Melanomas were enriched in “naive T-cell”, “effector memory T-cell”, “exhausted T-cell”, “resting Treg T-cell” and “Th1-like” signatures, irrespective of their BRAF, NF1 or RAS mutational status. Somatic mutations in IDO1 and HLA-DRA were frequent and could be involved in hindering patient response to ICI therapies. We finally analyzed transcriptome profiles of ICI-treated patients and associated their response with high levels of IFNγ, Merck18, CD274, CD8, and low levels of myeloid-derived suppressor cells (MDSCs), cancer-associated fibroblasts (CAFs) and M2 macrophages, irrespective of their TMB status. Overall, our findings highlight the importance of pre-existing T-cell immunity in ICI therapeutic outcomes in skin melanoma and suggest that TMBlow patients could also benefit from such therapies.Skin melanoma cells are tightly interconnected with their tumor microenvironment (TME), which influences their initiation, progression, and sensitivity/resistance to therapeutic interventions. An immune-active TME favors patient response to immune checkpoint inhibition (ICI), but not all patients respond to therapy. Here, we assessed differential gene expression in primary and metastatic tumors from the TCGA-SKCM dataset, compared to normal skin samples from the GTEx project and validated key findings across 4 independent GEO datasets, as well as using immunohistochemistry in independent patient cohorts. We focused our attention on examining the expression of various immune receptors, immune-cell fractions, immune-related signatures and mutational signatures across cutaneous melanomas with diverse tumor mutation burdens (TMB). Globally, the expression of most immunoreceptors correlated with patient survival, but did not differ between TMB BRAF, NRAS and NF1  is the ecosystem surrounding a tumor and includes the extracellular matrix, blood vessels and stromal cells. It also encompasses a diverse number of immune cells, such as dendritic cells (DCs), neutrophils, natural killer (NK) cells, T-cells and B-cells, as well as immunosuppressors, including myeloid-derived suppressor cells (MDSCs), regulatory T (Treg) cells, tumor-associated macrophages (TAMs) or cancer-associated fibroblasts (CAFs). All these, constitute an ecosystem where they interact with the tumor cells bidirectionally, modulating the malignant phenotype . An immuThe tumor’s relationship with immune cells within the TME can remarkably influence cancer cell proliferation, progression, and metastasis . This unTIGIT, IDO1 and LAG3, other than PD-1, PD-L1/2 and CTLA-4, associate with the patients’ overall and disease-free survival, but not with the TMB, corroborating that immunogenicity in these tumors is affected by other factors as well. In addition, we found that skin melanomas are significantly enriched in the “naive T-cell”, “effector memory T-cell”, “exhausted T-cell”, “resting Treg T-cell” and “Th1-like” signatures, irrespective of their BRAF, NF1 and RAS mutational status. We also show that despite the similar immune-cell fractions between TMBhigh and TMBlow tumors, the first have a higher ratio of M1/M2 macrophages. Our data further support that somatic mutations in IDO1 and HLA-DRA are frequent and could be involved in hindering patient response to ICI therapies. We finally provide evidence that TMB alone is not the best predictor of immunotherapy response and therefore, anti-PD-1/anti-CTLA-4 monotherapy or combination ICI therapy could also be applied to TMBlow patients.Here, we delved into the expression of a group of activating and inhibitory immune receptors in the TME of skin melanoma patients with diverse TMB. We also examined immune-related signatures, fractions of immune-cells and mutational signatures across tumors with a low or high TMB. Our results indicate that elevated expression levels of https://portal.gdc.cancer.gov/). Apart from one matched blood sample from the TCGA cohort that was used as control, we included normal skin samples from the GTEx project  for differential gene expression analysis, totaling to 557 controls. TCGA and GTEx samples were re-aligned to the hg38 genome and were processed using a uniform bioinformatic pipeline, to eliminate batch effects.We extracted whole exome and RNA-seq data from the TCGA-SKCM dataset, containing 461 primary and metastatic skin melanoma samples, in total. All data, including patient clinicopathological information and MAF files, were assessed from GDC Data Portal  enrichment analysis for the top 250 up- and down-regulated genes in primary (or metastatic) skin melanomas, respectively, using Enrichment Analysis Visualization Appyter. Similar gene sets from GO analysis were clustered together using Uniform Manifold Approximation and Projection (UMAP)   and the PD-1, PD-L1/2, CTLA-4, TIGIT, IDO1/2 and other prospective immunoreceptors . To calculate each gene’s expression, we used one-way ANOVA, and the disease state  as variable to calculate differential expression. The expression data were first log2(TPM+1) transformed for differential analysis and the |log2FC| was defined as median (skin melanoma) - median , as explained before  repository were analyzed for subsequent validation of the top deregulated genes in primary (or metastatic) melanomas against their adjacent normal skin samples, or between primary and metastatic melanomas, depending on the study. In specific, we obtained microarray data from the studies with the following GEO accession numbers: GSE8401, containing 31 primary and 52 metastatic melanomas , 26; GSE samples ; GSE4651 samples ; and GSE samples . P-valuemut, n=147), NF1 mutants , RAS hotspot mutants  and triple-wild type  tumors, using GEPIA2 ; effector T-cells ; effector memory T-cells ; central memory T-cells ; resident memory T-cells ; exhausted T-cells ; resting Tregs ; effector Tregs ; and Th1-like cells . The |log2FC>1| and p<0.01 (ANOVA) were used to assess differences with statistical significance between groups. Principal component analysis (PCA) was used to automatically perform dimensionality reduction on data from the TCGA-SKCM dataset and normal suprapubic skin (not exposed to the sun), based on the expression of these signatures in the samples. The expression of specific immune-checkpoints was also explored individually across the different molecular or immune subtypes  , as well as between BRAF hotspot mutants (BRAFg GEPIA2 . The siglow”). The upper quartile involved tumors with an increased rate of mutation, i.e., >30 total mutations/Mb or >20 non-synonymous mutations/MB (“TMBhigh”). Among the TMBhigh subgroup, 18 tumors with >81 total mutations/Mb were considered as “extremely hypermutated”. The rest 50% of samples was termed “TMB-intermediate” . Tumor stratification based on their TMB (synonymous and non-synonymous mutations) was also reflected on their neoantigen burden, being significantly higher among TMBhigh tumors  versus TMBint  and TMBlow tumors . Maftools  mutations/Mb or <5.14 non-synonymous mutations/MB  and 23 immunoinhibitors  across TMBhigh, TMBint and TMBlow melanoma tumors.The mutation rate was then correlated with the expression of either activating or inhibitory immune receptors within each TMB subgroup. In specific, we compared the expression of 49 immunostimulators (https://cancer.sanger.ac.uk/signatures/). Each signature’s contribution was calculated separately for primary and metastatic skin melanomas. Cancer driver mutations were identified using IntOGen  and doublet base substitutions (DBS) using SigProfiler’s MatrixGenerator and Extractor, as previously described in detail , 33. SBS IntOGen .We analyzed each tumor’s cell type fraction by extracting data from the Cancer Immunome Database (TCIA) . The abshttps://www.proteinatlas.org/) , rinsed in Tris-buffered saline (TBS) and subjected to heat-induced epitope retrieval (HIER) using a pressure boiler. Sections were then incubated overnight at 4°C with mouse monoclonal antibodies (mAbs) against IDO1 , PD-1 , PD-L1, a marker specific for T-cells, B-cells and tumor cells , LAG3 , the cytotoxic T-cell markers CD8A The image used in Figures 1-3 has part labels; however, the description is missing in the caption. Could you clarify this? Provide revised files if necessary.and CD8B , and the Treg-specific marker FOXP3 . The UltraVision LP HRP polymer®, Ultra V Block and DAB quanto substrate system®  were used for detection. Finally, slides were rinsed in tap water, counterstained with hematoxylin, dehydrated in grade ethanol and coverslipped. Slides were then independently assessed by two observers. Sections of hyper-reactive tonsils were used as positive controls for anti-PD-L1 and anti-CD8 staining and preimmune rabbit serum as a negative control for nonspecific staining. Protein staining was scored as 2+ (high (>75% positive cells) or medium (50-75% positive cells) staining), l+  and 0 (staining not detected or <5% positive cells) with strong, medium, weak or negative intensity. The percentage (%) of TILs (200x magnification) in the was also scored. Slide scanning was performed on a VENTANA iScan HT slide scanner v1.1.1 (Roche).An independent cohort of 11 skin melanoma samples from the Human Protein Atlas (as.org/)  and tissIDO1, CXCL10, CXCL9, HLA-DRA, STAT1 and IFNG  and gene expression was measured in log2(FPKM-UQ+1) values using the UCSC Xena platform (http://mupit.icm.jhu.edu/) was used to map the SNVs on the crystal structure of each protein, in 3D (hg38).We evaluated gene expression along with the detection of SNVs and Indels across an IFN-γ-related signature, composed of and IFNG . We alsoplatform . MuPIT Ihigh and TMBlow tumors (ranging from 0-10) based on the expression of immunomodulators, effector T-cells, effector memory T-cells and immunosuppressors. Their immunophenotypes were visualized using immunophenograms, as previously described  (2(TPM+1) values) were downloaded and normalized towards the control samples. Each gene was normalized by subtracting the expression value in the reference control samples. Higher TIDE values indicate that the patient has higher potentials of tumor immune evasion and is, therefore, less likely to benefit from the corresponding immune-checkpoint blockade. The IFNG values indicate the IFNγ response biomarkers of IFNγ, ACAT1, IDO1, CXCL10, CXCL9 and HLA-DRA. From the analysis we also deduced the expression of CD274 (PD-L1), the average expression from CD8A and CD8B genes, the levels of cytotoxic T-lymphocytes, each patient’s dysfunction of the tumor, exclusion potential of the tumor, as well as the Pearson’s correlation between gene expression and MDSCs, CAFs and M1/M2 TAMs.Tumor Immune Dysfunction and Exclusion  , 44 was rd.edu/) , Hugo etrd.edu/) , Nathansrd.edu/) , Prat etrd.edu/) , Lauss erd.edu/) , Riaz etrd.edu/)  and Giderd.edu/) ]. Pre-trhigh and TMBlow tumors, were assessed using the nonparametric Mann-Whitney test. Gene expression (log2(TPM+1) values were profiled using violin plots across different pathological stages of the tumors. Multivariate analysis of variance (MANOVA) with the F statistic was used to estimate differences across the different stages. We used Kaplan-Meier curves to plot overall and disease-free survival in patients with high or low expression of immune checkpoints or different multi-gene signatures, using the median expression as cut-off. The log-rank test with HR and 95% CI was used for analysis. Adjusted p-values <0.05 were considered statistically significant. Correlations between each patient’s TIL or TMB load and the expression of immune receptors were assessed using Pearson’s test. All statistical analyses were performed using GraphPad Prism v9.0.0.121. Clusters of similar GO terms were computed using the Leiden algorithm (Differences in gene expression between high and low activating (or inhibitory) immune receptor-expressing tumors or between TMBlgorithm  