Fragaria × ananassa) fruits are rich in ascorbic acid (AsA) and anthocyanin, which are essential antioxidants for human health. However, the underlying regulatory mechanism of these antioxidant accumulation, especially AsA accumulation in strawberry fruits, remains largely unknown. In this study, we identified FaAKR23 was a regulator of AsA and anthocyanin accumulation. We transiently expressed FaAKR23 in strawberry fruits and conducted metabolic and molecular analyses to explore the role of FaAKR23 in AsA and anthocyanin accumulation. Transient silencing of FaAKR23 (FaAKR23-RNAi) in strawberry fruits significantly decreased the AsA and anthocyanin contents compared with control . Correspondingly, expression of some structural genes and regulatory factors involved in these two antioxidants’ accumulation was dramatically repressed. In addition, transcriptome analysis of EV-RNAi and FaAKR23-RNAi fruits suggested that FaAKR23 was also involved in starch and sucrose metabolism as well as plant–pathogen interaction. Overall, these results not only provide the coordinated regulatory function of FaAKR23 on AsA and anthocyanin accumulation but also offer a promising candidate gene for strawberry breeding with high antioxidants.Strawberry ( Fragaria × ananassa), a worldwide fruit, has excellent commercial value and health benefits [Strawberry  pathway is the major pathway for AsA biosynthesis in higher plants [d-mannose pyrophosphorylase (GMP), GDP-d-mannose-3′,5′-epimerase (GME), GDP-l-galactose phosphorylase (GGP), l-galactose-1-phosphate phosphatase (GPP), l-galactose dehydrogenase , and l-galactono-1,4-lactone dehydrogenase  [d-GalUA) pathway is another validated AsA biosynthetic pathway in plants; it was first identified in strawberry plants [d-GalUA reductase  that catalyzes a key step in the d-GalUA pathway [FaGalUR in Arabidopsis, potato, and tomato led to a 2~3-fold increase in AsA content in the transgenic plant leaves and fruits compared with control [FaGalUR plays a significant role in AsA biosynthesis and can be used as a target to improve AsA content in strawberry fruits by genome editing [AsA is an important water-soluble antioxidant that is indispensable for eliminating reactive oxygen species (ROS), thereby keeping cell redox levels stable and maintaining normal activities in plants. The r plants ,4, whichy plants . In 2003 pathway . Overexp control ,7,8. The editing ,10.FaMYB10, FaGAMYB, FaMYB9/11, FabHLH3, FaTTG1, FaRAV1, FaMADS1a, FaMADS9, FaRAP, and FaRIF [Anthocyanin is the major pigment contributing to strawberry fruit coloring, which influences the commodity and nutritive value of strawberries. Moreover, anthocyanin is important antioxidant in plants, and it plays a key role in exogenous stress tolerance . The biond FaRIF ,19,20,21nd FaRIF ,23,24,25AKR1A performs the function of glucuronolactone reductase; knockout AKR1A induces the lack of AsA in mice [AKR1A might participate in AsA biosynthesis in mice. In plants, AKRs plays an important role in the accumulation of antioxidants and stress tolerance [FaGalUR is an important enzyme in the d-GalUA pathway for AsA synthesis [MsALR, in alfalfa; the heterologous expression of MsALR in tobacco significantly improved its tolerance to herbicide and redox damage caused by heavy metals [PsAK1R or OsAKR1 prominently increased herbicide resistance [2O2 level, elevating the chlorophyll content, and, finally, enhancing salt tolerance [Prunus persica), expression of the aldo-keto reductase gene PpAKR1 was induced by ABA, oxidative stress, low temperature, and salt stress; furthermore, PpAKR1 enhanced salt resistance [Aldo-keto reductases (AKRs) are a superfamily of NADH-dependent oxidoreductases, including aldehyde reductase, aldose reductase and xylose reductase . AKRs ar in mice , which iolerance ,29; for ynthesis . Oberschsistance , and theolerance . In peacsistance .FaAKR23 was identified as the most dramatically increased FaAKR gene during fruit development and ripening in cultivated strawberries [FaAKR23 is a regulator of AsA and anthocyanin accumulation in strawberry fruits. The results not only provide the coordinated regulatory function of FaAKR23 on AsA and anthocyanin accumulation but also offer a promising candidate gene for strawberry breeding with high antioxidants.According to our previous work, wberries . HoweverF. × ananassa cv. ‘Sweet Charlie’) were grown in a greenhouse under natural sunlight conditions at the Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China. Fruit ripening stages were classified based on days post-anthesis (DPAs), including small green (SG), big green (BG), white (W), turning (T), and red (R) [Octoploid strawberries ( red (R) . The colhttps://www.rosaceae.org, accessed on 1 January 2022), and the reported AKR proteins in Arabidopsis thaliana, Oryza sativa L., and Prunus persica were acquired from NCBI. The phylogenetic tree was constructed via the neighbor-joining method in MEGA version 7.0 software with 1000 bootstrap replicates. The protein sequence alignment was performed using Clustal X version 2.1 with default parameters [Based on our previous study , the FaArameters  The sequFaAKR23 without a stop codon was amplified by PCR, cloned into the pCAMBIA-super1300 vector, and then transformed into Agrobacterium tumefaciens strain GV3101. Agrobacteria cultures were resuspended in infiltration buffer to a final OD600 = 0.6 and then were infiltrated into epidermal cells of 4- to 6-week-old tobacco leaves. The infiltrated tobacco leaves were incubated at 23 ℃ under an 8 h/16 h light/dark cycle for 48–72 h; after that, the leaves were subjected to green fluorescent protein fluorescence observation using a confocal laser scanning microscope (Nikon A1). The primers used for cloning are listed in The full-length coding sequence (CDS) of FaAKR23. The full-length FaAKR23 CDS was PCR amplified from the strawberry genome and recombined into the pCAMBIA-super1300 vector. To construct the FaAKR23-RNAi vector, two fragments of the FaAKR23 CDS were inserted into vector pFGC5941 in the opposite orientation on either side of the chalcone synthase A intron. FaAKR23-OE, FaAKR23-RNAi, and empty vectors (EV-OE and EV-RNAi) were introduced into the Agrobacterium tumefaciens strain GV3101, and the agrobacterium cells were cultured at 28 °C in LB medium. The agrobacterium cultures were resuspended in infiltration buffer (10 mM MES (pH 5.6), 10 mM MgCl2, and 100 µM acetosyringone) and adjusted to OD600 = 0.8. The agrobacterium suspension was injected in the octoploid fruits at the BG stage, with consistent growth status when they became hygrophanous. After 7 days, these injected fruits were collected and frozen in liquid nitrogen immediately and then stored at −80 °C for further analysis. For each individual experiment, three biological repeats were performed; each repeat consisted of 10 fruit individuals. The primers used in vector construction are provided in The overexpression vector pCAMBIA-super1300 and RNAi vector pFGC5941 were used to overexpress and silence FaAKR23 or empty vector was ground to powder under liquid nitrogen. Approximately 1 g powder was mixed with 5 mL methanol-0.05% HCl and then extracted at 4 °C under dark for 12 h. The supernatant was collected by centrifugation for further analysis. Anthocyanin content was determined by the pH-differential method using buffer solutions of sodium acetate  and potassium chloride , and the absorbance was read at 530 and 700 nm, respectively [Strawberry fruits transient expression of ectively .w/v) metaphosphoric acid and 2 mM EDTA, chilled on ice for 20 min, and centrifuged at 13,000 rpm for 20 min at 4 °C. The 15 µL filtered supernatant was injected into the HPLC system. The mobile phase consisted of 0.1 M sodium acetate/acetic acid buffer and 5% methanol (pH 5.8), and AsA was detected using a C18 5 µm 25 × 0.46 column. Total AsA was calculated by comparison with the standard curve using the peak areas at 254 nm [The total AsA content of strawberry fruits was determined by high-performance liquid chromatography (HPLC) (Waters 2695). Briefly, 5 g frozen samples in powder were suspended in 2%  according to the manufacturer’s instructions. The first-strand cDNA was synthesized from 1 μg total RNA with EasyScript® First-Strand cDNA Synthesis SuperMix . The qRT-PCR was performed on a CFX96TM Real-Time System  using TB GreenTM Premix Ex TaqTM II . FaCHP1 was used as the internal reference to standardize the transcript level [−ΔΔCt method. Three biological replicates were repeated. The primers are listed in Total RNA was extracted from strawberry fruits using an E.Z.N.A.pt level . RelativFaAKR23-RNAi using the E.Z.N.A.® Total RNA Kit . RNA concentration and purity were measured using Nano Drop 2000 . RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system . Sequencing was performed on an Illumina genome analyzer at the Biomarker Technologies Corporation. Genes with an adjusted FDR of <0.01, as determined by DESeq2, were considered differentially expressed [http://www.ncbi.nlm.nih.gov/sra, accessed on 15 August 2022).Total RNA was extracted from the strawberry fruits of EV-RNAi and xpressed . Three it-tests .Statistical analysis was performed with GraphPad Prism (version 8.0); statistically significant differences between samples were calculated using two-tailed Student’s FaAKR23 was the highest in receptacles and that its expression levels increased by 142-fold from the green fruit stage to the red fruit stage, followed by the FaAKR24, which is the homologous gene of FaGalUR [FaAKR23. To analyze the evolutionary relationship among these reported AKR members in Arabidopsis thaliana, Oryza sativa L., Prunus persica, and Fragaria × ananassa [In our previous work, we identified that the transcript level of  FaGalUR . In thisananassa ,38,39,40FaAKR23, we firstly examined the FaAKR23 transcription levels in different tissues of octoploid strawberry F. × ananassa cv. ‘Sweet Charlie’  tag, driven by the CaMV 35S promoter. We transiently expressed the FaAKR23-GFP fusion protein in tobacco leaf cells. Visualization of the fused protein showed that FaAKR23 has a dual localization in both nucleus and cytoplasm D.FaAKR23 in strawberry fruit ripening and quality formation, we transiently expressed FaAKR23 in octoploid strawberry fruits via agroinfiltration. The visual phenotype suggested that FaAKR23 was involved in anthocyanin accumulation  using EV-RNAi and FaAKR23-RNAi fruits. FDR < 0.01 and fold change ≥ 2 were set as the threshold for significantly differential expression  classification of differentially expressed genes (DEGs) in EV-RNAi and ormation . Furtheregulated . These rThe AKR superfamily is a large enzyme group of oxidoreductases that mainly use NADP(H) as a cofactor; this group is widely distributed in animals, plants, yeast, and bacteria and regulates cell oxidation reduction under multiple stress conditions. Although AKRs play numerous roles in the metabolism of steroids, sugars, and other carbonyls, many AKR superfamily members remain uncharacterized .FaAKR genes were identified from the genome and divided into 20 subgroups [FaGalUR (FaAKR24) has been characterized and proven to play an important role in AsA biosynthesis [FaGalUR in Arabidopsis, potato, and tomato leads to a 2~3-fold increase in AsA content in