Pseudomonas fluorescens strain SS101 (PfSS101) followed by site-directed mutagenesis previously suggested that sulfur assimilation may play an important role in growth promotion and induced systemic resistance in Arabidopsis. Here, we investigated the effects of sulfur metabolism in PfSS101 on growth, defense, and shoot metabolomes of Arabidopsis and the Brassica crop, Broccoli. Root tips of seedlings of Arabidopsis and two Broccoli cultivars were treated with PfSS101 or with a mutant disrupted in the adenylsulfate reductase cysH, a key gene in cysteine and methionine biosynthesis. Phenotyping of plants treated with wild-type PfSS101 or its cysH mutant revealed that sulfur assimilation in PfSS101 was associated with enhanced growth of Arabidopsis but with a reduction in shoot biomass of two Broccoli cultivars. Untargeted metabolomics revealed that cysH-mediated sulfur assimilation in PfSS101 had significant effects on shoot chemistry of Arabidopsis, in particular on chain elongation of aliphatic glucosinolates (GLSs) and on indole metabolites, including camalexin and the growth hormone indole-3-acetic acid. In Broccoli, PfSS101 sulfur assimilation significantly upregulated the relative abundance of several shoot metabolites, in particular, indolic GLSs and phenylpropanoids. These metabolome changes in Broccoli plants coincided with PfSS101-mediated suppression of leaf infections by Xanthomonas campestris. Our study showed the metabolic interconnectedness of plants and their root-associated microbiota.Genome-wide analysis of plant-growth-promoting  Pseudomonas is an abundant member of the plant microbiome, particularly of the rhizosphere. Various studies have shown that different strains of root-associated Pseudomonas species can promote plant growth, alter root architecture, and induce systemic resistance [Pseudomonas fluorescens strain SS101 (PfSS101) that were associated with growth promotion and induced systemic resistance in Arabidopsis [Pseudomonas syringae pv. tomato (Pst). Subsequent validation by site-directed mutagenesis and genetic complementation of specific mutants in PfSS101demonstrated that the phosphogluconate dehydratase gene edd, the response regulator gene colR, and the adenylsulfate reductase gene cysH play important roles in plant growth promotion, alteration of root architecture, and induced systemic resistance (ISR). cysH is a key gene involved in sulfur assimilation and in the biosynthesis of the amino acids cysteine and methionine. Transcriptome analysis of Arabidopsis further revealed that biosynthetic processes associated with sulfur compounds (in particular cysteine and GLSs) were the most significantly enriched in seedlings treated with PfSS101 when compared to control plants and to plants treated with the cysH mutant [PfSS101 modulates sulfur metabolism in Arabidopsis, confirming and extending earlier results from Meldau et al. [Arabidopsis by different Bacillus strains.The genus sistance ,2,3. By bidopsis . Twenty-H mutant . These ru et al.  and Azizu et al.  who attr42−) taken up by roots from the environment is turned into adenosine 5′-phosphosulfate (APS) and subsequently reduced to sulfite (SO32−) and then to sulfide (S2−). The condensation of S2− and an amino acid derivative O-acetylserine results in cysteine. In plants, this key amino acid is incorporated into proteins and glutathione or serves as the sulfur donor of methionine and sulfur-containing secondary metabolites [Pseudomonas fluorescens strain SS101 (PfSS101) increased the levels of glucosinolates (GLSs), coumarins, flavonoids, and camalexin in shoots and roots of Arabidopsis [Arabidopsis mutants, that the sulfur-containing indolic compound camalexin is associated with the growth-promoting activity of Pseudomonas sp. CH267. In summary, the above-mentioned studies indicate that rhizobacteria can influence their host metabolism. However, if and to what extent a particular bacterial trait influences host metabolism, growth, and tolerance to biotic stresses remains largely elusive.In plants, sulfate (SOabolites . Previoubidopsis . In a rebidopsis  elegantlcysH mutation) in Pf SS101 on the shoot metabolome of Arabidopsis. To that end, we adopted a non-targeted comparative metabolomics approach to assess the differences between the shoot metabolomes of Arabidopsis seedlings treated with either PfSS101 or cysH-mutant 20H12. Furthermore, we investigated if strain PfSS101 can induce similar phenotypic and metabolome changes in shoots of the related Brassicaceous crop plant Broccoli, Brassica oleracea var. italica, and if these changes are associated with sulfur metabolism. For Broccoli, two cultivars were selected based on their contrasting levels of GLSs  and thereby possible differences in sulfur needs. The PfSS101-mediated phenotypic responses investigated for the Broccoli cultivars included root and shoot growth as well as induced systemic resistance against two pathovars of Xanthomonas campestris, an important bacterial leaf pathogen of Broccoli and other cruciferous crops [The aim of the present study was to investigate the effects of sulfur metabolism  were surface-sterilized as previously described [Brassica oleracea var. italica) cultivars, namely Coronado and Malibu, were kindly provided by Bejo Seeds . Cultivar Coronado has higher levels of specific GLSs  than cultivar Malibu. Surface sterilization of the Broccoli seeds was performed by immersing 2–3 g of seeds for 30 min in 30 mL of 1% (v/v) sodium hypochlorite supplemented with 0.1% (v/v) of Tween 20 followed by three washes with ample sterile distilled water. For the Arabidopsis assays, sterile seeds were sown on 90-mm-diameter Petri dishes containing 20 mL half-strength Murashige and Skoog (MS) agar medium, containing 0.5% sucrose (w/v) and 1.2% plant agar (w/v). For the Broccoli assays, five sterile seeds were sown on 140-mm-diameter petri dishes containing 50 mL of half-strength MS agar. The plates were then placed in a climate chamber maintained at 21 °C/21 °C day/night; 180 µmol light m−2s−1, 16 h light/8 h dark cycle, and 70% relative humidity. After a week of Arabidopsis growth and five days of Broccoli growth, roots of each seedling were inoculated with a 2 µL of bacterial suspension (~109 cells mL−1). After inoculation, plants were placed back in the same growth chamber until harvest.Seeds of escribed . Seeds oPseudomonas fluorescens strain SS101 (PfSS101 (Taxonomy ID: 1038924)) was originally isolated from the wheat rhizosphere [cysH (adenylylsulfate reductase)-mutant 20H12 of PfSS101 was generated by site-directed mutagenesis as described by Cheng et al. [PfSS101 and mutant 20H12 were cultured in King’s medium B (KB) at 25 °C for 16 h. Broccoli leaf pathogens Xanthomonas campestris pv. armoraciae (Xca) and Xanthomonas campestris pv. campestris (Xcc) were kindly provided by Bejo Seeds and were cultured in Luria Bertani (LB) medium  at 25 °C for 16 h. Bacterial cells were collected by centrifugation, washed three times and resuspended in with 10 mM MgSO4 to a final density of 109 cells mL−1 (OD600 = 1.0).zosphere . cysH (aPfSS101 or the cysH mutant and grown for 11 days. The first true leaves of the primed plants were then pierced and 2 µL (109 cells mL−1) of inoculum of the bacterial pathogens Xca and Xcc was applied. Ten days after pathogen challenge, a 0–5 ordinal scale that scores the migration of the lesion from the inoculation spot to other parts of the shoot was used  to inhibit fungal growth and incubated for 3 days at 25 °C. Colonies with the typical phenotypes of Xca and Xcc were counted and expressed as colony-forming units (cfu) mg−1 of shoot fresh weight.The roots of the Broccoli plants were treated with was used . Scale: Arabidopsis and Broccoli were harvested at 11 days after root treatment with buffer (control), PfSS101, or mutant 20H12. For each plant species/cultivar X rhizobacteria combination, four biological replicates were used with 10 Arabidopsis and five Broccoli seedlings per replicate. In brief, shoots were snap frozen in liquid nitrogen and ground to fine powder under continuous cooling and kept at −80 °C until further use. To extract semi-polar secondary metabolites, 300 µL of 99.89% methanol containing 0.13% (v/v) formic acid was added to 100 mg plant material in 2 mL round bottom Eppendorf tubes, mixed, and sonicated for 15 min followed by centrifugation for 15 min at 20,000× g. The supernatants were transferred to 96-well filter plates , vacuum filtrated to the 96-deep-well autosampler plates  using a Genesis Workstation .Shoots of v/v) acetonitrile in 0.1% (v/v) formic acid (FA) in water at a flow rate of 0.19 mL per min. Then, 5 µL of each extract was injected and compounds were separated on a Luna C18 column  maintained at 40 °C [m/z 200) in a range of m/z 95–1350. Subsequent MS/MS experiments for identification of selected metabolites were performed with separate positive or negative electrospray ionization at a normalized collision energy of 27 and a mass resolution of 17,500. The ionization voltage was optimized at 3.5 kV for positive mode and 2.5 kV for negative mode; capillary temperature was set at 250 °C; the auxiliary gas heater temperature was set to 220 °C; and sheath gas, auxiliary gas and the sweep gas flow were optimized at 36, 10, and 1 arbitrary units, respectively. Automatic gain control was set a 3e6 and the injection time at 100 ms. External mass calibration with formic acid clusters was performed in both positive and negative ionization modes before each sample series.An UltiMate 3000 U-HPLC system  was employed to create a 45-min linear gradient of 5–35% (at 40 °C . The detp < 0.05) and fold changes more or less than 2.0, either increased or decreased, were applied to identify mass signals that were significantly changed in bacteria-treated compared to control samples. Then, we performed a bootstrapping analysis to remove duplicate metabolites between the two ionization modes (positive and negative) incorporating retention time and mass relationship between two metabolites. Data transformation and scaling were performed in GeneMaths XT . Transformed and scaled values were used for hierarchical cluster analysis using Pearson’s correlation coefficient and the unweighted pair group method with arithmetic mean (UPGMA). Baseline-corrected mass features of the raw LC–MS data were extracted and aligned using Metalign software 3.0. . The masArabidopsis and Broccoli using a similar LC–MS system and identical chromatographic conditions . If moll et al.  were pril et al.  and Metll et al. .p < 0.05. For the disease severity data, beta regression analysis was employed to examine the interaction effect of two independent variables (Rhizobacteria and Broccoli cultivars) on disease severity of the two bacterial pathogens using (“betareg”) package in R [Changes in shoot biomass and pathogen incidence between treatments were analyzed with R Studio software (Version 3.6.1) following similar methodology used in our previous studies ,8,17. Thage in R . The corArabidopsis roots with PfSS101 and the cysH-mutant 20H12 led to a significant increase in total plant biomass by 62.8% ± 4.6 and 35.1% ± 4.8, respectively  and changed root architecture by reducing the length of the primary root and enhancing lateral root formation. In contrast, the cysH-mutant 20H12 neither affected neither root biomass