Our results indicate that age-specific HPV vaccine responsiveness is mostly due to quantitative differences of immune cell precursors rather than qualitative defects in B cells. In addition, our results indicate that adults also have a good humoral immunogenic profile, suggesting that their inclusion in catch-up programmes is desirable.Adult vaccination programs are receiving increasing attention however, little is known regarding the impact of age on the maintenance of the immune response. We investigated this issue in the context of a human papillomavirus (HPV) vaccination program collecting real-world data on the durability of humoral immunity in 315 female subjects stratified according to vaccination age (adolescents and adults) and sampled at early or late time points after the last vaccine dose. HPV-specific IgGs, but not memory B cells, were induced and maintained at higher levels in subjects vaccinated during adolescence. Nonetheless, antibody functions waned over time to a similar degree in adolescents and adults. To shed light on this phenomena, we analyzed quantitative and qualitative properties of lymphocytes. Similar biochemical features were observed between B-cell subsets from individuals belonging to the two age groups. Long term humoral responses toward vaccines administered at an earlier age were comparably maintained between adolescents and adults. The percentages of naïve B and CD4 However, the increase in life expectancy and the overall large number of older individuals in the Western population have highlighted the need of protecting the latter age group2. Adult immunization may nonetheless pose some challenges from the immunological point of view4.Immunization programs for the pediatric population are usually well established, reaching coverage levels higher than those of adults5. In this context, it is crucial to fully understand the kinetics of vaccine immunogenicity and the duration of immune responses as a function of age of immunization, especially for those vaccines intended to protect the adult population. HPV vaccination represents an ideal scenario, being routinely administered to adolescents and in catch-up programmes for adults.Questions such as what is the best age to be immunized, the frequency of booster doses and whether age influences the declining rate of immune responses, are well addressed for pediatric vaccines, while for adults they are mostly based on empirical observations6 has been widely used for more than a decade, demonstrating very high levels of efficacy8 and relying mainly on neutralizing antibodies11. Infection with high-risk HPV types, mainly HPV-16 and HPV-18, is associated with the occurrence of ano-genital and oropharyngeal precancerous lesions and cancers, and infection with low-risk HPV-6 and HPV-11 is a common cause of genital warts. These conditions, which represent a relevant disease burden worldwide, can be prevented by vaccination. After an initial focus on adolescents, current HPV vaccination campaigns also include adults. However, in general immune system development occurs in the first years of life until adolescence and the changes in the immune functions observed between teenagers and adults, suggest that similar interventions (e.g. immunization programs) may require adjustments depending on the target age group. Indeed, the same vaccine may have different outcomes depending on the age of the recipient.The aluminum hydroxyphosphate sulfate salt-adjuvanted quadrivalent vaccine  against the human papillomavirusIn this study we aimed to understand the impact of age on the maintenance of the immune response, by investigating different aspects of humoral immune responses at short and long time intervals subsequent to vaccination with 4vHPV in women of different age groups. Moreover, we also investigated the persistence of immunity in the adult population following childhood vaccination in the first year of life, by assessing antibody levels.To determine whether the age of vaccination influences the induction and the duration of the immune response to the 4vHPV vaccine, HPV-specific antibodies were measured 1–8 months (“Early cohort”) and 1–5 years (“Late cohort”) after immunization in female subjects stratified in two groups based on the age at the time of vaccination  after vaccination. The slope of the regression lines, which indicate how the variables (“months post-vaccination” and “IgG titers”) are related, was compared between adolescents and adults. As expected, in both age groups we observed a loss over time of vaccine-specific IgG , whose titers are higher in adolescents compared to adults. To assess whether this paradigm is confirmed also along adulthood, we compared humoral responses to the 4vHPV between young adults 18–26 y) and middle-aged adults (27–53 y) in the “Early cohort”  should be affected, and thus only HPV-specific- IgG responses should be reduced. To investigate this issue, we measured humoral responses toward the measles and oral polio (OPV) vaccines, which were given to both adolescents and adults in their first year of life. As shown in Fig. To investigate whether the age of vaccination affects the quantity and quality of plasma cells (PC) responsible for the secretion of circulating antibodies, we measured their frequency and metabolic properties, in the “Early cohort”. However, no significant differences were observed between adolescents and adults  in adolescents compared to young adults at short time-points after vaccination25 and the smallpox-vaccine26. In the latter context, children-recipients maintained humoral immunity for decades while the majority of adults-vaccinees have undetectable antibody levels within 10 years26. Nonetheless, it should be noted that in the case of 4vHPV vaccination, protective levels of binding and neutralizing IgG are maintained in adults of different age-groups (ranging from 18- to 45-year-old) for several years after vaccination27. This is particularly true for nAb, whose physiological drop over time has been shown to be age-independent19. Consistently with the concept that age of vaccination has mild effects on antibody quality, we observed subtle and scarce differences in antibody avidity between adolescents and adults. Similar findings have been observed in other studies comparing the avidity and affinity of IgG induced by inactivated influenza virus vaccines in subjects ranging from 18- to 100-year-old28 or by tick-borne encephalitis virus vaccines in subjects <30 y and >50 y29.Interestingly, the persistence of binding antibodies was superior in younger vaccine recipients. Although we cannot exclude that sexual debut occurring in adolescents may have boosted vaccine responses in this age-group, the increased humoral responses in younger vaccine recipients has been observed for other vaccines as well including the bivalent HPV-16/HPV-18 vaccine Cervarix30. This seems to be caused by low titers, avidity and neutralizing capacity of antibodies induced by natural infections32. It is thus very unlikely that the lower responses we observed in adults could be due to pre-existing IgGs that neutralize the VLPs and hamper vaccine immunogenicity. Rather, we would expect the inverse to occur, such as a higher immunogenicity in HPV seropositive women due to the boosting of pre-existing immunity. Nevertheless, this phenomenon does not seem to occur, or rather contributes marginally to humoral responses in adults .As we could not measure pre-vaccination HPV seroprevalence, we are unable to ascertain at which level pre-existing immunity may have influenced our results. However, a recent study has shown that vaccine-induced IgG levels are comparable between subjects that were HPV seropositive or seronegative at the time of vaccination33 showed that HPV-6-, HPV-11- and HPV-16-specific memory B cells measured 1 month after the last dose, were similar in 9–13-year-old and 16–26-year-old subjects vaccinated with the 4vHPV vaccine; in contrast to our observations however, the younger age group developed higher numbers of HPV-18-specific memory B cells. The frequencies of memory B cells observed in our study were in general higher than those reported by Smolen et al.33. Such an effect may be caused by methodological differences (i.e. at the level of Elispot procedures) between the two studies. Further differences may be accounted for by the fact that we collected samples at time points beyond 1 month after the last dose, and the fact that we also included subjects that were older than those enrolled by Smolen et al. Our study was performed in a real-world context, and thus we might have included also HPV-seropositive women. As their proportion is known to increase with age34, we cannot exclude that the higher frequency of HPV-specific memory B cells observed for some HPV vaccine types is due to a secondary response and thus proliferation of this cell subset. However, the HPV seroprevalence in the Italian unvaccinated women population is around 8%35, and this percentage is usually higher in younger compared to middle-aged adults37. Therefore, the relative low proportion of seropositive women in the unvaccinated Italian population and the similar percentages of HPV-specific memory B cells that we measured between 18–26 y and 27–53 y adults would argue against a major role of pre-existing memory B-cells in the peculiar age-specific pattern we observed, though further studies are needed to confirm this aspect. Consistently, in our cohort, adults displayed a higher percentages of memory B cells specific for HPV-11 and HPV-18 and not for HPV-6 and HPV-16 compared to adolescents. If this was due to a previous exposure, we would expect the inverse to occur, with adults presenting higher responses toward HPV-6 and HPV-16 since the latter are more than two times more prevalent than HPV-11 and HPV-1837.Surprisingly, we observed that vaccine-specific memory B cells were induced at similar, or even slightly higher levels in adults compared to adolescents. In agreement with our results, Smolen and colleagues38. Frasca and colleagues38 observed that age does not affect the percentages of vaccine-induced circulating PC. We also observed comparable PC frequencies between adolescents and adults in the “Early cohort”, although our analysis focused on bulk, and not antigen-specific, PC. In addition, we failed to find correlations between circulating PC and HPV-specific IgG. Thus, although further analyses are required, we could speculate that the lower long-term vaccine-specific IgG we observed in adults is not due to alterations of the circulating PC compartment. We would also tend to exclude age-related issues of PC replenishment by memory B cells, as vaccine-specific memory B lymphocytes were comparable among age groups and, more in general, circulating memory B cells and antibodies are thought to be independently