the transgenic plant leaves and fruits compared with the wild-type [FaAKR23 was the most extremely upregulated FaAKR gene during fruit development and ripening [FaAKR23 and FaGalUR (FaAKR24) were in a close phylogenetic relationship, and AtA6PR1 and AtA6PR2 were also located in the same cluster (AtA6PR1 and AtA6PR2 were proposed to be related to the aldose 6-phosphate reductase pathway, which is involved in sorbitol synthesis. Sorbitol is the main form of photosynthetically fixed carbon, translocated via the phloem from source to sink organs in plants [FaAKR23 might be involved in strawberry fruit ripening and quality formation.In octoploid strawberry, a total of 80 ubgroups . Among tynthesis . Overexpild-type ,7,8. Howripening  (Figure  cluster A. Previon plants ; consequFaAKR23 by transient expression in strawberry fruits and conducted metabolic and molecular analyses. The results demonstrated that FaAKR23 is indeed involved in AsA and anthocyanin accumulation through the regulation of the transcription level of downstream structural genes as well as some regulator factors of these two metabolic pathways. AsA content obviously decreased in the FaAKR23-RNAi fruits (FaGalUR, FaGGP, and FaGME (FaAKR23 might work as a transcription factor (ERF98 [ABI4 [bHLH59 [SlHZ24 [VTC1, GGP, and GME to promote or reduce AsA synthesis. More recently, in kiwifruit, ethylene response factor AcERF91 has been found to increase the expression of AcGGP3 [AceMYBS1 and AceGBF3 influence AsA biosynthesis by activating the transcription of AceGGP3 [FaAKR23 in AsA accumulation be further clarified.To confirm this deduction, we manipulated i fruits C. The RNnd FaGME A. Interen factor D. To datr (ERF98 ; ABI4 [498 [ABI4 , the bas [bHLH59 , the HD- [SlHZ24 , and SlN [SlHZ24  bind to f AcGGP3 , and tra AceGGP3 . ConsistFaAKR23-RNAi fruits (FaAKR23-RNAi transcriptome data and qRT-PCR analysis, the transcript levels of anthocyanin biosynthetic genes FaCHS, FaCHI, FaF3H, FaDFR, and FaANS were significantly repressed. Moreover, the expression of FaMYB10 was decreased in FaAKR23-RNAi fruits; it is a powerful and conserved regulator of anthocyanin biosynthesis in diploid and octoploid strawberry fruits [CHS1, CHI, DFR, F3H, and DFR individuals by forming an MYB–bHLH–WD complex, thereby regulating anthocyanin accumulation [FaRAV1, FaRAP, FaSnRK2.6, FaMADS9, FaRIF, FaMYB9/MYB11, and FaGAMYB [FaAKR23 and FaMYB10 results in anthocyanin accumulation or whether FaAKR23 regulates anthocyanin by directly binding to the promoter of FaMYB10.In addition, anthocyanin was also dramatically decreased in i fruits D, along i fruits B. In FaAy fruits ,13,14,21 FaGAMYB ,19,20,21 FaGAMYB ,23,24,25FaAKR23 is overexpressed in strawberry fruits, FaAKR23-OE fruits do not show higher content of either AsA or anthocyanin compared to EV-OE fruits (GGP or GalUR genes are overexpressed in plants, the AsA level did not increase as remarkably as that of gene expression; in some cases, AsA was only moderately promoted [FaMYB10, to improve the biosynthesis of the two antioxidants.Nevertheless, when E fruits . We specpromoted ,50. MoreFaWRKY transcription factors was significantly upregulated and that 3 FaWRKY members were downregulated in FaAKR23-RNAi transiently transformed fruits (WRKY genes compose one of the largest families of transcription factors in land plants and are characterized by the highly conserved WRKY domain; WRKY proteins have been reported to play key roles in regulating growth, development, and biotic and abiotic stresses [FaWRKY1 has been characterized and involved in mediating defense responses to the fungus Colletotrichum acutatum [FvPLA promoter to control fruit softening [FaAKR23 may play multiple roles in strawberry in addition to AsA and anthocyanin accumulation in fruits.Furthermore, RNA-seq data revealed that many DEGs were involved in plant–pathogen interaction, starch and sucrose metabolism, and the MAPK signaling pathway. We discovered here that the expression of 23 d fruits . WRKY gestresses ,52,53,54acutatum ; moreoveoftening . These rFaAKR23 that modulates downstream structural genes and regulatory factors should be addressed, and the interacting proteins that assist FaAKR23 in regulating AsA and anthocyanin content need to be further identified. In addition, whether plant hormone or abiotic stress acts as upstream signaling to regulate the expression of FaAKR23 and then is involved in AsA and anthocyanin accumulation needs further research evidence to be clarified. Overall, our study provides new clues about the regulatory mechanisms of AsA and anthocyanin accumulation and offers a promising candidate gene for strawberry breeding with high antioxidants.To sum up, the specific regulatory mechanism of FaAKR23 is a regulator of AsA and anthocyanin accumulation in strawberry fruits by metabolic and molecular analyses. From the transcriptome data and qRT-PCR analysis, the transcript levels of the AsA and anthocyanin metabolism-related genes were significantly regulated in the FaAKR23-RNAi fruits compared with EV-RNAi fruits. Moreover, FaAKR23 might be involved in the starch and sucrose metabolism and plant–pathogen interaction. Finally, this study provides new genetic clues for elucidating the regulation mechanism of AsA and anthocyanin accumulation. However, further research needs to be conducted on the specific molecular mechanism of FaAKR23-mediated AsA and anthocyanin accumulation in strawberry fruits.AsA and anthocyanin are crucial antioxidants that not only maintain the normal growth and development of plants but also contribute to human health and disease prevention. From this study, we demonstrate, for the first time, that


Malignant melanoma is an aggressive cancer of the skin and the leading cause of death from skin cancer. One major risk factor linked to melanoma development is exposure to UV radiation. However, the sharp increase in melanoma cases cannot be explained only by more UV exposure. Identifying additional modifiable social-environmental risk factors for melanoma beyond UV exposure would greatly impact public health initiatives and the methods of patient outreach and education. Recent studies have shown the link between melanoma and alcohol consumption. This perspective review paper aims to understand the mechanisms underlying ethanol’s ability to induce human cancers, including melanoma.Although cancer mortality has declined among the general population, the incidence of melanoma continues to rise. While identifying high-risk cohorts with genetic risk factors improves public health initiatives and clinical care management, recognizing modifiable risk factors such as social-environmental risk factors would also affect the methods of patient outreach and education. One major modifiable social-environmental risk factor associated with melanoma is ultraviolet (UV) radiation. However, not all forms of melanoma are correlated with sun exposure or occur in sun-exposed areas. Additionally, UV exposure is rarely associated with tumor progression. Another social-environmental factor, pregnancy, does not explain the sharply increased incidence of melanoma. Recent studies have demonstrated that alcohol consumption is positively linked with an increased risk of cancers, including melanoma. This perspective review paper summarizes epidemiological data correlating melanoma incidence with alcohol consumption, describes the biochemical mechanisms of ethanol metabolism, and discusses how ethanol and ethanol metabolites contribute to human cancer, including melanoma. Cutaneous melanoma is an aggressive malignancy of the skin and a significant public health concern. Melanoma incidence continues to rise globally, placing a greater burden on industrialized countries ,2,3,4,5.CDKN2A), CDK4, BRCA1-associated protein-1 (BAP1), protection of telomeres 1 (POT1), and telomerase reverse transcriptase (TERT) . We. We43]. We conducted a literature search in Medline through Pubmed for epidemiological studies from 1977 to 29 July 2022. We designed the search strategy using the following keywords “melanoma” AND  OR  without restrictions on the geographic area, language, or publication status. The whole search is outlined in Two authors (T.Y. and Z.Z.) independently screened the titles and abstracts of all retrieved records. Potentially relevant titles and abstracts were recorded, and the full-text articles were screened for final eligibility. Disagreements were resolved through discussion with a third author (M.F.).#C6, C9, C21, C25, C27). Alcohol consumption is positively correlated with melanoma incidence in 15 studies , negatively correlated in two studies , and not correlated in 12 studies .The association between consumption and melanoma incidence was first suggested by Williams et al. in 1977 from the Third National Cancer Survey of 7518 patients with invasive cancer, including melanoma . SubsequC14, where no association was found between alcohol consumption and uveal melanoma. More studies are needed to assess the relationship between non-cutaneous melanoma and alcohol consumption.Most studies assessed cutaneous but not non-cutaneous melanoma. Because non-cutaneous melanoma is not related to UV exposure, the potential risk factors of non-cutaneous melanoma may include other social-environmental risk factors such as alcohol consumption. However, no studies assessed the link between alcohol consumption and mucosal melanoma. Uveal melanoma was assessed only in #While some cohort and case-control studies shown in #M1), who showed that alcohol consumption was positively associated with melanoma risk (pooled relative risks (pRR) = 1.20; p = 0.006) by analyzing 14 case-control and two cohort studies of 6251 melanoma cases [#M2) reported a pooled analysis of eight melanoma case-control studies in women [p < 0.05). Bagnardi et al. (#M3) investigated 572 studies on the effect of alcohol on 23 cancer types [#M4) used three large prospective cohort studies that followed 210,252 participants from the United States for 18.3 years [Ptrend < 0.04). Interestingly, they also assessed 835 melanoma in situ cases and found a stronger association than invasive melanoma . Recently, Mehta et al. reported a strong association between alcohol consumption and melanoma risk  by analyzing five reports [#M2), which weighted 52.7% of their study. Similarly, Gandini et al. analyzed 20 independent studies  and reported that alcohol intake was moderately associated with melanoma risk (summary relative risk (SRR) = 1.29 for the highest category vs. the lowest category of alcohol intake and SRR = 1.95 for cumulative intake) [M2), a meta-analysis by Rivera et al. (#M4), and a cohort study by Allen et al. (#C21) contributing to 80% of the controls; therefore, we excluded two meta-analyses studies by Metha et al. and Gandini et al. in The first report came from Rota et al. in 2014 (ma cases . The folin women . After aer types . While c.3 years . They fo reports . However intake) . HoweverCollectively, these four meta-analyses indicate a moderate detrimental effect of alcohol consumption on melanoma incidence.