and poinWe initially detected the significantly deregulated genes, having a broad distribution across all chromosomes, in primary and metastatic skin melanoma Table S1On the other hand, the top 250 down-regulated genes were enriched in regulation of extrinsic apoptotic signaling pathway; positive regulation of protein localization to cell periphery; maintenance of protein location in nucleus; response to cytokine; positive regulation of protein localization to plasma membrane; regulation of protein localization to plasma membrane; ribosome biogenesis; and positive regulation of NF-kappaB transcription factor activity, among other GO-BP terms. They were also enriched in cytoskeleton-nuclear membrane anchor activity; chloride channel inhibitor activity; nucleoside-diphosphatase activity; chloride channel regulator activity, among other GO-MF terms, as well as in (cytosolic) large ribosomal subunit; melanosome membrane; and chitosome; pigment granule membrane, among other GO-CC terms , LAG3, HAVCR2, TIGIT, ILT2, ILT4, ADORA2A, IDO1 and IDO2 had better overall survival versus low-expressing patients. What’s more, patients with higher levels of CD8, VISTA, PD-L2, LAG3, ADORA2A, IDO1, IDO2 and ILT2 had markedly improved disease-free survival, suggesting that their expression can be used as a prognostic marker, with high levels being favorable in melanoma , IDO1 , LAG3  and ADORA2A  in skin melanomas, irrespective of their mutation rate.The TIL load is a predictive biomarker for patient response to anti-PD1/PD-L1 immunotherapy . We hypoCD274 (PD-L1) expression correlated significantly with the rest immune checkpoints (apart from VTCN1) in skin melanoma compared to normal skin (not exposed to the sun), especially with PD-L2, ILT2, HAVCR2 and TIGIT . Interestingly, we found a primary tumor to associate with SBS4 (tobacco smoking) and a metastatic tumor to associate with SBS17b. The latter signature is of unkown aetiology, but previous studies have associated it with 5FU chemotherapy treatment and to damage inflicted by reactive oxygen species (POLE/POLD1 mutated tumors (SBS10b), they have been shown to have a higher number of neoantigens and infiltrating lymphocytes p*Cp(C/G)>T and (C/T)p*Cp(A/G)>T mutations, whereas the latter, of transversions, A>G and (A/G)p*C>T mutations Figure 3 species . As expe species –69. As rphocytes .We also found a substantial variation in the number of doublet base substitutions (DBS) . Among these, we identified a high percentage of DBS1 and ID13, both due to exposure to UV light. DBS1 is mainly composed of CC>TT on the untranscribed strands of genes indicative of damage to cytosine and repair by transcription-coupled nucleotide excision repair (TC-NER), and it associates with SBS7a/SBS7b , 72. ID1TTN , BRAF , XIRP2 , THSD7B , USH2A , NRAS , RLN  and TPTE , among others, having a lower mutation frequency , among which, BRAF dominated ; 5p15.33 ; 6p24.3 ; 7q34 ; 8q24.21 ; 9p21.3 (47.47% deletion and 52.53% loss); 11q23.3 ; 10q23.31 ; 4q34.3 ; 9p23 , 6q26  and 1p22.1  among others (Copy number variations (CNVs) were also observed across all tumor samples, irrespective of their TMB status. In addition, we did not detect any difference in the intra-tumoral genomic heterogeneity between TMBH scores . CNVs weg others Figure 3g others .CD274 (PD-L1) along with other immunoinhibitors and immunostimulators, across TMBhigh, TMBint and TMBlow skin melanomas. Globally, we found that the expression of most immunoreceptors does not differ across the three TMB subgroups (p>0.05) (CD274 expressed higher in TMBhigh tumors (p<0.05), but still without any significant correlation with the TMB (Pearson’s rho (r)=0.052, p=0.372). We also noted differences in the expression of TNFSF18, KDR and ENTPD1, which were lower in TMBhigh tumors (p<0.05) but also did not correlate significantly with the TMB . In contrast, the expression of TNFSF9 was marginally higher in TMBlow melanomas (p=0.06) and correlated negatively with the TMB . A few other correlations we could note, were between TNFSF9 and TMB , NT5E and TMB , as well as between MICA and TMB . Paradoxially, however, the expression of several well-known inhibitory receptors, including CTLA-4, PDCD1 (PD-1), TIGIT, IDO1, LAG3, ADORA2A and VTCN1, was similar between TMBhigh and TMBlow tumors, corroborating that in general, the expression of immune checkpoints and TMB are independent biomarkers in skin melanoma. This finding was further supported by our IHC data, showing that PD-1, PD-L1, IDO1 and LAG3 protein levels are also similar across melanomas of differential TMB status ; it was higher in the stroma than in the parenchyma of primary tumors, but this percentage decreased in the metastatic cases. Taken together, these data suggest that TMB is not the only factor that affects immunogenicity. In fact, other factors apart from high PD-L1 expression, seem to also affect immunogenicity in skin melanoma and therefore, prevent TMBlow patients to benefit from ICI therapies. These include high levels of IFNγ, CD8 and GZMA/PRF1 , ashigh and TMBint tumors, both having a higher ratio of M1/M2 macrophages compared to TMBlow tumors. In addition, the CD8+ T-cells/Tregs ratio was similar between the three TMB subgroups  . In specific, these contained 28 missense mutations, 1 stop gained and 1 splice acceptor variant in IDO1 , followed by NF1  and PTEN . Finally, we detected a smaller number of somatic mutations in B2M , IFNGR1 (4 missense and two 3’ UTR variants), IFNGR2 , JAK1 , JAK2 (2 frameshift and 6 missense mutations) and IRF1 (1 missense and one 3’UTR variant) (NRAS mutations (linked with high NRAS expression) and the inactivating NF1 mutations (linked with decreased NF1 expression), all the other mutations were randomly distributed across all melanoma tumors, irrespective of their gene expression  Table S7Tumor immune evasion is based on the infiltration of dysfunctional T-cells in the tumor, but also the prevention of T-cell infiltration into the TME. TIDE scores predict better patient response to anti-PD-1 and anti-CTLA-4 therapies, compared to TMB and PD-L1, and can be used to predict longer patient overall survival . Using 7high, TMBint and TMBlow patients treated with anti-PD1 or anti-CTLA-4 alone, a combination of both immune checkpoint inhibitors, or any of them. Interestingly, our analysis revealed similar IPS scores across all TMB subtypes , but we also defined as TMBint those tumors with a mutational burden between 7.42 and 30 mut/Mb.High TMB was initially noted to correlate with response to anti-CTLA-4 immunotherapy in melanoma , 86. Durhigh skin melanomas correlate with high levels of IFNγ, CD8+ T-cells in the TME, cancer neoepitopes, as well as high expression of PD-L1 and further immune receptors. In addition, TMBhigh patients experience longer survival and greater response rates after ICI therapy, compared to TMBlow ones , defined by the expression of granzyme A and perforin 1, both secreted by effector cytotoxic CD8+ T-cells and NK cells against their target cells . In contrast, TNFSF18, KDR and ENTPD1 showed lower expression levels among TMBhigh tumors, and also did not correlate with the TMB. Collectively, these observations strongly indicate that immunogenicity in these tumors is affected by other factors as well, other than the TMB, and therefore, TMBlow patients could also benefit from ICI therapies.By stratifying patients based on their TMB, we found that those having a higher mutation rate (>30 mut/Mb) did not express higher In the KEYNOTE-002 study, pembrolizumab (anti-PD-1) was established as a new standard treatment after progression on ipilimumab (anti-CTLA-4) and other therapies . A year Our findings also corroborate that the expression of immune checkpoints and the quantification of the mutational burden seem to be independent predictive biomarkers of ICI therapy in melanoma patients. These results are in line with recent reports mentioning that PD-L1 expression and TMB have non-overlapping effects on the response rate to PD-1/PD-L1 inhibitors and can thus, be used to categorize the immunologic subtypes of different tumor types , 96. In We also show that, apart from CTLA-4 and PD-1, there are many other immune receptors expressed by T-cells, which influence the TME and act as checkpoints, negatively regulating immune responses in skin melanoma , 98. As high skin melanomas, providing evidence that patients with a high number of immunogenic mutations have an increased survival. Indeed, the lymphocytic score associated with better survival in these patients, in agreement with previous reports . Other signatures involving inhibitory cells (effector Treg T-cell and resting Treg T-cell signatures), or helper T-cells (Th1-like cell signature), were also higher in skin melanoma, underlying the intricate immunological reactions taking place within the tumor’s microenvironment. Looking deeper into the fractions of immune-cells within the TME however, we did not observe significant differences between TMBPOLE/POLD1 mutations (SBS10b) and tobacco smoking (SBS4).Notably, different genomic events and the immune microenvironment in skin melanoma seem to orchestrate the patients’ resistance to ICI therapies or their relapse . FrameshIFNG and other IFN-γ-related genes in skin melanoma, and questioned whether their presence associates with gene expression. Our data reveal that IDO1 and HLA-DRA are frequently mutated in skin melanoma, but these mutations do not seem to associate with their gene expression. Nevertheless, the frequency of the somatic mutations that we detected both in IDO1 and HLA-DRA, suggests that these are common events taking place in skin melanoma and could be involved in hindering patient response to ICI therapies. Their contribution to immune evasion and resistance to ICI therapies, could take place in parallel with other well-known mutations in BRAF, NRAS, NF1, PTEN and B2M, as well as in other genes involved in the IFN-γ signaling pathway, being critical in mediating antitumor immunity (Together with granzyme B and perforin, IFN-γ acts as a cytotoxic cytokine that initiates apoptosis in tumor cells , 106. IFimmunity .We finally showed that non-responders to anti-PD-1 and/or anti-CTLA-4 ICI therapies have lower IFNG, Merck18, CD274 and CD8 scores, and lower dysfunction of the tumor. In addition, they have higher exclusion potential of the tumor and higher levels in the immune suppressive MDSCs, CAFs and M2 macrophages, compared to ICI-responders. The latter cell types, on their own and cooperatively, induce an immune-suppressive TME that prevents anti-tumor cytotoxic and Th1-directed T-cell activities, mainly through the release of cytokines, chemokines, and other soluble mediators . In addilow patients.Overall, we highlight the associations between various immune receptors, TMB, TILs, patient survival and their response to ICI therapies. Taken together, our data highlight the importance of pre-existing T-cell immunity in the therapeutic outcome. They also corroborate that the expression of most immunoreceptors and TMB are independent biomarkers in predicting treatment response in skin melanoma and that ICI therapies could also be applied to TMBThe original contributions presented in the study are included in the article/GG and AZ acquired data and analyzed them. AZ developed the methodology, analyzed data and interpreted them. AZ wrote the manuscript and supervised the study. GG and AZ critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.We would like to acknowledge TCGA, ICGC, HPA, GDAC and TCIA for providing the genetic and clinical data of the skin melanoma patients that were analyzed in this study. We would also like to acknowledge assistance with extracting mutational signatures from Dr. Ilias Georgakopoulos-Soares.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.