regulated40. More in-depth investigations are needed to assess any potential age-related defect during the generation of long-lived non-circulating plasma cells (LLPC). The comparable responses toward childhood vaccines that we observed in the two age-groups would exclude an age-dependent decline of this compartment (i.e. previously established LLPC), while it is more plausible that their induction is altered with increasing age.Of note, the phenomenon of similar frequencies of memory B cells despite different antibody levels between two distinct age groups has been observed also for other vaccines41. It has been proposed that a reduction in the number of vaccine-specific PC (and thus of naïve B cells primed by the vaccine antigen), and not a reduction of secreted IgG by PC, explains the lower humoral responses with advancing age42. Despite the observation that the B-cell compartment was shifted toward memory phenotypes in our cohorts of adult individuals, the frequency of naïve B cells did not correlate with HPV-specific IgG. This might also be due to the fact that we did not measure absolute numbers of naïve B cells, but their percentage. In addition, it has been proposed that increasing age is associated with a lower proliferative capacity of B cells41, thus suggesting that the higher numbers of vaccine-specific PC observed by others in young recipients42 may be due to both higher precursor numbers and their increased ability to generate antibody-secreting cells. So far, few promising studies have characterized age-associated metabolic changes at the B-cell level43. However, in this respect, we did not observe any difference between adolescents and young adults. Nonetheless, biochemical perturbations could occur later in life and thus be undetectable in our cohorts which did not include elderly individuals.The numbers of naïve B cells decreases with age+ T cells and HPV-specific IgG. Naïve CD4+ T cells will differentiate into different types of effectors, including T follicular helper (TFH) cells, upon interaction with their cognate antigen. Such CD4+ T cells are important for the generation and maturation of antibody-secreting cells45. The interaction between naïve B cells and TFH cells occurs within the germinal centers43 and is defective in aging, leading to a reduced humoral responses47. Therefore, lower numbers of naïve CD4+ T cells could result in lower humoral responses.Finally, we observed a correlation between naïve CD4This study has some limitations. In particular, the lack of data about HPV natural exposure prior to vaccination and/or sampling and about behavioral attitudes  may hamper result interpretation. Nonetheless, the study was intended to collect real-world data, and all the biases that could have affected our results will be, most likely, also present at a population level. This possibly makes our findings generalizable within the HPV-vaccine context.49 who, together with adolescents, constitute a population often affected by VPI outbreaks51. Furthermore, adults can be a source of contagion for more fragile individuals (e.g. newborns or elderly)52. As a consequence, a growing number of countries is implementing vaccine campaigns for adults. In this study, we observed that adults, although displaying lower responses than adolescents, present with detectable and well maintained humoral responses in particular with respect to memory B cells after HPV vaccination. Our study provides encouraging results of vaccination programs aimed at embracing all age groups. Nonetheless, when applicable, vaccinations at younger age (e.g. adolescence) are still the gold standard to achieve high personal protection levels. In particular, our data strongly support the large-scale implementation of HPV immunization programs for teenagers.In conclusion, modern vaccinology recognizes the importance of immunization programs for young and middle-aged adults, as the major burden of vaccine-preventable infections (VPIs) is in adultsn = 174) was tested at 1–8 months (median of 2 months) after the third vaccine dose and is defined as “Early cohort”, while a second subgroup (n = 141) was tested at 1–5 years (median of 4 years) after the third vaccine dose and is defined as “Late cohort”. In the “Early cohort”, 117 were adolescents at the time of vaccination  and 57 were adults . In the “Late cohort”, 70 were adolescents at the time of vaccination  and 71 were adults .This observational, cross-sectional study was conducted in the framework of an organized vaccination program in Italy. We evaluated and compared the immune responses to the 4vHPV vaccine  in 315 female subjects. One subgroup  where HPV vaccine was offered by organized vaccination (10–14 years old) and implementation (older ages) programs. Girls and women who had previously completed the vaccination schedule were invited by letter to participate in the study, which consisted in blood sampling, to measure the persistence of the immune response elicited by the vaccination. Exclusion criteria were fever and other symptoms of active infection in the three weeks prior to blood collection, presence of chronic inflammatory diseases, immunosuppressive therapy, and pregnancy. The study was approved by the Ethics Committee of Padova University-Hospital (Protocol N° 2413P) and conducted according to the principles expressed in the Declaration of Helsinki. All participants, or their parents for minors, signed a written informed consent form.A control cohort of 24 unvaccinated women with unknown previous history of HPV infection (age range: 22–40-year-old) was recruited and samples analyzed to determine the cut-off values of immunogenicity assays.53 and serum separator tubes (for serum specimens). Not all the assays were performed on all subjects. The number of individuals tested for each analysis is specified in the Figure legends.Peripheral blood mononuclear cells (PBMCs), plasma and sera were purified from whole blood for subsequent analyses by Ficoll-Paque  density gradient centrifugation (for PBMCs and plasma specimens)54 in 293TT cells53. The 293TT cell line was kindly provided by John T. Schiller  and then grown in Dulbecco’s modified Eagle’s Medium (DMEM) (Gibco Life Technologies) containing 10% FBS, 1% antibiotic-antimicotic solution, 1% glutamax, 1% non-essential aminoacids , and 250 μg/mL hygromycin B (Sigma-Aldrich). For each HPV type-specific VLP, 293TT cells (7 × 106) were seeded in 75 cm2 flasks and incubated 16–18 h at 37 °C up to 50% confluence. Cells were lipofected  with 20 μg of plasmid DNA, according to manufacturer’s instructions, which were incubated at 37 °C and then collected 48 h later. The cells were washed in Dulbecco’s phosphate buffered saline (D-PBS) containing 9.5 mM MgCl2, transferred to new eppendorf tubes and centrifuged at 2000 rpm  for 7 min. The cellular pellet was resuspended in D-PBS (1.5× the cellular volume) and then lysed with Triton X-100 (1/20 of the volume), 0.1% Benzonase  0.1% Plasmid Safe , and 1 M ammonium sulfate (pH 9) (1/40 of the volume) at 37 °C for 24 h. The cell lysate was added with 0.17 M NaCl, incubated on ice for 20 min. and centrifuged at 10,000 rpm at 4 °C. The supernatant was left on ice for two hours and VLPs were purified by means of an iodixanol gradient (Sigma–Aldrich) at 50,000 rpm for 3.5 h . Fractions were collected from the bottom of the tube, read for protein concentration , and stored at −80 °C until use. The fractions were analyzed by SDS-PAGE, silver staining  and western blot using mouse anti-L1 primary IgG antibodies , and an anti-mouse HRP-secondary antibody (Abcam). For each type-specific VLP, all of the fractions positive for L1 derived from several transfections and purifications procedures were pooled together to obtain a single type-specific VLP stock to be used for all immunological assays . The protein concentration of each VLP stock was measured by BCA and disposable aliquots (25 μg) were stored at −80 °C until use. All of the plasticware used in these procedures was siliconized .Plasmids p6sheLLr+, p16sheLL, and p18sheLL, containing L1/L2 genes of HPV6, 16 and 18 were kindly provided by dr. John T. Schiller , whereas plasmid p11L1h containing the L1 gene of HPV11 was kindly provided by dr. Martin Müller . The preparation and purification of HPV type-specific virus like particles (VLPs) was done according to the protocol of Buck et al.55. PsVs were obtained after transfection of cells with equal amounts of p16shell, p18shell, p31shell, or p45shell plasmids and the reporter pYSEAP plasmid. The neutralization assays were done on cells seeded in 96-well plates at a concentration of 30,000/well in assay buffer , grown for 2–5 h at 37 °C and incubated with HPV PsVs alone or previously mixed with sera  in duplicate wells. After 72 h, SEAP expression was measured in cell supernatants using the SEAP Reporter Gene Assay. The neutralizing titer was determined as the reciprocal of the final dilution of serum that yielded <50% of mean RLU measured with PsVs alone and reported as ED50 (effective dose producing 50% response). The limit of quantification of the assay was set at 40 ED50. Samples with neutralizing titers equal to or higher than 40 ED50 were considered positive, whereas samples with neutralizing titers lower than 40 ED50 were assigned a value of 1 for the purpose of geometric mean titer (GMT) calculation.The preparation and purification of HPV type-specific pseudovirions (PsVs) encapsidating a SEAP reporter plasmid and the neutralization assays on serum samples were done in 293TT cells53. Plates were coated with 100 μl/well of each HPV type-specific VLP at 4 °C for 18–20 h, washed and incubated with 1% bovine serum albumin  in PBS for 1 h and then with serially diluted (1:2 dilution) plasma samples (50 μl) (duplicate wells) for 2 h at 37 °C. After extensive washing, immunecomplexes were detected by incubation with a goat anti-human IgG-HRP  for 1 h and addition of ABTS substrate (Thermo Fisher Scientific) for 30 min at 37 °C. In each plate, blank , negative controls (wells with a pool of plasma of unvaccinated subjects), and positive controls (wells with a pool of plasma of vaccinated subjects) were included. Optical density (OD) at 620 nm was determined with an automatic plate reader and each OD value was subtracted from the blank56. Titers were determined based on the reciprocal of the dilutions interpolating the cut-off value. For each type-specific VLP, the cut-off values were 0.374 for HPV-16; 0.346 for HPV-18; 0.388 for HPV-6; 0.346 for HPV-1153, and were calculated as the mean +3 standard deviations of the OD values of the control cohort.HPV type-specific IgG titers were measured by ELISAMeasles- and OPV- specific antibody presence was determined on plasma samples using commercially available kits  according to the manufacturers’ instructions.58. Based on the ELISA IgG titers, selected plasma