#C1, C2, C3, C4, C6, C10, C13, C14, C15, C16, C18, C19, C20, C21, C22, C23, C25, C26, C28, C29). While seven studies showed no correlations , 13 studies clearly showed alcohol dose-dependent effects .Alcohol beverages can have both beneficial and detrimental impacts on human health, which could be influenced by ethanol amounts . Therefo#C15) conducted a case-control study by recruiting 502 newly diagnosed patients with melanoma and 565 controls from outpatient clinics and computed ORs for melanoma and alcohol intake using logistic regression analysis [P for trend = 0.003). They also analyzed the relationship between melanoma and the number of alcoholic beverages and found that individuals who consumed ≥ 1.4 drinks per week had an increased risk for melanoma (OR = 1.55). While the data were not shown, they also mentioned that the magnitude of melanoma risk with increasing alcohol consumption was greater among women than men.For example, Millen et al.  followed 59,575 White postmenopausal women for over 10 years and examined the relationship between melanoma and alcohol consumption using Cox proportional hazards models [Pglobal = 0.0013). Lifetime alcohol consumption was also positively associated with melanoma risk (p = 0.0046).A prospective cohort study by Kubo et al. (s models . Clinica#C26) examined cancer incidence in 124,193 men and women from 1978 to 1985. They found that moderate (one to two drinks per day) or heavy (≥three drinks per day) alcohol drinking was associated with an increased risk of five cancers, including melanoma but not 12 other cancer types [p < 0.001) or 2.2 (p < 0.001) for moderate or heavy drinkers, respectively, compared to lifelong alcohol abstainers. Interestingly, light drinking (<one drink per day) increased the risk of only two cancers: melanoma  and breast cancer .A retrospective cohort study by Klatsky et al. (er types . The HR #M1) noted a dose-risk relationship between alcohol consumption and melanoma risk, with pRRs of melanoma of 1.10 for light drinking  and 1.18 for moderate-heavy alcohol drinking  [#M2) could not find a dose-response association from his pooled analysis [#M3) is similar to that by Rota et al. While pRRs were 1.11 and 1.20 for light  and moderate  drinkers, they could not evaluate the effect of heavy drinking  on the risk of melanoma due to limited data [Meta-analysis studies in alcohol) . Howeveranalysis . The treted data .#M4) conducted a combined dose-dependent analysis and showed an alcohol dose-dependent association with melanoma [On the other hand, Rivera et al.  by plotting each country’s data and calculating spearman’s rank correlation coefficient for melanoma incidence and mortality with alcohol consumption [R = 0.59; p < 0.001).We also analyzed the World Health Organization (WHO) database globally and reported a strong positive correlation between alcohol consumption amount and melanoma incidence , were more likely to develop blisters , and had a higher frequency of analgesic use after sunburn  [#C6, C8, C12, C13, C15, C16, C17, C18, C19, C20, C23, C25). Among them, eight studies clearly showed UV-independent alcohol effects on melanoma .Several studies provided the relationship between alcohol consumption and UV exposure, a significant risk factor for melanoma. Warthan et al. analyzed 56 sunburned beachgoers and reported that alcohol drinkers had a greater body surface area sunburned than non-drinkers  . Mukamal= 0.007) . Therefo#M1) showed that the pRR of melanoma from alcohol drinking decreased from 1.25 to 1.15 after adjusting for UV exposure [Meta-analysis studies in exposure . This daexposure .#M2) demonstrated a positive correlation between alcohol intake and melanoma incidence after adjustment for sun exposure  [Pinteraction = 0.043): Among those who never used tanning devices, alcohol consumption was positively associated with melanoma  whereas this association was not observed for those who ever used tanning devices . They further assessed the influence of anatomic sites and found that alcohol drinkers have an increased risk for trunk melanoma  but not other sites, such as head, neck, and extremities, which are more sun-exposed [#M4) also showed a stronger association between alcohol consumption and melanoma in UV-protected skin (trunk) compared to UV-exposed skin  (1.73 vs. 1.03 HRs) [On the other hand, Miura et al. (1.1–1.5) . They fu-exposed . Rivera .03 HRs) .In conclusion, many epidemiological studies demonstrated an association between alcohol consumption and melanoma. However, they do not necessarily indicate a causal relationship. The association seems to be alcohol dose-dependent and UV-independent, although alcohol and UV could work synergistically. Genetic predispositions and geographical influences may also add to the observed variance.The following section will explore how alcohol consumption can contribute to melanoma development and progression.Absorption of orally administered alcohol beverages depends on ethanol concentration, blood flow, rate of ingestion and gastric emptying, beverage type, food intake, and the irritant properties of ethanol . When inThe putative benefits from alcohol intake come from various factors, including ethanol amount and ingredients . For exa2O2, hydroxide ion (OHˉ), and peroxide ion (O2ˉ)) [However, the alcohol-mediated benefits disappear and are overridden by toxicity following heavy drinking and chronic consumption. In addition to the harmful intermediate metabolite AcAH, cytochrome p450 2E1 (CYP2E1) is induced and activated in response to high doses of ethanol, resulting in increased ROS ) . IARC can (O2ˉ)) . Ethanoln (O2ˉ)) ,90. AcAHn (O2ˉ)) ,92,93.This chapter will review the roles of ethanol or AcAH in cellular biology and speculate on potential mechanisms connecting ethanol or AcAH to melanoma initiation and progression.Ethanol and/or AcAH induce oxidative stress, DNA damage, and lipid peroxidation, which activate protein kinases and signaling pathways implicated in glycolysis, fatty acid oxidation, inflammation, differentiation, angiogenesis, and metastasis, thereby creating a favorable microenvironment for tumor initiation and progression .Both ethanol and AcAH promote oxidative stress. After ethanol uptake, ADH-catalyzed reactions in the cytosol and ALDH2-mediated reactions in the mitochondria reduce an NAD+/NADH redox ratio , regenerAcAH is a highly reactive metabolite and a mutagen. AcAH-mediated DNA damage includes adduct formation, double-strand breaks, point mutations, DNA-DNA cross-links, sister chromatid exchanges, and chromosomal aberrations . AcAH biAnother mutagenic effect of ethanol and/or AcAH is mediated by CYP2E1 induction, resulting in increased ROS generation. ROS-induced lipid peroxidation products such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) are genotoxic, thus generating mutagenic DNA adducts . In addiThe abovementioned changes activate multiple signal transduction mechanisms, such as cAMP/PKA signaling ,103,104,Lastly, ethanol and its metabolites damage various types of progenitor/stem cells, such as embryonic stem cells and tumor-initiating cells, impairing cell differentiation and genomic stability, leading to cellular aging and carcinogenesis ,121.Most research on alcohol consumption is centered around its effects on the liver and gastrointestinal tract but rarely on the skin ,123. NonCutibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis possess ADH to convert ethanol to AcAH [However, it is crucial to consider ethanol’s direct effects on skin cells. For a long time, it has been known that ingested ethanol is secreted by the eccrine glands of human skin ,130, wit to AcAH . Therefo to AcAH ,135,136  to AcAH ,138,139 In addition to oxidative metabolism, a smaller fraction of ethanol undergoes a non-oxidative route of metabolism , similar to genetic changes of human melanoma for CDKN2A, encoding p16INK4a and p19ARF [Strickland et al. reported that treating C3H/HeNCr mice with UV light and topical ethanol application (25% in water) thrice weekly for about 30 weeks induced primary cutaneous melanoma in 20 to 30% of the mice . The freh tumors , which oh tumors . BRAF angression . Active ignaling . Furtherd p19ARF .While UV is more frequently associated with the development of non-melanoma skin cancer than melanoma, it is unclear how the combination of ethanol and UV induced a relatively equal number of melanoma tumors compared to squamous cell carcinoma . As ethaConsidering these data collectively, it is likely that such signaling pathways are critical components of ethanol-induced carcinogenesis in some cancers, including melanoma.w/v) ethanol for >4 weeks) inhibited spontaneous and experimental metastases [Several studies have reported the biological effects of chronic alcohol consumption on melanoma progression and metastasis. Tan et al. found that ethanol-treated B16F10 melanoma tumors exhibited enhanced angiogenesis through increased vascular endothelial growth factor (VEGF) expression, contributing to tumor progression . On the tastases ,177. Thetastases , implicaSeveral mechanisms could be involved in alcohol-mediated melanoma progression and metastasis. For example, ethanol disturbs mitochondrial dynamics  by increAnother consequence of chronic and acute alcohol consumption is interference with immune cell numbers and function, which may facilitate melanoma progression and metastasis ,186. B16Ethanol can also upregulate the expression of nerve growth factor receptor (NGFR/CD271) through NF-κB signaling in human melanoma cells . NGFR exTogether, the accumulated data suggest a link between chronic alcohol consumption and melanoma progression. Further studies are required to ascertain these findings, especially in human melanoma patients, and elucidate the underlying mechanisms and biology. In addition to the factors mentioned above, the role of other potential factors such as genetic instability, metabolic rewiring, and skin microbiota need to be determined for a comprehensive understanding of alcohol-associated melanoma progression.Despite greater public health and UV protective efforts, melanoma incidence has continued to increase, warranting further exploration into alternative, modifiable social-environmental risk factors that may be implicated in melanoma pathogenesis. In addition to UV exposure, ethanol and AcAH have each been found to cause genetic changes associated with carcinogenesis. Specifically, ethanol and AcAH’s carcinogenic activity is tied to their ability to induce various forms of DNA damage and signaling activation. However, the particular role of these adducts and pathways in melanoma initiation remains unclear. Recent epidemiology studies revealed a positive correlation between alcohol and melanoma incidence. It seems clear that chronic alcohol drinking produces persistent and complex changes in skin cell components, metabolic activities, and signaling networks that lead to a progressive pathological transition in physiological responses. Equally important, chronic alcohol consumption leads to disturbances in ethanol metabolism, an inflammatory milieu, and an immunosuppressive state that contributes to melanoma initiation, progression, and metastasis. These effects of alcohol could be synergistic with or independent of UV exposure. Understanding the mechanism underlying the causal relationship between alcohol consumption and melanoma initiation is still in its infancy. Further research is needed to determine the roles of ethanol and AcAH in melanoma development.