To what extent are nonnicotine e-cigarettes used, and what flavors, brands, and ingredients are most common?This cross-sectional study of 6131 US residents aged 13 to 40 years found that 25.9% had ever used a nonnicotine e-cigarette, 16.7% had used one in the past 30 days, and 12.4% had used one in the past 7 days; 18.8% had ever co-used nonnicotine and nicotine e-cigarettes. The most-used flavors were sweet, dessert, or candy flavors; fruit flavors; and mint or menthol flavors; most common ingredients were tetrahydrocannabinol, cannabidiol, melatonin, caffeine, and essential oils.These findings suggest that a significant proportion of US residents are using nonnicotine flavored e-cigarettes marketed with unsupported health claims, which warrants further research, regulation, and prevention. This cross-sectional study evaluates nonnicotine e-cigarette use patterns, including common flavors, brands, and ingredients. Nonnicotine e-cigarettes contain chemicals, flavorants, and solvents that have known health harms and/or have not been proven safe for inhalation.To evaluate nonnicotine e-cigarette use patterns, including common flavors, brands, and ingredients.This cross-sectional study included a convenience sample of US residents aged 13 to 40 years who completed an online survey in November and December 2021. Quota sampling was used for an equal proportion of participants aged 13 to 17 years, 18 to 20 years, and 21 to 40 years, balanced for sex, race, and ethnicity per the latest US Census.Nonnicotine e-cigarette use ; co-use with nicotine e-cigarettes; age at first try; and flavors, brands, and ingredients used.Overall, 6131 participants  completed the survey (55.1% completion rate). Among all participants, 1590 (25.9%) had ever used a nonnicotine e-cigarette, 1021 (16.7%) used one in the past 30 days, and 760 (12.4%) used one in the past 7 days. By age group, 227 of 1630 participants aged 13 to 17 years (13.9%), 497 of 2033 participants aged 18 to 20 years (24.4%), 399 of 1041 participants aged 21 to 24 years (38.3%), and 467 of 1427 participants aged 25 to 40 years (32.7%) had ever used nonnicotine e-cigarettes. Among 1590 participants who had ever used a nonnicotine e-cigarette, 549 (34.5%) had used one more than 10 times; 1017 (63.9%) finished 1 nonnicotine e-cigarette in less than 1 week. Co-use of nonnicotine with nicotine e-cigarettes was reported by 1155 participants (18.8%), 1363 (22.2%) exclusively used nicotine e-cigarettes, and 431 (7.0%) exclusively used nonnicotine e-cigarettes. Most-used flavors were sweet, dessert, or candy (578 [36.3%]); fruit (532 [33.4%]); and mint or menthol (321 [20.2%]); similar flavor patterns were observed for the top 2 flavors among those who used nonnicotine e-cigarettes in the past 30 days, followed by combinations of coffee, alcohol, flower, plant, and mint or menthol flavors by age group. Participants most reported using tetrahydrocannabinol (587 [36.9%]), cannabidiol (537 [33.7%]), melatonin (438 [27.5%]), caffeine (428 [26.9%]), and essential oils (364 [22.9%]) in their nonnicotine e-cigarettes.In this study of adolescents, young adults, and adults, a sizeable proportion reported having used nonnicotine e-cigarettes and co-using them with nicotine e-cigarettes. Surveillance studies should further assess nonnicotine e-cigarette use patterns and regulations, and prevention should be developed to address youth appeal, unsubstantiated health claims, and possible health harms. Research also shows that cannabis use in e-cigarettes is common among youth.8 While e-cigarettes containing tetrahydrocannabinol (THC) and cannabidiol (CBD) have been on the market for some years, other nonnicotine e-cigarette products have more recently entered the market. However, we lack information about the use of these other nonnicotine e-cigarettes.10 These products state that they do not contain nicotine; however, the inhaled liquid and/or resulting aerosol contain essential oils, melatonin, caffeine, herbs and herbal supplements, vitamins, flavoring chemicals, and solvents.12Millions of adolescents,14 are found on the US Food and Drug Administration’s (FDA) list of harmful and potentially harmful constituents in tobacco products.15 Aldehydes may increase cancer risk,16 flavorants are associated with inflammation and cell dysfunction in in vitro studies,18 and propylene glycol and vegetable glycerin can disrupt cell metabolism and airway function.20 Using cannabis is associated with risks of addiction and adverse effects on brain development, especially among youth,21 and recent data indicate that inhaled cannabis in e-cigarettes has significantly greater psychological and physiological effects compared with its traditional smoked form.22 As in the case of nicotine e-cigarettes, where independent chemical assessments have identified more chemical compounds with toxic health effects than nicotine e-cigarettes claim,23 the overall negative effect of harmful or potentially harmful ingredients in nonnicotine e-cigarettes remains unknown.Several health and safety concerns that apply to nicotine e-cigarettes may translate to nonnicotine e-cigarettes. Ingredients reported in nonnicotine e-cigarettes (based on websites and packaging) are also present in nicotine e-cigarettes. Some of these ingredients, such as propylene glycol, vegetable glycerin, aldehydes, volatile organic compounds, polycyclic aromatic hydrocarbons, metals, silicate particles, and other heavy metals,24 device types,26 patterns of use,28 and health effects of using e-cigarettes with nicotine34 and cannabis.22 However, few studies address nonnicotine e-cigarettes, including usage patterns; preferred brands, flavors, and other ingredients; and co-use with nicotine e-cigarettes. Given health concerns regarding nonnicotine e-cigarettes, it is important to understand the extent to which nonnicotine e-cigarettes are being used as well as what brands, flavors, and ingredients are used most. This study examines nonnicotine e-cigarette use among a national online sample of adolescents, young adults, and adults and identifies the specific brands, flavors, and ingredients being used. In addition, given that we know that nicotine e-cigarette use is common, we assess the extent to which people are co-using nonnicotine and nicotine e-cigarettes. Findings from this study can guide further surveillance of nonnicotine e-cigarette use and concurrent use of nonnicotine and nicotine e-cigarettes; identify areas for research on ingredients, flavor types, and health effects from using nonnicotine e-cigarettes; inform targeted and specific prevention messages aimed at reducing use of nonnicotine e-cigarettes; and guide regulation of nonnicotine e-cigarettes that make unsubstantiated health claims or market to youth.Research has focused on prevalence,AAPOR) reporting guideline by describing the study sample, reporting survey completion rate, and following procedures to maintain data quality.41In this cross-sectional study, we conducted a national, anonymous online survey from November 17 to December 15, 2021. Participants were recruited by Qualtrics from its network of online research panels; interested panelists provided online consent (for those 18 years and older) or assent (for those younger than 18 years) before beginning the self-administered survey. Per the institutional review board, we were granted a waiver of parental consent for those under age 18 years because the surveys were anonymous, to maintain privacy of participant use patterns and prevent breach of confidentiality regarding potential self-incriminating or antisocial behavior, and to avoid parental coercion. The institutional review board at Stanford University approved the study protocol. This study followed the American Association for Public Opinion Research  with the following sampling quotas: 1:1:1 ratio of participants aged 13 to 17 years (adolescents), 18 to 20 years , and 21 to 40 years (young adults and adults) as well as self-reported sex, race, and ethnicity that approximated the US Census. These age groups allow us to address our research questions among a range of people from early adolescence through young adulthood  to adults who have passed the phase of brain development and are no longer at heightened risk for addiction.38Survey items were developed and pilot tested with 42 adolescents, young adults, and adults. Sample sociodemographic information included self-reported age, sex , gender and sexual orientation , androgynous, or asexual], not listed above [please specify], and prefer not to say), race and ethnicity classified by investigators , and financial comfort .40 all participants first viewed a description of nonnicotine e-cigarettes .” There were 18 randomly presented answer choices: (1) flowers ; (2) essential oils ; (3) other plant extract ; (4) spice ; (5) coffee or any related flavor ; (6) tea; (7) mint; (8) menthol; (9) ice; (10) wintergreen; (11) fruit ; (12) sweets or dessert flavors ; (13) alcohol ; (14) candy; (15) other beverage ; (16) unflavored; (17) “don’t know or don’t remember”; and (18) other, please specify (text entry option). Any use of flavor types mint, menthol, ice, or wintergreen was collapsed into mint or menthol flavor type, and flavor types of sweets and candy were collapsed into sweet, dessert, or candy. Next, we asked participants to identify flavors used in the past 30 days and the flavor used most in the past 30 days.P < .05.Descriptive analyses were performed to ascertain nonnicotine e-cigarette usage  across sociodemographic factors and co-use with nicotine e-cigarettes. Using a multivariate test of means, we assessed whether the mean age of trying nonnicotine e-cigarettes was equal to that of nicotine e-cigarettes. Next, among those who ever used nonnicotine e-cigarettes, we summarized use-related behaviors and self-reported ingredients by age group . eTable 1 in the Per the AAPOR reporting guidelines, we report that 6131 of 11 118 people who clicked on the survey link completed the entire survey . Among the 11 118 responders, 4306 clicked the survey link after their age, sex, and race and ethnicity quota had been filled and were therefore not included in the final study sample. We further excluded 681 people for not meeting quality and attention checks. The final sample included 6131 participants who completed the entire survey.Participants’ mean age was 21.9 ; 3454 (56.3%) self-reported as female, 1573 (25.7%) as LGBTQ+, 318 (5.2%) as Asian, Native Hawaiian, or Pacific Islander non-Hispanic, 841 (13.7%) as Black or African American non-Hispanic, 1143 (18.6%) as Hispanic or Latino, 3162 (51.6%) as White non-Hispanic, 250 (4.1%) as other or multiracial non-Hispanic (including American Indian and Alaska Native non-Hispanic and participants identifying as >1 race non-Hispanic), and 417 (6.8%) preferred not to say .Among all participants, 1590 (25.9%) had ever used a nonnicotine e-cigarette; 1021 (16.7%) had used a nonnicotine e-cigarette in the past 30 days, and 760 (12.4%) used a nonnicotine e-cigarette in the past 7 days. Among all participants who had ever used a nonnicotine e-cigarette, 549 (34.5%) had used such devices more than 10 times, 294 (18.5%) had used such devices more than 20 times, and 82 (5.2%) had used them more than 100 times. Overall, 469 (29.5%) reported using within 5 minutes of waking up, 605 (38.1%) used it as recently as on the day of the survey, 1017 (63.9%) took less than 1 week to finish 1 nonnicotine e-cigarette, and 1186 (74.6%) were likely to use a nonnicotine e-cigarette again in the next 6 months .Examining nonnicotine e-cigarette use by age group, 227 of 1630 participants aged 13 to 17 years (13.9%), 497 of 2033 aged 18 to 20 years (24.4%), 399 of 1041 aged 21 to 24 years (38.3%), and 467 of 1427 aged 25 to 40 years (32.7%) had ever used nonnicotine e-cigarettes. In the past 30 days, 113 of those aged 13 to 17 years (6.9%); 265 of those aged 18 to 20 years (13.0%), 281 of those aged 21 to 24 years (27.0%), and 362 of those aged 25 to 40 years (25.4%) had used nonnicotine e-cigarettes. Among those younger than age 24, between 16% and 20% reported using more than 20 times, between 27% and 30% reported using within 5 minutes of waking up, between 30% and 40% used as recently as on the day of the survey, and between 66% and 77% were likely to use a nonnicotine e-cigarette again in the next 6 months. Furthermore, between 42% and 67% of nonnicotine e-cigarette ever users younger than 24 years took less than 1 week to finish 1 nonnicotine e-cigarette. Additional information about nonnicotine vaping behaviors among past 30-day users of nonnicotine e-cigarettes is available in eTable 1 in the P = .004), pod- or cartridge-based devices , and other e-cigarettes .In our sample, 2536 participants (41.3%) had ever used a nicotine e-cigarette, 1705 (27.8%) had used in the past 30 days, and 1346 (21.9%) had used in the past 7 days . OverallIn the total sample, participants most commonly reported having ever used the following ingredients in nonnicotine e-cigarettes: THC (587 [36.9%]), CBD (537 [33.7%]), melatonin (438 [27.5%]), caffeine (428 [26.9%]), and essential oils (364 [22.9%]), in that order . Among pAs seen in eTable 2 in the Among those who ever used a nonnicotine e-cigarette, 578 (36.3%) reported ever having used sweet, dessert, or candy flavors; 532 (33.4%) used fruit flavors; and 321 (20.2%) used mint or menthol flavors, closely followed by coffee, flower, and essential oil flavors . Among n10 or on just cannabis (THC or CBD) e-cigarette use.22 Our findings show that nonnicotine e-cigarettes are commonly used across all age groups, including in the past 30 and past 7 days, soon after waking, and as recently as on the day of the survey. Furthermore, there was substantial co-use of nonnicotine and nicotine e-cigarettes, with participants