Phellodendron amurense Rupr., a species of Rutaceae, is a nationally protected and valuable medicinal plant. It is generally considered to be dioecious. With the discovery of monoecious P. amurense, the phenomenon that its sex development is regulated by epigenetics has been revealed, but the way epigenetics affects the sex differentiation of P. amurense is still unclear. In this study, we investigated the effect of DNA methylation on the sexual development of P. amurense. The young inflorescences of male plants were treated with the demethylation agent 5-azaC, and the induced female flowers were obtained. The induced female flowers’ morphological functions and transcriptome levels were close to those of normally developed plants. Genes associated with the development of female flowers were studied by comparing the differences in transcriptome levels between the male and female flowers. Referring to sex-related genes reported in other plants, 188 candidate genes related to the development of female flowers were obtained, including sex-regulating genes, genes related to the formation and development of sexual organs, genes related to biochemical pathways, and hormone-related genes. RPP0W, PAL3, MCM2, MCM6, SUP, PIN1, AINTEGUMENTA, AINTEGUMENTA-LIKE6, AGL11, SEUSS, SHI-RELATED SEQUENCE 5, and ESR2 were preliminarily considered the key genes for female flower development. This study has demonstrated that epigenetics was involved in the sex regulation of P. amurense, with DNA methylation as one of its regulatory modes. Moreover, some candidate genes related to the sexual differentiation of P. amurense were obtained with analysis. These results are of great significance for further exploring the mechanism of sex differentiation of P. amurense and studying of sex differentiation of plants. As the sex organ of plants, floral organs play an important role in plant reproduction. Flower development begins with flower bud differentiation. During the development of flower buds, the meristems differentiate into different flower organ primordia first and then develop into corresponding flower organs. Flower buds can be divided into bisexual flowers and unisexual flowers according to the development of pistils and stamens, and unisexual flowers can be divided into female and male flowers according to the abortion of stamens or pistils. Genetic factors, environmental conditions, hormones, and other factors can regulate the sex differentiation in flowers. Among them, epigenetics has been widely considered to play a crucial role in plant sexual differentiation. DNA methylation is one of the most intensively studied modes of epigenetic regulation ,2.ARR17 gene is silenced by male-specific DNA methylation . T. T219]. After treatment with 5-azaC, some male flower buds were induced to transform into female flowers with complete structure and function, while the remaining male flowers had no change in structure and function, and there were no other variation types. Induced female flowers were generated in about 70% of male individuals treated, and induced female flower buds were generated in 7–50% of inflorescence treated among the male individuals in which induced female flowers were generated. Induced female flowers were completely identified as natural female flowers in morphology, structure, and function, characterized in that stamens degenerated to different degrees and ovaries could develop into fruits with seeds .The 15 cDNA libraries constructed from all samples were sequenced on an Illumina high-throughput sequencing platform. After filtering the adaptors and low-quality sequences, 335,043,541 high-quality clean reads were generated from the 15 cDNA libraries. The resulting assembly consisted of 263,986 transcripts with an N50 value of 2180 bp. Removing redundant sequences resulted in a total of 86,610 unigenes with an N50 value of 1790 bp. The length distribution of unigenes ranged from 301 to 16,091 bp, nearly 33% of which were 300–500 bp long, approximately 50% of which were also distributed between 500 and 2000 bp, while the remaining 17% were clustered under a size distribution of ≥2000 bp.A total of 52,231 unigenes (60.30%) were annotated in at least one of the Nr, GO, and KEGG databases . There wCitrus sinensis (25.4%). Unigenes were also matched significantly with Citrus clementina (20.8%) and Citrus unshiu (20.4%); additionally, a small number of unigenes matched with Vitis vinifera (3.0%) and Quercus variabilis (1.6%). The result of Nr annotation suggested P. amurense was more similar to C. sinensis, Citrus clementina, and Citrus unshiu, which was consistent with the fact that those four species all belong to Rutaceae . Among the biological processes category, a total of 22,827 unigenes were categorized into 26 functional groups, and “cellular processes” (GO: 0009987) and “metabolic processes” (GO: 0008152) were the predominant groups. Within the cellular components category, a total of 25,399 unigenes were categorized into five functional groups, and cellular anatomical entry (GO: 0110165), intracellular (GO: 0005622), and protein-containing complex (GO: 0032991) were the most overrepresented groups. For the molecular function category, a total of 14,535 unigenes were categorized into 12 functional groups, and a large proportion of unigenes were clustered into binding (GO: 0005488) and catalytic activity (GO: 0003824) .As the annotation of the KEGG pathway database has suggested, a total of 17,184 unigenes were assigned to 5 main categories, including Cellular Process (6655), Environmental Information Processing (5223), Genetic Information Processing , Metabolism , and Organismal System (9580) .Differential expression analysis was conducted between female and male flowers (F vs. M) to acquire information on DEGs between the two samples. A total of 16,600 DEGs (19.16%) were identified between F and M, including 7107 up-regulated genes and 9493 down-regulated genes in F compared with M.p-value < 0.05) and the top 30 GO terms distribution for DEGs are shown in The KEGG pathways in which DEGs enriched significantly -related genes IAA27 and SAU50, ethylene (ETH)-related gene AIL5, and gibberellin (GA)-related gene G3OX3. The male-specific genes included stamen development gene GNL2, pollen maturation and pollen tube growth gene AGL104, pollen early development gene ZAT3, male gametophyte development gene MMD1, pectinesterase-related gene PEI, and ATPase-related gene V-type proton ATPase subunit G1.Some DEGs with female or male-specific expressions might strongly contribute to sexual differentiation. The female-specific genes included early development genes of female organs TOZ19 and genes involved in plant sex regulation, such as RPP0W, PAL3, MCM6, MCM2, and ABA2, had higher expression in F, while male-determining gene GPAT3 and the sex regulatory gene GAST1 had higher expression in M.Among A-group DEGs, the sex-determining gene PIN1, PHABULOSA (PHB), AGL11, AINTEGUMENTA (ANT), AINTEGUMENTA-LIKE6 (AIL6), SUP, SHI-RELATED SEQUENCE 5 (SRS5), and SEUSS. Furthermore, many genes related to female organ subsequent development were up-regulated in F compared with M, including stigma differentiation genes HEC3 and SPATULA; carpel development genes YABBY2, YABBY1, and WOX9; female gamete development genes PROLIFERA and ATH1; and other pistil development genes SHOOT MERISTEMLESS, FIL, RADIALIS-like 1 (RL1), and RADIALIS-like 2 (RL2). Additionally, flowering time genes CRY1 and COL2 were higher in F. Meanwhile, some of the highly expressed genes in M associated with tapetum/pollen and other male organ early development genes included BAM2, FLOWERING LOCUS D (FD), ZAT3, and HMG-CoA. In addition, anther/pollen-related genes MYBS3, APG, MAC1, HXK5, LIM2, AGL104, GAUTE, CALS5, and other male subsequent development genes GNL2 showed higher expression in M.Within B-group DEGs, F strongly expressed a few genes responsible for stigma/ovule and other female organ primordia compared with M. These genes included MET1; RdDM (RNA silencing and RNA-directed DNA methylation) pathway-related gene AGO16; the gene encoding sodium pyruvate cotransporter BASS2; sugar and lipid metabolic genes G3P, KPYC1, and TPS; and flavonoid, xylogen, and other chemical metabolic genes FL3H, F3PH, and MYBC were up-regulated in F. Meanwhile, the gene encoding a zinc finger protein C3H18; histone lysine methylase-related gene ATXR6; sugar metabolic genes PEI, PEL, GGAP2, and PLDA1; flavonoid, polyphenol, and other chemical synthesis genes CCOAMT, LAR, and MYB111; and cell cycle pathway-related genes CDKF4 and IBS1 were higher in M.For C-group DEGs, methylase-related gene ARFs, IAAs, TIR1, LAX3, and SAU50), GA-related genes , cytokinin (CTK)-related genes , abscisic acid (ABA)-related genes (ABF2 and ALDO3), jasmonic acid (JA)-related genes (FAD8 and AOS), ETH-related genes (ETR1 and AIL5), and a