samples were diluted in PBS-1% BSA to obtain an OD of 1 ± 0.5, added  to 96-well plates pre-coated with each HPV type-specific VLP, as described above, for 2 h at 37 °C and washed. Negative and positive controls were included in each plate. Plates were then incubated with 50 μl/well of 0.5–5 M GuHCl for each sample (that removes the low avidity antibodies while high avidity antibodies remain bound) for 15 min at room temperature under gentle agitation. Untreated samples and control wells were incubated with PBS alone. After extensive washing, immune complexes were detected as described above. For each sample, the mean OD of duplicates were calculated and percentage of binding was determined as [(treated OD/untreated OD) × 100]. The avidity index (AI) is the extrapolated molar concentration (M) of GuHCl required to reduce the absorbance of the untreated, control well by 50%.The avidity of antigen-specific IgGs was evaluated on selected samples with high IgG titers by a modified ELISA53. Thawed PBMCs were diluted in RPMI 1640 medium containing 10% FBS (0.5 × 106/ml), seeded in 24-well plates , and grown under unstimulated or stimulated conditions with ODN (3 μg/ml), S. aureus Cowan I (1:10.000), and Pokeweed (0.1 μg/ml) at 37 °C for 5 days. Ninety-six-well plates  were pre-washed with 70% ethanol for 2 min and water, and then pre-adsorbed, at 4 °C for 20–24 h, with 100 μl of an anti-human IgG capture antibody  to quantify the total number of B cells secreting IgG, or with 100 μl of each type-specific VLP (12.5 μg/ml) to quantify the number of HPV type-specific B cells. After extensive washing with PBS, 200 μl of unstimulated and stimulated PBMCs (1.25 × 106/ml) were seeded in each HPV type-specific plate (duplicate wells), whereas 200, 100 and 50 μl of unstimulated and stimulated PBMCs (6.25 × 104/ml) were added (duplicate wells) to the total IgG B-cell Elispot wells. In all plates, blank wells containing 200 μl of culture medium alone were included. After incubation at 37 °C for 2 h, plates were washed and processed with an anti-human-IgG-biotin detection antibody provided by the kit. Spots were counted using an Elispot reader . The percentage of each HPV type-specific memory B cells was calculated as [antigen-specific B cells (SFC/106 PBMC) / total memory B cells (SFC/106 PBMC)] × 100].B-cell Elispot assays were performed using a commercial “Human IgG B-cell Elispot” kit + T lymphocytes were analyzed by flow cytometry. PBMCs were surface stained in the dark for 15 min at room temperature with directly conjugated monoclonal antibodies to identify different B- and T- cell subsets (Supplementary Table TM-ThermoFisher Scientic); APC-αCD27 and VioBlue®-αCD38  and APC/Cyanine7-αCD19 . Non-viable cells were eliminated from the analysis using LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific). Naïve CD4+ T cells were identified as CD27+ CD45RA+, naïve B cells as CD38- CD27-, memory B cells as CD27+ CD38- and plasma cells as CD38highCD27+  for 20 min at 37 °C or 5 μM CellROX Green Reagent  for 30 min at 37 °C, respectively60. Additional stains were used as described above to characterize the metabolic profile of distinct B-cell subsets in each assay. Data were acquired using a BD FACSCanto II flow cytometer  and analyzed with FlowJo software (BD Biosciences).To determine glucose uptake, mitochondrial membrane potential, neutral lipid (NL) content or reactive oxygen species (ROS) production, PBMCs were incubated with 50 μM 2′-amino)-2-deoxyglucose (2-NBDG) for 20 min at 37 °C, 25 nM tetramethylrhodamine, methyl ester, perchlorate (TMRM) for 30 min at 37 °C, 10 μM 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-P-values less than 0.05 were considered to be statistically significant. Statistical analysis was performed using the softwares R61 and Rstudio62 with the packages Tidyverse63 Scales64.Mann–Whitney test was used to compare the difference between two independent groups, while correlations were analyzed by Spearman’s rank test. A simple linear regression was used to estimate the decay (slope) of immune responses (y or Log(y)) as a function of the months post-vaccination (x) for each cohort and age group. The equations of the regression lines have to be intended as y = q + mx or Log(y) = q + mx, with q indicating the intercept and m the slope. Regression lines were compared using ANCOVA. Further information on research design is available in the Supplementary MaterialREPORTING SUMMARY


Eb4859 produced the thermoset of highest tensile strength, 19 ± 7 MPa, amongst the set of varied crosslinkers. The formulation featuring UDM (HTM:UDM:tbutysty) offered ease of processing and was seemingly the easiest to print. Investigations of reactive diluent showed that styrene produced the thermoset of the highest extent of cure and the overall highest tensile strength, 25 ± 5 MPa, while tbutylSty produced the thermoset with the greatest Tan-δ Tg, 231 °C. HTM was synthesized, formulated with diluents, crosslinkers and initiators. The HTM resins were then 3D printed to produce thermosets of Tg values greater than 200 °C. The polymer properties were evaluated and a structure–reactivity relationship was discussed.Vat photopolymerization additive manufacturing (Vat AM) technologies have found niche industrial use being able to produce personalized parts in moderate quantity. However, Vat AM lacks in its ability to produce parts of satisfactory thermal and mechanical properties for structural applications. The purpose of this investigation was to develop high-performance resins with glass transition temperatures (Tg) above 200 °C for Vat AM, evaluate the properties of the produced thermosets and establish a structure–property relationship of the thermosets produced. Herein, we have developed SLA-type resins that feature bio-derived monomer hesperetin trimethacrylate (HTM) synthesized from the flavonone hesperetin. Diluents 4-acryloyl morpholine, styrene, 4-methyl styrene and 4-tert butylstyrene (tbutylsty) were photocured with HTM as the monomer and all produced thermosets with Tg values above 200 °C. Investigations of suitable crosslinkers urethane dimethacrylate, the vinyl ester CN 151 and Ebecryl 4859 (Eb4859) showed that each crosslinker displayed different benefits when formulated with HTM as the monomer and tbutylSty as the diluent (HTM:crosslinker:tbutylSty with mass ratio 2:1:2). The crosslinker CN 151 produced the thermoset of greatest onset of thermal decomposition temperature (T Additive manufacturing has been propelled to the forefront of research efforts in the US armed forces due to its ability to produce materials in a timely manner, the ability to produce parts of unique geometry difficult to produce by any other means, and the possibility of on-demand manufacturing at the warfighters’ fingertips ,2,3. SucAlthough AM as a general technology has garnered much attention and variable success, the AM method vat photopolymerization has only garnered limited use in niche industry applications such as molds for corrective dentistry, jewelry making, and hearing aid production . Yet, pr2 while having glass transition temperatures in excess of 120 °C [AM methods that employ photopolymerization, namely vat photopolymerization and ink jet technologies, feature amongst the highest resolutions compared to other methods of AM. As many customizable printers exist that are affordable and user friendly, vat photopolymerization has garnered more attention for research investigations than ink jet technologies ,11,12,13f 120 °C . Zhou etf 120 °C . Hegde ef 120 °C . Hegde eAs outlined above, moderate success has been displayed towards the desired high-temperature thermoset, with Tg values greater than 200 °C and degradation temperatures above 350 °C. Herein, our chosen strategy is the development of high-performance materials by way of synthesis of suitable methacrylate monomer sourced from biobased materials. Often found within nature are scaffolds of complex structure of specific stereochemistry difficult to synthesize by simple synthetic means. Efforts towards sustainability have reinvigorated the development of acrylates, methacrylates and vinyl esters sourced from nature ,32,33,34As the goal was to produce high-performance polymers with Tg values above 200 °C, we envisioned the synthesis and the 3D printing of hesperetin monomer to allow for the development of high-temperature thermosets. Hesperetin is a flavonoid belonging to the subgroup flavanone due to its carbonyl substitution in its fused ring system. Hesperetin can be extracted from various citrus fruits such as lemons, grapefruits and oranges . As mentAll chemicals were used as received without further purification unless otherwise stated. Hesperetin was purchased from Cayman Chemicals (≥98% purity)  and was utilized as the core structure for the synthesis of bio-derived methacrylate, hesperetin trimethacrylate (HTM). Methacryloyl chloride  was used as the acid chloride for the installation of the polymerizable methacrylate substitution. The solvent dichloromethane  was employed as the reaction solvent along with in house distilled water. KOH  was employed as base in superstoichiometric amounts. Tetrabutyl ammonium bromide  was used as the phase transfer catalyst. Hydroquinone  was employed as a free-radical inhibitor following work-up of desired product. 4-Acryloyl morpholine , styrene , 4-tert butylstyrene , 4-methylstyrene  and 4-acetoxystyrene  were used as reactive diluents for blending of resins. Isobornyl acrylate (IBOA), isobornyl methacrylate (IBOM) and Ebecryl 4859 were obtained from Allnex as free samples. The vinyl ester CN151 was obtained from Sartomer. The urethane dimethacrylate (UDM) was purchased from sigma Aldrich. Phenylbis  phosphine oxide (BAPO)  and Trigonox 239-A  were used as photoinitiator and thermal initiator, respectively, for the free radical polymerizations. Chemical 1H and 13C NMR spectra were recorded on 400 MHz (100 MHz for 13C NMR) Bruker NMR spectrometer. Data from NMR spectroscopy are reported as chemical shifts (δ ppm). 