As for many solid cancers, nasopharyngeal cancer (NPC) interacts with the immune system. In this retrospective study, immune features of NPC were explored and assessed against Epstein-Barr virus status, clinical stage, and survival. Specific immune phenotypes were identified based on presence and distribution of CD8+ T-cells: i.e., “inflamed”, “excluded”, and “deserted” NPC, which carried important prognostic information. Presence and distribution of CD207+ cells, likely representing antigen-presenting dendritic cells, were demonstrated, suggesting a potential for immune cell targeting. Gene expression revealed differences in immune profiles between NPC and control tissue as well as between subgroups of NPC based on CD8 expression (high vs. low). Taken together, the observations may be of relevance to prognostication of NPC as well as for explorations into the field of immunotherapy.n = 48) were classified into “inflamed”, “excluded”, and “deserted” phenotypes. In addition, CD8+ T-cells and CD207+ DCs were quantified. The data were analyzed in relation to Epstein–Barr virus-encoded small RNA (EBER), Epstein-Barr virus (EBV) DNA, and survival. Separately, data on gene expression from a public database were analyzed. 61.7% of NPC lesions were “inflamed”, 29.8% were “excluded”, and 8.5% were “deserted”. While CD8+ cells were present in cancer cell areas and in surrounding stroma, CD207+ cells were observed largely in cancer cell areas. High CD8+ T-cell presence was associated with EBV+ disease, but no such pattern was observed for CD207+ DCs. There was a difference in disease-free survival in favor of “inflamed” over “excluded” NPC. Gene expression analysis revealed differences between NPC and control tissue  as well as between subgroups of NPC based on CD8 expression (high vs. low). In conclusion, NPC lesions are heterogeneous with regard to distribution of CD8+ T-cells and CD207+ DCs. NPC can be classified into immune phenotypes that carry prognostic information. CD207+ DCs may represent a target for immunotherapy with potential to facilitate the antigen cross-presentation necessary to execute cytotoxic T-lymphocyte responses.Nasopharyngeal cancer (NPC) features intralesional immune cells, but data are lacking on presence/distribution of T-cells and dendritic cells (DCs). Based on intralesional distribution of lymphocytes, a series of NPC biopsies ( There is a need for new prognostic options and treatment principles for nasopharyngeal cancer (NPC), a malignancy frequently associated with Epstein–Barr virus (EBV), and measures targeting the immune system may offer such possibilities. However, this requires detailed knowledge about the cancer and its local microenvironment, particularly on the presence and distribution of intralesional immune cells and their targets and functions. In a recent study by Wang et al., the importance of the immune status of NPC was underscored: focusing on immune checkpoints PD-L1 and B7-H4 on tumor cells and PD-L1, B7-H3, B7-H4, IDO-1, VISTA, ICOS, and OX40 on intralesional immune cells, specific signatures were demonstrated to predict survival .With regard to interactions between the immune system and NPC, tissue-infiltrating lymphocytes (TILs) are of importance. The density/distribution of TILs has been investigated and identified as an independent positive prognostic factor . ArguablIn this study, biopsy material from patients with NPC, of which we have previously reported data on intralesional levels of EBV DNA and survival , were reFor all 48 patients, EBER slides were retrieved and new slides were produced for visualization of CD8+ and CD207+ cells, respectively. One individual was excluded from the analysis due to insufficient material for immunohistochemistry : this patient was incidentally also the only one lacking follow-up. For the remaining 47 patients, it was possible to perform all but three analyses. Two of these were related to a case of a heavily EBER-positive tumor with dense infiltrates of lymphocytes, making a distinction of nontumor-infiltrated surrounding stroma  too unreliable. The third was an analysis of CD207 where areas of surrounding stroma were lacking.In Immune phenotypes of the NPC lesions were estimated based on qualitative assessment of the presence and distribution of lymphocytes on slides stained for hematoxylin, cytokeratin, and CD8. Most lesions (61.7%) were of an immune “inflamed” phenotype (lymphocytes infiltrating cancer cell areas). Immune “excluded” (lymphocytes in the surrounding stroma but not infiltrating cancer cell areas) and “deserted” phenotypes (no lymphocytes in either cancer cell areas or the surrounding stroma) were less frequent: 29.8% and 8.5%, respectively. In whole biopsies , areas eligible for analysis of CD8 and CD207, respectively, were 14.8 (6.5–28.7) mm2 and 12.3 (5.5–22.4) mm2. The frequencies of pixels representing CD8 and CD207, respectively, were 2.17% (0.73–3.18) and 0.15% (0.040–0.58). There was no correlation between the CD8 and CD207 ratios. The morphological distribution patterns of CD8 and CD207 are depicted in p = 0.034, higher frequency for “inflamed”), “inflamed” and “deserted” , as well as “excluded” and “deserted” . No such differences were observed for ratios of pixels representing CD207  (cancer cells and surrounding stroma. The frequencies of pixels representing CD207 were 0.25% (0.040–1.39) for areas of cancer cells and 0.030% (0–0.065) for surrounding stroma, representing an 8-fold difference (p < 0.0001)  b. For qu 0.0001) c. There p = 0.0090)  and one featured an advanced local lesion (stage IVA). There was no difference in disease-specific survival (DSS) between immune phenotypes, but a there was a statistically significant difference in disease-free survival (DFS) between the “inflamed” and “excluded” phenotype ( 0.0090) , the latn = 4) and that cases of stage I disease (n = 4) were all of the “inflamed” phenotype, there was a difference between stage I–III and IV disease with regard to phenotypes (p = 0.042). No statistically significant associations were observed for other selected stage combinations or T-stage differences. There were no statistically significant differences between stage or stage combinations, and CD8+ or CD207+, either for whole biopsies or for selected regions.In line with the findings that cases with a “deserted” phenotype were all stage IV disease at diagnosis (cf. -negative NPC lesions (p = 0.0065) and for EBV DNA-positive cf. -negative NPC lesions (p = 0.028). In contrast, there were no differences in CD207 ratios for whole biopsies (n = 12) aggregated as “deserted” > “excluded” > “inflamed”: “Inflamed” vs. “excluded” (p = 0.0045), “inflamed” vs. “deserted” (p < 0.0001), and “excluded” vs. “deserted” (p = 0.043). For intralesional EBV DNA (present or not present), a difference was shown between “inflamed” vs. either “excluded”  or “deserted” , while there was no such difference for “excluded” vs. “deserted”. A similar pattern was seen for quantitative EBV DNA data .The CD8 ratios for whole biopsies a were hibiopsies b and neiDNA data , where acf. both CD8+ high and low NPC, higher fibroblasts score in CD8+ low NPC cf. control, and a significant increase in macrophages for CD8+ high NPC cf. controls -activated cells were increased in CD8+ high NPC cf. controls. In contrast, signatures for the naïve and memory B cells were significantly lower in both NPC groups cf. controls  database, including mRNA profiles of 31 NPC samples and 10 control nasopharyngeal samples, were in silico immunoprofiled and further assessed based on CD8+ T cell-related transcripts. Utilization of the CD8+ T-cell signatures, based on cell profiling scores from Puram et al. and Newman et al. via CIBERSORTX ,18, enabcontrols a. On concontrols b. The DCAs the CD8A gene is the most representative functional gene associated with CD8+ T cells , furtherGene expression data analysis showed that 48% (15/31) of the NPC cases displayed an interferon signature ,23 in cocf. EBV-negative NPC. In contrast, no such differences were observed for CD207. Gene expression analysis revealed differences between NPC and control tissue  as well as between subgroups of NPC based on CD8 expression (high vs. low). Taken together, the observations may be of relevance to prognostication of NPC as well as for explorations into the field of immunotherapy.In this study, primary biopsies from patients with NPC were analyzed. A marked inter- and intraindividual variation was observed with regard to growth pattern of cancer cells as well as to presentation of cytokeratin, EBERs, and immune cells. Based on presence and distribution of lymphocytes as established for other cancers , the lesThe notion that head and neck cancer lesions, including NPC, are heterogeneous in nature , was coIn this study, as previously suggested for other cancers ,25, the cf. the surrounding stroma. However, while CD207+ DCs were present in close relation to cancer cells, this was apparently not enough to induce a meaningful immunological response targeting cancer antigens, which would prevent NPC from occurring or to be killed off. We suggest that the DC presence in NPC represents treatment possibilities and that the C-lectin receptor CD207 and potentially other pattern recognition receptors known to facilitate cross-presentation of antigen [The DC is specialized in antigen presentation and for a successful adaptive immune response to occur; either by the immune system through its own capacity or facilitated by “vaccinations”, this cell type needs to be present and active. Through this study, as CD207 is considered a selective DC marker ,29, our  antigen ,5 may becf. controls), albeit with marked heterogeneity between NPC samples. When comparing high vs. low CD8 vs. controls, high fibroblasts scores for CD8 low NPC were revealed. Furthermore, a high activated Natural Killer (NK) cell profile for CD8 high NPC was observed. (DCs could not be determined for NPC cf. control tissue.) Taken together, our observations suggest that there are immunological subsets of NPC and distinctions between NPC rich in CD8 (likely representing an “inflamed” phenotype) and low in CD8 (“excluded” and particularly “deserted” phenotypes). However, the gene expression data are difficult to put into a greater context given the lack of synchronous morphological assessment and clinical data, reflecting a general problem with gene expression data extracted from public databases.The interplay between the immune system and cancer cells in NPC is not sufficient to eradicate the disease despite the fact that antigens are present and that key immune cells such as DCs and T-cells are available. More information is needed, and the present gene expression analysis, performed on available transcriptional data from EBV-positive NPC and control tissue ,18, highIn a previous study, we explored the association between intralesional EBV DNA and survival . When thThe study was of a retrospective design and involved an analysis of formalin-fixated paraffin-embedded (FFPE) primary tumor tissue from a well-defined population of 48 patients with NPC diagnosed between 2001 and 2015. Data from this material have been reported previously focusing on intralesional EBV DNA and survival . ThroughPatient characteristics have been previously reported . Briefly2O2. Sections were then incubated with an anti-CD207  or anti-CD8  primary antibody for 1 h at room temperature. After subsequent incubation with polymer/HRP-linked secondary antibodies for 30 min, the immunoreactivity was visualized using diaminobenzidine 3.3 (DAB) HRP chromogen (resulting in a brown opaque staining). Next, sections were incubated with Dako Double Stain Blocking Reagent (Dako/Agilent) to prevent additional binding of secondary antibodies to the first primary antibody. Sections were then incubated with an anti-cytokeratin primary antibody . This second-round immunoreactivity was also visualized with a polymer/HRP-linked secondary antibody , but the visualization was performed with Vina Green HRP chromogen . Finally, sections were counterstained with Mayer’s hematoxylin, air-dried, and mounted with Pertex . In addition to the protocols above, previously stained slides for EBER evaluation (EBER-ISH for EBER1 and EBER2) were retrieved [A modified double-staining immunohistochemistry protocol  was applied in order to simultaneously identify cytokeratin-positive cancer cells together with CD207+ DCs or CD8+ T cells. Briefly, 4-µm rehydrated sections were subjected to double-staining immunohistochemistry in the automated Autostainer IHC-robot (Dako/Agilent) using the EnVision Doublestain System kit K5361 (Dako/Agilent). Prior to immunohistochemistry, an antigen retrieval procedure was performed in a PT-link machine for heat-induced epitope retrieval (HIER) using low pH (pH 6) retrieval buffer. Endogenous peroxidase activity was blocked with HBased on overall lymphocyte presence, and with special regards to CD8+ T-cell distribution , the lesions were classified according to immune phenotypes defined as “inflamed” (lymphocytes infiltrating cancer cell areas), “excluded” , and “deserted” (no or exceedingly few lymphocytes both in cancer cell areas and the surrounding stroma). In cases of highly heterogeneous lesions, the dominating pattern was chosen for the classification. This assessment was performed temporarily blinded to clinical data (J.S.N) and verified totally blinded by a second observer (S.S.): EBER slides were not used as support in the analysis.Slides stained for CD8 and CD207 were reviewed in a standardized manner utilizing cytokeratin  with EBER slides as support. Through digital image dissection, nonrelevant regions were excluded. These comprised physical artefacts , normal epithelium, germinal centers, and gland structures. The dissections were performed on consecutive slides for CD8 and CD207, enabling a consequent and representative exclusion strategy. The Positive Pixel 9-algorithm (Aperio Technologies) was used to automatically segment out tissue background area as well as positive staining  by color threshold values, and then the ratio of the total analyzed tissue area with staining-positive pixels was calculated. Accordingly, this ratio represented the quantities of CD8 and CD207 in the target area. Thresholds to define staining (chromogen) positivity were the same for CD8 and CD207.2 after exclusion of nonrelevant regions as defined above) were selected to represent areas of cancer cells and areas of surrounding stroma (in direct proximity to cancer cells). CD207 positivity was quantitated for both locations. However, CD8 positivity was quantitated for the surrounding stroma only. The latter was due to a very high lymphocyte presence, which in combination with a variable degree of lymphocyte infiltration, rendered it impossible to quantitate CD8 within cancer cell areas in an objective and robust manner. When the presence of the marker (CD8 and DC207) was different between various areas of the tumor, the area with more pronounced presence was chosen. The selected areas were assessed for each marker following the same digital image quantification process as described above. All quantification procedures were performed blinded to the clinical data.In addition, areas (minimum 0.1 mmhttps://www.ncbi.nlm.nih.gov/geo/: accession number GSE12452) [n = 10), comprising 4 cases of nasopharyngeal tissue adjacent to but separated from NPC lesions and 6 cases of nasopharyngeal tissue from non-NPC subjects, and NPC tissue . Normalized mRNA data, generated with the Affymetrix Human Genome U133 Plus 2.0 Array, were obtained from the GEO database (SE12452) ,16. The Utilizing CIBERSORTX , the gen2-tests were performed to explore associations between CD8 and CD207  and EBER (present or not), EBV DNA (present or not), immune phenotypes, and stage. Correlations between CD8 and CD207 ratios and between these ratios and EBV-DNA were explored using the Spearman test. Comparisons of ratios for CD207 between areas of cancer cells and surrounding stroma was explored using the Wilcoxon Signed Ranks test. Survival, i.e., DSS and DFS in relation to CD8 and CD207, respectively, as well as to immune phenotypes, was described using Kaplan–Meyer curves, and significance levels were determined by the log-rank test. A Cox regression analysis was not performed due to the restricted sample size. The explicit p-value was provided when the p-value was <0.05 but >0.0001, whereas lower values were provided as <0.0001.For the principal data set subjected to digital imaging, statistical analyses were performed using SPSS version 25 . Data were presented as medians with interquartile ranges (IQR). For comparisons of CD8 and CD207 ratios, respectively, between immune phenotypes of NPC and between tumor stages, an analysis of variance  was followed by the Mann Whitney U-test. For comparisons of EBV DNA load between immune phenotypes, the Kruskal–Wallis test was followed by the Mann Whitney U-test. χp-values less than 0.01 were considered statistically significant. For gene expression data analyzed in silico, statistical analyses were performed using GraphPad version 8.4.3 . The intensity fractions obtained were analyzed for differences in the immune cell populations using the two-way ANOVA, Tukey method.CD8A-correlated gene markers and the interferon signatures were examined using the limma multigroup analysis (edgeR) . A PearsIn conclusion, NPC lesions are heterogeneous with regard to the presence and distribution of cancer cells as well as immune cells (CD8+ T-cells and CD207+ DCs). In addition, there are immune-related differences between subgroups of NPC based on CD8 expression (high vs. low). Arguably, this nature, in addition to a variable presence of EBV-associated antigen, must be taken into account when prognostic information is examined and candidate treatment targets are explored. With regard to distribution of lymphocytes (notably CD8+ T-cells), NPC may be classified into “inflamed”, “excluded”, and “deserted” immune phenotypes, and these subsets carry prognostic information that is easily accessible and that can be linked to EBV status. CD207+ DCs, likely representing the myeloid CD1c+ subtype, are present in NPC lesions. Intralesional DCs may represent a possibility for immunotherapy, and CD207 may be a specific target based on its ability to facilitate a cross-presentation of antigens necessary to produce antigen-specific cytotoxic T-cell responses.


Tau and amyloid beta (Aβ) are the prime suspects for driving pathology in Alzheimer’s disease (AD) and, as such, have become the focus of therapeutic development. Recent research, however, shows that these proteins have been highly conserved throughout evolution and may have crucial, physiological roles. Such functions may be lost during AD progression or be unintentionally disrupted by tau- or Aβ-targeting therapies. Tau has been revealed to be more than a simple stabiliser of microtubules, reported to play a role in a range of biological processes including myelination, glucose metabolism, axonal transport, microtubule dynamics, iron homeostasis, neurogenesis, motor function, learning and memory, neuronal excitability, and DNA protection. Aβ is similarly multifunctional, and is proposed to regulate learning and memory, angiogenesis, neurogenesis, repair leaks in the blood–brain barrier, promote recovery from injury, and act as an antimicrobial peptide and tumour suppressor. This review will discuss potential physiological roles of tau and Aβ, highlighting how changes to these functions may contribute to pathology, as well as the implications for therapeutic development. We propose that a balanced consideration of both the physiological and pathological roles of tau and Aβ will be essential for the design of safe and effective therapeutics. Alzheimer’s disease (AD) is a terminal neurodegenerative disorder associated with severe progressive dementia . The disSince the identification of microtubule-associated protein tau (MAPT) and amyloid beta (Aβ) as the components of NFTs and extracellular plaques, respectively, research has primarily focused on the toxic roles these proteins play in AD pathogenesis , 241. AcIn this review, we discuss the emerging evidence for key physiological roles of tau and Aβ. The toxic roles of these proteins have been extensively reviewed elsewhere , 241, anAPP gene on chromosome 21  are expressed in a variety of tissues throughout the body 3)). WhilMAPT-like gene has been found in lampreys, hagfish, and sharks, pinpointing an origin over 550 million years ago  is a common feature of AD . One poscontrols . Taken tcontrols . Interescontrols . Once agIncreasing evidence demonstrates that the levels of both tau and Aβ proteins increase following stroke , spinal BACE1 knockout or inhibition improves recovery after sciatic nerve injury [BACE1−/− mice have impaired remyelination after sciatic nerve lesion [BACE1−/− and APP−/− mice have an increased risk of mortality following cerebral ischaemia, indicating that this is likely a consequence of Aβ loss [BACE1−/− mice compared to wild-type mice [1-40 to BACE1−/− mice after CCI, demonstrating a potential role for Aβ in recovery [worsened outcome, demonstrating a hormetic role for Aβ [Attempts to clarify the function of increased Aβ after injury have produced conflicting results and it is often difficult to separate specific effects of Aβ from functions of APP, which is known to be upregulated in axons after injury and may have protective functions . Whilst e injury  or TBI [e injury , others e lesion , 111 ande lesion  or TBI [e lesion . Interes Aβ loss . Mannix ype mice . A follorecovery . In the e for Aβ  Fig. 4)BACE1 knoe for Aβ .MAPT−/− mice suffer worse functional outcome after sciatic nerve injury [MAPT−/− mice are resistant to some functional and cognitive deficits after TBI, although this depends on whether the injury was repetitive [. found that following experimental stroke, MAPT−/− mice were protected from neurological deficits and excitotoxic brain damage [Studies exploring the role of tau after injury have also been contradictory. e injury  and EAE e injury , reminispetitive  and whetpetitive . A studyn damage . The autn damage . Whilst n damage . TherefoMitochondrial dysfunction is an early and important pathogenic feature of AD , and botMAPT gene, interacts with peroxiredoxin 6, an antioxidant enzyme that protects cells from oxidative damage [MAPT may also impact the function of saitohin. Therefore, caution may be required when utilising tau- or Aβ-targeting therapies, to avoid potential exacerbation of oxidative damage.Many studies have found an association between mitochondrial Aβ deposition and oxidative stress , 193. Hoe damage . Whilst . found that tau knockdown reduced mitochondrial mobility and increased the number of abnormal mitochondria [Tau has also been proposed to regulate mitochondrial function. Sapir et alchondria . A recenchondria . Howeverchondria  and mouschondria . These sIn 2002, Robinson and Bishop proposed that Aβ is normally produced to “bind toxic solutes” with the formation of amyloid plaques being “an efficient means of presenting these toxins to phagocytes” . Since tChlamydia pneumoniae and Borrelia burgdorferi, can increase production and deposition of Aβ in vitro and in vivo, where microbial DNA can be found associated with amyloid plaques , iewed in ). Intereiewed in . Interesiewed in . It is iiewed in , it is piewed in , and thiiewed in .. found that Aβ exhibits antimicrobial action of equal or greater potency than LL-37  against eight common microbes [Candida albicans, Gram-negative, and Gram-positive bacteria [, Kumar et al. found that Aβ overexpression significantly increases survival in 5xFAD mice infected with S. Typhimurium, and in C. elegans infected with C. albicans and S. Typhimurium [APP−/− mice show a trend for increased mortality after infection [The increased expression of Aβ after infection is proposed to be an innate immune response, and there is evidence that Aβ can act as an antimicrobial peptide (AMP) (reviewed in ). Sosciamicrobes . Applicamicrobes  in cell bacteria , 236 in himurium . The authimurium . Alternahimurium . Interesnfection  and a confection , althougnfection .A number of groups have postulated that iron dyshomeostasis could contribute to AD and other neurodegenerative diseases , with irAPP−/− neurons retain more iron [APP−/− mice show vulnerability to oxidative damage from dietary iron [. found that MAPT−/− mice exhibit age-dependent iron accumulation associated with neurodegeneration, cognitive deficits, and parkinsonian-like motor deficits [MAPT−/− mice with the iron chelator clioquinol [MAPT−/− mouse model [Whilst the role of Aβ in regulating iron homeostasis is unclear, APP has been proposed to have iron-export ferroxidase activity and interacts with, and stabilises, a major iron transport protein, ferroportin , 63. Indary iron . Tau mayary iron . Lei et deficits . These dioquinol . Interesioquinol , and theioquinol . Taken tse model , potenti, tau readily binds RNA [Whilst the majority of tau protein is located in the cytoplasm, there is clear evidence for nuclear association of tau in cell lines , 234, prinds RNA  and DNA inds RNA , 275. Tainds RNA , 275. Tainds RNA , raisingMAPT−/− mice show increased DNA fragmentation under physiological conditions and are highly susceptible to DNA breakage after hyperthermic stress [MAPT−/− mice also show delayed repair of double-strand breaks after heat shock which was especially evident in the CA1 hippocampal subfield [Neurons continuously face the harmful effects of oxidative stress and evidence points to a key role for tau in preventing DNA damage and promoting repair . Cellulac stress . MAPT−/−subfield . CautionMAPT mutations consistently show chromosomal aberrations, including breakages, gaps, aneuploidies, and translocations [MAPT mutations could induce dysfunction via loss of function, potentially through disruption of the mitotic spindle [MAPT−/− neurons, hallmarks of normal pericentromeric heterochromatin (clustering of H3K9me3 and HP1α) are also disrupted [Cells from individuals with ocations . Interes spindle . In MAPTisrupted , 169, inrepressing transcription of a number of genes implicated in neuronal function [In addition to maintaining genomic integrity, tau may also regulate transcription of genes involved in neuronal function. A recent paper reported that shRNA knockdown of tau in neurons greatly reduced both mRNA and protein levels of VGLUT1 . Overexpfunction , with tafunction . Additiofunction  or reprefunction  this pro1-42 can regulate transcription, including upregulation of APP and insulin-like growth factor receptors [Whilst the APP-processing product AICD is frequently associated with the nucleus (reviewed in ), a nucleceptors , 13. Howeceptors  and increceptors . The facMAPT mutation have an increased risk of developing cancer [An increasing number of studies have reported a striking inverse association between AD and many types of cancer (reviewed in ), with og cancer . Togethe. suggest that Aβ could also indirectly suppress tumour formation through intercepting oncogenic viruses, or via scavenging free metal ions, restricting availability of micronutrients required for cell proliferation [BACE1 expression in invasive ductal carcinoma, indicating that reduced levels of Aβ could potentially increase the proliferation rate of this aggressive tumour [increased levels of APP have been found in a number of cancers, with worse outcomes associated with higher APP levels . Furthe. showed that tau expression levels vary in different cancer types and high tau expression levels correlate with increased patient survival [MAPT knockdown enhanced cell growth and invasion and suggested that tau may be a tumour-suppressive protein in clear cell renal cell carcinoma [MAPT mutations increase the risk of developing cancer suggests that tau mutations may result in loss of a tumour-suppressive function [MAPT expression correlates with poor prognosis and taxane (a microtubule-stabilising drug) resistance in gastric cancer [As microtubules are crucial components of the mitotic spindle and regulate the cell cycle , it seemsurvival . Anotherarcinoma . The finfunction . On the c cancer . It appeDiabetes is a well-established risk factor for developing AD . Insulin. reported that MAPT−/− mice exhibit insulin resistance in the hippocampus, suggesting that tau plays a normal role in the cellular response to insulin, a function that may be lost in AD [MAPT−/− mice have been shown to develop pancreatic β cell dysfunction and glucose intolerance [increased glucose metabolism, but large accumulations of hyperphosphorylated tau associated with Aβ deposition correlated instead with hypometabolism, suggestive of a hormetic function for tau (Fig. MAPT H1 haplotype is associated with increased glucose intolerance in humans [Marciniak et alst in AD . The autst in AD . Tau is st in AD , and MAPolerance . A recenolerance . This sttau Fig. , or Aβ-mn humans . Taken tBACE1−/− mice show impaired insulin expression in the pancreas [BACE1−/− mice have enhanced insulin sensitivity [Whilst Aβ has been proposed to dysregulate insulin signalling , there ipancreas  and siRNpancreas . Howeversitivity . Notablysitivity , indicatThe functions of tau and Aβ are influenced by their location. Shifts within the cell, expression in different cells or locations within the body, can alter the roles that these proteins play, whether they are beneficial or harmful, and how therapeutic treatments will influence function.Different isoforms, aggregation status, and post-translational modifications can dramatically alter the function of tau and Aβ.The concentrations of tau and Aβ are crucial for regulating physiological versus pathological function. This “hormetic nature”, where too much or too little protein causes functional deficits, raises the likelihood of a “therapeutic sweet spot” where the physiologically optimal concentrations lie, outside of which may result in damage Fig. .AD is likely a combination of both gain- and loss-of-function phenotypes. Physiological responses can become hijacked to become toxic, detrimental functions can appear, or normal functions can be lost during disease-related changes.Contradictory findings are common in the literature. The potential for developmental compensation in constitutive knockouts, as well as experimental differences in factors such as age, sex, and environment, will all impact outcome. Future studies testing multiple timepoints, acute versus constitutive knockdown, different genetic backgrounds, mixed-sex cohorts, and increasing comparisons between animal and human tissue will greatly clarify genuine phenotypes from experimental artefacts.From molecular interactions with DNA to influencing complex behaviour, the physiological roles of tau Fig.  and Aβ  are multWith the above in mind, a meticulous approach to therapeutic development is crucial. Therapies must balance potentially detrimental effects of targeting tau Table  and Aβ  with the


Phenols and tocopherols decreased 1.2-fold in ‘Chandler’ and 1.3-fold in ‘Howard’. Using multivariate analysis, samples were discriminated in three groups according with their oxidative levels. Increases of volatiles in oil and kernel were associated with higher oxidative levels. Pentanal, 2-methylpropanal, hexanal, (E)-2-pentenal, 3-octanone, octanal, (Z)-2-penten-1-ol, hexanol, (E)-2-octenal, 1-octen-3-ol, benzaldehyde, -2,4-nonadienal, and hexanoic acid in kernels were adequate at distinguishing oxidation levels and as oxidative markers in walnuts. Kernel volatiles is a useful measurement for walnut oxidation during storage without any prior fat extraction.Kernel oxidation susceptibility and pellicle darkening are among the biggest concerns regarding walnut quality. Monitoring oxidation is crucial to preserve quality from production to consumption. Chemical oxidation parameters , fatty acid profile, tocopherols, phenols, and volatiles in ‘Chandler’ and ‘Howard’ kernels were studied at different time points during 28 weeks of storage to evaluate potential oxidation markers. During storage, peroxide value, UV absorbances, and volatiles concentration increased; oxidative stability, phenols, and tocopherols decreased, while fatty acid profile was unaffected. ‘Chandler’ had a lower peroxide value, K Juglans regia L.) belongs to the Juglandaceae family that originated in Persia, with production and consumption spread throughout the world [Walnut , free fatty acidity (FFA), or sensory ,11. HoweJuglans regia—‘Chandler’ and ‘Howard’) were harvested at full maturity (onset of hull split) from commercial plantations at Wheatland, California, USA. Without the hull, the fruits were dried at 30 ± 2 °C in a day’s time using a commercial air dryer and then unshelled mechanically.Walnut fruits . γ-tocopherol, δ-tocopherol, α-tocopherol, α-tocopherol acetate, and refined olive oil were purchased from Sigma-Aldrich . Volatile standards were purchased from Sigma-Aldrich . The water was obtained from a Milli-Q-Plus water purification system .Walnuts (18 kg) were stored in four (4.5 kg of kernels each) open transparent containers (65 cm × 50 cm × 30 cm) in temperature-controlled chambers (25 ± 2 °C) at 50% RH exposed to fluorescent light for seven months. Samples of 1.5 kg of kernels were collected for oil extraction and analysis every week during the first month of storage and every four weeks for the following six months. Kernels (1 kg) were sampled every week during the first four weeks of the study and every four weeks for the following six months of storage for a totality of 11 sampling points. Oil was extracted within 3 h after each sampling.w/w) of moisture content, sprinkled with fresh water, to ensure uniformity of extraction conditions within samples [Walnut kernels were conditioned to reach 3%  were grounded using a Cuisinart Mini-Prep Plus® food processor , model DLC-2A., weighed in a 600 mL beaker and dried in the oven at 105 °C until constant weight. After the sample reached room temperature in a desiccator the weight of the dry paste was registered. Each sample was analyzed in triplicates.The total fat content was obtained by Soxhlet standard extraction mode of the Buchi extraction system  using the previously dried ground walnuts with n-hexane for 50 extraction cycles and 30 minutes rinsing232, K268) (Ch 5-91(09)). Each treatment was analyzed in triplicates.Standard methods of American Oil Chemistry Society  were useA Rancimat apparatus 617  was used to measure oxidative stability. Oil samples (3 g) and ground kernels (0.5 g) were weighed into test tubes and oxidized at 110 °C with 20 l/h of airflow. Each treatment was analyzed in triplicates.Individual kernel color was evaluated following the Dried Fruit Association (DFA) guidelines . DFA is The fatty acid composition of oil samples was determined as methyl esters by gas chromatography/mass spectrometry according to Tapia et al. . Aliquotg force, 5 min, Beckman GS-15R). Samples were stored at −20 °C to allow the separation between oil and organic phase. The organic extract was filtered  and analyzed using UPLC-DAD. A blank followed by a standard mix  was run after every fifteen samples. The analysis was performed on an Agilent 1290 Infinity II LC system with a diode-array detector using an Agilent ZORBAX Eclipse Plus C18 column . The mobile phase was methanol:water (96:4) with 20 µL injection volume and flow rate of 1.0 mL/min. The total run time was 12 min with DAD signal recorded at 292 nm. Tocopherols were identified by their retention time in comparison with the standards. Each sample was analyzed in triplicates. Extraction was performed according to Gimeno et al.  with somg for 10 min. Then, 0.4 g of the previously combined extract  and 1 g of oil  are extracted using 20 mL in two extraction (10 + 10 mL) of MeOH:H2O:HCOOH (80:20:0.1), with 2 min vortex followed by 5 min ultrasound and centrifugation at 2000× g for 10 min. The supernatant was collected, and the solid residues were re-extracted a second