first trying nonnicotine e-cigarettes on average at a younger age than nicotine e-cigarettes. Overall, the most common ingredients used in nonnicotine e-cigarettes were THC, CBD, melatonin, and caffeine. Cloudy and Vitaminvape were the most popular and commonly used nonnicotine e-cigarette brands. The most-used flavor types among all participants in the past 30 days were sweet, dessert, or candy; fruit; caffeine; alcohol; flower; and mint or menthol flavors.This study extends the limited literature on nonnicotine e-cigarette products, which has largely focused on similarities with nicotine e-cigarette marketing, on toxic effects from what the industry refers to as vitamin vaping,26 nearly all brands of nonnicotine e-cigarettes resemble disposable-type devices. Second, similar to findings that adolescent nicotine e-cigarette users report using multiple device types,26 we found substantial co-use of nonnicotine and nicotine e-cigarettes. Third, similar to nicotine e-cigarette studies,42 many users and especially young adults do not know what ingredients or flavors they are using in their nonnicotine e-cigarettes.43 Finally, the most commonly used flavors in nonnicotine e-cigarettes in the past 30 days were fruit and sweet, dessert, candy flavors, similar to most commonly used flavors in nicotine1 and cannabis44 e-cigarettes.Although no prior data on the specifics of nonnicotine e-cigarette use are, to our knowledge, available, findings from studies on nicotine e-cigarette products and use patterns can inform and support our findings. First, like findings that disposable nicotine e-cigarettes are now the most widely used nicotine e-cigarette,20 There may be risks in using nonnicotine e-cigarettes alone and additive risk to those co-using with nicotine e-cigarettes. Several nonnicotine e-cigarette companies seem to acknowledge harms and suggest that people should not inhale the aerosol into their lungs .45 It remains unknown whether users of these devices are using these products as intended. Second, the FDA has raised concerns in warning letters to nonnicotine e-cigarette companies and public advisories, noting that while several ingredients may be “generally recognized as safe” and thus approved as dietary supplements for ingestion, they may be harmful when inhaled in aerosolized form and cannot be legally marketed as dietary supplements.47 Third, nonnicotine e-cigarette companies appear to be targeting adolescents and young adults by marketing their products with unproven health benefits, such as an improved immune system, weight loss, better sleep, and more energy,48 which may appeal to adolescents’ and young adults’ needs to appear trendy, environmentally friendly, and tech-savvy and to prioritize self-care. Finally, nonnicotine e-cigarettes are currently not regulated as either tobacco products49 or dietary supplements.50 With aggressive marketing targeting young people and gaps in regulation, the popularity and use of nonnicotine e-cigarettes can be expected to increase dramatically, just as nicotine e-cigarette use grew exponentially between 2015 and 2019.51 Thus, rapid surveillance of nonnicotine e-cigarette use is needed to accurately and continually estimate use patterns and health harms associated with these products as well as to develop regulatory solutions. In so doing, it is important to ask about specific brand names, flavors, and ingredients, given that evidence suggests that asking about specific vaping devices and e-cigarette content is more sensitive than asking about general use.39Our study highlights several issues of concern to researchers and policy makers. First, although nonnicotine e-cigarettes share many of the same ingredients as nicotine e-cigarettes, including several associated with known health harms, the health effects of aerosolizing and inhaling chemicals such as essential oils, vitamins, caffeine, and other herbs remain unknown.This study has several limitations. First, we report findings from a convenience sample, and thus the rates of use provided in our study are restricted to our sample and may not be generalizable. Second, self-reported information about participants’ use of products may be subject to recall-related errors. Third, participants were not asked about specific products used within each brand, which may have helped to identify which ingredients participants used instead of a broader question about ingredients used. Fourth, we cannot ascertain whether participants understood the difference between CBD and THC or if they misclassified their cannabis use.In this study, a sizeable number of adolescents, young adults, and adults reported having used nonnicotine e-cigarettes and co-using them with nicotine e-cigarettes. Despite unproven health and wellness claims made about these products, their safety is unknown and may present health harms from inhaling flavorants and other constituents. National surveillance studies should be conducted to further understand nonnicotine e-cigarette patterns and to develop regulations that address possible health harms of nonnicotine e-cigarettes. Our findings also suggest that the FDA should enforce against nonnicotine e-cigarettes that are making unsubstantiated dietary or other unauthorized health claims and targeting adolescent and young adult customers.


Triboelectric nanogenerators (TENGs) stand out as an attractive form of technology for the efficient harvest of mechanical energy and the powering of wearable devices due to their light weight, simplicity, high power density, and efficient vibration energy scavenging capabilities. However, the requirement for micro/nanostructures and/or complex and expensive instruments hinders their cheap mass production, thus limiting their practical applications. By using a simple, cost-effective, fast spray-coating process, we develop high-performance UV-curable triboelectric coatings for large-scale energy harvesting. The effect of different formulations and coating compositions on the triboelectric output is investigated to design triboelectric coatings with high output performance. The TENG based on a hybrid coating exhibits high output performance of 54.5 μA current, 1228.9 V voltage, 163.6 nC transferred charge and 3.51 mW output power. Moreover, the hybrid coatings show good long-term output stability. All the results indicate that the designed triboelectric coatings show great potential for large-scale energy harvesting with the advantages of cost-effectiveness, fast fabrication, easy mass production and long-term stability. The rapid development of flexible functional materials and advanced fabrication technologies has led to wearable devices being widely used in our daily lives, allowing us to monitor our health status and achieve individual eHealth ,2,3. Wea2 [To push the TENGs into practical applications, many research advances have been made to improve the output performance of the devices, including surface modification ,29, stru2 . Saqib a2 . Wang an2 . Kong an2 . By addiIn order to benefit the most from this energy-harvesting technology for wearable devices, we developed an organic coating that can be used for high-output TENGs via a simple painting process. Different formulations and coating compositions were evaluated to design high-performance triboelectric coatings. The TENG with 1:1 mixture of DFHMA and BA showed the highest output performance of 54.5 μA current, 1228.9 V voltage, 163.6 nC transferred charge, and 3.51 mW output power, as well as good long-term stability, and it can be used for large-scale energy harvesting through a fast and cost-effective spray-coating and a UV-curing process.Trimethylolpropane triacrylate (TMPTA) and tripropylene glycol diacrylate (TPGDA) was obtained from Shanghai Guang Yi Chemical Co., Ltd. (Shanghai China). Methyl methacrylate (MMA) was obtained from Tianjin Zhiyuan Chemical Co., Ltd. . n-butyl acrylate (BA), 2-Hydroxy-2-methylpropiophenone (HMPP), and Phenylbisphosphineoxide (XBPO) were obtained from Shanghai Macklin Biochemical Technology Co., Ltd. . Dodecafluoroheptyl methacrylate (DFHMA) and 2,2,3,4,4,4-Hexafluorobutyl methacrylate (HFBMA) were obtained from Shanghai Qinba Chemical Co., Ltd. . Fluororesin was purchased from Shanghai Yitu Industrial Co., Ltd. . Nylon-11 was purchased from Tianjin Heowns Biochemical Technology Co., Ltd. . Polytetrafluoroethylene (PTFE) film, Kapton film, poly (PET) film and copper foil tape were purchased from a local market. All other chemicals were of analytical grade and used without further treatment.The UV-curable resin contains 80 wt % oligomers  and 20 wt % UV monomers (TPGDA and TMPTA with a weight ratio of 3:1). The coating solution was prepared by mixing the UV-curable resin with 3 wt % HMPP and 1 wt % XBPO in xylene. After spray-coating the as-prepared solutions onto the PET substrate, they were cured under UV light at room temperature for 3–5 min to obtain the triboelectric coatings. The coatings with different formulations are referred to as HFBMA, DFHMA, Fluororesin, MMA, BA, and DFHMA + BA, respectively.A mixture of 9.37 g dichloromethane, 8.63 g anhydrous formic acid and 2 g nylon-11 was mixed for 2 h at room temperature to obtain a clear solution. As substrates, Kapton films were cut into 4 cm × 4 cm and cleaned with ethanol, acetone, and deionized water. After that, a Nylon-11 solution was spin-coated onto Kapton films at 500 rpm for 5s and 3000 rpm for 30 s. Then, the Nylon-11/Kapton layers were annealed at 80 °C to evaporate residual solvents. Thereafter, copper foil tapes were attached to Kapton on the opposite side of Nylon-11 as the bottom electrode.To fabricate the coating TENGs, UV-curable triboelectric coatings with different formulations were prepared directly onto 4 cm × 4 cm copper foil tapes, working as one of the friction layers. To test the effects of the coating composition on triboelectric output, the mass ratio between the DFHMA oligomer and UV monomers was changed from 4:1 to 2:1, 1;1, 1:2, and 1:4. Afterwards, the UV-curable triboelectric coating and Nylon-11 friction layer were assembled into a triboelectric generator for output performance characterization.To investigate the output performance of the TENG, an IVCL17-56 motor was used to periodically press and release the device. Short circuit current is measured by SR570 low-noise current amplifier  and the output voltage is measured by NI 9215 . Data were collected using LabVIEW programs .Among the polymers used in coatings, acrylic resins based on acrylate and methacrylate monomers dominate due to their excellent durability, weather resistance, gloss retention, adhesion, abrasion, and thermal resistance. So, in this work we chose four different acrylic monomers to develop an organic coating that can be used for triboelectricity harvesting. A commercial Fluororesin and PTFE film were also used as comparison. As mentioned in To obtain high output performance triboelectric coatings, we fabricated TENGs using triboelectric coatings with different formulations, including HFBMA, DFHMA, Fluororesin, MMA, BA, and DFHMA + BA. As a comparison, a commercial PTFE film based TENG was also fabricated. Nylon-11 spin-coated on Kapton film was used as another friction layer to construct TENGs. When the TENG was periodically pressed and released, Nylon-11 rubbed against the coating and generated positive charges on the Nylon-11 surface and negative charges on the surface of coating, according to the Triboelectric Series. As shown in 2 and 92.0 μC/m2, showing the same trend as short circuit current, output voltage and transferred charge. In order to determine the TENGs’ effective electric power, resistors are connected as external loads. The instantaneous current drops as the load resistance increases (ncreases e and thencreases f. The ouΦ DFHMA oligomers + (1 − Φ) UV monomers, where Φ is the mass ratio of DFHMA oligomers to UV monomers. TENG output performance with different Φ was evaluated and shown in Φ. This is primarily because DFHMA oligomers contain a large number of fluorine groups, while fluorine is the most electronegative element due to its strong ability to attract electrons. Increasing means adding more fluorine, which is beneficial to the output performance. This is also confirmed by the results of TENGs’ effective electric power (Φ = 80% is the highest, which is consistent with previous results.Along with coating formulations, we also examined the coating composition in relation to the triboelectric output. Specifically, the mass ratio between DFHMA oligomers and UV monomers was modulated as 4:1 to 2:1, 1:1, 1:2, and 1:4. To simplify the discussion, the composition of triboelectric coating is defined as ic power c,d. It cAs part of our efforts to design a high-output triboelectric coating, we investigated how mixing two components would affect its performance. A TENG with a 1:1 mixture of DFHMA and BA was fabricated and examined. As shown in As a triboelectric coating, robustness plays a crucial role in practical applications besides outputting high performance. To fully evaluate our triboelectric coating, we carried out a long-term output stability experiment for the TENG with hybrid composition coatings. As shown in In summary, we produced high-performance triboelectric coatings by using a simple, cost-effective, fast spray-coating process. In addition, the effect of different formulations and coating compositions on the triboelectric output was fully investigated. The hybrid composition coatings showed the best output performance as well as good long-term output stability. The hybrid composition-based TENG exhibited an output power approximately 87 times higher than that of the commercial PTFE film-based TENG, which opens up an exciting opportunity to improve the output performance of coating-based TENGs towards large-scale energy harvesting.