nor exerted major effects on root architecture (Treatment of PfSS101 and mutant 20H12 on plant growth was different from that observed for Arabidopsis. There was a significant interaction effect between the Broccoli cultivars and the rhizobacteria (wild type/mutant) for the shoot, the root, and total biomass , and significant increases in root biomass   showed significant reduction in disease severity, whereas treatment with mutant 20H12 was ineffective  cut-off of >2 and significance p-values of <0.05 in ANOVA were used to identify metabolites that were altered significantly by the treatment of the plants root with PfSS101 or its cysH mutant and used to compute principal component analysis (PCA) and hierarchical cluster analysis (HCA).Non-targeted LC–MS-based metabolite profiling was applied to investigate the impact of the Arabidopsis shoots in either positive or negative ionization mode, 128 (18%) metabolites were significantly different between the PfSS101 and its cysH-mutant treatments. Abundance and fold changes of each detected plant metabolite are shown in Pseudomonas (PfSS101 or 20H12)-treated and untreated plants. The HCA plot (PfSS101 or 20H12 as compared to the controls (PfSS101 and include the short-chain (C-4) isoleucine-derived aliphatic 2-methylbutyl GLS (glucocleomin), amino acids, and derivatives such as arginine and N-acetyl-L-tyrosine, and fatty acyl glycosides such as pantothenic acid-glucoside. Cluster 2 comprises metabolites that were depleted in plants treated with both PfSS101 and the mutant 20H12, and the depletion was more pronounced on plants treated with PfSS101. Some of the identified metabolites in this cluster include an amino acid and derivatives thereof such as glutamine and N-(1-deoxy-1-fructosyl) proline, 5-oxoproline, fatty acyl glycosides, and O-feruloylquinic acid. Metabolites that were increased by the PfSS101 treatment are shown in Cluster 4 and include a long-chain (C-8) aliphatic-GLS, i.e., 8-methylthiooctyl glucosinolate, a hydroxycinnamic acid glucuronide, i.e., sinapic acid-O-glucuronide, the medium-chained keto acid oxodecanoic acid, and the coumarin scopolin (see cluster 4). Cluster 5 represents metabolites that were enhanced in both PfSS101 and 20H12 treatments and include the long-chain (C-8) aliphatic-GLS 8-methylsulfinyloctyl glucosinolate (glucohirsutin), the hydroxycinnamic acid amide coumaroylagmatine and stress-associated alkaloids such as camalexin (20.5-fold up in PfSS101 and 17.3-fold up in 20H12), and indole-3-acetic acid (IAA) (8.4-fold in PfSS101 and 6.9-fold in 20H12). The second principal component (PC2) explained 27.9% of the total variance and includes metabolites that were mainly accumulated in plants treated with the 20H12 (Cluster 3) relative to the other treatments. Here, 4-methylsulfinylbutyl GLS (glucoraphanin) and 5-methylsulfinylpentyl GLS , middle-chained  aliphatic-GLSs, displayed 20H12-specific accumulation.From a total of 725 metabolites detected in HCA plot b showed Arabidopsis shoots by PfSS101 and mutant 20H12 are enhanced levels of the plant growth hormone IAA and the stress-associated alkaloid camalexin, while the most prominent differences in shoot metabolome involve the medium-chain keto acid oxodecanoic acid, and the sulfur-containing compounds 8-methylthiooctyl, 4-methylsulfinylbutyl, and 5-methylsulfinylpentyl GLS; these were all less enhanced in plants colonized by the cysH mutant compared to plants colonized by wild-type PfSS101.In conclusion, the most prominent similarities in shoot metabolome changes induced in PfSS101 further enhanced the levels of the indole GLS glucobrassicin by 5- and 4-fold, respectively, whereas the levels of the other two aliphatic GLSs were not significantly changed upon PfSS101 treatment  and thereby possible differences in sulfur needs. Our metabolome analysis confirmed that cultivar Coronado indeed has higher levels of these three GLSs  than cultivar Malibu . Treatmereatment . Treatme PfSS101 .cysH mutation in PfSS101 did have substantial impact on the overall Broccoli shoot metabolism . The HCA plot . Cluster 5, the largest cluster of PfSS101-enhanced metabolites, comprised metabolites associated with plant-defense-related biosynthesis of phenolic compounds such as the flavonoids kaempferol-di/tri-(feruloyl/ caffeoyl/ coumaroyl) glycosides, quercetin-tri-coumaroyl glycoside, rutin, hydroxycinnamates caffeic acid, ferulic acid, feruloylquinic acid, sinapic acid, chlorogenic acid, neocuscutoside C, and their derivatives, and resveratrol sulfoglucoside. In addition, some phenolic glucosides including ginnalin B, hydroxybenzaldehyde diglycoside, and hydrojuglone glucoside, as well as antioxidants such as ascorbic acid (vitamin C) and dehydroascorbic acid also belonged to this metabolite cluster that was enhanced in the PfSS101 treatment. Among the identified GLSs in cluster 5, the indole-GLS glucobrassicin and its derivatives showed significant increases in the PfSS101 treatment.The tabolism . DetaileHCA plot b showed PfSS101 and includes some amino acids and derivatives, allantoic acid, and benzylpenicilloic acid, as well as 3-sulfolactaldehyde. PC2 explained 30.6% of the total variation and was associated with metabolites that were intrinsically different in abundance between the two Broccoli cultivars  and clusters 9 and 11 (high in Coronado)). Furthermore, metabolite clusters 7, 9, and 11 showed higher accumulation in plants treated with PfSS101 when compared to mutant 20H12. Metabolites in both Broccoli cultivars that were similarly decreased by PfSS101 and cysH mutant, as compared to the control, grouped together in cluster 2 and included phenylalanine and tryptophan, which are the building blocks for phenylpropanoid and indole-GLS biosynthesis, respectively. In addition, malic acid was most abundant (1.8-fold) in Malibu (cluster 7) while the level of the aliphatic-GLS glucoiberverin was much higher (92-fold) in Coronado (cluster 11). The third PC explained 9.2% of the total variation (not shown) and was associated with metabolites that accumulated (cluster 4) or decreased in Malibu (cluster 8) treated with 20H12. In summary, the cysH mutation in PfSS101 specifically affected the abundance of metabolites in Broccoli shoots associated with flavonoid, hydroxycinnamate, and indole-GLS biosynthesis.Cluster 1 encompasses metabolites that were reduced in plants inoculated with PfSS101 promoted growth and altered root architecture of Arabidopsis, induced systemic resistance (ISR) and, based on targeted metabolomics, enhanced GLS levels in both roots and shoots [PfSS101, we then identified the adenylsulfate reductase gene cysH as one of the key genes associated with growth promotion and ISR in Arabidopsis [cysH is involved in sulfur assimilation and the biosynthesis of cysteine and methionine. Results from Cheng et al. [Arabidopsis in a concentration-dependent manner and triggered ISR against the bacterial leaf pathogen Pseudomonas syringae. Cysteine can enter the GLS biosynthesis pathway of Arabidopsis by different routes including (i) direct donation of reduced sulfur needed for GLS biosynthesis, (ii) incorporation of cysteine into methionine that, through a series of side-chain elongations, S-glycosylation, and other secondary modifications, ends up in the GLS pool, and (iii) conjugation of cysteine, glutamate, and glycine to form glutathione (GSH) [Arabidopsis upon root treatment with PfSS101 and provided several new findings. These include higher levels of two octyl GLS forms, i.e., 8-methylthio and 8-methylsulfinyl, in plants colonized by PfSS101. Furthermore, the isoleucine-derived short-chain (C-4) aliphatic 2-methylbutyl GLS showed significant reduction in plants treated with PfSS101. In Arabidopsis, side-chain elongation of aliphatic-GLS is catalyzed by the MAM1/MAM3 proteins, which condense 2-oxo acids and acetyl-CoA to extend the alkane C chain [PfSS101. Interestingly, transcriptome analysis from our previous study revealed that plants treated with PfSS101 showed significantly higher expression of both MAM1 and MAM3 genes when compared with plants treated with the cysH mutant or compared with untreated plants [PfSS101-treated plants, cysH-mutant-treated Arabidopsis seedlings contained lower levels of oxodecanoic acid and the long-chain octyl GLSs, and higher levels of the short-chain 4-methylsulfinylbutyl glucosinolate (C4) and 5-methylsulfinylpentyl glucosinolate (C5). Collectively these results indicate that the cysH gene of PfSS101 affects chain elongation of aliphatic-GLSs in leaves of PfSS101-treated Arabidopsis. Sulfur is essential for plant growth and plays an important role in plant defense against fungal pathogens and insect pests. We previously showed that the rhizobacterium d shoots . By screbidopsis . cysH isg et al.  further ne (GSH)  which inne (GSH) . The res C chain ,22. Oxod-chain C- aliphatiPfSS101 promoted growth and altered root architecture of Arabidopsis, induced systemic resistance (ISR) and, based on targeted metabolomics, enhanced GLS levels in both roots and shoots [PfSS101, we then identified the adenylsulfate reductase gene cysH as one of the key genes associated with growth promotion and ISR in Arabidopsis [cysH is involved in sulfur assimilation and the biosynthesis of cysteine and methionine. Results from Cheng et al. [Arabidopsis in a concentration-dependent manner and triggered ISR against the bacterial leaf pathogen Pseudomonas syringae. Cysteine can enter the GLS biosynthesis pathway of Arabidopsis by different routes including (i) direct donation of reduced sulfur needed for GLS biosynthesis, (ii) incorporation of cysteine into methionine that, through a series of side-chain elongations, S-glycosylation, and other secondary modifications, ends up in the GLS pool, and (iii) conjugation of cysteine, glutamate, and glycine to form glutathione (GSH) [Arabidopsis upon root treatment with PfSS101 and provided several new findings. These include higher levels of two octyl GLS forms, i.e., 8-methylthio and 8-methylsulfinyl, in plants colonized by PfSS101. Furthermore, the isoleucine-derived short-chain (C-4) aliphatic 2-methylbutyl GLS showed significant reduction in plants treated with PfSS101. In Arabidopsis, side-chain elongation of aliphatic-GLS is catalyzed by the MAM1/MAM3 proteins, which condense 2-oxo acids and acetyl-CoA to extend the alkane C chain [PfSS101. Interestingly, transcriptome analysis from our previous study revealed that plants treated with PfSS101 showed significantly higher expression of both MAM1 and MAM3 genes when compared with plants treated with the cysH mutant or compared with untreated plants [PfSS101-treated plants, cysH-mutant-treated Arabidopsis seedlings contained lower levels of oxodecanoic acid and the long-chain octyl GLSs, and higher levels of the short-chain 4-methylsulfinylbutyl glucosinolate (C4) and 5-methylsulfinylpentyl glucosinolate (C5). Collectively these results indicate that the cysH gene of PfSS101 affects chain elongation of aliphatic-GLSs in leaves of PfSS101-treated Arabidopsis. Sulfur is essential for plant growth and plays an important role in plant defense against fungal pathogens and insect pests. We previously showed that the rhizobacterium d shoots . By screbidopsis . cysH isg et al.  further ne (GSH)  which inne (GSH) . The res C chain ,22. Oxod-chain C- aliphaticysH-mediated