brassinosteroid (BR)-related gene (BRI1). Genes with higher expression in M included CTK-related genes (ARR9 and CKX7), ABA-related genes , ETH-related genes , and a JA-related gene (LOX4).As for D-group DEGs, up-regulated genes in F included IAA-related genes  cassette occurring in nearly all females and never in males, potentially regulated the sexual differentiation in Fragaria [MCM2 and MCM6, two genes involved in the cell cycle pathway, had much higher expression in the gynoecious line than those in weak female cucumbers and were considered key candidate genes of sexual differentiation in cucumbers [SRS5 had higher expression in early gynoecious inflorescence buds than monoecious plants in Jatropha curcas, which was considered a candidate regulator of the sex determination [In teration . RPP0W, Fragaria . MCM2 anucumbers . SRS5 hamination .P. amurense, ANT, AIL6, SEUSS, AGL11, SUP, PIN1, and ESR2 might promote the initiation of female floral organ primordia. ANT and its paralog AIL6 are two key regulators of flower development, including floral organ initiation, identity specification, growth, and patterning. On the one hand, they are important for establishing the flower primordia, such as ovule and female gametophyte in Arabidopsis. On the other hand, they participate in subsequent female flower development by regulating target genes including SPATULA, YABBY, SEP3, PHB, AS1, REV, and FIL and genes associated with hormones identified in P. amurense [SEUSS cooperates with ANT in a partially redundant manner to regulate the expression of downstream genes critical for the formation of ovules [AGL11 participates in the early development of ovules, which was demonstrated in many species, including Arabidopsis thaliana and Punica granatum [SUP and PIN1 were demonstrated to be involved with reproductive phase transition and female flower transition by promoting the abortion of male flower primordia in Jatropha curcas [SUP plays a role in maintaining the boundaries between stamens and carpels and regulating the development of outer ovule integument in Arabidopsis [ESR2 is a member of transcription factor BOL/DRNL/ESR2/SOB, which is expressed at the very early stages in aerial organ formation and has been proposed to be a marker for organ founder cells [P. amurense. It is worth mentioning that PAL3, MCM2, MCM6, AGL11, ANT, AIL6, SRS5, ESR2, and SUP have shown differential expression during our research aimed at early flowers (the result is unpublished), which could make the results of this study more convincing to some degree. Moreover, CYC2CL, RL1, RL2, PROLIFERA, FBP2, YABBY1, YABBY2, FIL, AS1, and PHB might be involved in subsequent female flower development in P. amurense. Two Cyc2CL transcripts (Cyc2CL-1 and Cyc2CL-2) regulate petal development and stamen abortion and are important for the ray floret (without stamens) development in the chrysanthemum [RL-like gene, also named RAD, was one of the shared genes in both pathways leading to reversion from male to hermaphrodite flowers in hexaploid D. kaki and ectopic overexpression of DkRAD in model plants resulting in hypergrowth of the gynoecium [PROLIFERA is necessary for megagametophyte and embryo development [FBP2, a MADS-box gene involved in flower development, represents the same E function as SEP3 in Arabidopsis [YABBY genes participate in ovule development. Among YABBY genes, YABBY2 plays a critical role in ovule development and is expressed in different ovule development stages, while YABBY1 has the highest expression in the Megaspore Mother Cell stage [FIL, AS1, PHB, and YABBY1 interact with ANT and SEUSS to regulate ovule development and promote organ polarity. For example, ANT combined with the YABBY gene FIL to promote organ polarity by up-regulating the expression of the adaxial-specifying HD-ZIP gene PHABULOSA. The SEUSS/LUG coregulator complex physically interacts with YABBY1 and/or ANT to regulate adaxial identity genes PHB and REV to promote ovule and carpel growth [ANT and SEUSS as initiation factors in P. amurense. Herein, a simple model of female flower development in P. amurense is proposed as a reference for future research  and female-related genes  were selected during the previous research of bark in young P. amurense [GPAT3, ASHR3, ARAD1, and GDSL) and the female-related gene BRI1 were consistent with the results in this study, suggesting that the genes above might play a role in early sex differentiation from the young stage of P. amurense.Male-related genes  and male flowers were identified by comparing female and male flowers. Further, 188 candidate genes of female flower development were screened out, including sex-regulatory genes, genes related to the formation and development of sexual organs, genes related to biochemical pathways, and hormone-related genes. Combined with gene functions and qRT-PCR analysis, RPP0W, PAL3, MCM2, MCM6, SUP, ANT, AIL6, AGL11, SEUSS, SRS5, and ESR2 were preliminarily considered to be the key genes for female flower development.Male flowers of