time as described above. The extracts were combined and membrane-filtered through cellulose filters . Total phenolic compounds from the extracts above were quantified by the Folin−Ciocalteu method [Phenols from kernel and oil were extracted using a method adapted from conditions described in previous studies . A doublSolid phase microextraction (SPME) was used to extract walnut volatile compounds. Sample of oil or ground kernel (3.0 ± 0.1 g), with 4-methyl-2-pentanol as internal standard (2.5 mg/kg), was analysed in a 20 mL glass vial and with a PTFE/silicone septum . After 10 min equilibration time at 40 °C, a solid-phase microextraction (SPME) fiber  was exposed to the sample headspace for 40 min. The volatile compounds were analysed using a GC system (Agilent Technologies) that comprise an autosampler (Agilent PAL RSI 85) with 45 positions, a gas chromatograph (GC Agilent 7820A), and a mass spectrometer (Agilent 5977B) with an electron impact source and a quadrupole analyzer. Compounds were separated using a Supelcowax 10  and helium at a flow rate of 1 mL/min as carrier gas. GC oven temperature started at 40 °C, after 10 min ramped at 3 °C/min to the final temperature of 200 °C. A blank followed by a standard mix was run after every ten samples. The data were recorded and analysed using Agilent MassHunter Qualitative Analysis. The volatile compounds were identified using the NIST 08 Mass Spectral Library and via comparison with the retention time and mass spectrum of their respective standards. A representation of a chromatogram from walnut kernel and oil volatile analysis with retention times is shown in p ≤ 0.05. The heatmap of volatile compounds was generated using the normalized volatile concentration, obtained by the ratio of each volatile concentration and the sum of volatile compounds from the same sample. The significant chemical data were then related to one another using multivariate analysis. Principal component analysis (PCA) was applied to evaluate the chemical composition of both cultivars present in the study. Hierarchical cluster analysis (HCA) and partial least square regression discriminant analysis (PLS-DA) were applied to identify their characteristic properties, and to know which are associated with oxidative stability. Data analysis were performed by SPSS version 27.0 software  and graphs were obtained using SigmaPlot version 12.5 .All data were tested by analysis of variance (ANOVA), and significant storage effects for each cultivar were followed by Tukey’s multiple comparison test at 232) and 268 nm (K268). Moisture is an essential factor in the quality of nuts, influencing their physical and chemical properties [p < 0.000). Compared with ‘Chandler’, ‘Howard’ had a higher initial moisture content, experienced a sharp decrease within the first week, and had lower moisture content throughout the 28 weeks.Walnuts have one of the highest fat content among nuts and foods . Fat conoperties . The moi−1 of oleic acid for both cultivars during storage, likely due to the drying process and storage conditions that reduced moisture content and inactivated lipases and other enzymes activity [Hydrolysis of triacylglycerols (TAG) is due to the lipolysis (by lipases) or hydrothermal activity, resulting in the release of free fatty acids and hydrolytic rancidity . Thus, Factivity ,23.p < 0.000) and was higher in the oil extracted from ‘Howard’ than it was from ‘Chandler’ (p < 0.000). In ‘Chandler’, PV ranged from 1.0 to 4.3 mEq O2kg−1 while in ‘Howard’, PV ranged from 1.8 to 6.4 mEq O2kg−1 between 0–28 weeks. PV of 3 mEq O2kg−1 is the maximum limit of acceptable quality of shelled walnuts [2kg−1 after 20 weeks of storage, ‘Chandler’ reached the same value after 24 weeks. According to the recognized PV limit of acceptable quality in kernels, ‘Chandler’ can be stored no longer than six months and ‘Howard’ no longer than four months under our studied storage condition.PV measures the concentration of primary oxidation products (hydroperoxides) in fats and oils . As oxid walnuts . ‘Howard232 determines the peroxides present via the conjugated double bonds of the hydroperoxides. Both cultivars had similar ranges of K232, though values from ‘Chandler’ were consistently lower than those from ‘Howard’. K232 values of ‘Chandler’ ranged from 0.95 to 3.08 while ‘Howard’ ranged from 1.13 to 3.2. K268 measures the conjugated trienes from more advanced oxidation processes. Same as K232 values, K268 values were higher in ‘Howard’ than they were in ‘Chandler’ at all time points (p < 0.000). K268 values of ‘Chandler’ ranged from 0.07 to 0.23 while ‘Howard’ ranged from 0.13 to 0.29. As oxidation progresses during storage, increasing K232 and K268 trends were observed in both cultivars. KOxidative stability gives an estimation of food susceptibility to oxidation; it can be used to provide information on the hypothetical stability of a product as it generally correlates with shelf life . Walnut p < 0.000). Kernel oxidative stability ranged from 13.4 to 6 h in ‘Chandler’ and from 11.7 to 2.5 h in ‘Howard’. Kernel oxidative stability significantly decreased when PV was close to the recognized parameters of acceptable quality of shelled walnuts of 3 O2kg−1 (2.6 O2 kg−1 in ‘Chandler’ at week 20 and 3.4 O2 kg−1 in ‘Howard’ at week 16). Differences between cultivars in PV, K268, and kernel oxidative stability were significant before storage, showing that these methods can be used to predict samples susceptibility to oxidation and to evaluate storage time and conditions. As we recently demonstrated [Kernel oxidative stability even though presented much higher values; it followed the same trend as oil oxidative stability between cultivars and during storage. ‘Chandler’ demonstrated higher kernel oxidative stability than ‘Howard’  and Howard (213.29 mg kg−1) [Tocopherols are natural antioxidants that inhibit lipid oxidation in vegetable oils , and wal−1 and decreased to 10,466 µg g−1, while ‘Howard’ started at 11,511 µg g−1 and decreased to 8325 µg g−1. The total phenolic concentration found in the present study is within the same range as those previously reported for ‘Chandler’ and ‘Howard’ grown in Spain, Greece, and Turkey and for ‘Chandler’ stored at 20 °C for 12 months [Total phenolic content of walnuts can be a useful criterion for overall walnut quality evaluation due to their contribution to the color, taste, flavor characteristics, and overall health benefits . Total p2 months ,30,37,39Contrary from the phenols in kernels, ‘Howard’ oil had a higher initial phenolic concentration than the oil from ‘Chandler’. During storage, as expected, phenols are used up to fend off oxidation, ‘Chandler’ decrease oil phenol concentration by −1.5-fold, similarly to kernels. However, ‘Howard’ demonstrated higher losses with −10.2-fold oil phenol concentration after 7 months of storage. Phenol decreased more rapidly in oil than in kernel, and in ‘Howard’ than in ‘Chandler’. Walnut oil presented much lower phenol concentration compared to kernels, due to their low oil solubility [(E)-2-hexenal; and linolenic acid is a precursor of -2,4-heptadienal, (E)-2-heptenal, and 2-methylpropanal. The oxidative degradation rate of linolenic acid is much faster than that of linoleic acid and oleic acid [−1 in ‘Chandler’ and from 3.8 to 105.72 mg kg−1 in ‘Howard’. As presented in (E)-2-pentenal > 2-pentyl-furan as the volatiles with highest concentrations, while ‘Howard’ presented 1-penten-3-ol > 1-hexanol > hexanal > 2-pentyl-furan > 1-pentanol with the highest concentrations. The hierarchy of the volatiles with highest concentration also changed during storage. Specifically, ‘Chandler’ presented hexanal > 1-pentanol > pentanal > 1-octen-3-ol > 2-pentyl-furan with the highest concentrations after 28 weeks, while ‘Howard’ presented hexanal > 2-pentyl-furan > 1-pentanol > 1-octen-3-ol > (E)-2-octenal as the volatiles with highest concentrations after the same period. Contrary to previous studies on walnut cultivars from Ukraine, China, and Chile [When lipid matrix of walnuts is exposed to heat and light, the hydrogen atom of double bond is extracted, and alkyl radicals are formed. These free radicals react with oxygen; peroxy radicals are formed by removing hydrogen atoms from another unsaturated fatty acids, leading to the formation of primary oxidation products called hydroperoxides by the mechanisms of initiation, propagation, and termination . These peic acid . Due to eic acid . At the eic acid , signifind Chile , 1-octennd Chile ,46 was n(E)-2-hexenal, two volatiles included in this study. Moreover, walnuts are known for their prevalence of 13-LOXs compared to those observed in other nuts, such as almonds [The detection of rancidity off-flavors depends on the type and degree of oxidation. In previous studies, hexanal was monitored to assess the oxidation degree in nut samples ,47,48. I almonds ,54. Ther-2,4-Heptadienal, 1-octanol, (E)-2-decenal, and -2,4-nonadienal were not detected in the oil , ethyl acetate (ester), and 1-octen-3-one (ketone) were identified in the oil extracted from kernels after storage.  the oil . These v the oil  than in 232, and K236 are higher in ‘Howard’ than in ‘Chandler’. Using PCA and HCA, we aimed to distinguish different oxidation levels and understand which chemical analysis and compounds are better related to kernel oxidative stability. These compounds might be better indicators of kernel oxidative stability during storage.The results shown previously indicate that the chemical composition of walnut kernel and oil were affected by the storage. Moreover, oxidative stability, PV, K232, K268, kernel OS, oil OS), pellicle darkness, kernel and oil phenols, fatty acid profile , and volatiles classes from kernel and oil (p < 0.001) given by Bartlett’s test of sphericity. Ketones, esters, and PUFA present in the oil show values lower than 0.3 in the correlation matrix with all other values. This may be an indication than these variables can be further reduced in the analysis, since they are not correlated with other measurements included in this manuscript to describe walnut oxidation. The loading plot of PCA  to explore the relative variability within the different walnut samples. PCA was generated with a correlation matrix of 66 observation from both walnut cultivars against 24 independent variables . The results indicated that two discriminant functions were optimal to build the model for discriminating samples. The scores and correlation loadings obtained for the first two latent components of the calculated PLS-DA model were combined in a plot (As demonstrated in the loading plot from PCA A, increan a plot . The firTo identify the specific volatiles that most contribute for the group separation, a stepwise method was applied. In twenty-one steps, thirteen volatile compounds that contribute for discrimination between oxidation groups were established . We founWalnuts are susceptible to oxidative rancidity due to high fat content and proportion of unsaturated fatty acids. Under the same storage condition, oil extracted from ‘Howard’ kernels had higher values of PV, UV absorbances, as well as lower phenols, tocopherols, and MUFA, comparing to oil extracted from ‘Chandler’. Higher concentrations of phenols and tocopherols in ‘Chandler’ likely contribute to their stability comparing to ‘Howard’.PCA, HCA, and PLS-DA were successfully employed to assess and compare the utility of different oxidation methods, including volatiles in kernels and oil. Kernel volatile compounds can distinguish samples with different storage time for ‘Chandler’ and ‘Howard’. This work shows that volatile measurements of the kernels can be applied to help growers and processors to assess walnuts level of oxidation at harvest and during storage without oil extraction. Future work needs to investigate the potential utility of these parameters as oxidation markers in walnut kernels under different and practical storage scenarios as well as their acceptable limits and relation to their sensory attributes.