Renin-angiotensin system (RAS), as a critical system for controlling body fluid and hemostasis, contains peptides and receptors, including angiotensin 1-7 (Ang 1-7) and Mas receptor (MasR). Ang 1-7 implements its function via MasR. Ang II is another peptide in RAS that performs its actions via two Ang II type 1 and 2 receptors (AT1R and AT2R). The functions of AT2R and MasR are very similar, and both have a vasodilation effect, while AT1R has a vasoconstriction role. MasR affects many mechanisms in the brain, heart, blood vessels, kidney, lung, endocrine, reproductive, skeletal muscle, and liver and probably acts like a paracrine hormone in these organs. The effect of Ang 1-7 in the kidney is complex according to the hydroelectrolyte status, the renal sympathetic nervous system, and the activity level of the RAS. The MasR expression and function seem more complex than Ang II receptors and have interacted with Ang II receptors and many other factors, including sex hormones. Also, pathological conditions including hypertension, diabetes, and ischemia-reperfusion could change MasR expression and function. In this review, we consider the role of sex differences in MasR expression and functions in the renal system under physiological and pathological conditions. The classic renin-angiotensin system (RAS) structure begins from renin secretion via juxtaglomerular cells in the kidney. Renin converts angiotensinogen to angiotensin (Ang) I. Then Ang-converting enzyme (ACE) converts Ang I to Ang II in the lungs. In parallel, ACE2 converts Ang II to Ang 1-7 . EvidencThere are heterodimerization and functional interactions between MasR and AT1R or AT2R .Previous studies indicate that treatment with RAS inhibitors increases ACE2 expression or action in numerous organs, including the heart –26, kidnHowever, the role of sex differences in MasR expression and activity is still challenging in the literature. In this review, we consider a variety of information databases such as PubMed, Google Scholar, and Scopus to specify the role of the sex hormone estrogen on the expression and function of the Ang 1-7/MasR axis.MasR is a G-protein recognized in 1986 during the investigation of tumors , 37, andLike other RAS receptors, the MasR expression depends on various factors and pathological conditions such as sex hormones, diabetes, hypertension, etc. MasR expression is different between the sexes in the renal system. The mRNA levels of the MasR were higher in adult female rats' kidneys than in males , 49. OneIn the renal ischemia/reperfusion (I/R) model, intrarenal MasR expression increased , 60. NumFrom a physiological point of view, the MasR function is more complex and does not have complete transparency. On the one hand, MasR is known as the Ang 1-7 specific receptor, and on the other hand, its interaction with RAS receptors created an unforeseen situation. That is difficult to accurately identify its exact function because it is affected by various parameters such as bradykinin, gender, hypertension, etc. In the following, we mention some of Ang 1-7 and MasR's effects on renal functions under several normal and pathological conditions.In isolated renal arteries, Ang 1-7 did not affect vascular function, but following Ang II infusion, it decreased vasoconstriction . In MasRCross-talk mechanisms between the MasR, AT1R, AT2R, bradykinin B2, and vasopressin V2 receptors control water transportation in the kidney , 74, 75.In male and female Wistar rats, simultaneous AT1R, AT2R, and MasR blocking, compared with only AT1R, AT2R blocking, Ang 1-7 infusion increased RBF response in males but not in female animals. So this finding indicated that renal vascular response to Ang 1-7 may not be exerted by MasR alone in males , 92. SomThere are sex differences in hemodynamics parameters to graded Ang II administration in 2K1C hypertensive rats with co-blockade AT1R and MasR. So RBF increased significantly in males than females when AT1R and MasR were blocked . Also, iAVE0991 treatment in the renal I/R model in male C57BL/6 Wild-type or Mas−/− mice promoted renoprotective effects and decreased infiltration of leukocytes in the kidney . Karimi ACE2/Ang 1-7/MasR axis is a vasodilator pathway in RAS, and this pathway's response varies in physiological and pathological conditions and gender. Gaining a more profound knowledge about MasR function in different situations could help develop therapeutic agents for treating hypertension and chronic kidney disease.


Diabetes mellitus is the most common endocrine disturbance in inherited mitochondrial diseases. It is essential to increase awareness of the correct diagnosis and treatment of diabetes in these patients and screen for the condition in family members, as diabetes might appear with distinctive clinical features, complications and at different ages of onset. The severity of mitochondrial-related diabetes is likely to manifest on a large scale of phenotypes depending on the location of the mutation and whether the number of affected mitochondria copies (heteroplasmy) reaches a critical threshold. Regarding diabetes treatment, the first-choice treatment for type 2 diabetes (T2D), metformin, is not recommended because of the risk of lactic acidosis. The preferred treatment for diabetes in patients with mitochondrial disorders is SGLT-2i and mitochondrial GLP-1-related substances. The tight relationship between mitochondrial dysfunction, reduced glucose-stimulated insulin secretion (GSIS), and diabetes development in human patients is acknowledged. However, despite the well-characterized role of mitochondria in GSIS, there is a relative lack of data in humans implicating mitochondrial dysfunction as a primary defect in T2D. Our recent studies have provided data supporting the significant role of the mitochondrial respiratory-chain enzyme, cytochrome c oxidase (COX), in regulating GSIS in a rodent model of T2D, the Cohen diabetic sensitive (CDs) rat. The nutritionally induced diabetic CDs rat demonstrates several features of mitochondrial diseases: markedly reduced COX activity in several tissues, increased reactive oxygen production, decreased ATP generation, and increased lactate dehydrogenase expression in islets. Moreover, our data demonstrate that reduced islet-COX activity precedes the onset of diabetes, suggesting that islet-COX deficiency is the primary defect causing diabetes in this model. This review examines the possibility of including T2D as a primary mitochondrial-related disease. Understanding the critical interdependence between diabetes and mitochondrial dysfunction, centering on the role of COX, may open novel avenues to diagnose and treat diabetes in patients with mitochondrial diseases and mitochondrial dysfunction in diabetic patients.  The absorption of blood glucose into the liver, fat, and skeletal muscle cells is dependent on insulin, a peptide hormone produced by β-cells in pancreatic islets . It is aPancreatic β-cells are sensitive glucose sensors that adjust precise quantities of insulin release to variations in blood glucose levels critical to ensure adequate entry of glucose and homeostasis. The process of glucose-stimulated insulin secretion (GSIS) is exceptionally dependent on mitochondrial function  3,10,11,15,16,17ATP channels and stimulate insulin secretion (2) [+ and FADH electron donors, β-cells acquire unique shuttles, the malate–aspartate shuttle and the glycerophosphate shuttle [ATP production in β-cells is crucial for stimulus-secretion coupling, a cascade of molecular events encompassing the initial sensing and transport of glucose to β-cells, triggering the exocytosis of insulin. GSIS requires increased mitochondrial ATP generation up to the metabolic threshold required for robust insulin release ,15,16,17ecretion . Glucosetion 2) ,15,16,17tion (2) ,18,19,20 ,15,16,1tion (2) ,29,30,31tion (2) ,33. To r shuttle ,34.p < 0.01) between CDs islet-COX activity and GSIS, implicating a critical role of islet-COX activity as a modulator of GSIS [Mitochondrial dysfunction in T2D: Despite the well-characterized role of mitochondria in β-cell stimulus-secretion coupling, there is a relative scarcity of data in humans implicating mitochondrial dysfunction in the pathogenesis of T2D. Observation in T2D patients supports the link between reduced ATP generation by the mitochondria and β-cell dysfunction. Abnormal mitochondrial morphology and reduced GSIS are found in β-cells from postmortem T2D patients ,37,38,39 of GSIS .Human mitochondrial DNA (mtDNA) is a circular, double-stranded, supercoiled molecule that encodes 37 genes, essential for OXPHOS and mitochondrial protein synthesis . There aMitochondria function relies on their structure and the organization of OXPHOS complexes. MRC complexes are arranged in the inner mitochondrial membrane as a “respirasome” ,57. The 2+-activated dephosphorylation, leading to monomerization and NDUFA4 moving from complex I to COX. The “active state” induces higher rates of COX activity and ATP synthesis but increases ROS formation, decreasing mitochondrial efficiency [2 = 0.9691, p < 0.001) and inversely with the elevation in blood glucose levels , supporting the notion that islet-COX activity could be a major metabolic regulator of β-cell function [COX plays a vital role in regulating mitochondrial respiration and OXPHOS to adjust ATP production to specific-cellular energy requirements, depending on nutritional or environmental stimuli. However, COX activity in pancreatic β-cells probably differs from other cell types, as it requires firm and tight regulation to ensure that the rate of insulin secretion closely mirrors the change in blood glucose levels. β-cells are totally dependent on OXPHOS for ATP production and cannot accelerate glycolysis to compensate for decreased OXPHOS due to repressed lactate dehydrogenase expression. COX adapts to changing environmental conditions ,20. Propficiency ,73. Exceficiency ,52,68,74ficiency . In a refunction . Moreovefunction The presence of β-cell dysfunction as an early defining event in the onset and progress of T2D is currently the predominantly accepted view ,15,22. HWe examine whether mitochondrial dysfunction could precede hyperglycemia promoting diabetes development in an inbred, lean model of nutritionally induced diabetes, the Cohen diabetic sensitive (CDs) rat ,47. The We also demonstrate a direct link between inhibition of COX activity and insulin secretion by incubating isolated islets with the COX-specific inhibitor, potassium cyanide, and demonstrating an apparent dose-dependent reduction in insulin secretion in isolated islets exposed to different doses of potassium cyanide .Interestingly, we also found that reduced islet-COX activity and GSIS were highly associated with the pancreatic inflammatory process featured by peri-islet infiltration of fat and activated macrophages releasing the proinflammatory cytokine interleukin 1-beta (IL1-β). IL1-β dose-dependently diminishes GSIS by substantially increasing nitrite levels ,45. The 2 = 0.9691, p < 0.001) and inversely with the elevation in blood glucose levels , suggesting that islet-COX activity could be a significant metabolic sensor in pancreatic β-cells. Our studies concluded that: (1) islet-COX activity is directly associated with GSIS; (2) pancreatic inflammation reduced GSIS via NO production triggered by IL-1β secreted from peri-islet-infiltrating macrophages; (3) COX activity was markedly reduced in hyperglycemic CDs due to a pre-existing inborn (primary) impairment in mitochondrial respiratory chain dysfunction setting the initial islet-COX activity at ≤46% of baseline, thus enabling an almost complete inhibition of islet-COX activity, diminished GSIS and increased blood glucose levels.In a recent study, we linked reduced mitochondrial COX activity in islets, insulin secretion, and glucose homeostasis. We demonstrated that hyperglycemia was initiated when islet-COX activity decreased below 46% of baseline, identifying a novel islet-COX activity threshold required to sustain normoglycemia . We demoDiabetes mellitus is not a classical mitochondrial disease . T2D lacIn conclusion, T2D is highly dependent on the mitochondria and as such could be considered a mitochondrial-associated disease. However, the question of whether it is a primary mitochondrial disease remains open. Diabetes mellitus is part of the pathologies of the mitochondrial disease exhibiting distinctive clinical features and complications that are not found in classical diabetes ,84. It i