SIRT1 plays critical roles in regulating and integrating cellular energy metabolism, response to stress, and circadian rhythm by modulating epigenetic and transcriptional regulation, mitochondrial homeostasis, proteostasis, telomere maintenance, inflammation, and the response to hypoxia. SIRT1 expression and activity decrease with aging, and enhancing its activity extends life span in various organisms, including mammals, and improves many age-related diseases, including cancer, metabolic, cardiovascular, neurodegenerative, respiratory, musculoskeletal, and renal diseases, but the opposite, that is, aggravation of various diseases, such as some cancers and neurodegenerative diseases, has also been reported. Accordingly, many natural and synthetic SIRT1 activators and inhibitors have been developed. Known SIRT1 activators of natural origin are mainly polyphenols. Nonetheless, various classes of non-polyphenolic monoterpenoids have been identified as inducers of SIRT1 expression and/or activity. This narrative review discusses current information on the evidence that supports the role of those compounds as SIRT1 activators and their potential both as tools for research and as pharmaceuticals for therapeutic application in age-related diseases.Sirtuin 1 (SIRT) is a class III, NAD The 20th century brought about an unprecedented improvement in living conditions for a large fraction of the world population, which, along with the development of many new drugs, namely anti-microbial drugs, including vaccines, resulted in a significant reduction of childhood mortality and extension of longevity. Even though such improvements are highly unevenly distributed throughout the world, the outcomes are still impressive worldwide. Many diseases, however, are still incurable, and the increased lifespan is not yet accompanied by a parallel increase in health span. Indeed, as aging is the major risk factor for many diseases, increased longevity is associated with a higher incidence and thus prevalence of most chronic diseases from cardiovascular, neurodegenerative, metabolic, musculoskeletal, kidney, and respiratory diseases to cancer. These diseases pose a huge burden on individuals as well as on societies with increasing, soon-to-be unbearable costs ,2.Regardless of controversies concerning the definition and understanding of aging and its relation with disease, a consensus exists as to the need of urgent measures to promote healthier aging . Among p+-dependent deacylating enzymes, are of special interest. SIRTs, which in humans comprise seven members (SIRT1-7), are class III histone deacetylases (HDAC) that differ from other classes of the same family in that the deacetylation reaction occurs in two steps that consume nicotinamide adenine dinucleotide (NAD+), resulting in the formation of the deacetylated protein, nicotinamide, and 2′-O-acetyl-ADP-ribose [Among those, sirtuins (SIRTs), a family of NADrs SIRT1-, are clars SIRT1-, are claP-ribose ,9.+/NADH ratio, increase the ability of cells to cope with stress conditions. Therefore, SIRTs play fundamental roles in many cellular processes that link energy metabolism and stress responses, being involved in epigenetic and transcriptional regulation, mitochondrial homeostasis, proteostasis, telomere maintenance, inflammation, and the response to hypoxia. These processes regulate crucial cell functions from DNA repair to proliferation, survival, differentiation, and circadian rhythms, all of which are critically dysregulated in aging and age-related diseases [SIRTs act as nutrient and metabolic sensors that, by responding to changes in the NADdiseases ,10,11,12diseases ,10,13,14The discovery of small molecule activators and inhibitors of SIRTs, particularly of SIRT1, further incremented research on the pathophysiologic roles of these enzymes and their potential therapeutic applications ,16,17 evHere, we review and discuss current information on the evidence that supports the role of natural, non-polyphenolic, monoterpene compounds as SIRT1 activators and their potential both as tools for research and as pharmaceuticals for therapeutic applications in aging and related diseases. We will also highlight some controversies and hurdles that still persist about SIRT1 and its potential to slow down aging and age-related diseases.Silent Information Regulator 2 (Sir2) gene and the first NAD+-dependent deacetylase [Saccaharomyces cerevisiae, in response to stress conditions, namely to calorie restriction [Among the mammalian SIRTs, SIRT1 is the most studied, as it was the first to be identified as the mammalian ortholog of Sir2, a protein encoded by the cetylase ,28 foundtriction . Shortlytriction ,31,32.Besides longevity extension, SIRT1 has been attributed many beneficial effects in a variety of age-related diseases ranging from metabolic disturbances, such as obesity and diabetes mellitus, especially type 2, as well as cardiovascular, neurodegenerative, pulmonary, renal, musculoskeletal, autoimmune, and skin diseases to cancer ,39,40,41Dysregulation of these mechanisms is associated with aging and related diseases, and increased SIRT1 expression and/or activity has been shown to improve life- and health-span, at least in part, by modulating those mechanisms ,43,44. I+ levels, which in turn determine SIRT1 activity. NAD+ production depends on the expression and activity of the enzyme nicotinamide phosphoribosyltransferase (NAMPT or visfatin), which is rhythmically induced by the core complex BMAL1:CLOCK. Furthermore, SIRT1 associates with BMAL1:CLOCK at the NAMPT promoter, contributing to the rhythmic circadian expression of this enzyme and, therefore, to circadian oscillations of NAD+ levels and its own activity [The interaction between SIRT1 and the clock system is complex. On one hand, SIRT1 and Per2 are involved in an antagonistic crosstalk in which SIRT1 deacetylates histone (H) 4 K16 in the Per2 promoter, decreasing its expression, while Per2 binds to the Sirt1 promoter, preventing BMAL1:CLOCK-induced transcription ,46. On tactivity . The relactivity ,49,50,51+ levels [SIRT1 and the clock system are also important regulators of the cellular response to stress by reciprocally modulating NF-κB. This transcription factor is the central regulator of the cellular response to stress, inflammation, and immune responses and plays a significant role in cell senescence, aging, and age-related diseases ,53,54. S+ levels ,57. More+ levels .On the other hand, inflammatory signaling pathways and the circadian rhythm system are also intertwined, regulating each other in a complex manner. Inflammatory cytokines whose expression is induced by NF-κB, namely Interleukin (IL)-1β, have been shown to disrupt the rhythmic expression of clock genes and clock-controlled catabolic pathways relevant in age-related degenerative diseases. In response to acute and chronic inflammation, NF-κB was shown to directly interact with BMAL1:CLOCK, driving the complex away from the promoter regions of its primary target genes, namely Cry, Per, and Rev-erb genes, thus repressing the negative feedback loop of the circadian clock ,60,61. TConversely, clock proteins were also found to regulate NF-κB activation and transcriptional activity. On one hand, CLOCK was shown to directly associate with NF-κB at the promoters of its target genes, increasing their transcription, probably by promoting RelA/p65 acetylation directly or through recruitment of other histone acetyltransferases. BMAL1 instead downregulates the CLOCK-dependent increase in NF-κB activity, likely by associating with CLOCK and driving it away from its interaction with NF-κB . Thus, tThe interdependence of SIRT1, circadian clock components and NF-κB, highlights the physiologic relevance of their coordinated interaction so that metabolism, circadian rhythms, and the ability to cope with stress can be adequately integrated to maintain homeostasis. In turn, disruption of this balance is associated with aging and related diseases. Conversely, restoring SIRT1 expression and activity. which are decreased in aging and disease, is an attractive strategy to increase life- and health-span ,16,17.5H8 [5H8), monoterpenoids (C10H16), sesquiterpenoids (C15H24), diterpenoids (C20H32), sesterpenoids (C25H40), triterpenoids (C30H48), tetraterpenoids or carotenoids (C40H64), and polyterpenoids ((C5H8)n) [Terpenoids, also known as isoprenoids, are a family of natural products, particularly secondary metabolites, comprising a diversity of structures . This fa5H8 . The ter5H8 . Accordi(C5H8)n) . MonoterMonoterpenes are the main components of essential oils and have been ascribed a diverse range of pharmacological activities . NeverthEx vivo studies using normal rat hearts treated with bakuchiol increased SIRT1 protein levels and, consequently, PGC-1α protein levels. Moreover, pre-treatment with bakuchiol counteracted the decrease of SIRT1 and PGC-1α protein levels induced by IR in rat hearts. As downstream effects, bakuchiol increased the activity of succinate dehydrogenase (complex II), cytochrome c oxidase (complex IV), and superoxide dismutase (SOD) as well as mitochondrial redox potential, whereas it decreased H2O2 formation and malondialdehyde content. Moreover, bakuchiol increased the levels of the anti-apoptotic factor, Bcl-2, and decreased the levels of the pro-apoptotic factors, Bax-2, and cleaved Caspase 3, thereby exhibiting cardioprotective effects against myocardial IRI. These results were confirmed using a known SIRT1 inhibitor, sirtinol, in IR-injured hearts and a SIRT1 small interfering RNA (siRNA) in IR-injured cardiomyocytes, respectively. These studies showed that decreasing SIRT1 activity and expression were both efficient in abolishing the protective effects of bakuchiol, suggesting that this terpenoid attenuates IRI by activating the SIRT1/PGC-1α pathway [Bakuchiol  is a mon pathway . Corrobo pathway . Overall pathway ,74.