1H NMR spectroscopy spectra include the corresponding integration values. Coupling constants (j) are reported in hertz (Hz). Standard abbreviations indicating multiplicity were utilized as follows: s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).Nuclear magnetic resonance spectroscopy was used to confirm the identity of the synthesized monomer. −1 to 4500 cm−1. A total of 32 scans were performed at room temperature. The reactive C=C double bonds of interest includes the peak at 930–958 cm−1 corresponding to the stretching of the (meth)acrylate C=C double bonds of HTM and the EB4859, UDM, CN151 crosslinkers. The other peaks of interest include 889–924 cm−1 corresponding to C=C bond stretching of styrene and its derivatives, 4-methylstyrene and 4-tert butylstyrene. Internal references were used corresponding to each compound analyzed as follows: 1713–1775 cm−1 ester peaks of methacrylates, 686–712 cm−1 aromatic stretching modes of styrene. Equation (1) is used to calculate the extent of cure of the individual monomers used based on the area (A) of the C=C peak after cure relative to the unreacted peak area (A0).AC=C0  is the absorbance of the peak of interest and the Astd0, is the absorbance of the corresponding internal standard peak. Uncured and cured refer to the uncured resin formulation and the post-cured thermoset, respectively. The overall extent of cure is calculated based on the mole fraction of the crosslinker and reactive diluent and the functionality level  of the crosslinker and is shown in Equation (2).Infrared (IR) spectroscopic absorbance was recorded on a Nicollet Is50 spectrometer. Fourier Transform Attenuated Total Reflection (FT-ATR) spectroscopy was used to obtain the IR absorbance signature of the synthesized HTM monomer and to also calculate the extent of cure based on the consumption of the diluent C=C and (meth)acrylate groups. The uncured resins and after post-cure thermosets, were scanned using FT-ATR in the range of 500 cmThe absorbance of HTM in solution was recorded using a Perkin Elmer 850+ UV/Vis spectrophotometer.The analyte of choice was dissolved in acetonitrile (MeCN), or reactive diluent as solvent. Serial dilution was performed to obtain the desired dilute solutions and scans were performed from 600 to 250 nm in increments of 1 nm. Absorbance Vs Wavelength was plotted and extinction coefficient also known as molar absorptivity were obtained.Resins were formulated by dissolution of HTM and initiator package into reactive diluent of choice. Resin formulations were placed in a planetary mixer to ensure sufficient mixing. Resin formulations were mixed in the mixer until a homogeneous solution was produced. As HTM and BAPO were sufficiently soluble in all diluents employed, no additional heating was necessary for dissolution.−1 and 100 to 1 s−1. Four points per decade were recorded to investigate shear behavior. All resins were Newtonian (no shear thinning was observed) and thus only viscosity is reported.Viscosity (cP) was determined for all 3D printing resin formulations, neat crosslinkers and neat reactive diluents of choice. Rheological investigations were conducted using an TA Instrument AR 2000 Rheometer with 40 mm parallel plate geometry and Peltier temperature control at 25 °C. A gap of 1000 microns was used and the viscosity for each sample was evaluated using a steady state flow procedure. The shear rate was increased in a logarithmic progression from 1 to 100 sd is the cure depth, Dp is the depth of penetration, Ec is the critical energy necessary for polymerization and Emax the exposure of a given surface area over a specific amount of time. Experimentally, a cylindrical vessel was filled with the resin formulation, which included HTM, diluent and cure package. The formulation was irradiated from the top of the cylindrical vessel at a given intensity for a duration of thirty seconds. The gelled material was removed and measured. Plots were made of Cd vs. ln(Emax) wherein the slope was Dp and the x-intercept was Ec. The Dymax BlueWave MX-150 Series LED spot curing system equipped with a wavelength of 405 nm and a 5 mm diameter spot cure optical fiber was used for this working curve experiment.In an effort to investigate photochemical curing behavior, working curves were plotted employing Jacob’s equation ,38,39, and cure package of 1.5 wt% BAPO and 1.1 wt% trigonox. A modified window pane method was used wherein 32 squares of light exposure and squares 1 to 33 were irradiated to different degrees. For this investigation and all subsequent 3D printing AnyCubic photon DLP/LCD screen 3D printer was used. The VAT was filled with the necessary HTM formulation, the build plate was removed. The material was 3D printed using the necessary STL file. After 3D printing, the resin was poured off and the modified window pane was rinsed with isopropyl alcohol or ethanol, the part was dried and the individual squares were measured for plotting in the working curve.Photocuring was accomplished employing a Dymax metal halide flood lamp. Resin formulations were thoroughly mixed, then poured in a two part metal mold with mirror like metallic backing. Glass was used to cover the front of the unreacted resin acting as a UV filter and to ensure samples did not curl or distort upon curing. Formulations were irradiated on one side for two minutes before being demolded, flipped over and irradiated on the opposite side for an additional two minutes. After initial cure, samples were post-cured for two hours at 150 °C, one hour at 180 °C and thirty minutes at 205 °C, with a heating rate of 5 °C/min unless otherwise stated.2. Working curves for HTM SLA resins were used to determine exposure time. Parts were printed with 50 micron layer heights, a bottom layer of 45 to 60 s and normal layers of 25 to 35 s depending on the specific resins employed.SLA/LCD additive manufacturing was accomplished using an AnyCubic Photon LCD/DLP 3D printer which uses a pixel wavelength of 405 nm. The light intensity was measured employing a Dymax radiometer. Light intensity was measured to be 13.5 mW/cmThermomechanical properties of flavone thermosets were measured by dynamic mechanical analysis (DMA). DMA was performed on a TA Instruments 3000 in single cantilever geometry. Thermosets were prepared with approximate dimensions of 63.5 mm × 8.2 mm × 1.5 mm (length × width × thickness). Test runs were conducted at a 1 Hz frequency, amplitude and 2 °C/min temperature ramp rate from 0 °C to 275 °C. Thermosets were tested twice minimally. Glass transition temperatures (Tg) listed are generally taken from the maximum of the Tan δ peak. Tan δ  and storage moduli reported are a result of the average of the samples tested. Loss modulus Tg is also displayed, taken as the temperature that corresponds to the maximum peak, and is given in units of °C.0) and peak indicating the maximum derivative of weight loss (Td) of the formed thermoset were measured using a TA Instruments Q500 Thermogravimetric Analyzer (TGA). Sample preparation included weighing between 5 and 10 mg of cured sample in a platinum pan and placing it appropriately in the Q500. The system parameters included heating to 600 °C at a heating rate of 10 °C min−1 under constant flow of N2 .Thermogravimetric properties including initial decomposition temperature  in H2O (100 mL) and allowed to cool to 0 °C. The KOHaq solution was allowed to come to room temperature followed by dropwise addition into the reaction mixture. Addition of KOHaq resulted in a dark brown homogeneous translucent mixture. The reaction mixture was allowed to stir at 0 °C for 5 min before the dropwise addition of methacryloyl chloride . Methacryloyl chloride was added over 10 min. The reaction was allowed to stir at 0 °C for 5 h. After five hours, the reaction mixture was diluted with H2O (400 mL), the organic and aqueous layers were separated and the aqueous phase extracted with dichloromethane (3 × 40 mL). The organic layers were combined and washed with saturated NaHCO3(aq) (100 mL) then H2O (3 × 133 mL). The organic phase was dried over anhydrous MgSO4, then filtered, and the crude mixture was concentrated in vacuo in the presence of silica gel (~100 g). The free-flowing yellow dried powder was vacuum filtered in a coarse-grained filter funnel. The filter cake was washed with dichloromethane (600 mL), which caused the liberation of a bright yellow filtrate. Two further washings of DCM was conducted until no additional color was liberated within the filtrate. The filtrates were combined and hydroquinone (20 mg) was added as an inhibitor. The dichloromethane was removed under reduced pressure at (30 °C) and the bright yellow viscous liquid was put in a vacuum oven. After oven drying, a pale yellow solid was obtained, which was characterized via 1H NMR spectroscopy confirming the desired product. The desired product was afforded as a pale yellow solid  with approximately 500 ppm hydroquinone as inhibitor.Hesperetin trimethacrylate (HTM) was synthesized from a modified procedure from the literature . Scheme 1H NMR and 13C NMR spectra of HTM, respectively. Both display the expected trimethyl groups in the aliphatic region corresponding to the terminal methyl groups of the methacrylate. Additionally noticeable in the proton spectrum are the signature chemical shifts indicating the cyclic core of the flavone at 2.68 , 2.9  and 5.33 ppm .−1 as well as the keynote peaks of the methacrylate double bond (C=C) at 943 cm−1.FTIR-ATR spectrum of HTM is displayed in UV/Vis spectroscopy was employed to determine the absorbance of HTM and compare to the absorbance of BAPO the photoinitiator of choice. HTM was synthesized in one step in a biphasic mixture employing tetrabutyl ammonium bromide as the phase transfer catalyst. Increased reaction scale caused complications with extraction and separation. Thus, HTM was synthesized on twenty gram scales. However, multiple batches were easily synthesized in succession, allowing for the synthesis of hundreds of grams of HTM. The quality of the product was the same from batch to batch according the NMR.N-vinylpyrollidone, and styrene. IBOA and IBOM have the added benefit of being biobased as it is derived from pine resin and thus will increase the biocontent of the formulated resin [To ensure respectable curing of HTM to form a high-performance thermoset, allow for ease of processing, and allow for 3D printing of HTM via vat photopolymerization, a suitable diluent needed to be employed. As the desire was to obtain high-performance properties, namely Tg above 200 °C, a suitable diluent needed to be identified. In order to produce thermosets of desired thermal properties diluents of rigid structure, low viscosity and diluents in which HTM would have respectable solubility was desired. Initial investigations were centered on diluents known to produce copolymers yielding high-performance thermosets. Such diluents have in common either aromatic rings, cyclic structures, or rigid bicyclic moieties. These diluents also contain polymerizable substituents. Such diluents include, isobornyl (meth) acrylate  4-acryloyl morpholine (4AM), ed resin . HTM wased resin ,17,41. RPost-cured 3D printed samples were analyzed via DMA and Tg was obtained. As we desire Tg values above 200 °C, we used DMA Tan-δ values as a down select criteria. d) as diluents such as 4-acetoxystyrene (AceSty), 4-methylstyrene (MeSty) and 4-tert butylstyrene (tbutylSty) in one to one formulations with HTM as the monomer, and thus HTM:Styd thermosets were made. d and HTM:Sty. All of the thermosets that featured styrene derivatives as diluents displayed Tg values above 200 °C. The Tg trend was as expected mirroring that of