sulfur metabolism in PfSS101 appeared to adversely affect shoot biomass, as in both cultivars root treatment with the cysH mutant resulted in higher overall biomass compared to wild-type PfSS101. The total biomass of both PfSS101-treated cultivars showed no significant changes relative to their non-treated controls, but PfSS101 significantly affected biomass allocation between shoot and roots with significant reductions of the shoot biomass and significant increases in the root biomass. In contrast to wild-type PfSS101, the cysH mutant significantly enhanced total biomass of cultivar Malibu. Also, the change in biomass allocation by the cysH mutant was not as apparent as it was for wild-type PfSS101. Collectively these results suggest that cysH-mediated sulfur assimilation in PfSS101 has a neutral to negative effect on Broccoli plant growth in a cultivar-dependent manner. In the Broccoli shoot metabolome, PfSS101 enhanced the accumulation of defense-associated metabolites, specifically those related to the phenolic pathway of flavonols, including kaempferol and quercetin glycosides and various hydroxycinnamates, such as caffeic acid and ferulic acid conjugates, as well as the antioxidant ascorbic acid and the indole-GLSs glucobrassicin, desulfoglucobrassicin (indolylmethyl-desulfoglucosinolate), and 4-methoxyglucobrassicin (4-methoxy-3-indolylmethyl glucosinolate). By contrast, the levels of these flavonoids, hydroxycinnamates, and indole-GLSs only showed slight to moderate increases in Broccoli seedlings treated with the cysH mutant. The enhanced level of glucobrassicin and its derivatives, and the concomitant lower shoot biomass in PfSS101-treated seedlings suggest that cysH-mediated sulfur assimilation in PfSS101 can modulate tryptophan metabolism in Broccoli towards the biosynthesis of indole-GLSs instead of the growth-promoting phytohormone IAA. Furthermore, the relatively high abundance of flavonoids in Broccoli plants treated with PfSS101 could impact auxin biosynthesis, transport [PfSS101 could pose a limitation of carbon and energy resources, leading to an adverse effect on plant growth. For Broccoli, ransport , and disransport , therebyPfSS101 adversely affected overall growth in Broccoli, it led to enhanced defense against Xca in a cultivar-specific manner. When roots of the cultivar Coronado were treated with PfSS101 and the leaves challenged with Xca, a significantly reduced disease severity was observed, whereas root treatment with cysH-mutant 20H12 was not effective. By contrast, in cultivar Malibu both PfSS101 and mutant 20H12 reduced disease severity caused by Xca. The extent of PfSS101-mediated changes in the Broccoli shoot metabolome, particularly towards defensive secondary metabolites such as flavonoids, hydroxycinnamates, lignin, iridoid glycosides, and indole-GLSs was substantial. Phenolic compounds can have direct or indirect inhibitory effect on bacterial pathogens, involving disruption of growth- and reproduction-related processes in the pathogens [Xcc in Brassica oleracea [PfSS101-mediated systemic resistance in Broccoli against Xca and to some extent against Xcc is likely related, at least partly, to the observed changes in these shoot secondary metabolites. These changes in the shoot metabolome are likely associated with pathogen defense and their impact is dependent on the pathovar (Xca/Xcc) of the pathogen.Although sulfur assimilation by athogens ,26, whilathogens ,28. Simioleracea  while 4-oleracea . Hence, cysH-mediated sulfur assimilation in PfSS101. For example, the level of ascorbic acid (vitamin C), an antioxidant element [PfSS101-treated seedlings as well, possibly by an upregulation of the L-galactose pathway of ascorbate biosynthesis or due to less oxidative stress. In addition, a significant increase of scopolin, a coumarin glycoside, was detected in our study. Recent work on Arabidopsis treated with growth-promoting Pseudomonas simiae strain WCS417 revealed enhanced levels of scopoletin in root exudates and showed that scopoletin is an iron-mobilizing phenolic compound with selective antimicrobial activity that shapes the root-associated microbial community [Pseudomonas strain PfSS101. Whether these coumarins also have antibacterial activity against the two Xanthomonas campestris pathovars of Broccoli tested in this study, will be the subject of future investigations to unravel the importance of coumarins in the observed ISR response.Interestingly, there were other metabolite changes observed in Broccoli shoots that appear to be disconnected from  element ,32, was ommunity . Our resPseudomonas exerts significant changes in host plant secondary metabolism, in particular chain elongation of aliphatic glucosinolates (GLSs) and indole metabolites, including camalexin and the growth hormone indole-3-acetic acid. In Broccoli, PfSS101 sulfur assimilation significantly upregulated the relative abundance of several shoot metabolites, in particular indolic GLSs and phenylpropanoids, metabolome changes that coincided with PfSS101-mediated suppression of leaf infections by Xanthomonas campestris. Our study provides evidence of the interconnectedness of the metabolism of the host and that of a member of their root-associated microbiota. Integration of metabolomics with transcriptomics data followed by targeted mutagenesis or silencing of specific genes or pathways in the host plant will be needed to validate the functional importance of the observed metabolome changes for plant growth and defense.In conclusion, our results showed, for the first time, that disruption of sulfur metabolism in root-colonizing


Background: Every year in Europe 60,000 women develop cervical cancer and 30,000 die from the disease. HPV vaccines are currently believed to constitute an important element of cervical cancer control strategy. The introduction of the HPV vaccine constitutes a shift in health policy and health promotion practice. The aim of this study was to investigate knowledge, beliefs and communication preferences of the Greek public with regards to the HPV vaccine.Methods: Six focus groups (5–8 participants) were conducted with unvaccinated teenage girls, young women and mothers of vaccine-eligible girls, recruited via schools and universities. Pre-focus group questionnaires consisted of: socio-demographic details and a knowledge questionnaire. The discussion guide was based on issues raised by relevant studies such as disease severity and personal risk perception, perceived benefits and barriers to vaccination, etc. Participants were also shown three types of stimuli namely, a leaflet, an expert interview and a documentary containing equivalent information on HPV and the HPV vaccine. Thematic analysis was used for analysis of the qualitative data.Findings: Findings revealed that participants had low to moderate knowledge levels on HPV and cervical cancer. Beliefs specific to the HPV vaccine as well as general beliefs about medicines and their use in everyday life were prevalent. The vaccination dilemma was exacerbated by the conflicting advice received by health professionals coupled with the difficulties participants experienced in evaluating relevant health information. Participants judged all stimuli according to content and format but the documentary was their preferred health information communication option.Conclusions: Findings could contribute to the understanding of health decision making with regards to preventive vaccination and help formulate recommendations for health policy adjustments. Every year in Europe 60,000 women develop cervical cancer (CC), the second most frequent cancer in women under 45, leading to 30,000 deaths  have always constituted a formal source of health information for the general public Logan,  however In Greece, healthcare providers recognized the HPV vaccine as an important medical innovation, even though had concerns about its safety, effectiveness and long-term impact. Hence they did not consistently recommend vaccination and sometimes advised against it  who are the main target population for HPV vaccination, mothers of vaccine-eligible girls who are likely to influence their children’s decision or make it for them and young women (18–26) who are within the age range for vaccination but more likely to decide this for themselves.To the best of our knowledge, there have been no other qualitative research studies to date in Greece since the release of the HPV vaccine in 2008 investigating knowledge, beliefs and communication preferences of critical segments of Greek teenage girls, young women and mothers in relation to the HPV vaccine.What is the level of knowledge of mothers, teenage girls and young women regarding HPV and the HPV vaccine?What beliefs do mothers, teenage girls and young women hold with regards to the HPV vaccine?How do mothers, teenage girls and young women perceive different modes of health information communication regarding HPV and the HPV vaccine and which is judged as the preferred mode?Research questions included:This was a cross-sectional study employing qualitative methodology. Specifically, qualitative data was gathered via focus group discussions. Some quantitative data was gathered via pre-focus group questionnaires in order to provide a reference frame within which the qualitative data would be analyzed and interpreted.Six focus groups (5–8 participants each) were conducted with teenage girls, mothers of vaccine-eligible girls and young women recruited from two major Greek cities; Athens and Thessaloniki. Participants responded to invitations funneled  through key institutions namely local schools and universities.Two focus group discussions were held with each of the following: (1) unvaccinated teenage girls between the ages of 12 and 17; (2) unvaccinated young women between the ages of 18 and 26; (3) mothers of unvaccinated girls between the ages of 12 and 17.Pre-focus group questionnaires consisted of questions on socio-demographic data and a knowledge scale. Socio-demographic questions included age, education, personal history of cervical cancer, HPV awareness and previous vaccine recommendation by a healthcare professional.The knowledge item scale  produced a leaflet titled ‘Get vaccinated today! Vaccination to prevent human papillomaviruses causing cervical cancer and other serious diseases’ and followed a question and answer format. It presented the main facts about HPV and the link to cancer, HPV virus transmission, treatment of the HPV infection, the HPV vaccine, including safety, efficacy, mode of delivery, and vaccination target age groups. The leaflet could be found in Obstetrics and Gynecology clinics.The Greek Organization of Child and Pediatric Gynecology  and sequence  as well as a participant number  accompanies every quote. This study adhered to the current Research Ethics guidelines as articulated in the European Commission’s ‘Ethics for Researchers’ (7th Framework) and the declaration of Helsinki.The sample included 13 mothers out of which 2 were high school graduates and 11 were higher education graduates. Also, 11 university students and finally 16 school students out of which 8 attended Gymnasium (low secondary school) and 8 attended Lyceum (upper secondary school). The vaccine was recommended by a health professional to 60% of the sample. A small portion of the sample (5%) had