Ralstonia pickettii T1 on the Escherichia coli surface using Pseudomonas OprF protein as a fusion partner by C-terminal deletion-fusion strategy. Surface display of depolymerase was confirmed by flow cytometry, immunofluorescence microscopy and whole cell hydrolase activity. For the application, depolymerase was used as an immobilized catalyst of enantioselective hydrolysis reaction for the first time. After 48 h, (R)-methyl mandelate was completely hydrolyzed, and (S)-mandelic acid was produced with over 99% enantiomeric excess. Our findings suggest that surface displayed depolymerase on E. coli can be used as an enantioselective biocatalyst.We have expressed extracellular poly(3-hydroxybutyrate) (PHB) depolymerase of  Polyhydroxyalkanoates (PHAs) are biodegradable polyesters, that accumulate intracellularly as a carbon and/or energy source in numerous bacteria, to provide renewable resources under nutrient limited conditions. In PHA-accumulating bacteria, many enzymes, including PHA synthase, phasin, epimerase, oligomer hydrolase and depolymerase, play a role in the biosynthesis and degradation of PHAs -4. Of thRalstonia pickettii T1 depolymerase on the surface of E. coli cells using truncated Pseudomonas OprF as an anchoring motif and demonstrate the biocatalytic application of this system to enantioselective hydrolysis of a racemic ester.Thus far, various surface display systems have been reported, with potential applications in the development of vaccines, bioadsorbents, biocatalysts, antibody libraries and biosensors -10. In aR. pickettii T1 depolymerase gene was amplified using primers 5’-gctctagagccacggcggggcccggtgc-3’ and 5-cccaagctttcatgg acaattgccgacg-3’, and inserted into the XbaI and HindIII sites of pTacOprF164PL and pTacOprF188PL, respectively. For the immunodetection, 6 histidine residues (underlined) were added to C-terminal of depolymerase gene, which was amplified using primers 5’-gctctagagccacggcggggcccggtgc-3’ and 5’-cccaagctttcaatggtgatgatggtgatgtggacaattgccgacg-3’, and cloned into the XbaI and HindIII sites of pTacOprF164PL and pTacOprF188PL to make pTacOprF164RDH and pTacOprF188RDH, respectively. Primers for the amplification of the R. pickettii T1 PHB depolymerase gene were designed based on the reported sequence (J04223) [To construct pTacOprF164RD and pTacOprF188RD, For detection of displayed depolymerase fused with 6 histidine residues, recombinant cells were labeled with mouse anti-His antibody  diluted 1:100, followed by rabbit anti-mouse IgG antibody conjugated fluorescein isothiocyanate (FITC) diluted 1:200. The cells were analyzed using a flow cytometer . For the visualization under fluorescence microscope, cells were stained with the mouse anti-His antibody diluted 1:1000 and rabbit anti-mouse IgG conjugated with FITC diluted 1:3000. The samples were visualized by confocal fluorescence microscopy .After collection by centrifugation at 5,590 ×g and 4°C and washing with distilled water, recombinant cells were further freeze-dried for 48 h with lyophilizer  for measuring whole cell enzyme activity and application of enantioselective biocatalyst. Hydrolytic activity of genetically immobilized depolymerase was assayed by spectrophotometric method as mentioned elsewhere . In ordeP. aeruginosa PaO1 OprF was selected for depolymerase display as it allows successful and stable display of proteins in an active form on the surface of E. coli and P. putida [To date, cell surface display systems have employed many enzymes including lipase, polyethylene terephthalate (PET) degrading enzyme (PETase), cyotochrom P450, amylase, dimeric bovine adrenodoxin and carboxymethylcellulase as target proteins , 20, 21.. putida , 22.E. coli (pTacOprF188E) and E. coli (pTacOprF188RDH) incubated with anti-His antibody followed by FITC-conjugated secondary antibody. As shown in t) and depolymerase, with 6 histidine residues, was expressed outside of the E. coli. In order to confirm surface display of depolymerase, recombinant cells were analyzed using immunofluorescence microscopy. E. coli XL-10 Gold (pTacOprF188RDH) labeled with anti-His antibody followed by binding of FITC-conjugated secondary antibody showed fluorescent spots, which demonstrates depolymerase fused with 6 histidine residues was successfully expressed on the cell surface . Meanwhile, E. coli XL-10 Gold cells harboring pTacOprF188E showed little fluorescence  obtained with 0.1 mM IPTG induction to a value of 100, the relative activities with 0.01 and 1 mM IPTG were 21.3 and 64.8, respectively. Little hydrolase activity was observed in supernatant fractions. These suggest that OprFS)-mandelic acid, obtained in 48 h were over 40% and 99%, respectively.For application in biocatalysts, the enantioselective resolution of racemic methyl mandelate was investigated. The scheme of this reaction is shown in E. coli cells displaying depolymerase have potential as industrial catalysts similar to other hydrolases.It is known that PHA depolymerase shows enantioselective behavior towards substrate in aqueous solutions, however, the main application of surface displayed depolymerase has been the hydrolysis of polymer or oligomer, composed of monomeric units of 3-hydroxyacid , 16, 17.E. coli and demonstrate the application of this system to the enantioselective biotransformation for the first time. Furthermore, improvement of the enzyme characteristics via methods such as immobilization, fusion technique and directed evolution, could result in significant increases in activity, selective binding affinity or other key characteristics of depolymerases. Thus, the results of our efforts can be successfully applied to the various fields including bioremediation, biocatalysis, immunoassay, whole cell microarrays and whole cell biosensors.In conclusion, we reported the cell surface display of PHB depolymerase on the cell surface of


The most common subtype of ovarian cancer (OC) is the high-grade serous ovarian carcinoma (HGSOC), accounting for 70%–80% of all OC deaths. Although HGSOC is a potentially immunogenic tumor, clinical studies assessing the effectiveness of inhibitors of programmed death protein and its ligand (PD-1/PD-L1) in OC patients so far showed only response rates <15%. However, recent studies revealed an interesting prognostic role of plasma PD-1/PD-L1 and other circulating immunoregulatory molecules, such as the B- and T-lymphocyte attenuator (BTLA), butyrophilin sub-family 3A/CD277 receptors (BTN3A), and butyrophilin sub-family 2 member A1 (BTN2A1), in several solid tumors. Since evidence showed the prognostic relevance of pretreatment serum CA125 levels in OC, the aim of our study was to investigate if soluble forms of inhibitory immune checkpoints can enhance prognostic power of CA125 in advanced HGSOC women. Using specific ELISA tests, we examined the circulating PD-1, PD-L1, pan-BTN3As, BTN3A1, BTN2A1, and BTLA levels in 100 advanced HGSOC patients before treatment, correlating them with baseline serum CA125, age at diagnosis, body mass index (BMI), and peritoneal carcinomatosis. A multivariate analysis revealed that plasma BTN3A1 ≤4.75 ng/ml , age at diagnosis ≤60 years  and absence of peritoneal carcinomatosis  were independent prognostic factors for a longer progression-free survival (PFS) (≥30 months) in advanced HGSOC women. However, further two-factor multivariate analyses highlighted that baseline serum CA125 levels >401 U/ml and each soluble protein above respective concentration cutoff were covariates associated with shorter PFS (<30 months) and unfavorable clinical outcome, suggesting that contemporary measurement of both biomarkers than CA125 only could strengthen prognostic power of serum CA125 in predicting PFS of advanced HGSOC women. Plasma PD-L1, PD-1, BTN3A1, pan-sBTN3As, BTN2A1, or BTLA levels could be helpful biomarkers to increase prognostic value of CA125. Ovarian cancer (OC) is the seventh most frequently diagnosed tumor and the eighth leading cause of cancer death in women worldwide, with a 5-year relative survival rate of 49% , 2.High-grade serous ovarian carcinoma (HGSOC) is the most recurrent subtype and represents 70%–80% of all OC deaths . UnfavorThe most investigated immune checkpoint receptor is the programmed cell death protein 1 (PD-1), with its ligands, PD-L1, and PD-L2 . AlthougOther immune checkpoints, including B- and T-lymphocyte attenuator (BTLA) , butyropRecently, new evidence showed that investigating the soluble forms of inhibitory immune checkpoints may allow to obtain useful information about the evolution of cancer by predicting survival of patients affected by various tumors. Therefore, these studies suggested their potential use as prognostic biomarkers , 24–26. Since evidence showed the potential prognostic relevance of pretreatment serum CA125 (Cancer antigen 125) levels in OC , the aimWe prospectively studied a cohort of 100 advanced HGSOC women enrolled at the two Sicilian hospital centers: “Sicilian Regional Center for the Prevention, Diagnosis and Treatment of Rare and Heredo-Familial Tumors” of the Section of Medical Oncology of University Hospital Policlinico “P. Giaccone” of Palermo  and Department of Gynecologic Oncology of the Hospital ARNAS Civico “Di Cristina Benfratelli” of Palermo .The study (Protocol “TIC-OC v.1.1”) was approved by the ethical committee  of the university-affiliated hospital A.O.U.P. “P. Giaccone” of Palermo .The information regarding the age at diagnosis, personal history, histological subtype, grading, and International Federation of Gynecology and Obstetrics (FIGO) stages were anonymously collected for all recruited patients Table 1 2) according to the current therapeutic strategies. Patients with Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) ≥3 were excluded from the study.From May 2018 to July 2021, blood specimens were prospectively harvested from 100 patients with histological diagnosis of advanced HGSOC (stage IIIB–IV) at baseline, before surgery , and starting first-line chemotherapy with Carboplatin AUC (area under the curve) 5 and Paclitaxel , was used to confirm the previously obtained data.Baseline peripheral blood specimens from untreated advanced HGSOC women were collected, processed for plasma isolation, and analyzed through specific enzyme-linked immunosorbent assays (ELISAs) as previously described , 11, 28 Since some discrepancies, concerning the performances, reproducibility, sensitivity and specificity, cross-reactivity, differences in quantification