To investigate the efficacy and safety of aerosol inhalation of recombinant human interferon α1b (IFNα1b) injection for noninfluenza viral pneumonia.One hundred sixty-four patients with noninfluenza viral pneumonia were divided into IFNα1b and control groups. The IFNα1b group received routine treatment + aerosol inhalation of recombinant human IFNα1b injection . The control group received routine treatment + IFN analog . Overall response rate (ORR) of five kinds clinical symptoms. Further outcomes were daily average score and the response rate of each of the symptoms above.P < 0.05). There were no significant differences of the ORRs for coughing, chest pain and respiratory rate between the two groups (P > 0.05). The incidence of adverse events was 6.5% (n = 5) in IFNα1b group and 3.5% (n = 3) in control group (P > 0.05).A total of 163 patients were included in the full analysis set (FAS) and 151 patients were included in the per-protocol set (PPS). After 7 days of treatment, ORR of clinical symptoms was higher in IFNα1b group than that in control group for both the FAS and PPS. Moreover, after 7 days of treatment, the daily score of three efficacy indexes including expectoration, respiratory rate, and pulmonary rales were improved. The ORRs for expectoration and pulmonary rales were higher in the IFNα1b group than in the control group (Aerosol inhalation of recombinant human IFNα1b is safe and it can improve the clinical symptoms of noninfluenza viral pneumonia. Streptococcus pneumoniae. Other pathogens include human metapneumovirus, respiratory syncytial virus, parainfluenza virus, coronavirus, and adenovirus [Pneumonia is the leading cause of death in children in developing countries and the elderly in developed countries. Among the pathogens which cause community-acquired pneumonia (CAP), viral pneumonia is attracting increasing attention. It occurs in approximately 200 million people each year worldwide, while adults and children each accounting for 50% of cases . In receenovirus . IFNα waenovirus , 8. Sakaenovirus .Interferons (IFNs) are a family of multifunctional cytokines with broad-spectrum antiviral, antiproliferative, and immunomodulatory activities. IFN has broad-spectrum antiviral and immunomodulatory effects and is classified as type I, type II or type III based on different binding receptors . Among tTherefore, we performed a multicenter, randomized controlled study to investigate the efficacy and safety of aerosol inhalation of recombinant human IFNα1b for noninfluenza viral pneumonia and to provide a basis for its rational use and dose in clinical practice.Guidelines for the Diagnosis and Treatment of Adults with Community-Acquired Pneumonia from the Respiratory Disease Branch of the Chinese Medical Association (2016); 2) clinical diagnosis of viral pneumonia; 3) negative tests for influenza viruses; 4) inpatients, within 5 days of onset; and 5) ability to receive aerosol inhalation.This is a multicenter, randomized, double-blind, placebo-controlled trial, which was approved by the Ethics Committee of Beijing Ditan Hospital Capital Medical University. Each subject was informed of the purpose of the study and potential benefits and risks and signed an informed consent before the study. Inclusion Criteria: The inclusion criteria were as follows: 1) men or women at least 14 years old with pneumonia diagnosed according to the Tuberculosis bacillus, Streptococcus pneumoniae, Legionella pneumoniae, Mycoplasma pneumoniae, or Chlamydia infection; 2) unequivocal evidence of bacterial infection, procalcitonin (PCT) > 1 μg/L; 3) use of antiviral drugs in the week before screening or potential need for another antiviral treatment during the study; 4) subjects who required mechanical ventilation; 5) unstable or active chronic lung disease, diabetes, tumor, or HIV infection; 6) severe liver or kidney dysfunction; 7) participating or participated in another clinical study during the 30 days before study treatment; 8) a history of IFN allergy or other IFN contraindications; 9) pregnant (positive urine or serum pregnancy test) or nursing women.The exclusion criteria were as follows: 1) unequivocal evidence of The patients were randomly divided into IFNα1b group and control group.The IFNα1b group was given routine treatment (antibiotics and antitussive and expectorant drugs) and aerosol inhalation of recombinant human IFNα1b injection , 50 μg × 2 injections, bid. The control group was given routine treatment and aerosol inhalation of IFN analog , 2 injections, bid. Standardized compressor nebulizers were used at all sites to administer treatment via the same nebulization process.Guiding Principles for Clinical Study of New Chinese Medicines, 0, no coughing, expectoration, chest pain, or pulmonary rales, and a normal respiratory rate; 1, mild; 2, severe.The primary outcome was the overall response rate (ORR) of five pneumonia-related symptoms, including coughing, expectoration, chest pain, pulmonary rales, and respiratory rate after treatment. The ORR of clinical symptoms (%) =  / pretreatment score × 100%. The clinical signs and symptoms were scored according to the The secondary efficacy measures were daily average score and the response rate of each of the five pneumonia-related symptoms, including coughing, expectoration, chest pain, pulmonary rales, and respiratory rate before and after treatment.The measured vital signs included pulse, blood pressure, body temperature, and respiratory rate. The items of physical examination included skin and mucous membranes, lymph nodes, head and neck, chest, abdomen, spine, musculoskeletal system, nervous system. The clinical laboratory tests included complete blood count and serum biochemical examination.p-value less than 0.05 was considered to be statistically significant. The full analysis set (FAS) was used to analyze primary outcome, and the per-protocol set (PPS) was used to analyze secondary outcomes. Moreover, a superiority test was performed to analyze primary efficacy measures. Fisher’s exact test was used to analyze the overall incidence of adverse events and the incidence of drug-related adverse events.SAS v9.3 was used for statistical analysis. A two-side P > 0.05). However, there were significant differences of gender composition between the two groups (P < 0.05) , or time of onset between the two groups .Identified Viral pathogens in the enrolled subjects were shown in Table P < 0.05) (Table P > 0.05)  decreased rapidly, especially the expectoration and respiratory rate Fig. . On daysP < 0.05) (Table P > 0.05).The response rates of expectoration and pulmonary rales were significantly higher in the IFNα1b group than that in the control group (5) Table . No signP > 0.05) for both the FAS and PPS  within 90 days of discharge due to complaints other than noninfluenza viral pneumonia. Fisher’s exact probability test showed no significant between-group difference (PS Table .Table 7P > 0.05) (Table P > 0.05) (Table The rate of antibiotic use was 93.42% in the IFNα1b group and 90.80% in the control group (P > 0.05). Physical examinations, including examinations of the skin and mucous membranes, lymph nodes, head and neck, chest, abdomen, spine, musculoskeletal system, and nervous system, revealed no clinically significant abnormal findings. In urine analysis, stool tests, electrocardiography, and blood oxygen saturation level measurements, few subjects showed clinically significant abnormalities, and no significant between-group differences were found (P > 0.05). At the end of the treatment, the frequency of abnormal blood and biochemical tests was 176 cases in the IFNα1b group and 158 cases in the control group (P > 0.05).No significant differences of the vital signs  of the 164 subjects were observed between the two groups  injection for noninfluenza viral pneumonia. It was found that aerosol inhalation of recombinant human IFNα1b could improve the ORRs of the clinical symptoms with our additional adverse events in noninfluenza viral pneumonia.This study found that the ORRs of primary efficacy measures, including coughing, expectoration, pulmonary rales, respiratory rate, and chest pain were higher in the IFNα1b group than that in the control group, which received routine symptomatic treatment alone, suggesting that aerosol inhalation of recombinant human INFα1b effectively improves the overall response rate of the disease. IFNα is a recognized immunomodulatory therapy to suppress viral replication by inhibiting basal transcription processes.Because of its antiviral effects, IFNα has been used in trials in combination with other antiviral agents to prevent and treat emerging and reemerging virus infections for which no approved drugs are available –18. HoweProphylactic or early therapeutic administration of IFNs during MERS-CoV infection in rhesus macaques provided significant protection . HoweverWhen used in the systemic therapy, IFNs are mostly administered by an intramuscular injection. The most frequent adverse effects are flu-like symptoms, especially increased body temperature. In current study, no significant difference of the adverse events was observed between the IFNα1b group and the control group, suggesting that aerosol inhalation of recombinant human IFNα1b injection was safe and well tolerated.There were also some limitations in this study. First the analysis of viral pathogens could be uncertain, and not all cases have positive pathogen results. Second, the specimen was collected from the upper respiratory tract, which might not be the same pathogen as the lower respiratory tract.In conclusion, aerosol inhalation of recombinant human IFNα1b combined with routine treatment is safe and well tolerated in the treatment of noninfluenza viral pneumonia. It significantly improves the ORRs of clinical symptoms and accelerates recovery compared with routine treatment.