(S)-(+)-carvone  is a monHinokitiol , also knPaeoniflorin  is a pinCollectively, these studies point out paeoniflorin as an inducer of SIRT1 expression.Belonging to the monoterpenoid class, iridoids are also characterized by the 10-carbon backbone in which a six-membered ring with an oxygen atom is fused to a cyclopentane ring, the iridane skeleton. These compounds are frequently combined with sugar moieties, forming glycosides. They are divided in four groups: non-glycoside or simple iridoids, iridoid glycosides, secoiridoids that result from cleavage of a bond in the cyclopentane ring, and bisiridoids, which result from the dimerization of iridoids and secoiridoids ,84.Genipin  is an agCatalpol  has beenSirt1 knockdown abolished geniposide-mediated anti-inflammatory effects in palmitic acid-treated cardiomyocytes and hearts of obese mice. Interestingly, the authors found that geniposide did not increase SIRT1 protein levels in cardiomyocytes with inhibition of Glucagon-Like Peptide-1 Receptor (GLP-R1) or Glp-1r deficiency, suggesting that activation of SIRT1 by geniposide is mediated by the GLP-R1 [Geniposide  is a wele GLP-R1  and thuse GLP-R1 .Another study showed that geniposide is able to reduce the development of STZ-induced diabetic nephropathy by inhibiting the NF-κB pathway . This prAnother study showed that geniposide alleviates inflammatory responses in colitis via the AMPK/SIRT1 signaling pathway. Accordingly, geniposide increased the protein levels of SIRT1 in both in vivo and in vitro models, namely dextran sodium sulfate (DSS)-induced acute colitis in mice and LPS+ATP-treated murine bone-marrow-derived macrophages and the Raw 264.7 macrophage cell line. Interestingly, association of geniposide with EX-527 attenuated its effects, whereas association with an SIRT1 activator, SRT-1720, reinforced the beneficial effects described in the models used. Moreover, the same results were obtained when SIRT1 was knocked-down or overexpressed in Raw 264.7 cells . AltogetGenipin-1-β-D-gentiobioside  has a chLoganin  is anoth2O2 in primary osteoblasts. This effect was associated to decreased apoptosis and protein levels of pro-inflammatory mediators, namely cyclooxygenase 2, inducible nitric oxide synthase, IL-1β, IL-6, and TNF-α, in relation with decreased NF-κB nuclear accumulation [Monotropein  was alsomulation .Picroside II  is anothLastly, sweroside  was descGentiopicroside  is a secOleuropein  is an es+ [Similar to oleuropein, its aglycone also increased SIRT1 protein levels in 6-month-old TgCRND8 mice and in the neuronal cell line, N2a, treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), in in vivo and in vitro models of Alzheimer’s disease, respectively. Interestingly, oleuropein aglycone also decreased PolyADP-ribosylated proteins, which are the result of poly(ADP-ribose) polymerase 1 (PARP1) activation, in both models. Moreover, this compound also decreased PARP1 mRNA and protein levels in 6-month-old TgCRND8 mice, suggesting that oleuropein aglycone attenuated neuronal damage, at least in part, via modulation of the PARP1/SIRT1 signaling pathway . PARP1-m+ , the incper se but other biomolecules that direct or indirectly regulate its expression. Among those, recent studies identified several non-coding RNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circ-RNAs), as modulators of SIRT1. Several micro RNAs (miRNAs) have been shown to decrease SIRT1 expression by targeting its mRNA both in disease models and in human diseased tissues compared to normal controls. miR-211 is one of those miRNAs recently shown to be downregulated in lesioned epidermis samples from vitiligo patients in relation with increased expression of a lncRNA, MALAT1, which sequesters miR-211, enhancing SIRT1 expression. In human keratinocytes, MALAT1-dependent miR-211 downregulation increased SIRT1 expression, which, in turn, promoted keratinocyte differentiation and protected the cells from UVB-induced DNA damage [Multiple studies showed that increasing SIRT1 expression with the consequent increase in its protein levels and activity or enhancing its activity by allosteric interaction are efficient ways of increasing life- and health-span and to reverse the signs and symptoms of many age-related diseases ,16,17,26A damage . AnotherA damage . These aA damage . InteresA damage , thus suOn the other hand, most studies evaluated the effects of monoterpenes on SIRT1 at the mRNA and protein levels but did not evaluate their effects on the purified enzyme activity. Therefore, further studies are required to determine whether and which monoterpenes can directly increase SIRT1 activity by allosteric modulation in a mechanism similar to that involving SIRT1 activation by another class of natural compounds, the polyphenols, as well as by synthetic compounds ,16,17.In this respect, the observation that (S)-(+)-carvone directly enhances SIRT1 activity in chemico  demonstrDespite the beneficial effects associated with SIRT1 activity, there are still controversies regarding its therapeutic potential. On one hand, there are conditions in which SIRT1 inhibition seems to be preferable, such as cancer and Huntington’s disease ,118,119.From another point of view, potential deleterious consequences of increasing SIRT1 expression and activity also need to be considered. Upregulation of SIRT1 has been associated with various cancers , but the


SPINK5 gene mutations is a hallmark of the disease. Antigen penetration through the defective skin and nonspecific inflammation provide a pro-T helper 2 (Th2) immune microenvironment in the disease. Therefore, Th2 cytokines are considered to be candidate therapeutic targets.Netherton syndrome is a rare, life-threatening autosomal recessive genetic disorder with no effective treatment yet. Skin barrier dysfunction caused by To evaluate the clinical responses of patients with Netherton syndrome to dupilumab, an IL-4Rα antagonist, and identify changes in the Th1/2/17 pathway activity, skin barrier defect protein LEKTI expression after treatment.Four children with severe Netherton syndrome (aged 2 y to 4 y and 6 m) who were treated with dupilumab from January to June 2022 were evaluated at baseline, and at 4, 8, 12, 16, and 20 weeks after the start of dupilumab administration. Treatment response was assessed using the Eczema Area and Severity Index (EASI), the Numerical Rating Scale (NRS), the Dermatology Life Quality Index (CDLQI), and the Dermatitis Family Impact-questionnaire (DFI). Blood eosinophil counts, serum IgE levels and inflammatory cytokines were measured. The immunotyping of Th1/2/17 cells was performed by flow cytometry and cytokine expressions in T cell subsets were analyzed by single-cell RNA sequencing. In addition, expression of the LEKTI in skin lesions was evaluated by immunohistochemical analysis.P = 0.029). The baseline percentage of Th2 cells  was significantly higher in patients than that in healthy controls (HC) (P < 0.0001); this percentage fell from 8.25% ± 0.75% to 4.02% ± 0.62% after 20 weeks dupilumab treatment. There was no noticeable change in LEKTI protein expression in skin lesions pre- and post-treatment. Two patients reported mild ocular adverse effects, but there were no severe adverse events.All four patients experienced clinical improvement, with significantly reduced EASI scores (by 75.0–83.9%) and NRS (by 87.5–90.0%) from baseline to 20 weeks of treatment. Improved quality of life scores were also seen for all patients, as measured by CDLQI and DFI. Serum IgE levels also fell by 75.6–86.9%. The serum Th2 cytokines IL-4, IL-5 and IL-13 were found at low level, with no significant changes during the treatment. However, Th2 cytokines expressed by T cells, especially IL-4, decreased at single-cell level after treatment (Dupilumab may be an effective and safe treatment option in a subset of pediatric patients with Netherton syndrome, especially in improving itch and the quality of life. These effects were achieved in part by suppression of the Th2-mediated inflammation. SPINK5 gene. The disorder is characterized by the classical triad symptoms of congenital ichthyosis, bamboo hair, and atopic diathesis with high serum IgE level  protein which leads to an unrestricted activity of kallikrein (KLK)-related peptidases . This will cause severe skin barrier breakdown and secondary inflammation , also known as Comel-Netherton syndrome, is a rare autosomal recessive disorder caused by germline loss-of-function mutations in gE level . In most56500, alammation .NS is a multisystemic disease for which a safe and effective treatment is not yet available. Several novel systematic treatments have been suggested in the recent literature, including immunoglobulins and biologicals that target specific inflammatory pathways . DupilumSPINK5 mutations, and decreased/absent LEKTI expression upon immunostaining analysis. Health controls (HCs) comprised eight age-matched subjects . Patients were evaluated, and blood was collected at baseline (before therapy), and at 4, 8, 12, 16 and 20 weeks after dupilumab administration. Skin biopsies were collected at baseline and 20 weeks. All parents provided written informed consent, and the study was approved by the Ethics Committee of the Children’s Hospital of Chongqing Medical University (2021-17).Four NS patients  from four unrelated Chinese families were enrolled. A diagnosis of NS was made based on clinical signs, All NS patients received a loading dose of subcutaneously administered dupilumab (400 mg), followed by 200 mg every 4 weeks for 20 weeks. During dupilumab treatment, patients were encouraged to continue the use of moisturizers, topical corticosteroids, or 1% Pimecrolimus, which were not monitored specifically.9 cell/L.The Children’s Dermatology Life Quality Index (CDLQI) and the Dermatitis Family Impact-questionnaire (DFI) were adopted for evaluation of quality of life, the Eczema Area and Severity Index (EASI), and the Numerical Rating Scale (NRS) were used at every visit for assessing changes in symptoms. Laboratory tests, including total serum immunoglobulin (Ig) E and eosinophil (ESO) counts, were conducted at every visit. A high level of serum IgE was defined as >165.0 IU/ml, and eosinophilia was defined as a blood eosinophil count >0.68×10β, IL-1α, TNF- α, TNF- β, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-12p70, IL-13, IL-17A, IL-23, and IL-31, were measured at baseline, 4 and 20 weeks after treatment with dupilumab by Luminex 200™ System (Millipore) based on manufacturer’s instructions. Serum samples were diluted 2 times and incubated for 3 hours with a mix of beads coupled to antibodies specific for each measured cytokine. Then a mixed secondary streptavidin-coupled antibodies was added for 1 hour followed by phycoerythrin (PE)-conjugated biotin after several washing steps. The signal intensity was measured by PE fluorescence and a standard curve was performed to identify the absolute concentration of each cytokine in tested samples.Multiple cytokines, including IL-1 https://cytosig.ccr.cancer.gov/) was employed in T cell subsets to evaluate the cytokine signaling activity, including IL-1A, IL-1B, IL-2, IL-4, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-21, and IL-22.The PBMCs from 4 NS patients at baseline and 20 weeks after dupilumab treatment were sequenced for single cells using the 10x Genomics scRNA-seq platform. Based on the single-cell transcriptomic data, Cytokine Signaling Analyzer  were isolated from blood samples using Ficoll-Hypaque  gradient centrifugation. To obtain Th1/2/17 cells, PBMCs were incubated for 30 min in with specific antibodies  in PBS containing 2% fetal bovine serum. Cells were examined immediately using a BD FACS-CantoII, and data were analyzed using FlowJo software . Cells were classified as follows: Th17 cells, CD3μm), and embedded in paraffin blocks. The slides were permeabilized with 0.5% H2O2, then stained with a SPINK5 polyclonal antibody  overnight at 4°C. A secondary biotinylated antibody  was performed, then reacted with diaminobenzidine (DAB) . After counterstaining with hematoxylin, images were captured under a digitalized brightfield microscope  and analyzed with ImageJ software (version 2.1.0). Average optical density (AOD) was used to evaluate immunofluorescence staining.After consent was obtained from the parents, skin biopsy was conducted in the ichthyosis lineariz circumflexa (ILC) lesions of patients 2 and 3. Four normal skin specimens obtained from routine surgery were served as controls. Skin sections were fixed, sectioned transversely . A two-tailed unpaired t-test was used for single comparisons, and the Wilcoxon matched pairs signed rank test or the Mann-Whitney test was used to assess the statistical significance of differences between groups .SPINK5 mutations in all patients.  