in the plasma and serum, and run temperature, were observed in other commercially available tests, specific ELISAs produced by the company DYNABIO S.A.  were used according to the previously described recommendations , 22. AllIn particular, these specific ELISAs were used because some assays were either not commercially available  or were not satisfactory . These ELISA tests not only showed very good performances but also established conditions for optimal determination of concentrations of the six markers in blood (use of plasma instead of serum), solving the problem regarding the differences in quantification of proteins between the plasma and serum, which are detected when using other commercial kits. All six used ELISA tests showed good linearity and a high specificity. The linearity for sPD-1 measurement in the test ranges from 0.01 to 5.00 ng/ml, for sPD-L1 from 0.02 to 2.00 ng/ml, for pan-sBTN3As from 0.10 to 8.00 ng/ml, for sBTN3A1 from 0.05 to 8.00 ng/ml, for sBTN2A1 from 0.03 to 2.00 ng/ml, and for sBTLA from 0.25 to 8.00 ng/ml, as shown in Specific details on the experimental protocol regarding the used ELISA assays are reported in An analysis by receiver operating characteristic (ROC) curves  was carrUsing specific ELISAs, we performed the measurement of plasma levels of sPD-1, sPD-L1, sBTN3A1, pan-sBTN3As, sBTN2A1, and sBTLA in 100 advanced HGSOC women, before surgery and of starting first-line chemotherapy.The optimal concentration cutoffs (Youden-index-associated criterion) to discriminate advanced HGSOC patients based on long (≥30 months) versus short PFS (<30 months) were determined for each circulating immune checkpoint through ROC analysis.The best concentration cutoffs were 2.48 ng/ml for sPD-1 , 0.42 ng/ml for sPD-L1 , 4.75 ng/ml for sBTN3A1 , 13.06 ng/ml for pan-sBTN3As , 5.59 ng/ml for sBTN2A1 , and 2.78 ng/ml for sBTLA . The same analysis also allowed to establish the most suitable thresholds of three different considered factors: age at diagnosis, baseline CA125, and BMI. Therefore, the optimal thresholds for age at diagnosis, CA125, and BMI were, respectively, the following: 60 years , 401 U/ml , and 25 kg/m2 .Using scatter plots by group, we graphically depicted the circulating levels of each immune checkpoint, ages at diagnosis, serum CA125 levels, and BMIs, dividing the advanced-stage HGSOC women into two groups at long versus short PFS based on each examined parameter Figure 1As shown, most of advanced HGSOC women with PFS <30 months had higher plasma levels of biomarkers (above specific thresholds) and age at diagnosis >60 years, CA125 >401 U/ml, and BMI >25.Since the clinical role of plasma immune checkpoints in predicting survival of advanced HGSOC women has yet to be elucidated, we carried out a Kaplan–Meier survival analysis in order to investigate the prognostic relevance of plasma sPD-1, sPD-L1, sBTN3A1, pan-sBTN3As, sBTN2A1, and sBTLA. The thresholds previously identified by ROC analysis allowed to distinguish advanced HGSOC patients on the basis of low and high plasma levels for each analyzed marker (below and above the specific cutoffs). Kaplan–Meier curves showed the relationship between plasma concentrations of immune checkpoints and PFS (Concentration cutoffs associated with favorable prognosis and longer PFS were determined for sPD-1 (≤2.48 ng/ml), sPD-L1 (≤0.42 ng/ml), sBTN3A1 (≤4.75 ng/ml), pan-sBTN3As (≤13.06 ng/ml), sBTN2A1 (≤5.59 ng/ml), and sBTLA (≤2.78 ng/ml) ; 40 versus 24 months for sPD-L1 ; 37 versus 21 months for sBTN3A1 ; 35 versus 21 months for pan-sBTN3As ; 32 versus 25 months for sBTN2A1 ; and 32 versus 24 months for sBTLA .In particular, women with lower baseline concentrations showed the following median PFS values than those with higher levels: 30 versus 24 months for sPD-1 , while a greater advantage in median PFS (14–16 months) was associated with baseline plasma concentrations of sPD-L1, sBTN3A1, and pan-sBTN3As below their specific thresholds.Furthermore, Kaplan–Meier analysis allowed to evaluate the association between PFS and serum CA125 levels, age at diagnosis, baseline BMI, or peritoneal carcinomatosis at onset  was associated with age at diagnosis ≤60 years, serum CA125 ≤401 U/ml, BMI ≤25, or absence of peritoneal carcinomatosis ; 32 versus 22 months for serum CA125  and for BMI , respectively; and 38 versus 17 months for peritoneal carcinomatosis . No peritoneal carcinosis at diagnosis showed a greater gain in PFS.Specifically, median PFS values for patients with age at diagnosis ≤60 years, serum CA125 ≤ 401 U/ml, BMI ≤25, or absence of peritoneal carcinosis compared to values above the specific thresholds were the following: 32 versus 19 months for age at diagnosis  individually correlated in a statistically significant way with baseline serum CA125 >401 U/ml levels, suggesting shorter PFS (<30 months) and poor prognosis ; 48 versus 24 months for sPD-L1  and sBTN3A1 , respectively; 45 versus 28 months for pan-sBTN3As ; 45 versus 19 months for sBTN2A1 ; and 45 versus 24 months for sBTLA .In particular, advanced HGSOC women from validation cohort with lower baseline concentrations of each soluble protein exhibited the following median PFS values compared to those with higher concentrations: 48 versus 28 months for sPD-1  or serum CA125 >401 U/ml . Women with BMI ≤25 or absence of peritoneal carcinosis showed higher median PFS values compared to those observed in women with opposite features: 45 versus 17 months for BMI  or absence of peritoneal carcinosis .Furthermore, an additional Kaplan–Meier analysis confirmed also for validation cohort that age at diagnosis ≤60 years, baseline serum CA125 ≤401 U/ml levels, BMI ≤25, or absence of peritoneal carcinomatosis were associated with a longer PFS Figure 3A further multivariate analysis conducted on validation cohort confirmed that low BTN3A1 concentrations (≤4.75 ng/ml) in plasma, age at diagnosis ≤60 years, and absence of peritoneal carcinosis are independent prognostic factors for a longer PFS in women with advanced HGSOC Table 3.Scientific research is continuously looking for new prognostic indicators able to predict patient survival, enhancing the therapy efficacy. Due to difficulty detected in early detection of OC, the identification of specific biomarkers could improve disease management and provide information helpful for predicting prognosis .Among the numerous investigated biomarkers, CA125, also known as carbohydrate antigen 125, often considered the “gold standard,” has proven to be the most significant indicator involved in screening, detection, management, and survival of OC . Serum CIn the last years, the ability of PD-L1 and PD-1 to act as a marker for clinical outcome was evaluated by several studies , which dRecently, several studies highlighted an association between poor prognosis and high plasma PD-1 and PD-L1 concentrations in different tumors , 26, 28,Furthermore, in recent years, our research group analyzed the plasma levels of other immunomodulatory proteins, such as BTLA and butyrophilins, in individuals with different cancers , 21, 22.Since several studies demonstrated the prognostic impact of pretreatment serum CA125 levels in predicting the optimal treatment strategy, clinical outcome, and survival in OC , our invA survival analysis by Kaplan–Meier curves highlighted that plasma concentrations of each immunoregulatory protein were inversely correlated with PFS of advanced HGSOC patients, allowing to divide them into two subgroups on the basis of a longer (≥30 months) versus shorter PFS (<30 months). A benefit in median PFS ranging from 6 to 16 months was observed when circulating levels of soluble proteins were below the specific concentration thresholds. This suggests that, in the future, sPD-1, sPD-L1, sBTN3A1, pan-sBTN3As, sBTN2A1, and sBTLA could act as useful biomarkers for predicting survival of women with advanced HGSOC, enabling to improve patient clinical management and adopt personalized therapeutic strategies for some patients.Additionally, our investigation also assessed the impact of age at diagnosis, serum CA125, baseline BMI, and peritoneal carcinomatosis at onset on survival of advanced HGSOC patients, suggesting the negative effect of age at diagnosis over 60 years, high serum CA125 levels (>401 U/ml), excess body weight (BMI > 25), or presence of peritoneal carcinomatosis on PFS.Furthermore, a multivariate analysis performed to study the impact of different baseline covariates  on PFS revealed that only the plasma concentration of sBTN3A1>4.75 ng/ml, age at diagnosis >60 years, and presence of peritoneal carcinomatosis were independent prognostic factors for a shorter PFS (<30 months) of advanced HGSOC women. This suggests that circulating sBTN3A1 levels, age at diagnosis, and presence/absence of peritoneal carcinomatosis rather than serum CA125 levels should be considered before starting the therapeutic treatment in advanced HGSOC patients.BTN3A1 showed a significant immunoregulatory function exerted through modulation of the anti-tumor immune response and activation of γδ T cells , 54. SinFinally, additional two-factor multivariate analyses highlighted that circulating levels of each immunomodulatory protein  were individually associated with serum CA125 levels, suggesting that contemporary measurement of both biomarkers than CA125 only could strengthen the prognostic power of serum CA125 in predicting PFS of advanced HGSOC women.Although this investigation provides significant and useful information to current knowledge in the field, it presents some potential limitations, including the relatively limited number of analyzed patients in the leading cohort, the lack of a sufficiently large validation set able to test each new putative biomarker , and the potential interference of the plasma matrix during the dosage in plasma of immune checkpoints through ELISA assays . In addition, a larger number of studies are needed to deeply investigate the releasing mechanisms  of the soluble form of each immunoregulatory protein from tumors and/or stromal cells.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was reviewed and approved by Comitato Etico Palermo 1, University-affiliated Hospital A.O.U.P. “P. Giaccone” of Palermo - (Protocol “TIC-OC v.1.1”). The patients/participants provided their written informed consent to participate in this study.Conceptualization: DF, JLI, AR, VB and VC. Sample collection and investigation: DF, CB, LRC, SC, MCDD, CF, MCL, LM, UR, SV, GP and VC. Experimental analysis: DF and JLI. Data curation and analysis: DF, CB and LRC, MCDD, SC, CF, MCL, LM and TDBR. Writing: DF, CB and LRC. Critical revision of the manuscript: DF, JLI, AR, VB, VC, SV and GP. Supervision: JLI, AR, VB and DF. The figures of the manuscript were conceived and designed by DF, CB, LRC, UR and TR. The tables were conceived and designed by DF, CB, LRC, UR, LM and TR. Literature data were acquired and analyzed by DF, CB, LRC, LM, UR and TR. All authors contributed to the article and approved the submitted version.All authors thank Dr. Chiara Drago for the language English revision. All authors thank the following nurses for the blood draws: Accurso Gaspare, Argentano Salvatore, Basile Anna, Curatolo Giovanna, D’Angelo Davide, Fricano Emanuela, Grizzanti Elisa, La Corte Carolina, Laganà Lucio, Marino Giuseppina, Martorana Eleonora, Pellerito Ilenia, and Sciortino Melania.JLI is cofounder of PanCa Therapeutics and PredicitngMed.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.


