genes in cancer, as well as down-regulated genes in neurodegenerative diseases and up-regulated genes in cancer . SubsequFurthermore, the intricate mechanisms underlying the effects of TDP-43 in neurodegenerative diseases can be elucidated from various perspectives, including point mutations within the genome, multiple splicing mutants, and phosphorylation proteins. However, it remains to be investigated whether TDP-43 mutations and truncations exist and hold significant roles in cancer. Additionally, given its role as a splicing factor, the phosphorylation status of TDP-43 directly impacts its splicing function, prompting inquiry into the presence of abnormal phosphorylation of TDP-43 in tumor cells. While exogenous overexpression of TDP-43 can lead to elevated expression of the 35 kD isoform, which may exert cytotoxic effects on cancer cells and induce cell death , furtherTDP-43 is expressed in almost all cell types and is highly conserved among different species. Increasing evidence confirms its crucial regulatory role in various developmental processes, including embryonic development, body fat metabolism, mammary gland development, the reproductive system and so on.Tardbp mRNA [in vitro, blastocysts with homozygous Tardbp deletion exhibit impaired outgrowth in the inner cell mass. Conversely, mice with heterozygous TDP-43 deletion display normal development and fertility, with no noticeable abnormalities up to 14 months of age and no changes in TDP-43 protein expression compared to wild-type mice [Tardbp knockout mice died between ED 3.5 and 8.5, heterozygous Tardbp mice showed no differences in TDP-43 expression or phenotypic changes, including body weight, growth rate, appearance, and fertility, and further displayed no gross tissue abnormalities up to the age of 6 months compared to control littermates [Tardbp mRNA and produced a nonfunctional truncated TDP-43 variant in mice after crossing with the CAG-Cre transgenic mouse line [Various studies utilizing knockout mice have provided compelling evidence for the crucial roles of TDP-43 in embryonic development. Researchers have previously constructed TDP-43 knockout mice by targeting the translation initiation site of exon 2 of dbp mRNA , with suype mice  also repuse line . In accoTardbp knockout mice remained stable across these models, indicating that TDP-43 possesses an inherent regulatory mechanism to mitigate the adverse effects of TDP-43 deletion.Collectively, these different mouse models demonstrate that homozygous loss of TDP-43 can result in embryo death during blastocyst implantation, confirming the necessity of TDP-43 expression in embryonic development. In addition, protein levels in heterozygous Regarding the mechanism by which TDP-43 safeguards embryonic development, a recent study conducted in mouse embryonic stem cells (mESCs) discovered that TDP-43 protects the embryonic genome by interacting with L1 open reading frame 1 protein (L1 ORF1p) .Tardbp mice with Rosa26-ErCre mice (which express Cre protein upon tamoxifen induction) to generate inducible Tardbp knockout mice. Upon tamoxifen induction, these conditional homozygous Tardbp knockout mice unexpectedly died by day 9, characterized by substantial fat loss and increased fatty acid consumption. Mechanistically, deletion of TDP-43 down-regulated the expression of obesity-associated gene Tbc1d1, leading to alterations in body fat metabolism [In addition to its vital role in embryonic development, the potential functions of TDP-43 in adult animals have been increasingly investigated. To overcome the issue of embryonic death caused by TDP-43 deletion, researchers have employed conditional knockout mice to study the physiological function of TDP-43. For example, Chiang et al. crossed floxed A315T mutant mice (ALS model) can restore bioenergetic balance and extend lifespan [Cyp8b1 by binding with the lncRNA lncLSTR, thus regulating systemic lipid metabolism in mice [Accumulating evidence supports the involvement of TDP-43 in fat metabolism. Stallings et al. demonstrated that TDP-43 regulates fat homeostasis and glucose metabolism . Coughlalifespan . Egawa elifespan . Li et a in mice . Studies in mice . In our Lipids serve as essential components of biological membranes, not only providing efficient energy storage for organisms, but also acting as signaling molecules/messengers in diverse developmental pathways , with thAcrv1, which codes for the sperm acrosomal protein SP-10, is strictly expressed