Dermatoscopical patterns were analysed in 100 patients: starburst pattern correlated with benign histology (26% SN (p = 0.004)), while multicomponent pattern correlated with atypical/malignant lesions ). Eighty-five lesions were subjected to fluorescence in situ hybridization (FISH): 34  were FISH-positive; 51  were FISH-negative (p = 0.0038). Discussion: This study confirmed predominant benign histology (82%) of paediatric spitzoid lesions, thus detecting 17% AST and 1% SM, highlighting the need for caution in handling spitzoid lesions. Conclusion: Until AST are considered potentially malignant proliferations and no reliable criteria are identified to distinguish them, the authors suggest a prudent approach, especially in children.Introduction: Spitzoid lesions are a wide tumour class comprising Spitz nevus (SN), atypical Spitz tumour (AST) and Spitz melanoma (SM). Materials and Methods: We conducted a single-centre-based retrospective survey on all histologically diagnosed spitzoid lesions of paediatric patients (1–18 years) of the last 10 years (2012–2022). Histopathological reports and electronic records of patients were used to retrieve relevant data regarding patients’ features, clinical and dermatoscopical aspects of lesions when recorded, and FISH tests when present. Results: Of 255 lesions, 82% were histologically benign, 17% atypical, 1% malignant. Clinically, 100% of SM were large (≥6 mm) and raised; AST were mainly large (63%), raised (98%), pink (95%). Small (≤5 mm), pigmented, flat lesions correlated with benign histology  ( Spitzoid lesions represent a challenging entity, as their spectrum ranges from benign Spitz nevi (SN) to malignant Spitz melanoma (SM), comprising also atypical spitzoid tumours (AST), which are spitzoid tumours of uncertain malignant potential and difficult to classify histologically ,2,3.Spitzoid lesions were first described by pathologist Sophie Spitz in 1948 and have been widely studied morphologically and biologically ever since. At first, the term “juvenile melanoma” was used by Dr. Spitz to define these lesions, typical of childhood, with both clinical and histopathological malignant aspects. However, after observing the generally benign biological behaviour of these lesions, the spitzoid class was soon regarded as completely benign. Cases of malignancy with metastatic disease were outright attributed to misdiagnoses of melanomas mimicking spitzoid lesions ,6. In thSpitzoid lesions are all histologically characterized by spindle cell and epithelioid cell melanocytic proliferations, as first described by Dr. Spitz, but while SN display benign histological features and SM display malignant histological features, AST present some atypical histological features in between SN and SM, yet insufficient to make a diagnosis of melanoma .In addition, recently, a distinction between “Spitz melanoma” and “Spitzoid melanoma” has been established. Though both present clinical and histopathological aspects reminiscent of Spitz nevi, “Spitz melanoma” presents typical melanoma driver mutations, while “Spitzoid melanoma” displays kinase fusions typifying Spitz tumours ,11.To ultimately distinguish spitzoid proliferations into biologically benign or malignant, sentinel lymph node biopsy (SLNB) was previously used, yet this practice is almost abandoned, as its prognostic and therapeutic utility is actually debated, in addition to its high morbidity ,12,13.Of note, spitzoid lesions present with an overall incidence of 1.4 to 7 cases per 100,000 person per year and characteristically develop in the paediatric age or in young adults, although all age groups can be affected. Almost 40% of Spitz lesions appear before the age of fifteen, and 77% appear before the age of thirty, while in older individuals, the presence of these lesions is quite uncommon ,15. By aClinically, Spitz nevi generally