are shown in the Treatment with dupilumab led to a significant improvement in clinical signs  at baseline. During treatment, serum IgE levels fell steadily in all patients (P < 0.0001) (P = 0.0021). The numbers of Th17 and Th1 cells, however, tended to increase slightly, but the changes had no statistically significance (P = 0.0128) (P = 0.029).  signaling in keratinocyte . UpregulIn present study, 4 pediatric NS patients presented with ILC phenotype, one of them displayed erythroderma at his early age. Immunophenotyping of PBMCs at the baseline revealed increased Th2 subset in all patients, but only slightly increased Th17 cell numbers. After dupilumab treatment, except for sustained symptomatic improvements, a significant decrease in both percentage  and absolute number of Th2 cells were observed, suggesting that the Th2 pathway activation contributed to the systemic inflammation in our cohort. This hypothesis is supported by increased IL-4 production in T cell subsets, which could in turn induces B cell proliferation, isotype switching and IgE production . What isWe further evaluated the LEKTI protein expression in skin lesions of the NS patients in pre-and-post dupilumab therapy. Unsurprisingly, there were no obvious changes in LEKTI expression were observed, indicating that the improvements in skin lesions by dupilumab mainly rely on suppression of downstream Th2-mediated inflammation, rather than to regulate or correct underlying LEKTI defects. In regarding to the drug safety, only two patients in this study have reported mild ocular symptoms, which similar to that in treatment of atopic dermatitis, in the entire 20-week treatment period. It goes without saying that dupilumab has accumulated a lot of experience and safety profiles in pediatric population, especially it has recently been approved for children up to 6 months of age . ContrarTaken together, our results suggest that dupilumab is a safe and effective treatment option for certain subtype of pediatric NS patients, especially with respect to in improving skin symptoms, itching and the quality of life. The possible mechanisms may involve the suppression of the Th2-mediated inflammation, but further studies with larger samples are needed.The study has a small number of recruited patients and the molecular features in skin lesion were not detected, which should be taken into account when drawing conclusions.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: GEO submission, accession number(GSE214985).The studies involving human participants were reviewed and approved by the Ethics Committee of the Children’s Hospital of Chongqing Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.Concept and design: XL, HW, SYu, and XB. SYa and XL had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Drafting of the manuscript: SYa, XL, HW and XB. All authors participate in patient enrollment and follow-up.


Caspase is a well-studied metazoan protease involved in programmed cell death and immunity in animals. Obviously, homologues of caspases with evolutionarily similar sequences and functions should exist in plants, and yet, they do not exist in plants. Plants contain structural homologues of caspases called metacaspases, which differ from animal caspases in a rather distinct way. Metacaspases, a family of cysteine proteases, play critical roles in programmed cell death during plant development and defense responses. Plant metacaspases are further subdivided into types I, II, and III. In the type I Arabidopsis MCs, AtMC1 and AtMC2 have similar structures, but antagonistically regulate hypersensitive response cell death upon immune receptor activation. This regulatory action is similar to caspase-1 inhibition by caspase-12 in animals. However, so far very little is known about the biological function of the other plant metacaspases. From the increased availability of genomic data, the number of metacaspases in the genomes of various plant species varies from 1 in green algae to 15 in Glycine max. It is implied that the functions of plant metacaspases will vary due to these diverse evolutions. This review is presented to comparatively analyze the evolution and function of plant metacaspases compared to caspases. Apoptosis is one of the programmed cell deaths that is crucial for tissue development and homeostasis . Program2+-dependent manner [Magnaporthe oryzae has two metacaspase genes, MoMca1 and MoMca2 [Momca1mca2 strain contains increased insoluble aggregates during vegetative growth. MoMca1 and MoMca2 promote the clearance of the insoluble aggregates in M. oryzae [Caspases are comprised of a pro-domain and a mature chain, which is folded into a caspase/hemoglobinase. After the removal of the pro-domain, caspases undergo proteolytic cleavage that releases two peptide fragments with the large subunit p20 (about 20 kDa) and small subunit p10 (about 10 kDa), respectively. Caspase-1, caspase-2, caspase-4, caspase-5, caspase-9, caspase-11, and caspase-12 also have amino-terminal pro-domains including caspase recruitment domains, or caspase activation and recruitment domains (CARDs), which are interaction motifs. Effector caspase-3, caspase-6, and caspase-7 contain short pro-domains and these caspases are activated by the initiator caspases A. Caspast manner . Yca1 had MoMca2 . It has d MoMca2 . Double . oryzae . Metacas+-dependent as a damage-induced plant immune defense [Guillardia theta algae. [G. theta metacaspases exhibit 1 type I (GtMC1), 1 type III (GtMC2), and 11 metacaspase-like types (GtMC3-GtMC14) [The caspase family is the only family of cysteine proteases with members in all kingdoms according to the MEROPS database ,16. Plan defense . Large l defense . Type IIa algae. . G. thet-GtMC14) . Type IIThe intracellular localization of caspase and metacaspase shows various distributions ,22. In pCasp2−/− primary skin fibroblasts are protected upon oxidant treatment [Caspase-1 can target and cleave the GATA4 transcription factor, which regulates cardiac cell fate . Mature reatment . A generreatment . Additioreatment ,36. It cTritium aestivum, type II metacaspase TaeMCAII has been detected in dynamic localizations [2+. Sieve elements are different from typical programmed cell death, but it can be expected that the function of metacaspases during development is determined by changes in their subcellular localization that are dependent on Ca2+. The yeast genome only has a single type I metacaspase, Yca1, which localizes in insoluble protein aggregates via its N-terminal pro-domain and promotes aggregate clearance . The losizations . The autizations . These rVitis rupestris L.) metacaspases, VrMC2 and VrMC5 have been identified as type I and type II metacaspases, respectively [Oryza sativa OsMC1, which is localized exclusively in the nucleus, has putative nuclear localization signals (NLS) in the N-terminal region [Two grapevine  and Guillardia theta (GtMC1) lack the N-terminal region completely [OsMC8 has a very short N-terminus extension, and it is not clear whether it actually functions as a pro-domain, but it is, nonetheless, included in type I . Similarmpletely . Thus, tVvMC6 is expressed in floral tissue and exhibits low expression patterns in other tissues. Function of VvMC6 might be flower formation and ovule development in grapes. Transcription levels of type I VvMC1, VvMC3, and VvMC4 increases during endosperm abortion in seedless grape. Thus, grape metacaspase function is involved in developmental regulation in various tissues. The function of type II AtMC4 and AtMC9 is involved in leaf senescence, because gene expression of these genes is highly up-regulated in senescence conditions [Arabidopsis is cell growth defect factor 1 (Cdf1), which promotes proapoptotic Bax-like cell death via enhancing reactive oxygen species (ROS) [Arabidopsis Cdf1-related gene Responsive to Senescence (CRS) increases gene expression of AtMC4 and AtMC9 during senescence [PhMC1-RNAi transgenic plants exhibit normal growth phenotypes, but flower senescence is enhanced [Metacaspases are known to play an important role in developmentally regulated PCD in plants ,47. The nditions . A putates (ROS) . Overexpnescence . It woulSIMC7 and type I SIMC2 exhibit a precise pattern which increased according to the maturation process of tomatoes  infection and more resistance to both P. syringae (empty vector control) and P. syringae (avrRpt2) than Col-0 WT plants [As previously reported, AtMC2 appears to have a role in hypersensitive cell death. In the case of type I  process C. The sinditions ,57. AlthT plants . AtMC1-mT plants ,56,57. AAtMC8 shows highly enhanced gene expression upon P. syringae and Sclerotinia sclerotiorum (cottony soft rot). AtMC7 and AtMC9 exhibit inhibited gene expression upon Phytophthora parasitica (soilborne pathogen). Gene expression of AtMC1 and AtMC5 is upregulated upon P. syringae, P. parasitica, and S. sclerotiorum , and Bactericera cockerelli (tomato/potato psyllid) infection  proteins ,62. For ructures . Previouructures . These rondition . The TP5ondition . Recentlondition . Physicaondition . In earlondition ,70. Thusatmc1 atg5 or atmc1 atg18 double mutants exhibit an additive effect on HR cell death suppression and negatively regulate senescence [Arabidopsis ATG6-GFP associates with AtMC1-Myc and AtMC2-Myc proteins (Petunia × hybrida autophagy PhATG6 silencing affects metacaspase 1 (PhMC1) gene expression [In plants, it has already been reported that their functions genetically interact between autophagy and metacaspase mutants ,71. The nescence . Howevernescence , coIP waproteins B. Interepression . Converspression . This suFrom an evolutionary point of view, the prediction of the function of metacaspase, a conformational analogue of caspase, will be somewhat similar. However, the distinction between functions such as initiator and effector of caspases is not yet clear. Regarding mutant phenotypes, metacaspase is also involved, such as in immunity and developmental PCD, in which caspase is involved. Further studies suggest that the function of metacaspases may be in the regulation of autophagy-related apoptosis in genetical and physical interactions. Knowledge of whether the functions of type I and type II are differentiated, and various approaches, are required to determine target proteins.