at the transcriptional level in the testes and round spermatids. Evidence has shown that the proximal promoter of Acrv1 is sufficient to maintain round spermatid-specific expression and acts as an insulator, preventing ectopic expression of Acrv1 in somatic cells [Acrv1 promoter via two GTGTGT-motifs, eventually inhibiting premature Acrv1 expression during spermatogenesis [Acrv1 gene transcription in a histone deacetylase-independent manner [Acrv1 ectopic expression [Precise regulation of cell type-specific gene transcription is crucial for spermatogenesis, where a multitude of testis-related genes are activated in a programmed spatiotemporal order. For example, ic cells . TDP-43 ogenesis . TDP-43 t manner . In somapression . These fSubsequent investigations have revealed that TDP-43 is expressed in both Sertoli cells and germ cells, indicating its potential adoption of multiple conformational states during different stages of spermatogenesis. Studies have shown that TDP-43 expression initiates in type B/intermediate spermatogonia, reaches its peak in preleptotene spermatocytes, disappears in leptotene and zygotene spermatocytes, reappears in pachytene spermatocytes and early round spermatids, and subsequently decreases in later spermatids . The varLactation, a highly distinctive feature of mammals, provides offspring with sufficient nutrition to ensure their survival. In earlier research, we found that TDP-43 is required for mammary gland development , 83.In our previous studies focused on identifying key post-transcriptional regulatory genes during lactation and exploring the impact of positive selection on species divergence, we conducted an evolutionary analysis of RNA-binding proteins (RBPs) across 15 mammalian species. Our analysis revealed that TDP-43 underwent positive selection within the mammalian lineage, after applying false discovery rate correction. Subsequently, we investigated the expression patterns of TDP-43 during different stages of mammary gland development in mice and observed high expression levels during pregnancy and early lactation. Notably, TDP-43 knockout in the mouse mammary gland resulted in a significant reduction in milk production, with subsequent starvation of the pups within three weeks postpartum. Furthermore, we collected human milk from lactating women and analyzed the expression level of TDP-43 in isolated RNA from the milk lipids. Remarkably, we found a positive correlation between TDP-43 expression and higher milk output, suggesting that TDP-43 mRNA expression may serve as a potential indicator of lactation in parturient women . FurtherSox2 alternative polyadenylation. Additionally, TDP-43 is also reported to regulate the expression of the Neat1 gene, repressing the formation of paraspeckles. Notably, during differentiation, TDP-43 is recruited to paraspeckles and facilitates the exit of ESCs from pluripotency and embryonic patterning [Stem cells are necessary for tissue development, homeostasis, and repair . Abnormatterning . Moreovetterning .Myolinc and regulates muscle-related gene expression, while knockdown of TDP-43 inhibits myogenic differentiation [TDP-43 is also implicated in various other developmental processes. Regarding myogenesis, Militello et al. found that TDP-43 binds to the muscle-enriched lncRNA ntiation . Furtherntiation . Li et antiation . In the ntiation . TDP-43 ntiation .Initially recognized as a protein associated with HIV, TDP-43 has been increasing implicated in the entry, replication, and latency of other viruses  beyond HHIV-1 gene expression [Malat1 and undergoes cleavage to form the TDP-35 isoform. This process promotes the production of IRF3-initiated antiviral type I interferons (IFNs) by preventing IRF3 proteasomal degradation, thereby enhancing host defense against viral infection [Multiple studies have provided evidence that TDP-43 acts as a protective factor in individuals during viral infections. Ou et al. first cloned and characterized TDP-43 in 1995, demonstrating its binding to the TAR sequence motifs of HIV-1 and inhibition of pression . Althougpression , more repression , 95. Addnfection .TDP-43 may also have negative effects on individuals. For example, during HBV infection, TDP-43 acts as a host factor that stimulates gene transcription by binding to the HBV core promoter, forms complexes with other proteins supporting the HBV life cycle, and inhibits pregenomic HBV RNA splicing, thereby promoting the production of HBV replicative