appear as single dome-shaped papules, flesh-coloured to pinkish or red in colour, due to the high vascularization and low melanin content; thus, almost 10% of lesions are pigmented with brown colour . The lesAccording to literature data, AST are typically larger in size (>10 mm) than SN, occur in older (>10 years) patients, more often on the trunk, and are characteristically asymmetric—irregular in colour and borders. Yet further characterization and analysis of these lesions is needed ,21.SM are generally hypopigmented or non-pigmented lesions, though also pigmented lesions with multiple colours with progressive growth have been described in the literature. Lesions usually reach relevant (>10 mm) dimensions and may display ulceration. Head and neck and limbs are frequent locations ,22.Histologically, SN are well delimited melanocytic lesions, mostly with a compound presentation, that can be located both at the dermo-epidermal junction and within the dermis, though some SN are limited to the dermo-epidermal junction with cytological features similar to the compound counterpart . EpithelThe cells tend to form uniform nests, vertically oriented, with a dermal component that matures with depth and, less markedly, towards the periphery; yet a pagetoid spread of single, isolated, melanocytes can be noted at the upper epidermal layers or at the dermo-epidermal junction.2) can be observed in the upper or mid part of the lesion, while deeper mitoses are rare, and atypical mitoses are absent [Typical mitosis , absence of Kamino bodies and ulceration. Although some grade of atypical features is observed in AST, criteria for diagnosing melanoma are not sufficiently met, placing AST in between SN and SM [AST are histologically characterized by at least one atypical feature among poor lateral delimitation, asymmetry, lack of dermal maturation, great downwards extension, plentiful isolated melanocytes in the upper dermis rather than forming nests, presence of mitoses in the dermis  of the last 10 years (2012–2022). Histopathological reports and electronic records of patients were used to retrieve all relevant data: histopathological diagnosis, age at diagnosis, sex, anatomical site of lesion, size of lesion, clinical aspect, dermatoscopical aspect if recorded and FISH test when present. All lesions were surgically excised with punch biopsy or with fusiform excision using a no. 15 scalpel blade. Then standard histological examination was carried out by two expert dermatopathologists trained in skin tumour diagnosis on formalin-fixed, paraffin-embedded tissue sections. Histologic sections were obtained dissecting the specimen perpendicular along its longest axis. Conventional histology using a routine staining procedure with haematoxylin and eosin (H/E) staining was applied, and immunohistochemical markers, including MIB-1, HMB-45, p16, S100 and Melan-A, were used as complementary analyses to the ultimate histopathological diagnosis.Lesions displaying overlapping histologic features with conventional Spitz nevus and spitzoid melanoma were defined as AST, a borderline spitzoid melanocytic lesion of uncertain malignant potential.Fluorescence in situ hybridization (FISH) conventional melanoma probes  with probes targeting four loci—cyclin D1 (CCND1) on 11q13 (Vysis LSI CCND1-Spectrum green), V-myb myeloblastosis viral oncogene homolog (MYB) on 6q23 (Vysis LSI MYB-Spectrum gold), ras responsive element binding protein 1 (RREB1) on 6p25 (Vysis LSI RREB1-Spectrum red) and centromeric enumeration probe control for chromosome 6 (Vysis LSI CEP6-Spectrum aqua) were performed in all histologically diagnosed AST and in an equal number of SN, matched for age and sex, as controls. The FISH test was considered positive for the following chromosomal abnormalities: greater than 38% CCND1 gain; greater than 29% RREB1 gain; greater than 55% relative gain of RREB/CEP6; and greater than 40% relative loss of MYB/CEP6.Patients with histologically atypical and malignant lesions were followed up for clinical evaluation of local recurrences and lymph node macro-metastases.2 test were used to analyse data statistically; p values < 0.01 were considered statistically significant.Fisher’s exact test and χp > 0.05). Specifically, patients 12 years or older had no more malignant lesions  than younger patients  (p > 0.05).Overall, 255 paediatric spitzoid lesions were included in the present study, with mean age of 7.9 