been informed by a health professional that they had precancerous cervix lesions.The mean HPV knowledge sample score was relatively low with a mean score 3.8 (SD = 1.94) out of a possible maximum score of 10, while mean knowledge scale scores differed across the three age groups .Table 1U-test analyses were performed and revealed significant differences between mothers and young women  as well as between young women and teenage girls . The percentage of correct and incorrect responses for each knowledge item is shown in Mann–Whitney The themes are individually analyzed and presented along with supporting representative themes .Figure The majority of participants were aware of the link between HPV and cervical cancer (cc). Specifically, that CC is a possible but not definite outcome of the HPV infection.The idea that a virus may cause cancer was surprising to a few participants in one of the mothers’ groups who had no prior knowledge of the HPV-CC link. After further discussion within the group this concept did not seem as outlandish as initially thought by participants who managed to think of other examples of benign or non-life threatening conditions evolving to cancer i.e. colon polyps to colon cancer.Cervical cancer was judged as a serious health issue which could have consequences on a woman’s reproductive ability. Moreover, participants were acutely aware of the importance of symptoms with regards to seeking medical help and benefits of early detection of CC. Also, they realized current medical advancement allows for the successful treatment of CC. Finally, they recognized the smear test and the HPV vaccine are both effective ways of CC prevention.Overall, young women and teenage girls were more knowledgeable than mothers on HPV and CC issues, a fact also observed from the quantitative data. However, all participants stated that they knew much more about other forms of cancer (i.e. breast cancer) compared to CC and much more about other sexually transmitted infections (i.e. HIV) compared to HPV. This is not surprising as these health issues either have received much more publicity and/or have been at the center of the public health arena for a longer period of time.Necessity beliefs were expressed by participants revolving around the idea that the vaccine is an important medical innovation, a powerful tool for all to use and the only vaccine that can protect against cancer. Great focus was given on the regret they would feel if they did not get vaccinated and during the course of their lives contracted an HPV infection. Furthermore, participants were aware that HPV vaccine can protect against the most frequently seen, high-risk strains of HPV and thought it was an effective means of CC prevention.As these were unvaccinated individuals, in their attempt to make a decision, they weighed their concerns about the risk of getting vaccinated against the benefits of protecting oneself from cancer. This negotiation involved an active search of information about the HPV types the vaccine could guard against and the side effects commonly experienced by vaccine users.All of the participants cited the novelty of the vaccine as the main barrier to vaccination. Specific concerns mainly included possible long-term consequences and short-term side effects. Other concerns expressed included the possibility that individuals have an allergy to an ingredient within the vaccine or have a weak immune system and do not respond to the vaccine in the desired way. Finally, the possibility that the virus manifests itself after it enters the body through the vaccine as if the person has been infected.The information communicated by health care professionals naturally influenced their beliefs about the HPV vaccine and their attitudes towards vaccination. Participants stated that they had received conflicting advice from healthcare professionals in terms of the vaccination recommendation, a fact which made them question the reliability of the vaccine itself. Their concerns were further reinforced by testimonials they had read or heard about, of individuals receiving the HPV vaccine and suffering serious side effects such as paralysis.The majority of participants voiced the opinion that healthcare professionals are eager to promote the administration of HPV vaccine and other medicines because they have an ulterior motive. Specifically, health care professionals gain financially from pharmaceutical companies and therefore medical decision making is essentially shaped by their product promotion than by strictly medical purposes. They also voiced concerns about the procedures pharmaceuticals followed to get a product from research to the market, the location and participants of clinical trials and wondered about the ethical issues that might arise and how these are being handled.Doubts were expressed solely in the mothers’ group about the way the state is recording and categorizing incidents of HPV vaccine side effects. Furthermore, participants had reservations about whether the appropriate state agency is feeding back this information to the pharmaceutical companies or to the health care professionals in a systematic manner. Most importantly, they had a desire to know the manner in which members of the public can report incidents related to the vaccine. This was especially salient due to the fact that the vaccine is not administered in the context of state vaccination effort but rather can be administered individually at a local pharmacy store without a follow-up. Finally, mothers wondered whether this information was ever made public through formal sources.This theme pertained to the advance of medical science, a continuous process during which the old becomes outdated and gives way to the new. There was a consensus among participants that a lot of medical recommendations once considered imperative were disputed sometime in the future. Older participants mentioned as an example the polio vaccination, which was converted into an injectable form and consequently lost its potency. Medical science itself is questioning medical innovations after consequences start to show. Participants’ indecisiveness with regards to the vaccination was enhanced by their concern that this medical innovation, namely the HPV vaccine, might not be the medical recommendation a few years from now.Another issue raised by participants was what they refer to as ‘over-medicalization of life’. Participants of all groups felt that every simple act of life in the present day and age has to be a cause for concern. Mothers regretted the fact that their daughters cannot enjoy the freedom and carelessness they themselves had enjoyed in their youth. On the other hand, young women and teenage girls expressed the notion that the complexity of this matter which should be simple and driven by emotion has instead become a series of calculated actions and a cause for concern and anxiety which definitely kills the magic of love affairs.Another issue raised by participants was the overuse of medicines. Participants thought that there is too much medication on offer and it cannot all be necessary. Participants were also quite critical about the urgency under which these vaccines are offered implying overuse by the medical community. Some participants provided counterarguments such as that medicine is evolving and life expectancy in the western world has been prolonged specifically due to the use of medication. However, others remained apprehensive about the use of medicines despite their widespread presence in everyday life.A small number of patients from the mothers and young women’s groups voiced the opinion that all medicines are addictive or harmful as they contain chemicals bound to be dangerous for the human body. They made a point of stating that they looked for a herbal or natural alternative to most medicines. With regards to vaccinations in particular, a minority of participants thought that they debilitate the human organism and prevent it from fighting independently. Finally, a minority of mothers expressed the wish to talk to naturopaths or homeopathic doctors about HPV vaccination. Specifically, listen to a debate of an expert ‘outside the system’ against a traditional western medicine doctor.Young women had made their own decision not to get vaccinated. Teenage participants reported that health decisions were always taken by the family upon the recommendation of a healthcare professional. In contrast, some mothers thought that their teenage daughters should be involved in the decision of the HPV vaccination. Others thought that under the law the decision should rest with them since their daughters did not have the knowledge or criteria on which to base a decision. During this part of the discussion, mothers displayed an array of negative emotions resulting from the cognitive process of decision making. Concern, fear and guilt were the most prominent emotions.The decision-making process was based on participants’ evaluation of risk. It is noteworthy that participants tried to make sense of the statistical figures they heard during the documentary and expert discussion. Some participants became confused when they tried to combine the information they heard from both stimuli. For example, in the documentary, it was mentioned that 500,000 women die of CC every year. And in the expert discussion, it was mentioned that 80% will have the virus but only 2% will actually get cancer. Also, the level of protection a condom can offer to women is approximately 70%. Some participants thought that since only 2% of women out of 80% will get cancer and if one considers the protection of condom use and cervical cancer screening, it is not really necessary to get the vaccine which does not even provide 100% protection from all HPV strains. Others thought this level of protection was satisfactory. It is noteworthy that the decision making concerning the vaccine was judged on facts the medical significance of which could not really be judged by participants, making an informed decision quite a challenge.Participants shared their stories with regards to advice they had received from health professionals. The majority of participants reported negative, conflicting or complex advice. Sometimes, mothers were told to wait until their daughters got a regular cycle. In other cases, participants had received a recommendation but only for one of the two vaccines. Other participants were told that the decision should be made by them because the vaccine is still very new and no conclusion with regards to its effectiveness can be drawn yet. Even when the advice was not conflicting, namely both the pediatrician’s and the gynecologist’s advice coincided, participants still could not come to a decision or delayed making the decision due to their own concerns. Conflicting advice led to second or third opinions that further complicated the decision-making process.Some participants evaluated HPV as a normal risk that all individuals take by embarking on a sex life however some stereotypes were also voiced. Specifically, some teenage participants thought that really promiscuous behavior puts individuals at risk while some young women thought that individuals who did not take precautions such as use a condom were most at risk.This theme only came up in the mothers’ groups. Mothers