Picornaviridae, Flaviviridae and Coronaviridae families, proteolytically process innate immune components and blunt their functions.As a frontline defense mechanism against viral infections, the innate immune system is the primary target of viral antagonism. A number of virulence factors encoded by viruses play roles in circumventing host defenses and augmenting viral replication. Among these factors are viral proteases, which are primarily responsible for maturation of viral proteins, but in addition cause proteolytic cleavage of cellular proteins involved in innate immune signaling. The study of these viral protease-mediated host cleavages has illuminated the intricacies of innate immune networks and yielded valuable insights into viral pathogenesis. In this review, we will provide a brief summary of how proteases of positive-strand RNA viruses, mainly from the  Human cells are equipped to defend themselves against viral infections. When encountered by a virus, they mount a rapid and potent immune response that creates a protective environment in the infected cell and alerts the neighboring cells to an ongoing viral assault ,2. A keyPicornaviridae, Flaviviridae, and Coronaviridae, followed by a brief description of key cellular proteins involved in innate immune signaling and their targeting by viral proteases.Viruses, on the other hand, must circumvent host defenses to complete their lifecycle . Therefopro and 3Cpro for enteroviruses, Lpro and 3Cpro for aphthoviruses, and 3Cpro for hepatoviruses and cardioviruses [pro and 3Cpro are cysteine proteases that adopt the chymotrypsin-like fold [pro adopts the papain-like fold [Most positive-strand RNA viruses encode one or more proteases, which play key roles in maturation of viral proteins and inhibition of host antiviral functions. The role of the viral protease activity in host antagonism has been most extensively studied in the context of picornaviruses, flaviviruses, and coronaviruses. Picornaviruses are small non-enveloped viruses with a genome size between 7.5 and 10 kb and can be grouped into 68 genera (as of July 2021) . The Entoviruses ,39. The ike fold ,41,42,43ike fold ,45.Flaviviridae family includes a number of clinically important viruses that mainly fall into two genera, Flavivirus and Hepacivirus. The various members of the genus Flavivirus include Zika virus (ZIKV), dengue virus (DENV), yellow fever virus (YF), West Nile virus (WNV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and tick-borne encephalitis virus (TBEV). These viruses are highly pathogenic to humans, mainly transmitted through mosquitoes and ticks, and prevalent in different parts of the world. The ~11 kb genome of flaviviruses encodes a polyprotein that is cleaved by host proteases and a single viral protease, NS2B-NS3pro, into 10 mature proteins [pro functions as an active enzyme only in the presence of the cofactor NS2B, which is an integral membrane protein of 14 kDa [pro and NS3-NS4Apro, to yield 10 mature proteins [pro, the main protease responsible for polyprotein processing, forms a noncovalent heterodimer consisting of a catalytic subunit NS3 and an activating cofactor NS4A [pro and NS3-NS4Apro are serine proteases with the chymotrypsin-like fold [The proteins ,49  [Coronaviridae family also contains endemic viruses, such as NL-63, OC-43, 229-E, and HKU-1, which exhibit seasonality and mostly cause mild respiratory disease [pro) and a 3C-like protease (3CLpro) [pro and 3CLpro are cysteine proteases, however, while PLpro adopts the papain-like fold [pro features the chymotrypsin-like fold [The S-CoV-2) ,65. ThisS-CoV-2) , and as S-CoV-2) . Before S-CoV-2) ,68 and MS-CoV-2) , made a  disease . Coronav disease . This la disease ,73. The (3CLpro) ,75  . The PRRpro and FMDV Lpro have been shown to cleave MDA5 [pro of several picornaviruses, such as Coxsackievirus-A6 (CV-A6), CV-A16, EV-D68, and EV-A71. However, this interaction does not involve MDA5 cleavage, but instead limits the downstream interaction of MDA5 with MAVS [Viruses target PRRs to evade innate immune surveillance. Due to their localization on the cell surface and in endosomal compartments, TLRs mostly remain shielded from exposure to viral proteases. The RLRs, on the other hand, reside in the cytoplasm and are frequently targeted by viral proteases. The Coxsackievirus B3 (CVB3) 2Aave MDA5 ,96. The ith MAVS ,97.pro of EV-A71, CVB3, PV, and EMCV has been reported to inhibit RIG-I [pro-mediated RIG-I inhibition. While Feng et al. reported that EV-A71 3Cpro cleaves RIG-I [pro acts by binding to N-terminal CARD domains of RIG-I and inhibiting the subsequent recruitment of MAVS [pro. It is possible that in an overexpression setting, the viral protease does not achieve high enough localized concentration required for RIG-I cleavage. Alternatively, the 3CD protease precursor formed during the polyprotein processing in infected cells may be the main protease responsible for cleaving RIG-I. There are examples in the literature where the protease precursors of picornaviruses, and not the mature protease, cleave a cellular protein [RIG-I is also targeted by viral proteases. The 3Cit RIG-I ,98. Confes RIG-I , Lei et  of MAVS . These c protein ,100.pro has been reported to cleave LGP2 [There is not much literature on LGP2 processing by viral proteases. The FMDV Lave LGP2 . As withUpon binding with PAMPs, the PRRs get activated and engage specific adaptor proteins, which serve as scaffolds for the assembly of large signaling complexes, called signalosomes. These signaling bodies then transduce signals to the downstream effector proteins. The TLRs signal via two major adapters, MyD88 and TRIF, leading to activation of transcription factors NF-κB and interferon-regulatory factors (IRFs) ,104,105.The RLRs signal through the adaptor protein, MAVS  ,112,113,Another protein, stimulator of IFN gene  ,123,124,pro [pro and 3Cpro of some picornaviruses, such as CVB, Seneca Valley virus (SVV), and FMDV, have also demonstrated the ability to cleave MAVS [pro precursor, 3ABCpro, to cleave MAVS [pro then carries out the MAVS cleavage. The mature 3Cpro alone does not localize to mitochondria and is therefore incapable of targeting MAVS. The HCV NS3-NS4Apro also cleaves MAVS, leading to suppression of the innate immune pathway [The adaptor proteins represent central signaling hubs of antiviral networks and are therefore an attractive target of virus-mediated proteolysis. MAVS is probably the most well characterized target of viral proteases. It is cleaved by a number of proteases from diverse virus families. Picornaviral proteases, including those of CVB3, PV, rhinoviruses, EV-D70, and EV-A71 ,95,130, pro ,132, Lprave MAVS ,134. Anoave MAVS . The tra pathway ,135,136.pro and 3Cpro of picornaviruses have been reported to process TRIF [pro cleaves TRIF at only one site [pro cleaves at two sites [pro, but not the mature 3Cpro, cleaves TRIF [pro [TRIF is also a common target of viral proteases. Both 2Aess TRIF ,137,138.ves TRIF . The TRIRIF [pro .pro to cleave the human STING protein [pro, although efficiently cleaves human STING, does not target the STING versions found in most other mammals, including non-human primates, possibly explaining the differential replication levels achieved by DENV in human versus other species [Several positive-strand RNA viruses employ their proteases to target the STING protein, authenticating the relevance of this DNA sensing adapter to RNA viruses. Many flaviviruses, such as ZIKV, DENV, WNV, and JEV, but not YFV, use their NS2B-NS3 protein ,142,143. protein . SimilarThe signaling cascades unleashed upon sensing of viral PAMPs in infected cells lead to production of proinflammatory cytokines and IFNs. Two major transcription factors that regulate induction of these genes are NF-κB and IRFs ,145. TheThe second group of transcription factors are IRFs that are primarily involved in transcription of IFN and IFN-stimulated genes (ISGs). Since the discovery of the first IRF, IRF1, in 1988 ,155, thepro to process IRF7 [pro cleaves IRF7 only at one site [pro processes the protein at two sites [pro [pro to cleave IRF3 [pro of PV, ECHO-1, and human rhinovirus B-14 cleaves the p65 subunit of the NF-κB complex, thereby shutting down the induction of proinflammatory genes [pro has been shown to inhibit IFN production by cleaving importin β1, a key nucleocytoplasmic transport receptor involved in nuclear import of IRF3 and NF-κB [IRFs and NF-κB are both targeted by viral proteases. EV-A71 and EV-D68 employ their 3Cess IRF7 ,169, theone site , EV-D68 tes [pro . A recenave IRF3 . The NF-ry genes . In addind NF-κB .The IFN family includes three main classes of related cytokines: Type I IFNs, type II IFN, and type III IFNs. Once released from virus-infected cells, they work in an autocrine and paracrine manner to create an antiviral state in both infected cells and the neighboring bystander cells. Interaction of IFNs with their receptors triggers activation of receptor tyrosine kinases JAK1 and TYK2, which in turn phosphorylate members of the STAT family of proteins, triggering their dimerization . STAT1 apro-dependent manner [pro limits the antiviral effect of IFN are unknown, the authors speculated that the protease might be cleaving ISGs responsible for inhibition of picornavirus replication. In another study, the EV-A71 3Cpro was shown to target IRF9 for proteolytic cleavage [pro to cleave STAT2 and inhibit the ISRE reporter activity [In addition to ablating the IFN production pathway, viral proteases also block events that happen after IFNs bind to their receptors on the cell surface. In an elegant study, Morrison et al. showed that several picornaviruses, such as EV-D70, HRV A16, and PV, are capable of replicating in type I IFN-treated cells in a 2At manner . While mcleavage , abrogatactivity .This review summarized the role of the viral protease activity in counteracting host defenses. Historically, the study of virus-mediated proteolysis of host proteins has relied on targeted molecular biology techniques, such as western blot. However, with terrific advances in systems biology approaches, it has now become possible to perform a global survey of protein cleavages in virus-infected cells ,179. Def