years, median age of 7 years, and male:female ratio of 1.1:1. Histologically, 82% were benign (210 SN), 17% atypical (43 AST), and 1% malignant (2 SM) . Featurep = 0.0385), head and neck were more associated with atypical/malignant histology (34% AST/SM) (p = 0.0005).Concerning the anatomic distribution of spitzoid lesions, lower limbs were most frequently involved , followed by upper limbs , head and neck , trunk , hands and feet  and perineum . Of SN, 35 were identified on the head and neck, 27 on the trunk and 148 on the limbs . Notablyp < 0.0001) ; while SN of the limbs were associated with pigmentation (62%)   and raised (100%); 50% were pink; AST were mainly large (63%), raised (98%) and pink (95%). Conversely, small (≤5 mm), pigmented and flat lesions were statistically associated with benign histology  ( 0.0001) . Head anOR 2.99) . InteresOR 2.99) . All of p < 0.0001); OR 66.50, 95% CI 25.97–170.27).Interestingly, flat lesions were statistically more associated with the presence of pigmentation (92% (55/60) pigmented; 8% (5/60) hypo-/non-pigmented) than raised lesions (32% (62/195) pigmented; 68% (133/195) hypo-/non-pigmented (p = 0.004). Conversely, a multicomponent pattern proved an association with atypical/malignant lesions (20 AST (56%) and 1 SM (50%), respectively; p = 0.0052). No other dermatoscopical pattern proved a statistical association with histology (p > 0.05) . The retrieved patterns are displayed in  > 0.05) ; 17% of  > 0.05) ; 17% of p = 0.0038; OR 4.04) , and 51 were FISH test negative . FISH test-positive lesions correlated with atypical histology (OR 4.04) .Regarding clinical follow-up and further management of patients, patients with AST were clinically evaluated every six months. SM were treated according to melanoma protocol. Among the numerous AST lesions analysed in this study, only 1 (2%) AST presented clinically evident lymph node macro-metastasis, and no clinical recurrences were reported.This single centre study comprised over 250 consecutive paediatric-specific spitzoid neoplasms of the last ten years, permitting us to obtain a wide case series of paediatric patients. Overall, literature data were confirmed. Paediatric spitzoid lesions were histologically benign in 82% of cases, though a relevant percentage of AST 17%) were retrieved, as well as 1% of SM, highlighting the need for utmost caution in the management of spitzoid neoplasms 7% were r,4,5,6,7.At present, the management of spitzoid lesions is controversial, and numerous succeeding guidelines have been developed by the IDS to help clinician handle morphological overlap between SN, AST and SM .In young children <12 years) a conservative approach is suggested in the management of spitzoid lesions, considering the rarity of malignant spitzoid lesions in young children and their utmost favourable prognosis 2 years a,10.Conversely, in a study by Lallas et al. conducted on 384 patients aged twelve years or older presenting spitzoid lesions, the risk of melanoma was around 13%. Notably, the risk increased with increasing age, reaching 50% risk of melanoma in patients aged 70 . ConsequHowever, it must be considered that IDS guidelines were developed relying on studies including mainly adult patients, with a mean age of 27.7 years. Thus, adult patients may represent a statistical bias, with underreported paediatric patients . On the p = 0.004; OR 27.32).Nevertheless, IDS guidelines further classify spitzoid lesions among young children; in patients <12 years, a symmetric starburst pattern can be handled conservatively, as it corresponds to Reed nevi . IDS 201Contrarily, a dermatoscopical multicomponent pattern and nodular spitzoid lesions should raise high suspicion in spitzoid paediatric lesions, being statistically associated with histologically atypical/malignant lesions  and demand utmost caution, as already suggested by Moscarella et al. .Nonetheless, it is worth noting that the malignant potential of AST has been widely discussed lately, as patients, especially paediatric patients, tend to have a favourable prognosis, even after positive SLNB ,4, whileNotably, in a cohort of 67 patients with AST, Ludgate et al. observed 47% positive for sentinel lymph nodes, yet of 57 patients subjected to SLNB, no patients presented recurrence of disease, and at 43.8 month follow-up, all of them were disease free ,33,34.However, the current definition of AST, as tumours of uncertain malignant potential, necessarily