in both groups were distinctly aware that sexual activity could expose their daughters to dangers such as the HPV virus. However, the majority of participants were undecided about the HPV vaccination. Still they stated that they used the HPV vaccine as leverage in discussion with their daughters to delay their sexual debut as much as possible. When participants came to realize that this was a common strategy used by many, they found it particularly entertaining. Using self-sarcasm, e.g. ‘we are awesome’ they admitted this was an agonizing attempt to protect their daughters from what is essentially an integral part of growing up. In their mind, the vaccine constituted a coming of age milestone; hence they tried to delay this decision as much as possible.Teenage participants stated that their source of information was mainly their family, friends, doctors and peer and that if they were to look for more it would be on the internet. Young women had heard about HPV at school or through their studies while others recalled a ministry of health campaign for the vaccine. Mothers had heard about HPV from the media or read about it on the Internet, while some had read leaflets in the health service. They had asked their own doctors (i.e. gynecologists), or their daughters’ doctors but also asked for the advice of relatives who happened to be health professionals of any kind.All participants stressed that they had information gaps around HPV and HPV vaccination. Among the issues participants from all groups would like to receive information on are: symptoms of HPV infection in women and men, pregnancy and HPV, the difference between the first dose of the HPV vaccine and subsequent booster doses, the possibility of administration after sexual debut, the possibility of a causal link between prostate cancer and HPV, ways of transmission, detection and cure, the vaccine’s active ingredient, the length of time needed for an infection to resolve itself, the possibility of being more prone to other strains of the virus if already infected by one strain, the role of men in transmission, the number of sexual partners that puts one at risk, HPV vaccination for boys, vaccination recommendations in other countries, precise recommended age of vaccination and the exact way the vaccine works against the virus.When asked what would sway their opinion in favor of the HPV vaccine participants highlighted that the media is a powerful source of information for the public concerning public health matters. However, they mentioned that the specific health issue as portrayed in the media rests on the motivation of fear over certainty that vaccination is the right decision.Mothers thought the HPV vaccination should be given by experts such as physicians in the context of a sexual health talk. Participants admitted the limitations they had in their roles as mothers due to the fact that they had no expert knowledge or authority in their daughters’ eyes to deliver such sensitive information.Young women and teenage girls thought recommendations should come from doctors but also from experts such as pharmacists. They believed that information about sexual health should be given at the end of primary school as well as junior high and during high school.Participants of all groups highlighted the danger of administering the vaccine outside the context of a sexual health talk. Furthermore, that it should be made clear that the vaccine cannot provide complete safety from all STDs, so that vaccinated individuals are not encouraged to engage in the risky sexual behavior.None of the participants had ever seen any of the stimuli so their reactions were spontaneous. Participants read and watched the stimuli attentively. They noticed inconsistencies, which reveals that they were very interested in the topic and did not receive the information passively. A summary of participants’ judgments on the stimuli format and content are shown in Teenage participants thought the level of information was appropriate and the content of the stimuli was equivalent. Judgments with regards to the expert interview revealed that teenage participants felt that the doctor was too confident in the way he was delivering the information. This came in sharp contrast with their own experience. Participants seemed to be a bit puzzled when they thought back to the contradictory information they had received. One participant said ‘I was surprised that he talked with such confidence  …  how is it possible for some to be so certain and others to … say something different’ . The fact that the doctor seemed so adamant, concerned them. Another participant said ‘He is so certain (about the vaccine), that it overpowers everything else’ . Participants felt that the leaflet was not very attractive and its title did not give away the topic. Also, they thought that it needed a better cover and a clearer aim. The documentary received only positive judgments i.e. this information is easier to follow in a documentary format due to its combined use of image and sound. When asked about other criteria such as format and structure, some participants thought the leaflet was more helpful as it had a definite structure and one could go over the information more than once. In contrast, some thought the discussion with the expert resembled reality more since the interaction between the journalists and the expert offered more information than the leaflet as the discussion was spontaneous and other topics could come up. However, the documentary was the preferred way of communication out of the three stimuli presented. The reasons given by participants were that they were able to put the HPV vaccine since they had a chance to ‘meet’ the researchers who developed it.Participants in the mothers’ groups said that the extract with the expert was informative. Moreover, they were surprised about some of the facts communicated such as that HPV can be transmitted even without full sexual intercourse or that 50% of youngsters have started to experiment sexually at the age of 14. They commented on the fact that the expert was certain and ‘he thought it natural that it (the vaccine) had to be done’ . Another criticism was that there was no indication of neither the time the vaccinations took place initially nor the statistical data available for Greece or Europe. The fact that the doctor appeared as an expert authority was not seen positively by some participants. The leaflet was judged as containing a satisfactory level of information. One criticism was that the issue of side effects is not touched upon. On the contrary, the issue of men is suddenly introduced in the last quite irrelevantly of any previous information. Overall, the documentary was judged positively. One aspect that caused negative criticism was the mass vaccination that it showed in Africa ‘I was scared by this mass vaccination. A little bit like guinea pigs’ . The documentary was judged as more convincing and impressive. However, still participants’ reservations concerning side effects were not addressed.Young women’s overall judgments of the stimuli were positive. With regards to the expert discussion, young women found a few inconsistencies i.e. that initially it was mentioned that the vaccine protects against HPV but later it was mentioned that this protection is not at 100%. Also, the expert was judged as not very convincing as it felt like ‘product promotion’ . Participants thought the documentary was more specific mentioning more information such as booster doses, etc. Moreover, participants pointed out that although the researcher who discovered the vaccine, did not ‘sell’ it as a unique truth or idealize it. Participants were quick to combine information and identify discrepancies in the information offered via the three stimuli. Specifically, the expert expressed the opinion that if a young girl gets vaccinated and gets smears then she will never get cancer. However, in the documentary, it was pointed out that other factors might be at play in the development of CC.The purpose of this study was to investigate the knowledge, beliefs and communication preferences of unvaccinated Greek adolescent girls, young women, and mothers of vaccine-eligible girls with regards to the HPV vaccine.This sample of non-vaccinated young women, teenagers and mothers whose daughters had not been vaccinated, had a low to moderate level of knowledge around issues relating to HPV and cervical cancer. Additionally, findings revealed a differentiation among the three groups. Specifically, young women had the highest knowledge score, followed by teenage girls, while mothers displayed the lowest level of knowledge. Nevertheless, all participants showed awareness that they lacked health literacy to assess health information on HPV and the HPV vaccine. The vaccination dilemma they experienced was further enhanced by the conflicting advice received by health care professionals. Indeed, participants realized the severity of cervical cancer, and they perceived the HPV vaccine as a powerful and essential weapon in the battle against it. However, they considered it to be a recent medical innovation and their concerns only fueled their fears concerning vaccination. Furthermore, in a conservative society such as the Greek one, the decision for vaccination was not seen as just a health-promoting decision but inadvertently symbolized young women’s sexual debut. Finally, participants expressed the belief that life nowadays is becoming increasingly over-medicalised due to the overpowering presence of medicine, which turns every simple act of life into something quite complex. Great distrust was shown towards the pharmaceutical industry as well as medicine in general, revealing a range of underpinning negative epistemological beliefs.Previous Greek studies .Furthermore, participants stated that they had received conflicting advice from healthcare professionals with regards to the HPV vaccine. According to Mårdby, Åkerlind, and Hedenrud  health cThe notion that different segments of the public might have difficulty understanding health statistics has been put forward by researchers a number of years earlier. According to Gigerenzer, Gaismaier, Kurz-Milcke, Schawartz, and Woloshin  health rStudies on consumers’ perceptions of health information in local media have revealed that participants think that news stories rely on sensationalism and emphatic headlines in order to attract attention and compete amongst themselves  further research should be conducted on the physicians’ role in the communication of health information and support should be provided in the form of guidance, information or resources (b) health information provided by the media should be based on well researched medical facts and presented by science journalists or science communication experts taking into account factors such as format, message framing and prevalent beliefs, (c) the public’s concerns should be investigated further through research and addressed via targeted public health campaigns and (d) sexual health education should be given special focus at schools, local communities and health care contexts. In conclusion, some of the study’s findings might be relevant for countries which have also adopted ‘on demand’ HPV vaccination schemes or are currently considering an HPV vaccination policy.