The impact of preweaning vaccination for bovine respiratory viruses on cattle health and subsequent bovine respiratory disease morbidity has been widely studied yet questions remain regarding the impact of these vaccines on host response and gene expression. Six randomly selected calves were vaccinated twice preweaning (T1 and T3) with a modified live vaccine for respiratory pathogens and 6 randomly selected calves were left unvaccinated. Whole blood samples were taken at first vaccination (T1), seven days later (T2), at revaccination and castration (T3), and at weaning (T4), and utilized for RNA isolation and sequencing. Serum from T3 and T4 was analyzed for antibodies to BRSV, BVDV1a, and BHV1. Sequenced RNA for all 48 samples was bioinformatically processed with a HISAT2/StringTie pipeline, utilizing reference guided assembly with the ARS-UCD1.2 bovine genome. Differentially expressed genes were identified through analyzing the impact of time across all calves, influence of vaccination across treatment groups at each timepoint, and the interaction of time and vaccination. Calves, regardless of vaccine administration, demonstrated an increase in gene expression over time related to specialized proresolving mediator production, lipid metabolism, and stimulation of immunoregulatory T-cells. Vaccination was associated with gene expression related to natural killer cell activity and helper T-cell differentiation, enriching for an upregulation in Th17-related gene expression, and downregulated genes involved in complement system activity and coagulation mechanisms. Type-1 interferon production was unaffected by the influence of vaccination nor time. To our knowledge, this is the first study to evaluate mechanisms of vaccination and development in healthy calves through RNA sequencing analysis. Vaccination remains one of the most important tools for controlling bovine respiratory disease (BRD) in beef calves . IncreasAlthough commercially available vaccines have been evaluated and approved by the USDA APHIS Center for Veterinary Biologics for purity, safety, potency, and efficacy, the requirements for efficacy studies are often quite different than the conditions under which the vaccine will be used in the field. While challenge studies can be very useful tools , they dovia time-course transcriptomics, and to pair those data with antibody titers and health records. These data will support exploration of associations and generation of hypotheses regarding the immune response to vaccination that may influence future research and use of vaccines in preweaned beef calves.Given this background, our objective was to explore differences in host gene expression in calves that were vaccinated preweaning with a modified live vaccine for respiratory pathogens or not In Vivo Experiments (ARRIVE) guidelines  and carried out in accordance with relevant IACUC and agency guidelines and regulations. This study was carried out in accordance with Animal Research: Reporting of Eighty-four bull calves were enrolled in a split plot design study to evaluate the impact of different management strategies on BRD morbidity, mortality, and performance . Animalsvia ear notch ELISA to evaluate PI status at T1; no PI positive calves were found. At T2, or 7 days post-vaccination (median age = 114 days), all calves were weighed and sampled. At T3 VAX calves were again administered (revaccinated with) 2 ml Pyramid 5 subcutaneously and NOVAX calves were given 2 ml 0.9% saline subcutaneously (median age = 183 days); all calves were castrated by knife with no analgesia on T3. All calves also received 5 ml of a multivalent clostridial bacterin-toxoid  subcutaneously at time point T1 and T3). All calves were handled so that no contact between vaccinated and non-vaccinated calves would occur. Calves were abruptly weaned at T4 (median age = 230 days) and entered the next phase of the study where they were kept in their original pastures in Mississippi for 3 days before being transported directly from Mississippi to Texas for backgrounding (DIRECT) or sent to an auction market where they stayed in a pen not in contact with other cattle for approximately 6 h, and then were transported for housing at an order buyer facility for 3 days prior to transport to Texas (AUCTION) Non-study calves from other sources were housed at the order buyer facility at the same time as the study calves, but they were not mixed with the study calves. In Texas , calves were kept in one of 12 pens corresponding to their original random assignment to whole and split plot treatments (n = 3 pens of each pair of treatments). Whole blood was collected from all calves into Tempus RNA blood tubes (Applied Biosystems) and into serum tubes via jugular venipuncture immediately prior to first vaccination (T1), seven days post-vaccination (T2), immediately prior to vaccine booster administration and castration (T3), and at time of abrupt weaning . Overall, there were 7 days between T1 and T2, 70 days between T2 and T3, and 47 days between T3 and T4.Calves were evaluated at four time points, described as T1, T2, T3, and T4. At T1, calves were vaccinated with 2 ml Pyramid 5  subcutaneously (VAX) or given 2 ml 0.9% saline subcutaneously (NOVAX) (median age = 107 days). Additionally, calves were tested via stratified random sampling within the calves that remained healthy throughout the study within each backgrounding pen which resulted in 1 calf selected per backgrounding pen . A total of 48 blood samples across the four time points were analyzed for whole blood transcriptomes. Metadata for all selected calves are found in Twelve calves that remained clinically unaffected by BRD during the cow-calf and backgrounding phases of production were selected for RNA sequencing n = 12).Serum collected at T3 and T4 was stored at −20°F before analysis at the University of Georgia's Athens Veterinary Diagnostic Laboratory. Serum neutralizing antibodies were assayed for bovine herpesvirus−1 (BHV-1), bovine viral diarrhea virus type 1a (BVDV1a), bovine respiratory syncytial virus (BRSV), and parainfluenza-3 virus (PI-3) per SOP # Ser013. Resulting titer levels for these antibodies are found in via generalized linear mixed effect models estimated via restricted pseudolikelihood with the Kenward-Rodgers adjustment for degrees of freedom in SAS 9.4. The model included vaccination status as a fixed effect and a random intercept for backgrounding pastures. Differences in least square means are reported and a cutoff of p ≤ 0.05 was used to determine significance.Differences in average daily gain between T1 and T4 were evaluated Total RNA isolation, quality control, sequencing library preparation, and sequencing was performed by the Texas A&M University Institute for Genome Sciences and Society . Total RNA was isolated with Tempus Spin RNA Isolation Kit (Applied Biosystems), based on manufacturer's instructions. Total RNA from each sample was analyzed for RNA concentration and integrity with a Qubit 2.0 Fluorometer (ThermoFisher) and an Agilent 2,200 Bioanalyzer (Agilent), respectively; all RNA samples were of high quality  and concentrations . Library preparation for mRNA was performed with the TruSeq Stranded mRNA Library Prep Kit (Illumina), following manufacturer's instruction. Paired-end sequencing for 150 base pair read fragments was performed on an Illumina NovaSeq 6000 analyzer  in one flow cell lane.GSE205004.Quality assessment of reads was performed with FastQC v0.11.9Gene-level count matrices were processed and analyzed in RStudio, using R v4.1.2. Samples were classified by vaccination group and time point, where raw gene counts were processed and filtered by procedures described by Chen et al. . Any genp-values were adjusted for false discovery rates (FDR) with the Benjamini-Hochberg method; genes were considered significantly expressed with an FDR ≥ 0.05. Pairwise comparisons for DEGs between each vaccination groups at every time point and within each vaccination group across each time point was performed with edgeR v3.36.0  across timepoints, vaccine groups, and the interactions between timepoints and vaccine group, where  v3.36.0 , 17, fitvia principal component analysis (PCA) was conducted with the Bioconductor package PCAtools v2.0.0,Heatmap, principal component, and clustering analyses were performed with all filtered and log2 count-per-million (log2CPM) values of TMM-normalized gene counts between all 48 samples. Heatmap and exploratory clustering analysis of samples, with respect to vaccination, time points, and individual IDs, were performed with the Bioconductor package pheatmap v1.0.12,p-values (FDR ≥ 0.05). Functional enrichment of DEGs were analyzed in three separate analyses: (1) DEGs shared between time points in both glmmSeq and QLF testing of vaccinated and non-vaccinated calves , (2) DEGs identified between vaccination groups across each time point by both glmmSeq–vaccination and QLF testing , removing DEGs identified by method #1, and (3) DEGs solely identified in glmmSeq analysis of Timepoint: Vaccine interactions; this approach allowed for the independent assessment of functional enrichment influenced by calf development  and vaccine administration. Enriched GO terms and pathways were evaluated for directionality (increased or decreased) based on log2 fold changes of associated DEGs. Clustering and visualization of enriched KEGG terms was performed with the embedded enrichment visualization tool within KOBAS-i, utilizing edge (correlation) thresholds of 0.40 and top n clusters set to 8; more information regarding the embedded enrichment visualization tool framework is provided by Bu et al.  terms, Reactome pathways, and KEGG pathways with KOBAS-i   between the VAX (model-adjusted least square mean 1.89 lbs) and NOVAX (model-adjusted least square mean 2.13 lbs) groups.Comparison of antibody titers indicated calves were likely naturally infected with BRSV and PI-3, because antibody titers to these agents increased between T3 and T4 in both vaccinated and non-vaccinated calves. Given the small number of calves evaluated, this somewhat clouds our ability to detect the effect of vaccination using serology of samples collected at only two timepoints. However, vaccinated calves appeared to respond well to MLV vaccination as indicated by the BVDV1a titer response . AverageRead mapping and alignment of the 48 transcriptomes to the ARS-UCD1.2 bovine reference genome resulted in an overall mapping rate average of 95.50% (s.d. = 0.96%). In total, gene-level alignment resulted in a total of 33,310 unique features, with a median library size of 41,089,614  . Pre-pron = 7) were highly similar in global gene expression prior to vaccine administration . Time of sampling (Timepoint) emerged as a considerable factor in determining distinction between groups (Vaccine) and individual calves (ID), as the majority of samples on the left side of the heatmap  were at time of vaccine boostering (T3) and weaning (T4). Several individuals  demonstrated high self-similarity in global gene expression between time points.Heatmap and unsupervised clustering analysis, seen in via PCA is found in r = 0.34, FDR < 0.10), Vaccine , and Timepoint . Two PCs, PC3 and PC4, accounting