Human adenoviruses cause disease at multiple mucosal sites, including the respiratory, gastrointestinal, and genitourinary tracts, and are common agents of conjunctivitis. One site of infection that has received sparse attention is the cornea, a transparent tissue and the window of the eye. While most adenovirus infections are self-limited, corneal inflammation (keratitis) due to adenovirus can persist or recur for months to years after infection, leading to reduced vision, discomfort, and light sensitivity. Topical corticosteroids effectively suppress late adenovirus keratitis but are associated with vision-threatening side effects. In this short review, we summarize current knowledge on infection of the cornea by adenoviruses, including corneal epithelial cell receptors and determinants of corneal tropism. We briefly discuss mechanisms of stromal keratitis due to adenovirus infection, and review an emerging therapy to mitigate adenovirus corneal infections based on evolving knowledge of corneal epithelial receptor usage. The human adenovirus (HAdV) is a ubiquitous pathogen that has contributed much to current knowledge of molecular biology, leading to critical revelations about the cell cycle and RNA splicing, among other discoveries . InfectiHAdV infections of the eye are common, and the most frequent cause of viral conjunctivitis ,27. HAdVConjunctival membrane formation and keratitis distinguish EKC from other ocular adenovirus infections ,34,35. T® [Animal models to study adenoviral pathogenesis have been limited by the specificity of HAdVs for human cells; mouse models can be used only to study innate immune responses to the virion ,40,41. T® , an epit® ,45.n = 92), Imre and colleagues showed typical adenovirus inclusions in 85% of corneal epithelial scrapings of studied patients [An abundance of evidence supports corneal epithelial cell infection by HAdVs. In a large clinical series of EKC cases (patients . Dawson patients . Maudgalpatients . Chodoshpatients . They fupatients , can reppatients .The tropism of particular HAdVs for corneal epithelial cells could account for the limited number of HAdV types associated with EKC . HoweverAs with all mucosa, the eye is bathed in secretions with antiviral components,  and the The primary ligand for adenovirus binding to its target cell is the trimeric fiber protein ,57. The Recent evidence has further clarified receptor-mediated entry of those types within HAdV-D associated with EKC, resulting in the first potential viral entry-based therapy for acute infection . As discvβ3, αvβ5, and αvβ1 [vβ1 and α3β1 [In the most widely cited paradigm for adenoviral entry , primaryand αvβ1 ,97,98 (aand α3β1 ,100, whiand α3β1  and autoand α3β1 , and leaand α3β1 ,104 and and α3β1 ,106. Theand α3β1 ,107,108.and α3β1 ,109. Recand α3β1 , was shoand α3β1 . To add and α3β1 .vβ5 can cluster in clathrin-enriched foci [Although the mechanistic connection between binding of the adenovirus fiber knob to a cell surface receptor and secondary aggregation of cell membrane integrins is relatively clear, the molecular events that connect integrin aggregation to endocytosis have not been clarified in corneal epithelial cells. Lee and coworkers recently showed that entry of HAdV-D37 occurs by a noncanonical clathrin-mediated entry pathway in both hTERT-immortalized human corneal epithelial cells, and in primary cultured human corneal epithelial cells . More sthed foci . Aggregahed foci , and clahed foci . These fIn summary, existing evidence implicates corneal epithelial cells as key to adenoviral corneal pathogenesis, with likely contributions from keratocytes and resident bone marrow derived cells in the corneal stroma to the immunopathology of EKC. Recent discoveries related to corneal epithelial cell entry by HAdV-Ds suggest hope for the development of an effective therapy to mitigate immunopathology in EKC.


Preference-based Health-Related Quality of Life (HRQL) is one of the most important indicators for calculating QALY  in a cost-effectiveness analysis. This study aimed to collect data on healthy individuals’ HRQL based on the preferences of Japanese people who had undergone a comprehensive health check-up, and to examine the influence of relevant factors, such as blood biochemical data and lifestyle behavior.We conducted a cross-sectional study targeting people who had undergone a comprehensive health check-up in 2015. Participants were asked to respond to a medical interview sheet. We then examined the utility value, as well as lifestyle habits such as alcohol intake, smoking, and exercise. HRQL was examined using EQ-5D-5L. Using a multiple regression analysis, we examined the influence of related factors, such as lifestyle and biochemical test data.r = − 0.045–0.113). The results of multiple regression analysis showed that significant facts were: being female, age (≧70 year-old), drinking alcohol (sometimes), activity (very often), and lack of sleep.We collected 2037 responses . The average preference-based health-related HRQL was 0.936 ± 0.087. A total of 1167 people (57.2%) responded that they were completely healthy. The biochemical test data that were recognized to correlate with HRQL were hemoglobin, total cholesterol, creatinine, all of which were weak (The HRQL of participants who had undergone a comprehensive health check-up was generally high, and only declined for those over 70 years of age. It is suggested that preference-based HRQL is related to physical activity, and that decrease of activity and lack of sleep leads to a decrease in HRQL. Preference-based Health-Related Quality of Life (HRQL) is one of the most important indicators, along with cost, for calculating QALY  in a cost-effectiveness analysis. Due to its application in the Health Technology Assessment (HTA) policy, its importance has increased. Since the HTA began in Japan in 2017, guidelines have been developed [EQ-5D was developed by the EuroQol Group, and two scoring algorithms, EQ-5D-3L  and EQ-5In Japan, it is common practice for healthy people to check their health status annually by participating in a comprehensive health check-up . This chWe targeted people who had undergone a comprehensive health check-up from January to March 2015, at the Clinic and Center for Preventive Medicine affiliated with St. Luke’s International Hospital in Tokyo. The hospital was located in the center of the capital, so the population surveyed was mostly urban residents. A total of 3800 participants had undergone the health check-up. Prior to the examination, medical interview sheets were sent to the subjects by mail. The sheets included a lifestyle survey and an EQ-5D-5L. These sheets were then collected on the day of the examination. Subjects had to be over 21 years of age and able to answer the medical interview sheet on their own.The EQ-5D-5L is a generic preference-based measure of HRQL developed by the EuroQol Group . The fivThrough self-report, the medical interview sheet confirmed whether or not the person was currently undergoing treatment for a disease . Furthermore, people were asked about the following lifestyle habits: alcohol intake, 3 levels, ,” level 2, “Sometimes (2-3 times a week),” level 3, “Habitually”); smoking, 3 levels ,” level 2, “Active (smoke on a daily basis),” level 3, “Former smoker”); lack of exercise, 2 levels ; physical activity, 4 levels ; exercise habit, 4 levels ; and sleep, 2 levels . Physical activity comprises subjective activity in daily life, while exercise habit comprises sweaty exercise for more than 20 min.In the comprehensive health checkup, body mass index (BMI), body fat, blood pressure was measured, and biochemical examination of blood was performed. The blood biochemical test examined the hemoglobin, blood glucose, HbA1c, low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol, neutral fat, thyroid-stimulating hormone (TSH), free thyroxine 4 (FT4), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), creatinine, and estimated glomerular filtration rate (EGFR). These blood biochemical examination findings were related to lifestyle-related diseases such as hypertension and heart disease. These series of examinations comprised the basic elements of the complete health checkup and were performed by the clinician, with the cost borne by the study subjects.A summary of HRQL scores was calculated by gender, age group  and lifestyle factors, and scores were then compared using variance analysis. To detect the influence of demographic factors and lifestyle factors on the HRQL scores, these variables were added to an analysis of variance (ANOVA). The influence of demographic characteristics and lifestyle factors on HRQL was determined using multiple regression analyses. Sex, age, drinking, smoking, physical activity and sleep, with the relevant dummy variables, were included as covariates in a multiple regression model with total HRQL score as the outcome. Exercise habit was not included in this model due to potential collinearity with physical activity. The significance level was set at 0.05. Statistical analyses were performed using STATA 14.0. This study was approved by the Ethics Committee of Niigata University of Health and Welfare  and the Institutional Review Board of St. Luke’s International Hospital (No.15-R086). Informed consent was obtained from all individual participants included in the study.2 and the average Body Fat was 24.3 ± 6.5%. Blood pressure and blood biochemical data were all within the standard value as average. Regarding self-reported chronic diseases, 300 people (14.7%) reported having high blood pressure, 276 (13.5%) reported having hyperlipidemia, 105 (5.1%) reported diabetes, 98 (4.8%) reported having uterine myoma, and 86 (4.2%) had gout.Table p < 0.001). There was no statistically significant difference regarding age group (p = 0.067).Table p < 0.001) for who drank alcohol “sometimes” (0.943 ± 0.076) and “habitually” (0.944 ± 0.079) (p < 0.001). Regarding lack of exercise, the highest mean scores were for “no” at 0.956 ± 0.069, with “yes” at 0.928 ± 0.092 (p < 0.001). A difference was also observed for daily activities, with a mean score of 0.967 ± 0.062 for “very often” in contrast to 0.912 ± 0.111 for “too little.” Finally, differences were found in exercise habit (p = 0.0011) and lack of sleep (p < 0.001).We further examined the relationship between lifestyle and HRQL score. Regarding alcohol intake, the mean score for “never” was 0.923 ± 0.098, while higher values were shown (p = 0.774), but differences were observed among age groups (p < 0.001), lack of exercise (p < 0.001), exercise habit (p < 0.001), and sleep patterns (p < 0.001).Data of the visual analogue scale (VAS) were also analyzed. There were no differences in gender (r = − 0.077), hemoglobin (r = 0.113), total cholesterol (r = − 0.045) and creatinine (r = 0.089), but the correlation was weak for all of them. BMI, body fat, HDL, GPT, and EGFR were also observed to have a correlation to VAS.Table p < 0.001). Moreover, subjects over 70 years of age had lower scores than those under 70 years of age . Regarding alcohol intake, both “sometimes”  and “habitually”  scored higher than “never.” Additionally, daily activities were indicated as factors causing a decrease, in contrast with “very often,” “usually” , “not so active” , and “too little” . Lack of sleep was also indicated as a factor that lowers HRQL scores .Finally, Table The results of this study demonstrated that the preference-based HRQL of healthy subjects who had undergone health checkups is related to demographic characteristics and lifestyle factors and is influenced by blood biochemical data. First, it was clear that the factor of aging did not lower the HRQL of people who live independently and receive health examinations. A medical check-up is a comprehensive test that uses blood biochemical tests to check for the onset of disease, so those who receive it are basically healthy. However, HRQL declines for the elderly over 70 years old. This is similar to the findings of Shiroiwa et al. , GermanyThere was no strong influence on the relationship between the health data collected by the comprehensive health check-up and HRQL indicated by EQ-5D-5L. It can be considered that blood biochemical data do not directly affect the five dimensions of mobility, self-care, usual activities, pain/discomfort, and anxiety/depression, as indicated by EQ-5D.The relationship between lifestyle and HRQL does have an impact, particularly alcohol intake, daily physical activity, exercise habit, and sleep patterns. Regarding alcohol intake, since people with drinking habits showed higher HRQL scores, this indicates that drinking does not lower HRQL. Drinking can be considered to have a positive influence on HRQL, since as indicated in several previous studies –19, modeOne of the limitations of this study is that the sample size is small compared to other studies targeting healthy subjects. Moreover, the study was only conducted in one hospital. Further research should clarify the relationship between chronic disease and HRQL scores, by increasing the sample size and conducting a longer-term survey. We would also like to reduce any possible bias that may accrue from including so few participating facilities by conducting a multi-facilities collaborative research project.In this study, we showed the HRQL of healthy subjects as indicated by EQ-5D-5L through a comprehensive health check-up, and clarified the relationship between demographic characteristics, blood biochemical data, and lifestyle behavior. It was shown that the factors leading to a decline of HRQL are being female and over 70 years of age, while factors that keep HRQL high are alcohol intake, high levels of physical activity in daily life, and adequate sleep. The results of this study will provide good suggestions for public health policies, including care prevention.Additional file 1.