Background: Despite the insupportable burden caused by the human papillomavirus (HPV) and high vaccine acceptability, vaccination programs are not currently available for men who have sex with men (MSM). We aimed to assess HPV infection by examining the willingness for vaccination among MSM and cost-effectiveness of the Chinese 2-valent HPV vaccine.Methods: We recruited MSM in Shenyang, China between July and December 2020 to conduct anal HPV testing and an online survey regarding HPV-related knowledge and vaccine acceptability. We performed a cost-effectiveness analysis to evaluate the incremental cost-effectiveness ratios (ICERs) of the Chinese 2-valent HPV vaccine.Results: A total of 234 participants completed the online survey; of those, 203 were successfully tested for HPV. The median age was 30 years [interquartile range (IQR): 23–38 years]. Most participants had at least undergraduate education . The acceptability rate for the free HPV vaccine was 57.7% (135/234). The prevalence of HPV types 16 and 18 was 14.9% (18/121) and 26.8% (22/82) in the willing and unwilling to vaccinate groups, respectively (P > 0.05). The prevalence of high-risk HPV among participants aged <30 and ≥50 years was 48.6 and 38.9%, respectively. Using the Chinese per capita gross domestic product (GDP) as a threshold, the Chinese 2-valent HPV vaccine would be a “very cost-effective” strategy, with an ICER value of USD 4,411. This evidence showed that the Chinese 2-valent HPV vaccine was more cost-effective than other imported vaccines.Conclusions: Targeted strategies should be utilized in MSM with different rates of vaccine acceptability. A pilot HPV vaccination program based on the Chinese 2-valent HPV vaccine for MSM is urgently warranted to reduce the burden of HPV and anal cancer. Men who have sex with men (MSM) are at a higher risk of infection with human papillomavirus (HPV) than females and heterosexual males with a global prevalence of 53.6 and 89.0% among human immunodeficiency virus-negative (HIV-negative) and HIV-positive MSM, respectively , 2. The Considering the heavy burden caused by HPV, there is high acceptability of vaccination against HPV among MSM. According to a recent systematic review, approximately half of MSM is willing to receive an HPV vaccine . A multiDespite the insupportable burden caused by HPV and strong willingness to effectively prevent infection with HPV, MSM are not included in the HPV prevention programs of most countries. Three licensed HPV vaccines  are currently available to prevent HPV-related disease  and haveWe sought to assess infection with HPV based on the different rates of willingness for vaccination among MSM and cost-effectiveness of the Chinese 2-valent HPV vaccine.We conducted a cross-sectional study between July and December 2020 to investigate the acceptability of the HPV vaccine and anal infection with HPV among MSM. This study was conducted through a voluntary counseling and testing clinic in The First Affiliated Hospital of China Medical University in Shenyang, China. Participants aged ≥18 years and willing to participate in the study were enrolled. All recruited participants completed an online questionnaire (duration: approximately 10 min) and, subsequently, anal specimens were collected. All participants were compensated with 50 RMB (approximately USD 7.77) for their time. In this study, we utilized the Checklist for Reporting Results of Cross-sectional Studies see .After receiving information regarding the content and protocol of the study, participants completed a self-administered online questionnaire on sociodemographic information , sexual behavioral characteristics, HPV-related knowledge, HPV vaccine acceptability, and use of pre-exposure prophylaxis (PrEP) for HIV , which was rotated left and right for 20 s. For consistency, a single technician collected all anal specimens. Collected specimens were stored in PreservCyt and at −20°C before transportation to Kaipu Medical Laboratory in Shenyang, China for polymerase chain reaction analysis and genotyping for the 23 types of HPV. These types of HPV were classified as high-risk HPV (HR-HPV) and low-risk HPV (LR-HPV). High-risk HPV are types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68; LR-HPV are types 6, 11, 42, 43, 44, 53, 73, 81, and 82. The 2-valent vaccine covers HPV types 16 and 18; the 4-valent vaccine covers HPV types 6, 11, 16, and 18; and the 9-valent vaccine covers HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58.We calculated the sample size using the Power Analysis and Sample Size (PASS) Version 15.0 software  based on a two-sided alpha of 0.05 and power of 0.8. Based on the results of previous studies on HPV vaccine acceptability among MSM, we set the estimated acceptability rate at 60%. The minimal sample size calculated with sufficient power was 194 participants. Considering the estimated 10% non-response rate, we calculated the expected sample size to be 214 participants.The cost-effectiveness analysis was reported according to the ISPOR Health Economic Evaluation Publication Guidelines-CHEERS Checklist and the HPV-FRAME reporting standards see , strategP-values <0.05 were considered statistically significant.For variables with a missing ratio <5% in the course of data processing, we imputed missing values using the mean and mode for continuous and categorical variables, respectively. In this study, there were no variables with a missing ratio >5%, which they would have been deleted. We used proportions for categorical variables, such as knowledge regarding HPV and vaccine acceptability. We used median and interquartile range (IQR) for the age of participants. We computed a score of HPV-related knowledge (range: 0–5) based on the above five questions . Knowledge regarding HPV was categorized into low (0), middle , and higOf note, 80.4% (234/291) of all eligible participants were included in this study. Among those, 93.2% (218/234) completed anal specimen collection and 86.8% (203/234) had available HPV testing results . The medIn the past 6 months, most participants played an only-insertive sex role  and had 1–2 male sex partners . Furthermore, 33 participants (14.1%) had female partners, and 71 participants (30.6%) self-reported that they used substances during or before sexual intercourse in the past 6 months .P < 0.05).The types of HPV were tested in a total of 218 anal swab samples; of those, 15 were excluded due to unsatisfactory sampling. The overall prevalence of anal HPV and HR-HPV was 59.6% (121/203) and 48.8% (99/203), respectively. We found a high prevalence of HR-HPV across all age subgroups of 203 MSM, ranging from 38.9 to 54.1% (data not shown). In the group of participants willing to receive a vaccine, the prevalence of HR-HPV was 41.3, 51.5, 57.1, and 36.4%, for MSM aged 18–29, 30–39, 40–49, and >50 years, respectively. Relatively higher prevalence of HR-HPV was observed in the group of participants not willing to receive a vaccine. No significant difference was found among the different age groups .The costs of four vaccination scenarios (three doses), namely 2-valent (Cecolin®), 2-valent (Cervarix®), 4-valent (Gardasil®), and 9-valent (Gardasil 9®) vaccines, were USD 18,595, USD 34,194, USD 46,499, and USD 74,749, respectively. According to China's per capita GDP in 2020, the 2-valent (Cecolin®) vaccination strategy was “very cost-effective.” Compared with those of other scenarios, the 2-valent (Cecolin®) vaccination scenario was more cost-effective  vaccine may be the most cost-effective strategy for preventing the development of anal cancer among MSM. This study provides additional evidence on the infection with HPV, demand for vaccination against HPV, and cost-effectiveness of the Chinese 2-valent HPV vaccine among MSM. This evidence presents the urgent need for a national pilot HPV vaccination program tailored to MSM. In addition, our results also provide a vital reference to most countries that have not launched HPV vaccination programs for MSM, particularly those with limited resources. The more economical Chinese 2-valent HPV vaccine may also provide another option for gender-neutral vaccination .The present findings revealed that the prevalence of HR-HPV among MSM in Shenyang, China was relatively higher (48.8%) compared with that reported in previous studies in Africa , 23 and Previous studies rarely investigated infection with HPV between MSM with different degrees of willingness to receive a vaccine against HPV. Notably, we found that the prevalence of anal HPV  was higher among MSM who were unwilling to receive a vaccine against HPV vs. those willing to receive a vaccine against HPV. The significantly higher prevalence among MSM who are unwilling to receive HPV vaccines was exclusively observed in the 2-valent HPV type subgroup, which may be explained by differences in behavior and awareness regarding protective measures. These findings also suggest that these individuals are at relatively higher risks of anal cancer caused by HPV. At present, MSM-orientated vaccination against HPV is not recommended in China. Therefore, improving the degree of willingness for vaccination against HPV may indirectly assist in avoiding infection with HR-HPV among MSM. Future strategies aimed at improving vaccine uptake and knowledge regarding HPV should focus on the corresponding MSM population to maximize the benefits.In previous studies, the age-specific prevalence of HPV among MSM was inconsistent. Most investigations reported a stably high prevalence of anal infection with HPV in all age groups , 31. TheWe identified potential facilitators of vaccination against HPV and factors associated with HR-HPV infection among MSM. As a result of education on self-protection and self-care , MSM witIn this study, we found that the 2-valent (Cecolin®) vaccine was the most cost-effective option for the vaccination of MSM compared with the 2-valent (Cervarix®), 4-valent (Gardasil®), and 9-valent (Gardasil 9®) vaccines. In 2020, the World Health Organization launched the first global strategy to accelerate the elimination of cervical cancer in 194 countries, including China . It meanThere are some limitations in this study. Firstly, selection bias due to the small sample and single enrollment method used in our study may limit the extrapolation of the present findings to the general MSM population. Secondly, a self-administered questionnaire was utilized in the present study; hence, recall bias with regard to characteristics of sexual behavior was inevitable. Thirdly, a causal conclusion on associations cannot be reached due to the cross-sectional trait of this study. Fourthly, given the limitations of our model of cost-effectiveness and the lower efficacy of vaccination among individuals infected with HR-HPV, the range of values for the ICER may not reflect the actual degree of uncertainty. Fifthly, HPV infection among MSM is more common in anal samples compared with oral and genital samples. Therefore, we only currently tested anal samples and correspondingly considered the cost-effectiveness on the prevention of anal cancer, which may underestimate the prevalence of HPV and the cost-effectiveness of vaccines. Thus, a comprehensive analysis based on anal, oral and genital sites results will be conducted in the next step. In addition, this study mainly aimed to appeal HPV vaccine accessibility among MSM in China and did not collect information toward vaccine hesitancy, which is also crucial to the subsequent real uptake of HPV vaccines in this population. Studies on HPV vaccine hesitancy among MSM are warranted in the future. Finally, given the effectiveness and cost after HPV vaccination, the vaccine is only recommended for individuals aged <45 years. However, individuals over 45 years of age also have the right and desire to vaccinate themselves, and may have self-vaccination behavior. Considering the life expectancy (74.7 years old), this population also has relatively long-term necessity of health care. Furthermore, based on the low cost of Chinese bivalent vaccines, we assumed that vaccination strategies are also cost-effective to include this group. Therefore, we included this population in study.Despite the limitations above, this was the first study on infection with HPV in combination with willingness of individuals for vaccination. The data underscore differentiated strategies for the prevention of HPV among MSM. We also assessed for the first time the cost-effectiveness of the Chinese HPV 2-valent vaccine vs. other imported vaccines among MSM. Furthermore, unlike in previous studies, the vaccine coverage was calculated based on vaccine acceptability using samples rather than estimation, which is closer to the natural condition in this population.MSM who are unwilling to receive a vaccine against HPV may suffer a heavier burden caused by HPV types 16 and 18. Therefore, there is a need for strategies aimed at improving knowledge regarding HPV among this population. The Chinese 2-valent vaccine against HPV provides a more economical option for the prevention of anal cancer, particularly in low- and middle-income countries with limited resources. A pilot HPV vaccination program for MSM in China is urgently warranted to reduce the prevalence of HPV and the associated anal cancer burden.