for 7.67 and 5.94% of total explained variance, respectively, demonstrated significant correlations with Timepoint but not Vaccine; PC3 demonstrated negative correlation with Timepoint  and ID  and PC4 demonstrated positive correlation with Timepoint  and Age , confounded by ID . Accounting for 5.74% of total explained variance, PC5 possessed significant negative correlation with Timepoint . While confounded by Pasture , PC10, accounting for 2.72% of total explained variance, possessed significant positive correlation with Vaccine . Notably, the strongest correlation found within this analysis was between PC11, accounting for 2.33% of total explained variance, and GR . The resulting pairwise plotting of PCs 1, 3, 4, 5, and 10 demonstrated relative overlapping of all samples at T1, with increasing dissimilarity of samples over time , possessed variance-driving genes which overlapped with glmmSeq–timepoint findings; BATF, EXTL2, PRDX2, RNF122, TIAM1, and TMCC3 were identified in both PC3-5 loadings plots and glmmSeq–timepoint analysis.Multidimensionality analysis and visualization of global gene expression patterns ver time . A biplover time . Genes dADAMTS12, ALOX15, ALOX5, CFL1, CPT1A, FBP1, FSCN1, IL5RA, LOC100297044 (CCL14), LOC615278 (TRIM39), LOC789732 (CD300C), MIF, OTUD7B, PEG10, PIKFYVE, PLP2, POLR2L, PPP2R1A, PRKCG, PYGM, TK1, and TP53. Shared DEGs from T1 vs. T4 comparisons enriched for 34 GO terms and 35 functional pathways. These GO terms were related to inflammatory response, cytokine-mediated signaling, magnesium ion binding, cellular response to oxidative stress, positive regulation of autophagy, T-cell co-stimulation, and actin/microtubule organization and development. Enriched pathways included the acquired immune system, interleukin signaling, cellular stress response, CD28 co-stimulation and signaling, and gap junction trafficking and regulation. These GO terms and pathways were primarily enriched by the following DEGs: ALOX15, CD80, HMGA1, HSPB8, IL17REL, IL5RA, LOC100297044 (CCL14), LOC533307 (LRRK2), LOC789732 (CD300LD), MAP3K8, NCF2, SLC7A11, TUBB, TUBB3, and ZC3H12A. Shared DEGs from T2 vs. T4 comparisons enriched for 94 GO terms and 29 functional pathways. These GO terms were related to inflammatory and cytokine-mediated response, specifically including interleukin-17 receptor activity, MHC class I protein complex binding, response to mercury and magnesium ions, antigenic stimuli and macrophage differentiation, and fatty acid metabolism and biosynthesis. Enriched pathways included cellular metabolism involving fructose, mannose, pyruvate, and lipid metabolism, cytokine-cytokine receptor interaction, and the biosynthesis of specialized proresolving mediators (SPMs), including SPMs derived from both docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). These GO terms and pathways were primarily enriched by the following DEGs: ALOX15, CEBPE, DECR2, FBP1, IL17REL, IL5RA, LOC100297044 (CCL14), LOC788694 (KLRC1), and SLC7A11. Visualization of the enriched KEGG pathway terms is found in ALOX15, IL5RA, IL17REL, LOC100297044 (CCL14), and SCL7A11) are found in Analysis of GO terms, KEGG pathways, and Reactome pathways of genes identified between glmmSeq–timepoints and edgeR QLF testing within both vaccination groups across time allowed for the assessment of enriched processes and pathways at three specific timepoint comparisons: (1) T1 vs. T3, (2) T1 vs. T4, and (3) T2 vs. T4 . Shared HEXDC; increased in Vaccinated) between vaccinated and non-vaccinated calves, therefore possessed no enriched GO terms nor pathways. Shared DEGs identified at T2 between vaccinated and non-vaccinated calves enriched for 139 GO terms and 61 functional pathways. These GO terms were related to immune response and regulation (increased in Vaccinated), T-cell activation (increased in Vaccinated), metal ion binding (increased in Vaccinated), positive transcriptional regulation and protein processing (increased in Vaccinated), cellular proliferation and maintenance (increased in Vaccinated), complement activity (decreased in Vaccinated), and apoptotic clearance and phagocytosis (decreased in Vaccinated). Enriched pathways included the immune system and cytokine signaling, including interleukin-37 signaling (increased in Vaccinated), complement and coagulation cascades (decreased in Vaccinated), enhanced transcriptional activity, largely involving RNA polymerase II (increased in Vaccinated), and vitamin B6 metabolism (decreased in Vaccinated). These GO terms and pathways were primarily enriched by the following DEGs: ARL4D, C3, CNOT4, GTF2A1, LOC785873 (TRIM26), POU2F1, PUS10, SMAD3, THBD, and ZBTB41. Visualization of the enriched KEGG pathways is found in A total of 435 genes were identified by glmmSeq-Vaccination to be differentially expressed , with 10AHSA2, BANP, C3, CACYBP, CCT2, DNAJA4, DNAJB1, DNAJB4, HIST1H3G, HSP90AB1, HSPA14, HSPA1A, HSPA4, HSPA6, HSPD1, HSPH1, KAT2A, MDM4, NUTF2, PTPRB, RAB11FIP3, RCHY1, SMAD3, STIP1, SYMPK, TOMM34, TRAF2, ZFAND2A, ZFP28, and ZNF473. Visualization of the enriched KEGG pathway terms is found in DNAJB1, DNAJB4, HSP90AB1, HSPA14, HSPA1A, HSPA4, HSPA6, HSPD1, HSPH1, and TRAF2) are found in Shared DEGs identified at T3 between vaccinated and non-vaccinated calves enriched for 71 GO terms and 25 functional pathways. These GO terms were related to neutrophil degranulation (increased in Vaccinated), antigen processing and presentation (increased in Vaccinated), ubiquitin protein binding and positive regulation (increased in Vaccinated), nuclear protein importing and response to protein folding (increased in Vaccinated), heat shock protein binding, specifically to Hsp70 and Hsp90 (increased in Vaccinated), T-cell activation (increased in Vaccinated), cellular response to interleukin-7 (increased in Vaccinated), and the positive regulation to ATPase activity (increased in Vaccinated). Enriched pathways included transcription activation (increased in Vaccinated), endocytosis and antigen processing and presentation (increased in Vaccinated), neutrophil degranulation (increased in Vaccinated), and cellular response to heat stress, including the regulation of HSF1-mediated heat shock response, Hsp90 chaperone cycle for steroid hormone receptors, and HSF1-dependent transactivation . These GO terms and pathways were primarily enriched by the following DEGs: AOC3, DCT, LOC100852061 (KIR2DS2), LOC101905165 (NKG2D), LOC112441504 (ULBP3), and LOXL4) is found in A total of 85 DEGs were identified by glmmSeq when evaluating the interaction between Vaccination and Timepoints, which enriched for 13 GO terms and six functional pathways . These GThe use of modified live viral (MLV) vaccines in beef cattle backgrounding and feeding operations remains one of the leading practices in managing risk of BRD in cattle populations . MultiplAlthough variable in terms of individual efficacy, several studies suggest that vaccinating herds of cattle with MLV vaccines reduces herd-level risk of BRD-associated morbidity and mortality and is associated with improved weight gain overtime  –27. Our via serology  are directly involved in the biosynthesis of SPMs,ALOX5, ALOX15, and HPGD); notably, HPGD was identified as a differentially expressed in glmmSeq – timepoint, NOVAX T1vT4, VAX T1vT3, and VAX T2vT3. These lipid molecules are profound regulators of both acute and chronic inflammation and are critical in promoting cellular clearance and tissue remodeling in response to respiratory disease  over time , HPGD, and IL5RA, have been identified as DEGs increased in expression at facility arrival in cattle that remain clinical healthy within high-risk populations, compared to cattle that develop BRD  on the gene expression of young calves, three connected mechanisms continually increased over time across all individuals: specialized proresolving mediator (SPM) biosynthesis, fatty acid and carbohydrate metabolism, and chemokine/cytokine mediated enhancement of acquired immunity. Specialized proresolving mediators consist of closely related classes of lipid mediators, derived from the lipoxygenation of arachidonic acid into LXA4   or from  disease –36. Receechanism . Crucialechanism –39; thisver time –44. Furtelop BRD –49. CollCLOCK, HDAC3, KIR3DL1, RACK1, and SNX17 to be key drivers of differences associated with vaccination, which are involved in regulating the activity and differentiation of T-cells and natural killer cells. CLOCK, in conjunction with BMAL1, is a circadian timekeeping protein which interacts with transcriptional regulators, which in turn upregulate genes such as HDAC3; this transcriptional network is responsible for the development and differentiation of Th17 cells , and the upregulation of T-cell-mediated immunity. Complement, a well-organized and highly regulated system of the immune system, is a critical component of host immunity for killing or neutralizing pathogens and maintaining immunological homeostasis . DAPK2 promotes cellular recruitment to sites of inflammation , DEGs identified in calves which received a MLV vaccination enriched for two major immune-related mechanisms-the downregulation of complement and coagulation cascades , two genes involved in T-cell development and inflammatory defense mechanisms, respectively, and a decrease in THBD and C3, involved in coagulation (via CD14-mediated chaperoning (HSPD1 initiates interferon-beta production through interactions with interferon regulatory factor 3 (IRF3)  identified DEGs and enriched immunological mechanisms involved in neutrophil degranulation, antigen processing and interleukin-7 response, T-cell activation, transcriptional activity, and heat shock protein activity and binding. At this time point in vaccinated calves compared to non-vaccinated calves, we again observed an increase in the expression of gulation , 83 and gulation . Both Hsgulation  and stimperoning . HSPD1 i3 (IRF3)  and is a3 (IRF3) . Further3 (IRF3) –94. Thisvia natural killer cells. These mechanisms were enriched by three key genes: LOC101905165 (NKGD2), LOC100852061 (KIR2DS2), and LOC112441504 (ULBP3). NKG2D serves as a costimulatory transmembrane receptor on natural killer cells, enhancing T-cell receptor activity and subsequent cytotoxic and gamma-delta T-cell function , accession number The animal study was reviewed and approved by Mississippi State University Animal Care and Use Committee (IACUC protocol #19-169).SC, AW, and BK conceived and designed the vaccination study funded by USDA. MS, AW, BK, and KH managed the live animal project and collected samples and metadata for analysis. MS and SC coordinated diagnostics, analyzed the data, and wrote the manuscript. SC, MS, AW, BK, and KH edited the manuscript and approved the final version. All authors agree to be accountable for the content of the work.This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2019-67015-29845 from the USDA National Institute of Food and Agriculture. Additionally, this project was supported in part by internal funds provided by Texas A&M AgriLife Research and Texas A&M University School of Veterinary Medicine and Biomedical Sciences.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture nor the internal supporters of this project.