E) to elucidate these primary structure characteristics. We investigated venom from ten species of “tiger” spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product’s diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization.Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMS CID experiments were performed using an electric potential to accelerate peptides/peptide fragment ions into a neutral gas that disrupts bonds to provide cleavage at the carbonyl-N backbone, resulting in Biemann fragments referred to as b and y ions 3+ charged states and greater, we collected data from the tryptic digests previously described . However, the ETcaD spectra of the [M + 2H]2+ displayed a very convoluted “hybrid” spectra of b, c, y, and z ions. Supplemental activation did not enhance the sequencing process, nor did it provide additional c and z ions. As in the ETD spectra, the reporter ions for isoAsp were not observed in the ETcaD experiments (data not shown). We tried this approach on ten other peptides of different residue makeup from these experiments with a mix of results (data not shown). However, many of the analyses yielded a similar assessment as to the one provided here. One last remark is the addition of <5 V of supplemental activation on classical ETD experiments on the [M + 3H]3+ ions (or ions of greater charge) did enhance the fragmentation up to 15% increase, but did not alter the quality of the fragmentation products, i.e., it did not form c and z ions that were not detected prior to the additional collision energy addition. Taken together, these data suggest that the use of supplemental activation is peptide specific and should be used with caution due to the formation of hybrid spectra that may convolute the experimental desire.Although ETD can deliver unique fragmentation patterns for de novo sequencing, there is a challenge in this application to normal proteomic workflows. As the precursor  400 m/z . Trypsin 400 m/z ,64. Ther 400 m/z . One solescribed  and alte + 3H]3+ A. All th–325 m/z B and a mm/z  -fibrinopeptide B human as a reference. To maximize ion resolution, all samples were processed for de novo sequencing and molecular mass characterization in “W-ion” optics mode (W-OpticsTM)  that provided FWHM >50,000 resolution. The following MS conditions were routinely employed: polarity ES+, capillary 2.80 kV, sampling cone 30, source offset 30, ToF 10.00 kV, pusher cycle time 124 μs and pusher frequency 8065 Hz, cone gas 600 L/h, source 125 °C, and desolvation 350 °C.Any aliquot of venom, toxin, or peptide digests were injected into a Waters UPLC AcquityTM) focused upon only those precursor ions that met strict selection criteria, i.e., signal intensity, charge state, and exact mass, allowing them to be selected preferentially for MS/MS. Routinely, four consecutive 0.3 s MS-MS scans were acquired: (1) CID on [M + 1H]1+ (CE Ramp 20–30 V), (2) CID on [M + 2H]2+ (CE Ramp 20–45 V), (3) electron transfer dissociation with supplemental activation (ETcaD) on [M + 2H]2+ , and (4) ETD on [M + 3H]3+ (wave velocity 600 m/s and wave height ~0.23 V). All ETD experiments were acquired with a discharge current of 24 mA after tuning the reagent ion 128 m/z  in the negative chemical ionization (nCI) source to ~1e5 intensity. Refill parameters were set as follows: refill time .To ensure efficient acquisition of all peptides and/or co-eluting compounds, all analyses for de novo sequencing were performed in data directed analysis (DDA−2 mbar (Ar), IMS gas pressure 1.0 mbar (N2), and a 7 V pulse adjusted as needed in subsequent experiments to maximize arrival time distribution (ATD). TWIMS was performed as described ,91. MS cCatecholamine A-differentiated (CAD) cells were maintained as previously described .VH of −100 mV.Whole-cell voltage-clamp recordings were performed as previously described  with theMass spectra and electrophysiological traces were processed as previously described  and all td’):td is the drift time of ion and c is a correction factor set-up for each instrument. Published CCS data (Ω) was then corrected by using a normalized CCS (Ω’) that accounted for reduced mass and charge state:Estimated CCS measurements were made by subtracting the instrumental offset and calculating the correct drift time  for fast inactivation was determined from an exponential curve and computed in two steps using the HEKA FitMaster V 2x90.5: (1) semi-logarithmic regression of x vs ln(y) and (2) abs(1/slope).The time constant (τDatabase: UniProt Knowledgebase under the accession number(s) C0HLN4.


To analyze the relationship between aortic measures and biometric parameters in a large cohort of consecutive patients undergoing computed tomography coronary angiography.1170 patients (717 men/453 women) performing computed tomography coronary angiography for coronary evaluation were retrospectively evaluated. Aortic diameters and areas were measured at reproducible anatomic landmarks, perpendicular to the axis of vessel, at the level of the aortic root (AoR), the sinotubular junction (STJ), and the tubular ascending aorta (TAo). Biometric parameters and cardiovascular risk factors were recorded.The average values of AoR, STJ, and TAo were 35.63 ± 5.00 mm, 30.56 ± 4.82 mm, 35.07 ± 5.84 mm. Hypertension was significantly associated with aortic dimensions.P < .001) and linearly increased with age. Low Spearman correlation coefficients were found and the correlation of TAo diameters with age displayed the highest values . Multiple linear regression analysis models were compared by R2. The best model used body surface area (BSA) and age as independent variables and TAo diameter as dependent variable .Aortic measures were significantly different between men and women  (In conclusion, in our population low correlation between aortic dimensions and biometric parameters highlights the difficulty of identifying normal ranges, as well as issues related to normalization using conventional biometric parameters. In this field, many imaging techniques have been employed for the assessment of aorta and its segments such as transthoracic echocardiography (TTE), transesophageal echocardiography, magnetic resonance imaging(MRI), computed tomography (CT), and conventional angiography. The most frequently used non-invasive technique in clinical practice is CT, as it is readily available and enables, thanks to the recent development of multidetector technology, the simultaneous evaluation of the aorta and coronaries.–4Accurate and reproducible measurements of aortic diameters are essential for the diagnosis, classification, and follow-up of aortic pathologies and to decide how/when to perform follow-up, prevention strategies, and select candidates to surgery.–12 while more recent studies used CT.–16Values for normal ranges have been first established by ultrasound–16 the correlation between aortic sizes and biometric parameters has been exploited by echocardiography or non-contrast electocardiogram (ECG) triggered CT in selected population .To date it has been very difficult to identify a single method able to provide the concept and the ranges of variability of normal ascending aorta. In previous studiesIn this study, we aimed to analyze in a large cohort of consecutive patients undergoing computed tomography coronary angiography (CTCA) the relationship between aortic dimensions and demographics, biometric parameters and risk factors, using different approaches.2–19 All patients underwent CTCA for coronary artery assessment which includes in the dataset ascending aorta. All patients gave informed consent for the investigation and the study was approved by the Institutional Ethics Committee .One thousand one hundred seventy (1170) consecutive patients, respecting the inclusion criteria mentioned below  and referred to CTCA for suspected coronary artery disease, were retrospectively evaluated. All demographics risk factors and patient relevant clinical data were prospectively gathered from medical records, such as age, gender, weight, height, and cardiovascular risk factors including family history of aortic disease, smoking status, diabetes, dyslipidemia, hypertension, and obesity. Dyslipidemia, diabetes, and hypertension were defined according to the current guidelines.Inclusion criteria are stable heart rate with sinus rhythm and ability to hold breath for at least 12 seconds. Exclusion criteria are known aortic disease, bicuspid aortic valve, previous coronary revascularization, previous acute myocardial infarction or severe heart failure, severe renal impairment (serum creatinine >2 mg/dL), atrial fibrillation, thyroid disorders, unstable clinical condition, known allergy to iodinated contrast agents and pregnancy.A dose of 5 mg atenolol was administered intravenously before the scan if the patients’ heart rate was >65 beats per minute. In addition, all patients received 0.8 mg of isosorbide dinitrate sublingually immediately before the scan.2.1All scans were performed on a 64-slice multidetector CT scanner . The angiographic study was preceded by an unenhanced acquisition to evaluate the distribution and amount of coronary calcium. The calcium score scan was performed using prospective ECG-gating with the following parameters: collimation 20 × 1.2 mm, gantry rotation time 330 ms, feed/rotation 4.8 mm, effective slice width 3 mm, increment 1.5 mm, kV 120, effective mAs 150.The CTCA was performed after intravenous administration of 80 to 100 mL of high iodine concentration contrast agent  at a rate of 4 to 5 mL/s followed by a 40 to 50 mL saline chaser at the same rate. A bolus-tracking technique was used to synchronize the arrival of contrast in the coronary arteries and the initiation of the scan. The following parameters were set: retrospective ECG-gating with prospective modulation of the dose, collimation 32 × 2 × 0.6 mm, gantry rotation time 330 ms, feed/rotation 3.84 mm (pitch 0.2), effective slice width 0.75 mm, reconstruction interval 0.4 mm, medium-smooth B30f reconstruction kernel, kV 120, mAs 700 to 900 (depending on patients’ features).The temporal windows for ECG-gated retrospective reconstructions were set at the end-diastolic  