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Board Committee of the First Affiliated Hospital of China Medical University in Shenyang, China. The patients/participants provided their written informed consent to participate in this study.Q-HH, J-JX, and HS conceived and designed the research study. Z-HY, Z-ZL, S-TC, Z-XC, and Q-HH collected the data for the study. Z-HY and Q-HH analyzed the data and interpreted the results. Z-HY wrote the first draft of the manuscript. Y-JJ, J-JX, Q-HH, and HS revised the manuscript. All authors have reviewed and approved the final manuscript.This work was funded by the Mega-Projects of National Science Research for the 13th Five-Year Plan (2017ZX10201101) and the National Natural Science Foundation of China (82073620).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Physiology is not only the most central aspect of biomedical life sciences, but also one of its largest areas. Being the “science of life” means that virtually every research concerning cells and animals also has a twist towards physiology. This makes this discipline so important that the Nobel Prize is awarded to “Medicine and Physiology”. In times of exponential growth of scientific knowledge and — owing to this — a massive diversification of research, smaller physiological meetings without a precise thematic focus are struggling. The diversity of the audience limits the number of qualified interaction partners. As a consequence, there has been a tendency towards highly specialised scientific meetings.The Europhysiology meeting series is the answer of Europe’s physiologists to this development. A pan-European meeting with an audience far beyond 1000 attendees and the reputation to attract the most famous speakers offers a key audience for each speciality and cutting-edge science at the highest level. The Europhysiology meeting series started in 2018 with a successful and very well received first meeting in London. Due to COVID-19, the 2020 meeting planned for Berlin had to be cancelled, but the meeting series will continue as agreed by the hosting societies.https://www.tivoli.dk) in the beautiful city of Copenhagen from September 16 to 18, 2022.In this year, 2022, another Europhysiology meeting will take place. Europhysiology 2022 will be an in-person event at the Tivoli Garden’s conference center (https://europhysiology2022.org/programme-16-sept/.- The programme is scientifically exciting and well balanced. The four hosting societies, The Physiological Society, The Scandinavian Physiological Society, The German Physiological Society, and the Federation of European Physiological Societies, have developed a programme that not only is diverse, but also showcases the most recent developments in science and a breadth of exciting topics. More information can be found at - The conference offers various opportunities for informal contact, scientific exchange, and networking. We all have missed the direct interaction with our peers during the COVID-19 pandemic. Finally, this option becomes available again with Europhysiology!- Europhysiology offers numerous possibilities for young researchers to increase their visibility. From a pre-symposium of the “Young Physiologists”, to sessions on career development and numerous awards. Poster and oral sessions shaped from selected abstracts offer ample possibilities for emerging researchers to present their work.- Focussed symposia in the main congress programme as well as pre-conference meetings of special interest groups in almost all areas of physiology assure that also more focused interactions are possible.København, never disappoints. It is a city not only easy to reach but also full of vibrant culture, good food, lasting impressions, and exciting history!- Finally, and aside from science, the cosmopolitan capital of Denmark, There are many reasons to attend this meeting.https://europhysiology2022.org/ until May 25th.Abstract to the meeting can be submitted under We look forward to welcoming you all, whatever your career stage, in Copenhagen at Europhysiology 2022. Let’s meet for real!


There is a lack of biomarkers for accurately prognosticating outcome in both human papillomavirus-related (HPV+) and tobacco- and alcohol-related (HPV−) oropharyngeal squamous cell carcinoma (OPSCC). The aims of this study were to i) develop and evaluate radiomic features within  and around tumor  on CT scans to predict HPV status; ii) investigate the prognostic value of the radiomic features for both HPV− and HPV+ patients, including within individual AJCC eighth edition-defined stage groups; and iii) develop and evaluate a clinicopathologic imaging nomogram involving radiomic, clinical, and pathologic factors for disease-free survival (DFS) prediction for HPV+ patients.CCF) with HPV+ OPSCC. We subdivided the TCIA cohort into training  and validation  based on an approximately 3:5 ratio for HPV status prediction. The top 15 radiomic features that were associated with HPV status were selected by the minimum redundancy–maximum relevance (MRMR) using ST and evaluated on SV. Using 3 of these 15 top HPV status-associated features, we created radiomic risk scores for both HPV+ (RRSHPV+) and HPV− patients (RRSHPV−) through a Cox regression model to predict DFS. RRSHPV+ was further externally validated on SCCF. Nomograms for the HPV+ population (Mp+RRS) were constructed. Both RRSHPV+ and Mp+RRS were used to prognosticate DFS for the AJCC eighth edition-defined stage I, stage II, and stage III patients separately.This retrospective study included 582 OPSCC patients, of which 462 were obtained from The Cancer Imaging Archive (TCIA) with available tumor segmentation and 120 were from Cleveland Clinic Foundation  the whole HPV+ population , ii) the AJCC eighth stage I population , and iii) the AJCC eighth stage II population . HPV+ nomogram Mp+RRS  was also prognostic of DFS .RRSCT-based radiomic signatures are associated with both HPV status and DFS in OPSCC patients. With additional validation, the radiomic signature and its corresponding nomogram could potentially be used for identifying HPV+ OPSCC patients who might be candidates for therapy deintensification. The rise in the incidence of human papillomavirus (HPV)-related cancers has caused a significant epidemiological shift  in orophTreatment of OPSCC patients is on the cusp of a paradigm shift; current clinical trials are geared toward reducing treatment toxicity for HPV+ patients without compromising survival outcomes since low-risk patients typically could benefit from a lower dose of radiotherapy or less invasive surgical operations (treatment deintensification) . HoweverOn the other hand, the unmet need for HPV− patients is to precisely identify patients at high risk of developing local or regional failure after treatment, patients who might be candidates for targeted treatment escalation . While tRadiomics is the process of computational extraction of large numbers of quantitative imaging features, such as texture features, from routine radiologic scans  for characterization of the disease . These fIn this study, we sought to explore the prognostic value of both intratumoral and peritumoral HPV status-associated radiomic features on CT scans and compared and combined them with clinical and pathologic factors on over 500 OPSCC patients. The prognostic radiomic biomarker for HPV+ OPSCC was validated both internally (237 patients) and externally (120 patients) on two different cohorts. We also evaluated the utility of the radiomic signature to prognosticate DFS within each individual AJCC eighth edition-defined stage group. Finally, this study also involved creation and validation of a clinicopathologic nomogram for estimating DFS for HPV+ OPSCC patients. n = 462) OPC-Radiomics cohort (n = 120). All patients had undergone pretreatment radiotherapy planning CT. TCIA is an open archive of deidentified cancer-specific medical images and associated clinical metadata accessible for public download (n = 6); ii) number of voxels within tumor is less than 200, which was deemed to be insufficient for feature extraction (n = 5); and iii) tumor mask contains normal brain tissue (n = 3). Following the patient exclusion criteria, 462 patients from the TCIA cohort and 120 patients from the CCF cohort were included for subsequent radiomic analysis. The flowchart for patient enrollment is illustrated in Two OPSCC cohorts were included in this study: The Cancer Imaging Archive  for the TCIA cohort . PrimaryAppendix E1, section 2. All feature values were transformed into new scores with a mean of 0 and a SD of 1 (z-score transformation). Both intratumoral and peritumoral feature extraction pipelines are now publicly available at https://github.com/ccipd.A total of 664 intratumoral and 1,485 peritumoral  radiomic features were extracted for all patients on all the compartments on a per-pixel basis. The feature sets for each study utilized included 16 gray-level intensity features , 40 intensity gradient-based features (quantifying intensity gradient variability), 52 gray-level co-occurrence matrix (GLCM) Haralick features  , 28 GaboT (180 patients: 100 HPV+ and 80 HPV−) and SV (282 patients: 237 HPV+ and 45 HPV−) in an approximately 3:5 ratio. For both HPV+ and HPV− patients, the ratio of non-censored patients in ST and SV was kept balanced. The clinical end points of interest for this study were HPV status and DFS. DFS was defined as the time interval from the radiotherapy end date to the date of either last follow-up (censored) or local, regional, distant failure and death (event), whichever happened first. The difference of continuous variables  between cohorts  was determined using the analysis of variance (ANOVA) and the association between categorical factors was estimated using the chi-square test.Within the TCIA cohort, 462 patients were randomly allocated to ST. To remove redundant features, all possible pairs of features in ST were tested for correlation by calculating the Spearman correlation coefficient (SCC). For any pair of features with SCC greater than 0.80, the feature with the higher Wilcoxon rank sum p-value was removed. A linear discriminant analysis (LDA) machine-learning classifier was subsequently trained in conjunction with the minimum redundancy–maximum relevance (MRMR) (t) that best discriminated between HPV+ vs. HPV− across all