the homopolymers of the diluents, HTM:MeSty (215 °C) < HTM:Sty (219 °C) < HTM:AceSty (224 °C) < HTM:tbutylSty (230 °C) and the homopolymers trend as follows: MeSty (97 °C) < styrene (100 °C) < AceSty (116 °C) < t-butylSty (127 °C) [We examined the properties of HTM copolymers with styrene derivatives. Formulations were mixed, cured and post-cured in a similar fashion as HTM:styrene employing styrene derivatives (Sty(127 °C) . EvidentTo aid in down select and ascertain ease of printing via vat photopolymerization, UV/Vis spectroscopy with HTM as the monomer were recorded and working curves plotted employing 405 nm light. Absorbance spectroscopy was recorded in the UV/Vis region of the spectrum of HTM and BAPO separately in the styrene derivatives of interest. d formulations were mixed in 1:1 mass ratio in a planetary mixer followed by the addition of the cure package trigonox (1.1 wt%) and BAPO (1.5 wt%). Further mixing was performed to ensure complete dissolution of all components. A 405 nm LED light source was used of known intensity to cure resin formulations and the cured layers were measured. The obtained data are plotted on a semi-logarithmic plot to obtain working curves. d formulations. Working curve of HTM:Styd formulations unveiled that styrene necessitated the greatest amount of energy to cure and upon curing achieved the greatest depth of penetration, 350 ± 40 microns with an Ec of 7900 ± 1400 (mW/cm2)∙s. The diluent t-butylSty allowed for the lowest depth of penetration with the lowest Ecrit, 222 ± 4 microns and 630 ± 50 (mW/cm2)∙s, respectively. The cure depth is a complex parameter that is affected by absorbers in the formulation, such as HTM and BAPO, but also the ability of the initiator to initiate polymerization. As HTM displays a non-zero absorbance at 405 nm, competitive absorbance occurs between HTM and BAPO the photoinitiator. HTM then reduces the amount of available photons and thus actively limits Dp. Absorbance spectra unveiled the increased absorbance of HTM in 4-tert-butylstyrene and 4-methylstyrene compared to that of styrene and 4-acetoxystyrene. This increased absorbance is likely the cause of lower penetration depth for the former diluents. Initiation of polymerization is also multifaceted. Initiation is affected by passive absorbers, such as inhibitors, which scavenge formed radicals, electronic interactions between diluent and monomers, and the inherent reactivity of diluents and HTM monomers towards polymerization. Additionally worth mentioning is the role of dissolved oxygen to inhibit polymerization. Values of Ecrit mirror that of the diluent inhibitor concentration. Thus, the formulation with the greatest amount of inhibitor has the highest Ecrit and is therefore more difficult to polymerize. Styrene is an exception to this trend. Styrene has the highest Ecrit even though the amount of inhibitor is least. The styrene electronics likely hinder polymerization compared to the Styd that contain alkyl substituents that have an increased electron density due to induction effects of the alkyl substituent. Additionally possible is that styrene could have a higher concentration of dissolved oxygen, but this is unlikely. The expected trend of inhibitor and Ecrit holds true for the remaining formulations. The diluent t-butylSty has the lowest inhibitor concentration of 100 ppm tertbutyl catechol and as follows the lowest Ecrit. Overall, Styd formulations feature cure depths near 300 microns and Ecrit on the order of 103 (mW/cm2)∙s. Critical energies of HTM:Styd formulations are far greater than what is generally exhibited in photocure systems. As acrylates have been shown to have Ec on the order of 101 (mW/cm2)∙s [d diluents are purified and oxygen content is either known or controlled would need to be conducted in order to elucidate a structure–reactivity relationship. This is outside of the scope of this manuscript.Absorbance of formulation components is known to affect the ability to photochemically polymerize said formulation as described in Jacobs’s equation. Jacob’s equation can be used to approximate the critical energy necessary for gelation (Ec) and the depth of penetration (Dp) of light irradiated through the surface of a photoreactive formulation ,43,45,46W/cm2)∙s ,43,45,46d diluents. All diluents have a significantly low viscosity on the order of 101 cP. As styrene is commonly used industrially, supply meets demand and thus styrene is cheapest amongst the set. Further, bulk prices of styrene drive the price per liter even lower. Styrene is more volatile than Styd considered in this investigation displayed by it having the highest vapor pressure of 5 mmHg at 25 °C. The vapor pressure of styrene is even more so problematic since it is an expected carcinogen. The next highest vapor pressure is that of 4-methylstyrene, 1.37 mmHg. Again this vapor pressure is problematic since the main toxicity is an aspiration hazard. Both 4-acetoxystyrene and 4-tert butylstyrene have vapor pressures below 1 mmHg. The cost of 4-acetoxystyrene is high compared to styrene and other Styd at 456 $/L. Benefits of 4-acetoxystyrene include its low vapor pressure of 0.0225 mmHg and mild toxicity concerns including acute oral toxicity, skin and eye irritation. The styrene derivative 4-tert-butylstyrene displays moderate cost 117 $/L, low vapor pressure (0.135 mmHg) with a main toxicity of eye and skin irritation, which are relatively minor compared to styrene itself. Most acrylates are generally skin and eye irritants as the methacrylate group itself is a known lachrymator. SLA-type photopolymerization employs an open vat of formulated resin. Although the vat is enclosed to limit ambient light from contacting solution surface and as a result initiating polymerization prematurely there is undoubtedly an amount of resin that is liberated from the solution at ambient conditions. Additionally worth consideration is post-processing which would expose the user to unreacted resin during cleanup and post-cure. Vat photopolymerization resins are known to be odorous. However, it is preferable to limit toxicity so as to not add danger to the operator or the surroundings. Due to its low vapor pressure, moderate toxicity and respectable cost as well as the highest Tg achieved in a binary blend with HTM of 230 °C, 4-tertbutylstyrene was the styrene derivative of choice to investigate crosslinker choice in HTM SLA resins.Along with the ability to produce thermosets with HTM monomers of Tg values greater than 200 °C and ease of polymerization via light initiation, other down select criteria considered for suitable diluent included diluent cost, viscosity, toxicity and vapor pressure. d vat photopolymerization resins. The crosslinkers under investigation include Eb4859, the aliphatic linear urethane dimethacrylate (UDM), and the vinyl ester of bisphenol A (CN151). This would allow for inferences, correlations and a structure–reactivity relationship of the employed crosslinkers, as both Eb4859 and UDM are urethane dimethacrylates, cyclo-aliphatic and linear aliphatic, respectively, and CN151 is a vinyl ester of bisphenol A. The crosslinkers are known to have lower Tg values 160 °C [d formulations. Again DMA was used as a down select criteria and the formulations were chosen based on their ability to afford a thermoset with Tg value greater than 200 °C. Initial investigations focused on different crosslinkers and styrene. Formulations that feature small-molecule crosslinkers and small-molecule diluents often produce brittle thermosets. Previous investigations unveiled the beneficial use of a suitable crosslinker in the production of polymers of greater strength. The use of the urethane dimethacrylate Ebecryl 4859 was beneficial when used to produce polymers of isosorbide methacrylate as the high-temperature biobased component and 4AM as the reactive diluent . Thus, ws 160 °C , 124 °C s 160 °C  and 138 s 160 °C ,45,46,47d (2:1:2). Resin formulations were then decided to investigate a structure reactivity–relationship of the crosslinker and the reactive diluent upon the polymer properties. Formulations HTM:Eb4859:Sty, HTM:Eb4859:MeSty, and HTM:Eb4859:tbutylSty were chosen in order to investigate the effect of the styrene diluent on the formed thermoset. The styrene, 4-methylstyrene and 4-tert butylstyrene all differ at the para position of the aromatic ring, with H, methyl or tertbutyl group, which affects electronics, molar volume and also alters the molecular weight and thus the moles of each diluent within the formulation. All of which could have bearing on the properties on the formed thermoset. Formulations HTM:UDM:tbutylSty and HTM:CN151:tbutylSty compared with HTM:Eb4859:tbutylSty, were chosen to investigate the influence of crosslinker on the properties of the formed thermoset. Again crosslinkers have different functionality as well as molecular weights. Based on the preliminary results featured above, 3D printed ternary HTM thermosets are expected to all feature Tg values above 200 °C.Using the knowledge gained from photocured thermosets we arrived at the formulations to investigate biobased vat photopolymerization 3D printing of HTM formulations. All formulations featured the following mass ratios HTM:crosslinker:StyResins were formulated and rheological properties were analyzed. All resin formulations were Newtonian fluids in the shear rate range investigated. 