intermediates, mRNAs, proteins, and virions .The above findings highlight the wide-ranging roles of TDP-43 throughout various stages of mammalian life, encompassing early development to aging  technology has revealed that TDP-43 can bind with more than 6 000 mRNA targets, representing about 30% of the entire transcriptome , 115, thchanisms . FurtherTDP-43 serves a critical function in the cellular stress response. Notably, the TDP-43 protein can sense intracellular signals, such as misfolding proteins Sup35, Pab1, and Pub1 . In ovarThe above studies emphasize the diverse and sometimes opposing functions of TDP-43 in various tissues and developmental stages, which may be attributed to the following factors:


Transplant recipients with CMV infection have a high risk of complications and mortality. This study aimed to better understand patient and CP knowledge of post-transplant CMV and identify how it can be improved.Fig 1). We report results from Phase 2 (September 2022–March 2023), which included an educational webinar with a pre- and post-webinar assessment comprising 4 questions on the risk, symptoms, treatment, and complications of CMV; participants (pts) self-reported their confidence rating pre- and post-webinar. Transplant recipients and CPs also completed a quantitative survey on CMV management and patient/CP burden. Pts were recruited via the PatientsLikeMe (PLM) website, email, or social media.The study included 2 research phases (Table 1). Patients primarily contacted their surgeon about CMV and sought information on the internet.The webinar was completed by 33 pts. There was a total of 86 and 121 correct responses to the 4 pre- and post-survey questions, respectively, indicating a significant improvement in understanding of CMV post-webinar as compared with before . Almost half (n=16 [48%]) of pts reported feeling “not at all confident” in recognizing CMV symptoms post-transplant pre-webinar; post-webinar, this was 0 pts. Twenty-eight transplant recipients  and 1 CP completed the quantitative survey. Half of recipients received a CMV diagnosis post-transplant and 16/28 reported having no awareness of their or their donor’s CMV status prior to transplant. Most (27/28) recipients reported a positive experience with their care team post-transplant. Half of recipients set goals with their care team or were provided resources to track them post-transplant. Recipients reported CMV medication changes and challenges with treatment (Although transplant recipients reported positive experiences with their healthcare team, the post-transplant care journey could be improved by better educating patients about CMV and CMV management. Aggregate-level learnings from the study with be shared on the PLM platform to reinforce the value of participation in research.Funding: Collaborative research study; primary funding from Takeda Pharmaceuticals U.S.A., Inc.Deepshikha Singh, MPH, PatientsLikeMe: Employee|PatientsLikeMe: Ownership Interest Mary C. Burke, MHA, PatientsLikeMe: Employee|PatientsLikeMe: Ownership Interest Subhara Raveendran, PhD, Ascendis Pharma: Employee|PatientsLikeMe: Ownership Interest Erlyn Rachelle Macarayan, PhD, PatientsLikeMe: Employee|PatientsLikeMe: Ownership Interest Maisha Razzaque, MS, PatientsLikeMe: Employee|PatientsLikeMe: Ownership Interest Genovefa Papanicolaou, MD, Allovir: Advisor/Consultant|Amplyx: Advisor/Consultant|Astellas: Advisor/Consultant|Cidara: Advisor/Consultant|CSL Behring: Advisor/Consultant|DSMC: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: institutional research support for clinical trials|MSD: Advisor/Consultant|Octapharma: Advisor/Consultant|Partners Rx: Advisor/Consultant|Shire/Takeda: institutional research support for clinical trials|Symbio: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Advisor/Consultant|Vera Pharma: Advisor/Consultant Macey L. Levan, PhD, Takeda Pharmaceuticals: Advisor/Consultant Maggie McCue, MS, RD, Takeda Pharmaceuticals U.S.A., Inc.: Employee Megan Gower, PharmD, Takeda Pharmaceuticals U.S.A., Inc.: Employee|Takeda Pharmaceuticals U.S.A., Inc.: Ownership Interest Daniele K. Gelone, PharmD, Abbott Laboratories: Ownership Interest|Ionis Pharmaceuticals: Ownership Interest|Johnson & Johnson: Ownership Interest|NovoNordisk: Ownership Interest|Pfizer: Ownership Interest|Regenxbio: Ownership Interest|Takeda Pharmaceuticals U.S.A., Inc.: Employee|Takeda Pharmaceuticals U.S.A., Inc.: Ownership Interest|Vertex Pharmaceuticals: Ownership Interest