iterations were identified from ST. An unadjusted p-value <0.05 (using two-sided Wilcoxon rank sum tests) was employed to indicate statistical significance. These features were then evaluated via the LDA classifier in terms of HPV status prediction on SV using the area under the receiver operating characteristic curve (AUC) metric.Since there were no HPV− patients in the CCF cohort, we used only the TCIA cohort for HPV status prediction. A machine learning classifier was first constructed using a combination of intratumoral and peritumoral features for the prediction of HPV status using Se (MRMR)  feature HPV+) and HPV− (RRSHPV−) categories. The least absolute shrinkage and selection operator (LASSO) method was applied to Ft using the cases in ST for both HPV+ and HPV− patients. After identifying the top ranked features, the corresponding LASSO coefficients were used for constructing risk classifiers for the HPV+ (RRSHPV+) and HPV− (RRSHPV−), respectively. Both RRSHPV+ and RRSHPV− were calculated for each patient via a linear combination of selected features that were weighted by corresponding coefficients:We also developed dedicated radiomic risk score classifiers for patients within the individual HPV+ (RRSn (ranging from 0 to 15) is the number of features selected by LASSO for HPV+ or HPV− patients, ix refers to the HPV status-associated feature value, and iβ is the corresponding weighted coefficient. The potential association of RRSHPV+ and RRSHPV− with DFS was first assessed in ST and then evaluated in SV. The prognostic ability of RRSHPV+ was further externally validated in SCCF. Patients were classified into high or low risk based on the median value of the RRSHPV+ or RRSHPV− in ST, which was then applied to SV and SCCF. Kaplan–Meier survival curves were used to visualize the survival rate for the high- and low-risk groups. At any given point on the survival curve, the probability that a patient in either the high-risk or low-risk group remains alive is presented  was averaged out by 10 cross-validation to minimize the error within ST. Constructions of RRSHPV+ and RRSHPV− were performed using in-house software implemented in the MATLAB R2019b platform (MathWorks).where resented . The logHPV+, RRSHPV−, and the individual clinicopathologic variables  on DFS was evaluated. Variables significant in univariate analysis were included for multivariable Cox proportional hazards analysis to investigate the relationships between the various covariates .Univariate Cox proportional hazards analysis on the effect of RRSHPV+ with existing clinical factors  and pathological staging factors , we constructed nomogram models for HPV+ patients comprising i) only the clinical factors (Mc) of gender, smoking, and drinking history; ii) only the pathologic staging factors (Mp) of AJCC eighth edition overall stage and N stage; and iii) both the pathologic T stage and the RRSHPV+ (Mp+RRS) using ST and validated them in SV and SCCF. The prognostic ability of Mp+RRS was compared against Mc and Mp for HPV+ populations in terms of the concordance index (C-index). The nomograms were validated using 1,000 bootstrap resampling to calculate C-index with confidence intervals. Calibration curve analysis was performed to compare the nomogram-predicted DFS with the actual DFS. Decision curve analysis was adopted to calculate the net benefit for Mp+RRS in comparison with Mc and Mp, for verification of the clinical usefulness of the nomogram , TCIA internal validation set (SV), and CCF external validation set (SCCF) are summarized in T, SV, and SCCF. All patients included in this study underwent radiotherapy. Eighty-seven of the 180 patients in ST (48.3%) and 145 of the 282 patients in SV (51.4%) were also treated with chemotherapy. The median DFS for HPV+ patients was 6.19 years in ST and 6.24 years in SV. The median DFS for HPV− patients was 1.27 years in ST and 1.55 years in SV. Thirty-three percent  and 33% (79/237) of HPV+ patients were not censored in ST and SV, respectively, while 77.5% (62/80) and 68.9% (31/45) of HPV− patients were not censored in ST and SV, respectively. There were 62 (18.4%) recurrences  for HPV+ patients and 64 (51.2%) recurrences  for HPV− patients.The clinical and pathologic characteristics of patients in the TCIA training set , while the median of Haralick correlation-info1 (p < 0.001) within the tumor. Both features were differentially expressed on CT scans between HPV+ and HPV− patients, and representative examples are illustrated via colormaps of the feature representations overlaid on the tumor areas or the annular ring regions around the tumor  and 0.87 , respectively. The corresponding AUCs on SV are 0.58  and 0.67 . When combining the intratumoral and peritumoral features, ROC analysis on ST yielded an accuracy of 0.79 and AUC of 0.84 , with a sensitivity of 0.89 and specificity of 0.68 when using a threshold of 0.4. For SV, we obtained an accuracy of 0.74 and an AUC of 0.70 , with a sensitivity of 0.78 and specificity of 0.53 when the same threshold from ST was applied. The areas under the curve (AUCs) for using intratumoral and peritumoral alone on SHPV+) and HPV− patients (RRSHPV−) across ST, SV, and SCCF are illustrated in HPV+ was constructed using two peritumoral and one intratumoral features, while RRSHPV− was constructed using three peritumoral features. The median RRSHPV+ value (−0.0809) in ST was used as the cutoff threshold for defining high- and low-risk groups, resulting in statistically significant DFS prediction by KM analysis in ST , SV , SCCF , and SV+SCCF  as illustrated in HPV− value (0.0076) in ST also resulted in a statistically significant prediction of DFS by Kaplan–Meier analysis in both the ST  and SV , as illustrated in A three-feature radiomic signature was identified as having the best prediction of DFS for both HPV+ and HPV− patients (HPV+) was further evaluated for the patients within the AJCC eighth edition-defined different stage groups in SV+SCCF. The HRs of predicting DFS using RRSHPV+ for stage I (p = 0.039), 3.61 , and 1.4 , respectively.The prognostic ability of the radiomic risk score  and T3 stage  remained independent prognostic factors for HPV+ patients in the Cox proportional hazards model , N1 stage , and N2 stage  were the independent prognostic factors in the multivariable Cox proportional hazards model.Results of the univariable analysis are shown in HPV+) were used to develop the radiomic nomogram Mp+RRS , while the C-index for the pathologic staging nomogram Mp was 0.62 (95% CI: 0.51–0.72), and that for the nomogram Mc using gender, smoking, and drinking history was 0.59 (95% CI: 0.49–0.69). When evaluated on SV+SCCF, Mp+RRS yielded a C-index of 0.59 (95% CI: 0.54–0.65) for DFS prediction, while for Mp and Mc, the C-indices were 0.59 (95% CI: 0.53–0.64) and 0.56 (95% CI: 0.51–0.61), respectively. The Kaplan–Meier survival curves for Mp+RRS in ST and in SV+SCCF are shown in p+RRS was significantly associated with DFS, independent of Mc and Mp in the multivariable analysis when evaluated on ST+SV+SCCF  for stage I, 2.07  for stage II, and 2.32  for stage III patients. The corresponding KM curves are shown in When evaluating the prognostic ability of MHPV+ OPSCC has better clinical prognosis and treatment response than the alcohol- and tobacco-related HPV− OPSCC . BecauseIn this work, we investigated the ability of both intratumoral and peritumoral radiomic biomarkers on CT scans to predict HPV status for a large cohort of 462 OPSCC patients. Additionally, we addressed the OPSCC prognosis prediction problem independently within the HPV+ and HPV− populations. The radiomic signature which was prognostic for the whole HPV+ population was also prognostic within the AJCC eighth edition stage I and stage II patients. Finally, the radiomic features were combined with pathologic staging factors to form a radiomic nomogram for individualized prognosis estimation for HPV+ OPSCC patients.via immunohistochemistry is the recommended test for determining HPV status. However, distinct populations of patients exist in whom the tumors overexpress p16 but are in fact negative for HPV-DNA or mRNA expression and vice versa , which yielded significant risk stratification based on DFS in the validation set for both HPV+ and HPV− populations. A previous study by Leijenaar et al. externally validated the prognostic value of intratumoral radiomic signatures in a larger cohort of 542 OPSCC  but did not consider the variation of the results by HPV status (p < 0.001) on the validation set. However, unlike our study, this study did not consider HPV status as an independent prognostic indicator for outcome prediction. The prognostic ability of the radiomic risk score (RRSHPV+) was also evaluated within each of the AJCC eighth edition-defined stage groups. Although various treatment deintensification strategies have been proposed by multiple clinical trials based on clinicopathologic factors, there is a lack of reliable biomarkers for risk-stratifying OPSCC patients within individual stage groups. In the HN002 trial, patients meeting criteria of either T1-T2 N1-N2b M0 or T3 N0 N-2b M0 (AJCC seventh ed.) with a ≤10 pack-year smoking history are selected as candidates for therapy deintensification , lymph node status (N), and cancer metastasis (M). However, these staging factors could not capture the intratumor heterogeneity, which has been shown to be a significant prognostic factor. The radiomic nomogram combined the pathologic staging information with the radiomic features extracted from the entire tumor on CT scans, enabling for robust pretreatment survival estimation. Combining the RRSHPV+ from experiment 2 with the pathologic T stage resulted in a nomogram that leads to a more individualized prognosis prediction. With refinement and improvement, this type of radiomic approach might guide more tailored treatment for patients with better survival outcome. Compared with risk stratification using only the conventional staging factors, the radiomic nomogram had an improved DFS estimation. A previous study by Fakhry et al. showed that a nomogram integrating clinicopathologic factors  could reliably predict progression-free survival , the Prostate Cancer Research Program , the Lung Cancer Research Program , and the Peer Reviewed Cancer Research Program (W81XWH-18-1-0404); the Kidney Precision Medicine Project (KPMP) Glue Grant; the Ohio Third Frontier Technology Validation Fund; the Clinical and Translational Science Collaborative of Cleveland (UL1TR0002548) from the National Center for Advancing Translational Sciences (NCATS) component of the National Institutes of Health and NIH roadmap for Medical Research; and the Wallace H. Coulter Foundation Program in the Department of Biomedical Engineering at Case Western Reserve University. KY was supported by the Computational Genomic Epidemiology of Cancer (CoGEC) Program at Case Comprehensive Cancer Center (T32CA094186).ResearchThe content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the U.S. Department of Veterans Affairs, the Department of Defense, or the United States Government.AM is an equity holder in Elucid Bioimaging and in Inspirata Inc. In addition, he has served as a scientific advisory board member for Inspirata Inc., AstraZeneca, Bristol Myers Squibb, and Merck. Currently, he serves on the advisory board of Aiforia Inc. He also has sponsored research agreements with Philips, AstraZeneca, Boehringer Ingelheim, and Bristol Myers Squibb. His technology has been licensed to Elucid Bioimaging. He is also involved in a NIH U24 grant with PathCore Inc. and three different R01 grants with Inspirata Inc. SK is a consultant for Merck and Regeneron, receives research support from Merck and Bristol Myers Squibb, and reports honoraria from UpToDate.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.