2, with depth of penetration ranging from 0.140 to 0.230 mm. All ternary formulations displayed Ec lower than that of the binary HTM:Styd formulations. The results indicate that employing the crosslinkers aided the polymerization because it resulted in a decrease in Ec relative to those found in In order to determine the feasibility of 3D printing the chosen formulation, a working curve was plotted using the AnyCubic Photon 3D printer. Anycubic Photon was chosen due to its ease of use and because its vat uses a fluorinated ethylene propylene film (FEP) instead of PDMS. Preliminary investigations unveiled that styrene was absorbed into the PDMS layer of Form 1+ vat, and thus an alternative printer was desired. Working curve of HTM SLA resins is displayed within the It was also found that formulations featuring Eb4859 displayed more variance than when UDM or CN151 crosslinkers were employed. Formulations consisting of Eb4859 featured a higher Ec compared to formulations that featured other crosslinkers. This could indicate that a higher inhibitor concentration is present in Eb4859 than in the other crosslinkers, but dissolved oxygen and molecular group effects (which hinder inherent reactivity) could also play a role. By way of processing, formulations featuring tbutylstyrene were easier to print indicated by the fact that fewer prints failed with tbutylstyrene relative to prints that featured styrene. The formulation HTM:UDM:tbutylSty was especially easy to print as entire batches that consisted of DMA bars and tensile specimen were able to be printed all together without complication. Due to the brittleness of CN151 formulations, removal from the build platform was problematic resulting in some broken samples.After understanding the rheological properties and investigating the printability HTM ternary resins, formulations were 3D printed on an Anycubic Photon 3D printer. Most commonly 3D printed samples undergo photochemical post-cure with mild heating. Using the Formlabs post-cure system DMA samples could only be heated to 80 °C. The temperature of 80 °C is far below that of the expected Tg. Due to this and the desire to produce 3D printed thermosets of similar Tg values as the photocured systems, the post-cure procedure remained the same as previously employed.d were determined in order to calculate the overall extent of cure . Turning attention to The extent of cure of the HTM 3D printed thermosets was investigated employing FTIR spectroscopy. The 3D printed post-cured samples were subjected to dynamic mechanical analysis to obtain thermomechanical properties and to ascertain the glass transition temperature. As desired, all 3D printed thermosets exhibited Tg values above 200 °C. The HTM:Eb4859:Sty was the thermoset with the lowest Tg but the highest storage modulus, 212.2 ± 0.4 °C and 3600 ± 200 MPa, respectively. HTM:Eb4859:Sty also had the highest extent of cure. The high extent of cure can contribute to the high storage modulus by reducing the amount of small-molecule plasticizer remaining in the network. All other thermosets displayed storage moduli near 3 GPa. The para substitution in 4-methylstyrene and 4-tert-butylstyrene adds rigidity to the thermosets and thereby increases the Tg, but has a reduced glassy modulus because of greater concentration of unreacted monomer. Thermoset HTM:Eb4859:tbutylSty displayed the lowest storage modulus of 2.8 ± 0.3 GPa. The low extent of cure of the tbutylSty likely played a role in the low storage modulus.The Tg values of all formulations were very similar to each other. The thermoset HTM:CN151:tbutylSty, although it had the lowest extent of cure, featured the highest Tan-δ Tg of 231 ± 0.1 °C. Yet, this formulation displayed a secondary loss modulus peak at ~200 °C, and if examining Tg via the loss modulus, it would be the lowest (140 °C) because of its lowest temperature corresponding to the global maximum in the loss modulus. When examining the storage modulus, HTM:CN151:tbutylSty is middle of the range. Interestingly, all thermosets featured a second low temperature maxima circa 40 °C in the loss modulus  with the0) above 300 °C. There is no low-temperature (<200 °C) decomposition and there is no trend in the T0 with the degree, indicating that the networks are relatively well cured. Focusing attention on thermosets with Eb4859 as the crosslinkers where the difference is with respect to the reactive diluent employed, it can be seen that T0 trend follows the alkyl substitution in the para position of the Styd reactive diluent. The thermoset featuring styrene as the reactive diluent displayed the lowest onset to decomposition, 309 ± 10 °C. The HTM:Eb4859:MeSty thermoset featured the next lowest T0, 312 ± 3 °C followed by the HTM:Eb4859:tbutylSty thermoset having the highest T0 amongst the set of thermosets that featured Eb4859 as the crosslinker, 320 ± 3 °C. This is contrary to what is seen in the literature wherein long aliphatic chains were displayed to reduce thermal properties [0 with reduced extent of cure and especially reduced extent of cure of the reactive diluents. This indicates that the trend in T0 in this series is driven by the size of short chain oligomers in the polymer, indicating that the styrene-based resins had the greatest concentrations of low molecular weight oligomers while tbutylSty-based resins had the lowest concentration of such lower weight oligomers. The relatively low char content for the styrene-based resin further supports this, indicating a high concentration of material is capable of volatizing before carbonization occurs. Comparing thermosets with the same crosslinker and the same reactive diluent, tbutylSty, thermoset HTM:UDM:tbutylSty which featured linear aliphatic urethane functionality (UDM) displayed the lowest onset of decomposition amongst the set, 313 ± 3 °C. The thermoset with the highest T0 was the crosslinker that featured aromatic substitution, CN151, with a T0 value of 352 ± 1 °C, as expected as aromatic vinyl esters have been shown to feature similar T0 values [d between 419 and 422 °C. The only exception being HTM:Eb4859:MeSty which displayed a significantly lower Td of 413 °C. Additionally worth mentioning is the percent char observed from the TGA of the thermosets ranging between 11% and 15%. Quite generally, (meth)acrylate thermosets feature percent char on the order of 101%. It is likely that the HTM flavone rigid bicyclic core is imparting some added thermal stability into the formed thermosets. This conjecture is under further investigation. In all, HTM thermosets display very good thermal stability comparative to ordinary petrochemical (meth)acrylate thermosets. Yet, HTM thermosets feature an elevated percent char at elevated temperatures surpassing that of ordinary petrochemical thermosets and HTM:CN151:tbutylSty displayed the highest T0 of 352 ± 1 °C.The thermal properties of the 3D printed HTM thermosets were characterized by thermal gravimetric analysis (TGA). Representative curves for each thermoset are present in the operties . In fact0 values . Most thLastly, the tensile properties of the 3D printed samples were tested and are displayed in The mechanical properties of HTM resins fall below the desired and expected thermoset properties. Lastovickova et al. displayed the high performance of ternary thermosets featuring isosorbide methacrylate which achieved a tensile strength of 42 ± 4 MPa with a Tg value of 214 ± 3 °C when printed on a Formlabs SLA 3D printer . They al0 value of 352 ± 1 °C, the two superior HTM resin formulations were determined to be HTM:Eb4859:Sty and HTM:Eb4859:tbutylSty due to their other favorable properties. HTM:Eb4859:tbutylSty displayed superior Tg values of 224 ± 2 °C, a tensile modulus of 3.8 ± 0.2 GPa, a room temperature storage modulus of 2.8 GPa, and an ultimate strength of 19 ± 7 MPa and viscosity of 113 ± 3 cP. However, HTM:Eb4859:Sty displayed the highest tensile strength of 25 ± 5 MPa, a room temperature storage modulus of 3.6 ± 0.2 GPa, a Tg value of 212.2 ± 0.4 °C and a low viscosity of 35 ± 1 cP. It is also worth mentioning that HTM:Eb4859:Sty thermosets achieved higher extent of cure compared to HTM:Eb4859:tbutylSty thermosets—85% and 75%, respectively. Albeit different diluents and crosslinkers have unique benefits when formulated with HTM, the HTM core consistently produced thermosets of high Tg values. In all, HTM SLA resins have proved to be printable by vat photopolymerization-type 3D printing on a commercial 3D printer, affording materials of high-performance thermal properties.The flavone monomer HTM was synthesized in one simple synthetic step using phase transfer catalysis from hesperetin as the starting material. Investigations unveiled that, when formulated with appropriate diluents, HTM afforded materials with Tg values greater than 200 °C. A series of high-temperature thermosets were unveiled featuring HTM as the monomer. Styrene derivatives proved to be advantageous diluents for the HTM monomer, affording resins for vat photopolymerization of low viscosity. Investigations unveiled that all styrene derivatives produced resins of low viscosity below 200 cP. 4-methylstyrene produced a resin formulation with the lowest viscosity, 23 cP. 4-tert butylstyrene displayed low toxicity, low vapor pressure and produced the 3D printed material with the highest Tg, 231 ± 0.1 (HTM:CN151:tbutylSty). Use of styrene as the diluent had the benefit of producing thermosets of highest overall cure, highest storage modulus and highest tensile strength. Additional crosslinkers proved useful in their ability to impart increased tensile strength within in thermosets. Urethane functionality proved beneficial over simple aromatic vinyl ester crosslinker CN151 with respect to tensile strength. Crosslinker Eb4859 produced thermosets of the highest tensile strength 25 ± 5 MPa (HTM:Eb4859:Sty). While CN151 had the benefit of increased resistance to thermal degradation with the highest T


WHO has launched an initiative aiming to eliminate cervical cancer as a public health problem. Elimination is a long-term target that needs long-lasting commitment. To support local authorities in implementing human papillomavirus (HPV) vaccination, we provide regional and country-specific estimates of cervical cancer burden and the projected impact of HPV vaccination among today's young girls who could develop cervical cancer if not vaccinated.The expected number of cervical cancer cases in the absence of vaccination among girls born between 2005 and 2014 was quantified by combining age-specific incidence rates from GLOBOCAN 2018 and cohort-specific mortality rates by age from UN demographic projections. Preventable cancers were estimated on the basis of HPV prevalence reduction attributable to vaccination and the relative contribution of each HPV type to cervical cancer incidence. We assessed the number of cervical cancer cases preventable through vaccines targeting HPV types 16 and 18, with and without cross-protection, and through vaccines targeting HPV types 16, 18, 31, 33, 45, 52, and 58.Globally, without vaccination, the burden of cervical cancer in these birth cohorts is expected to reach 11·6 million  cases by 2094. Approximately 75% of the burden will be concentrated in 25 countries mostly located in Africa and Asia, where the future number of cases is expected to increase manyfold, reaching 5·6 million (5·4–6·0) cases in Africa and 4·5 million (4·4–4·6) cases in Asia. Worldwide immunisation with an HPV vaccine targeted to HPV types 16 and 18, with cross-protection against HPV types 31, 33, and 45, could prevent about 8·7 million (8·5–9·0) cases.Detailed estimates of the increasing burden of cervical cancer and projected impact of HPV vaccination is of immediate relevance to public health decision makers. Shifting the focus of projections towards recently born girls who could develop cervical cancer if not vaccinated is fundamental to overcome stakeholders' hesitancy towards HPV vaccination.Bill & Melinda Gates Foundation, Canadian Institutes of Health Research. Approximately 570 000 new cases and 311 000 deaths from cervical cancer occurred in 2018. Cervical cancer is the fourth most common cancer in women worldwide and the greatest contributor to cancer-related mortality among women in sub-Saharan Africa.Infection with high-risk  types of human