and end-systolic  phases.2.2Image data were analyzed in consensus by 2 experienced operators on a dedicate offline workstation . Aortic diameters (technique used: inner-to-inner) and areas  were measured on diastolic dataset at conventional and reproducible anatomic landmarks, perpendicular to the axis of vessel. The aortic root (AoR) was measured as cusp-to-commissure in correspondence of the maximum diameter, the sinotubular junction (STJ) was measured at the narrowest level in the transition of AoR to the ascending aorta and the tubular ascending aorta (TAo) was taken at the level of the right pulmonary artery. Measurements are represented in Figure 2.3t-test or the Mann-Whitney test was chosen according to variables’ distribution. The 1-way analysis of variance or the Kruskal–Wallis test were used for comparison among 3 groups for parametric and non-parametric variables, respectively. In case of statistical significance, the Bonferroni post-hoc test was chosen. Inter-observer reproducibility was evaluated by intraclass correlation coefficient. Categorical variables were expressed as percentage and compared using the Chi-square test or the Fisher exact test. The Spearman correlation coefficient was used to analyze the relationship between aortic measurements and anthropometric features. Multiple linear regression analysis was performed in order to compare our data with published results.,6,7 Aortic sizes were used as dependent variables and biometric parameters such as age, body surface area (BSA), and height as independent variables. In each analysis, R2 was determined to give the proportion of the variability in the aortic size attributable to demographic variables. A P-value < .05 was considered for statistical significance.Statistical analysis was performed using R Core Team . Continuous variables were expressed as mean ± standard deviation or standard error. Data were tested for normality through the Shapiro–Wilk test. For comparison between 2 groups, the unpaired 3Demographic and clinical characteristics of the study population, stratified by gender, are reported in Table P = .001 for AoR and P < .001 for STJ and TAo; dyslipidemia: P = .024 for AoR, P = .019 for STJ and P = .033 for TAo; hypertension: P = .002 for AoR and P < .001 for STJ and TAo; obesity: P = .030 for AoR, P = .002 for STJ and P = .001 for TAo). Smoking status and diabetes were not significantly associated with aortic dimensions .Among cardiovascular risk factors, familiarity, dyslipidemia, hypertension, and obesity were significantly associated with aortic dimensions (familiarity: P < .001). Intraclass correlation coefficient was 0.971 P < .001 for AoR; 0.970 P < .001 for STJ and 0.976 P < .001 for TAo. Aortic measures were significantly larger in men as compared to women  (P < .001)  and group A versus group C (P < .001) but no statistically significance was found comparing group B versus group C (P = .625 for AoR and P = .112 for STJ). Instead, women presented statistical significance when comparing AoR and STJ in group B versus group C (P = .001 for AoR and P < .001 for STJ) and group A versus group C (P = .024 for AoR and P = .003 for STJ) but not group A versus group B (P = .141 for AoR and P = .057 for STJ). As for TAo diameters, a statistically significant difference was found between all groups (P < .001).Height, weight, body mass index, and BSA were significantly larger in men than in women (P = .49), but not in the other segments (P = .01 for STJ and P < .001 for TAo) .Indexing diameters by BSA eliminated the significant difference between men and women at the level of the AoR (o) Table . On the ρ = 0.372 for men and ρ = 0.373 for women, P < .001).There were weak correlations between the aortic diameters/areas and biometric parameters in both genders Table . TAo corR2 values  as compared to the model including age, gender, and height .Multiple linear regression analysis was performed using BSA and age as independent variables Table  and heigF = 34; M = 67) and the model including age, gender and BSA confirmed the previous results  as compared to the model including age, gender, and height .We conducted a sub-group analysis on patients without any risk factor , contrast media injection can be introduced in the scan protocol to obtain 3D angiograms, and, thanks to its high contrast resolution, it well delineates the blood flow and the vessel wall. CT is the preferred modality for the diagnosis, risk stratification, and management of aortic disease. Despite the use of ionizing radiations and the need for contrast media, it depicts with high spatial and geometric accuracy the aorta and its branches, well identifying both the lumen and the vessel wall as well as the presence of calcifications, atheroma, endoleaks, and dissection flaps.,27Many literature reports published reference values of aortic annulus, AoR, STJ, and TAo established by ultrasound. All imaging modalities entail inherent limitations and the use of ECG-gating during acquisition is essential to properly evaluate the aortic walls and avoid misdiagnosis. Due to intrinsic differences between methods, it is recommended that identical imaging technique should be used for serial measurements and the measure be taken on 3D data when available and using always the same technique.–30It is necessary to adopt standardized measurements to better assess changes in aortic size over time and avoid erroneous findings of arterial growth.4.2 Due to variations in size with patient age, gender, and body surface area, having a single diameter cutoff for abnormal diameter is frequently inaccurate. However, the traditionally accepted values for the upper limits of normal diameter for the sinuses of Valsava and the STJ are 4 cm and 3.6 cm for males and 3.6 cm and 3.2 cm for females respectively. Indeed, it is commonly acknowledged that several cardiovascular risk factors interfere with aortic size such as gender, age, cholesterol, or blood pressure.,24,32,33 Among these, hypertension influences the enlargement of more distal aortic segments while aortitis and genetic aortopathies may affect the proximal ascending aorta. The progression of aortic dilation with age is thought to be related to a higher collagen to elastin ratio, along with increased stiffness and pulse pressure.,11,32–34 For this reason, several authors reported aortic reference values evaluated in selected patients stratified, for example, by age and sex.,20–22,35 Nevertheless, the analyzed cohorts often comprise healthy subjects without coronary artery disease or cardiovascular risk factors.In healthy adults, aortic diameters do not usually exceed 40 mm and taper gradually downstream. this result likely reflects the increased risk of disease for men for a given age. The analysis in groups stratified according to age clearly indicate that aortic dimensions in men predominantly increase faster until 50 years and afterward more gradually while in women there is a turnaround with a slow increase until 50 years becoming noticeable later. These results can be interpreted, at least partly, in light of the protective effect of estrogens before menopause in women. In agreement with literature data,,23,24 all aortic diameters were significantly larger in men than women.According to these studies, in our population women were older, shorter, and weight less than men. As discussed by Nevsky et al,4.3 indexed values comparison denote a greater influence of BSA on AoR diameter while TAo is mainly influenced by height.In line with Muraru and colleagues,,13,16 the correlations between aortic dimensions and biometric parameters in our population provided low values both in male and female.As compared to the aforementioned studies,4.4R2 ranging from AoR to TAo. When the effect of age and risk factors was removed, an increased trend in R2 was observed both in male and female. In detail, the sub-analysis conducted in patients without risk factors, even if it constitutes a smaller sample, revealed that R2 values slightly increase. The highest correlations and R2 obtained for TAo underline that age and anthropometrical features have more influence on ascending aorta than AoR and STJ, reflecting a physiologic and mechanic cause. Moreover, in light of our results normal reference values and indexed parameters should be carefully considered when examining patients.Multiple linear regression analyses reflect this key point. In fact, aortic diameters were independently associated with age, gender, and BSA with a decreasing trend in 4.5Our study has some limitations: though patients were enrolled consecutively it is not a prospective study; we evaluated aortic measures only in 1 cardiac phase, in end-diastole, so the aortic distension during cardiac cycle was not examined. Moreover, we only focused on aortic size not analyzing its association with coronary artery disease.4.6There is currently the need to switch to investigational and observational studies and registries which should be able to use many more parameters and most probably other parameters. With this we mean that there is a need to introduce more complex assessment of available parameters and more parameters that are going to become what is currently defined as radiomics.5In conclusion, in our population CT aortic dimensions measured at AoR, STJ, and TAo showed low correlations with biometric parameters when we considered the potential influence of patients’ cardiovascular risk factors. This finding in a large cohort of consecutive patients highlights the difficulty of identifying normal ranges, as well as issues related to normalization using conventional biometric parameters.1. Guarantor of integrity of the entire study: Filippo Cademartiri.2. Study concept and design: Ernesto Forte, Carlo Cavaliere, Filippo Cademartiri.3. Literature research: Bruna Punzo.4. Clinical study: Erica Maffei, Filippo Cademartiri, Stefano Nistri.5. Experimental studies/data analysis: Erica Maffei, Filippo Cademartiri, Stefano Nistri.6. Statistical analysis: Ernesto Forte7. Manuscript preparation: Ernesto Forte, Bruna Punzo, Carlo Cavaliere.8. Manuscript editing: Marco Salvatore, Erica Maffei, Stefano Nistri.