papillomavirus (HPV) is a necessary cause of cervical cancer.The availability of effective prophylactic HPV vaccines, of validated HPV assays, and of effective treatment of cervical precancerous lesions and cervical cancer underpins the call for global action towards the elimination of cervical cancer as a public health problem launched by WHO in November, 2020.Evidence before this studyWHO has launched a global initiative aiming to eliminate cervical cancer as a public health problem. Scaling up in all countries of vaccination, screening, and precancer treatment, in addition to invasive cancer treatment and palliative care, underpins the global elimination strategy. We searched PubMed for studies published from Jan 1, 2000, to Nov 30, 2020, with the search terms “cervical cancer” or “human papillomavirus” or “HPV” and “elimination” and “model” or “modelling”. Four studies were retained out of 226 identified references. Findings from these studies suggest that widespread coverage of both human papillomavirus (HPV) vaccination and cervical screening has the potential to avert about 13 million cervical cancer cases by 2069. The reported findings also suggest that cervical cancer elimination might be attainable within a few decades in high-income countries but will take longer in low-income and middle-income countries (LMICs), and will be achieved towards the end of the 21st century in a number of sub-Saharan African countries where rates are currently the highest worldwide. However, by mid-2019, only 96 countries, mostly high income, had introduced HPV vaccination. The reported coverage of target birth cohorts is currently unequal throughout the world and, for many countries, often considerably lower than the recommended threshold of 90%. Therefore, worldwide efforts to bolster implementation and scale-up of vaccination programmes are essential, particularly considering that HPV vaccination is highly cost-effective, as recently confirmed by an updated global assessment done with the Papillomavirus Rapid Interface for Modelling and Economics.Added value of this studyAt a local level, public health decision makers need predictions targeted to today's girls and young women at an eligible age for HPV vaccination to inform and frame their vaccination plans. This analysis assesses the expected burden of cervical cancer in the absence of HPV vaccination and the impact of vaccination from the perspective of women born between 2005 and 2014, who could expect to be protected against cervical cancer during their lifespan. We have focused our attention on cohort-specific cervical cancer burden as it is an indicator of direct relevance to decision makers wanting to assess the risk to today's young girls at an eligible age for HPV vaccination. Public health authorities, particularly in LMICs, are facing multiple obstacles, such as vaccine supply shortage, budgetary constraints, and vaccine hesitancy, to implementing and scaling up HPV vaccination as a component of their national expanded programme of immunisation. Our regional and country-specific estimates could provide key stakeholders with clear information to reinforce commitment and overcome reluctance towards HPV vaccination.Implications of all the available evidenceRecent modelling exercises have shown that global cervical cancer elimination is a long-term target that will be achieved by scaling up vaccination, screening, and treatment of precancers and invasive cancers. Therefore, at a local level, to aim at elimination, it is essential to ensure long-lasting commitment to the allocation of adequate resources. Durable political and financial commitment towards HPV vaccination is effectively supported by the direct quantification of its impact on cervical cancer burden among today's girls and young women.Mathematical modelling exercises suggest that this goal might be attainable within a few decades in high-income countries, but will take longer in low-income and middle-income countries (LMICs), and might only be reached towards the end of this century in a number of sub-Saharan African countries where rates are currently the highest worldwide.cohort-specific mortality rates by age from the UN Department of Economic and Social Affairs Population Division, allowing for the competing risk of dying from any cause before being diagnosed with cervical cancer  in the absence of vaccination, along with the corresponding 95% uncertainty intervals (UIs), by combining age-specific incidence projections from GLOBOCAN and the The number of cervical cancer cases that would be preventable through vaccination between ages 15 and 79 years that would be reached between the years 2085 and 2094 in each country was then calculated by reducing the age-specific incidence of cervical cancer proportionally according to the relative contribution of each high-risk HPV type to cervical cancer incidence and assuming the elimination of HPV types targeted by the vaccine—ie, the highest HPV prevalence reduction achievable through a vaccination programme. The number of cervical cancer cases prevented in each country with suboptimal levels of HPV prevalence reduction attributable to vaccination was also estimated by ranging the prevalence reduction from 60% to 100% in all countries. Similar levels of prevalence reduction can result from different combinations of direct and indirect effects of specific characteristics of a vaccination programme , as well as population-specific sexual behaviour.As licensed HPV vaccines do not target all high-risk HPV types, the expected reduction of cervical cancer incidence attributable to vaccination in a given population is dependent on the distribution of HPV types in that population. The relative contributions of HPV types 16 and 18; types 31, 33, 45, 52, and 58; and other high-risk HPV types to the cervical cancer burden were derived from a published meta-analysis providing supra-national regional estimates. For each country, we used the corresponding regional estimate.UN Development Programme. HDI is a summary measure of average achievement in key dimensions of human development in a country . We assessed the correlation between the ratio of future-to-current number of cervical cancers cases and country-specific HDI using the Pearson's coefficient (r). To classify countries according to their contribution to the overall cervical cancer burden, countries were sorted by the expected number of cases and subsequently grouped into the following five categories: very high burden , high burden , medium burden , low burden , and very low burden . HDI was stratified into low–middle HDI (<0·70), high HDI (0·70–0·79), and very high HDI (≥0·80). All analyses were done with R (version 3.6.1).We present our results for the 185 countries included in GLOBOCAN by continent, according to their contribution to the expected absolute burden of cervical cancer among women born between 2005 and 2014 (hereafter referred to as burden) and grouped by their human development index (HDI) in the year 2018, as reported by the The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.Globally, in the absence of vaccination, we would expect 11·6 million (95% UI 11·4–12·0) cervical cancer cases to occur among women born between 2005 and 2014 . Africa When countries are grouped according to the expected burden of cervical cancer in the absence of vaccination, 51·3% of the overall expected burden  would affect birth cohorts in India, Nigeria, China, Tanzania, Indonesia, Uganda, Democratic Republic of the Congo, Ethiopia, and Kenya . Anotherr = –0·5) was observed between the ratio of future-to-current number of cervical cancers cases and country-specific HDI  cervical cancer cases  among women born between 2005 and 2014 . The larIn the appendix, we report detailed country-specific estimates, with 95% UIs, of the number of lifetime cervical cancer cases expected in the absence of HPV vaccination and potentially avoidable through vaccination at differing levels of vaccine effectiveness in the population . We alsoGlobally, in the absence of vaccination, 11·6 million cervical cancer cases are expected in all women born between 2005 and 2014 who are, or will be, eligible for HPV vaccination. In these birth cohorts, HPV vaccination  could prevent 8·7 million cases. 95% of all cases preventable through vaccination would be averted in Africa, Asia, and Latin America, 76% in 26 countries with an expected very high or high burden, and 70% in countries with low–middle HDI.Previous publications suggest that widespread coverage of both HPV vaccination and cervical cancer screening has the potential to avert about 13 million cervical cancer cases by 2069 worldwide.Approximately 50% of all cases are expected to occur before 60 years of age, with the average age at diagnosis higher in Africa, Asia, and Latin America. The relatively young age of occurrence of cervical cancer implies substantial loss of life-yearsoverall improved life expectancy and declining neonatal and child mortality.The global increase in the burden of cervical cancer is driven by the number of cases expected to occur in Africa, Latin America, and central and south Asia, and is due to an HPV prevalence reduction in the population is a directly observable measure of the population-level impact of HPV vaccination and results from the combined direct and indirect effect of specific characteristics of a vaccination programme as well as population-specific sexual behaviour. Of note, several different scenarios could lead to similar levels of prevalence reduction.Our approach to estimate the country-specific expected number of cervical cancers is designed to minimise the assumptions  underlying our projections. First, our projections are based on GLOBOCAN 2018 estimates, which are informed by the best available sources of cancer incidence and mortality data within a given country. Therefore, the validity of the national estimates depends on the degree of representativeness and the quality of source information.In conclusion, we have assessed the expected burden of cervical cancer in the absence of HPV vaccination and the impact of vaccination from the perspective of today's girls and young women at an eligible age for immunisation who could be protected against cervical cancer during their lifespan. As has been noted elsewhere, lifetime risk is a suitable indicator to inform public health targets,Readers can access the data used in this study from the links to public domain resources provided in the Methods. The code used to generate the reported estimates will be made available upon request to the corresponding author.ELF has received grants to his university and personal fees from Merck, outside of the submitted work. ELF has a patent “Methylation markers in cervical cancer” pending to his university. The other authors declare no competing interests.


Nasopharyngeal cancer (NPC) is known to be associated with Epstein–Barr virus (EBV). Pre-treatment and post-treatment detection of plasma cell-free EBV DNA has been shown to be useful as a diagnostic as well as a prognostic factor in NPC. On the other hand, the incidence of human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) is increasing. In contrast to cervical cancer, which is classically known to be an HPV-associated malignancy, HPV testing is not clinically applied for OPC, except for p16 immunostaining as a surrogate marker of HPV infection. One of the major characteristics of HPV-associated OPC is its association with a good prognosis compared with non-HPV-associated OPC. However, some patients still have a poor prognosis. Another characteristic of HPV-associated OPC is the distinct risk factor of high sexual activity. Establishing a biomarker for the prediction of the prognosis before and/or after initial treatment, as well as for diagnosis in populations at high risk, is of marked interest. With this background, HPV DNA detection in plasma and oral rinses has become an area of focus. In this review, the current significance of HPV DNA detection in plasma and oral rinse samples, as well as serum HPV antibody levels, is evaluated. In situ hybridization for EBV-encoded RNA (EBER) can clearly identify EBV-infected tumor cells, whereas elevation of the serum EBV titer has been useful as a diagnostic adjunct ,2. In adExtranodal natural killer/T-cell lymphoma, nasal type (ENKL) is another well-known EBV-associated head and neck malignancy. Lei et al. first reported that plasma EBV DNA was a useful tumor marker for the diagnosis, disease monitoring, and prediction of outcomes in patients with ENKL . Suzuki Head and neck cancer has typically been diagnosed in older patients, in association with the heavy use of tobacco and alcohol . On the With an increasing incidence of HPV-positive OPC, the projected annual number of OPC patients has surpassed that of cervical cancer patients since 2010 ,14. On tE6 and E7 viral oncogenes—have been found to have higher sensitivity than cytology but somewhat lower specificity in cervical cancer [From the perspective of cervical cancer screening, cytology-based screening programs have been implemented in many countries. On the other hand, various HPV-based screening methods—including HPV DNA tests by direct genomic detection or by amplification of a viral DNA fragment using polymerase chain reaction, and HPV mRNA tests to detect the expression of l cancer . Therefol cancer . Furtherl cancer . HoweverThe question of whether plasma/serum cell-free HPV DNA is useful as a biomarker of HPV-associated OPC, like EBV DNA in NPC, is of marked interest. Capone et al. first demonstrated the detection and quantitation of HPV DNA in the sera of four out of nine HPV-associated OPC patients . HoweverInterestingly, plasma HPV DNA reflects the tumor status, as observed in NPC, with Cao et al. demonstrating that serial measurements in 14 patients showed a rapid decline in HPV DNA, which became undetectable on the completion of radiotherapy, whereas the HPV DNA level increased to a detectable level at metastasis in three patients . SimilarAs mentioned before, cervical cancer is known to be associated with HPV. However, in contrast to OPC, there have been limited reports investigating the usefulness of plasma/serum HPV DNA in cervical cancer. In early reports, positivity was low—Pornthanakasem et al. showed that six (12%) out of 50 patients and Dong et al. showed 12 (7%) out of 175 patients tested positive ,26. ResuAs mentioned before, the association of HPV with cervical cancer is clinically applied to cervical screening with primary HPV testing. On the other hand, such a screening system has yet to be established for OPC, although the incidence of HPV-associated OPC has been increasing. From this perspective, oral rinses or saliva may be attractive samples due to the fact that their collection is non-invasive.Smith et al. reported in 2004 that the sensitivity and specificity of HPV DNA detection in oral rinse samples were 58% and 85%, respectively, when combining 67 OPC and 126 oral cancer patients . SeveralAs mentioned before, the quantitation of viral load may present clinical relevance in plasma. Similarly, some reports have investigated the HPV DNA viral load in oral rinse samples  32,36,4,436,44. Residual plasma EBV DNA after the completion of initial treatment is associated with a poorer prognosis in NPC patients. Similarly, as mentioned before, residual plasma HPV DNA could be a predictive indicator of a poorer prognosis in HPV-associated OPC patients. From a similar perspective, the dynamics of HPV DNA in oral rinses after treatment has been of marked interest. Chuang et al. reported that four of 20 patients with positive pre-treatment HPV DNA ultimately developed recurrence, and two of these four patients had HPV16-positive post-treatment salivary rinses, showing 50% sensitivity . On the p = 0.002). Overall survival was reduced among those with a post-treatment saliva HPV-positive status . Rettig et al. prospectively demonstrated that persistent HPV16 DNA was detected in oral rinse samples in five of 124 HPV-associated OPC patients, whereas it was detected in 67 of 124 patients in pre-treatment oral rinse samples [p < 0.001) and overall survival  than patients without persistent HPV16 DNA. Although the prognosis was not stated, Yoshida et al. also demonstrated that persistent HPV DNA in oral rises was only detected in one of eight HPV-associated OPC patients without clinically residual disease after treatment [Ahn et al. demonstrated that the sensitivity and specificity of pre-treatment HPV DNA detection in saliva in 83 OPC patients, including 72 HPV-positive and 11 HPV-negative patients, were 53% and 100%, respectively . Althoug samples . Furtherreatment . These rp < 0.001). Interestingly, the tumor-type HPV DNA load decreased rapidly during primary radiotherapy for most patients . On the other hand, the prevalence rates of tumor-type oral HPV DNA before and after primary surgical resection were 69% and 14%, respectively (p < 0.001). Furthermore, in the subset of patients who required adjuvant radiotherapy, the prevalence decreased from 70% to 38% after surgical resection and then decreased to 1% after radiotherapy (p < 0.001). Significantly lower 2-year overall survival  and recurrence-free survival  were observed among HPV-positive patients with persistent detection of tumor-type HPV in oral rinse samples after treatment than in those without detectable tumor-type HPV in oral rinse samples after treatment. These results suggest that HPV DNA in oral rinse samples rapidly decreases with treatment, and that the persistent detection of HPV DNA in oral rinse samples is associated with poorer survival and recurrence.A larger-scale prospective study was recently published by Fakhry et al. . Of theiAside from HPV DNA detection, many investigators have also been interested in serum HPV antibodies in HPV-associated cancer, including OPC, as described in a review by Mirghani . DahlstrTherefore, the association of pre- or post-treatment E6 antibody titer with the risk of recurrence has been investigated. With respect to pre-treatment serum E6 antibodies, conflicting reports have been published. Koslaova et al. and Fakhry et al. reported that being positive for E6 antibodies was associated with an increased risk of recurrence ,51. On tAlthough significant elevations of serum IgG and IgA antibodies against EBV viral capsid antigen (VCA) have been observed in NPC patients, no significant change was noted between patients in continuous remission and those with tumor recurrence . SimilarAnother well-known HPV-associated head and neck tumor is recurrent respiratory papillomatosis (RRP) . RRP is A preliminary report by Born et al. showed that 25 (93%) of 27 adult-onset RRP patients were positive for HPV DNA in oral rinses . FurtherOne of the interesting and important findings regarding RRP is that HPV DNA was detected in not only tumor tissue, but also in uninvolved sites in patients with active disease and in samples from patients in remission . TherefoThere are several possible options available in regard to HPV testing using blood or oral rinse samples . The maiAlthough it is known that HPV-associated OPC patients have a better prognosis than non-HPV-associated OPC patients, it is true that some HPV-associated OPC patients still show a poor prognosis. De-escalation of the treatment intensity for HPV-associated OPC has been vigorously investigated worldwide. However, de-escalated treatment should not be applied for patients with a poor prognosis. Based on the persistence of HPV-associated markers after treatment or the clearance of such markers, it may be appropriate to stratify treatment.With respect to RRP, no data have been made available as to whether the detection of HPV in oral rinse samples has a significant clinical role. Morphologically apparent RRP can be easily observed through an endoscopy. However, as post-treatment HPV DNA in oral rinses predicts the prognosis of HPV-associated OPC, HPV DNA in oral rinses in patients in remission may predict the recurrence of RRP.It is inevitable to discuss the various methods used to detect HPV DNA in plasma or oral rinse samples, and to measure the serum HPV titer obtained by various investigators. Recently, D’Souza et al. evaluated biomarker prevalence, and showed that oral rinse HPV testing had moderate-to poor sensitivity for HPV-associated OPC, whereas HPV E6 antibodies showed the best overall test performance . In that


We investigated the impact of earlier military combat on ability to cope with the COVID-19 pandemic in 379 male Vietnam veterans who responded to surveys in 1984, 1998, and 2020. Combat exposure was scored with a validated scale, contrasting lowest tertile (8-15) vs. medium/high (16-40). About one-fourth of veterans (26%) reported that their military experience made it easier to cope with the pandemic, while over half (59%) said it had no effect. Medium/high-combat veterans were more likely to report that their military experience made coping easier , but were less likely to report no effect of service on their coping than low-combat veterans . All 19 respondents (5%) who said military experience made coping more difficult were medium/high combat veterans. Military experience, and combat particularly, affected many of these veterans’ ability to cope with the pandemic decades after their service.