demands the highest attentiveness. Neither difficult-to-treat anatomical sites nor concerns about anaesthesiologic procedures should by any means constitute a therapeutic limitation in children . In paedRegarding the clinical application of cytogenetics, melanoma probes FISH technology recently demonstrated a high impact on melanocytic skin lesion analysis, permitting the rapid screening between melanomas and nevi ,37 but nIndeed, Gerami et al. proposed melanoma probe FISH to detect the copy number of genes RREB1, MYB, CCND1 and CEP6 . While tHowever, the majority of studies on the clinical applicability of FISH did not present long-term clinical follow up ,38,39,40p = 0.0038; OR 4.04)  , possiblRecently, FISH probes with greater specificity for spitzoid lesions than melanoma FISH probes were also developed . These FMolecular investigations represent promising complementary diagnostic tools to characterize spitzoid lesions and to predict their malignant potential, yet high costs and low availability have so far hindered real-world use in daily clinical practice. Universal, wider population studies would help to gain a deeper understanding and to standardize these techniques.Spitzoid lesions are highly difficult-to-manage lesions, especially in children, considering the complexity of their clinical, dermoscopical and histological differentiation ,2,3,4 InMore and more evidence is provided in the literature of the low biological aggressiveness of paediatric AST . Yet the


Since its initial report in Vietnam in early 2019, the African swine fever (ASF), a highly lethal and severe viral swine disease worldwide, continues to cause outbreaks in other Southeast Asian countries. This study analyzed and compared the genomic sequences of ASF viruses (ASFVs) during the first outbreak in Hung Yen (VN/HY/2019-ASFV1) and Quynh Phu provinces (VN/QP/2019-ASFV1) in Vietnam in 2019, and the subsequent outbreak in Hung Yen (VN/HY/2022-ASFV2) in 2022, to those of other ASFV strains. VN/HY/2019-ASFV1, VN/QP/2019-ASFV1, and VN/HY/2022-ASFV2 genomes were 189,113, 189,081, and 189,607 bp in length, encoding 196, 196, and 203 open reading frames (ORFs), respectively. VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1 shared a 99.91–99.99% average nucleotide identity with genotype II strains. Variations were identified in 28 ORFs in VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1 compared to 20 ASFV strains, and 16 ORFs in VN/HY/2022-ASFV2 compared to VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1. Vietnamese ASFV genomes were classified as IGR II variants between the I73R and I329L genes, with two copy tandem repeats between the A179L and A137R genes. A phylogenetic analysis based on the whole genomes of 27 ASFV strains indicated that the Vietnamese ASFV strains are genetically related to Estonia 2014, ASFV-SY18, and Russia/Odintsovo_02/14. These results reveal the complete genome sequences of ASFV circulating during the first outbreak in 2019, providing important insights into understanding the evolution, transmission, and genetic variation of ASFV in Vietnam. Asfaviridae) causes economic damage to farms due to stamping-out, social disruption due to a high mortality and quarantine, and pork import and export regulations [African swine fever (ASF) is a serious transboundary disease and highly infective among domestic pigs and wild boars. The ASF virus  as a viral hemorrhagic fever in domestic pigs; however, by the 1950s, it had spread to most African countries ,7,8. In Before the official report of the ASFV outbreak in Vietnam in 2019, clinical symptoms similar to ASF had occurred and were reported in all major pig farms in the northern and southern regions of Vietnam. Based on the temporal analysis of outbreak regions in China and considering the potential introduction of ASFV from the border regions of China, several ASF outbreaks occurred in the Yunnan and Guangxi provinces bordering Vietnam in southwestern China from October to December 2018 ,22. ReceIn this study, we collected ASFV-positive domestic pig tissues during the first 2019 outbreaks in Hung Yen and Quynh Phu provinces, northern Vietnam and whole blood samples in 2022 in Hung Yen and conducted NGS on these samples. We assembled the complete coding regions of the ASFV genomes from 2019 and 2022 in Vietnam. Notably, we obtained whole genomic sequences during the first outbreak in Vietnam directly from spleen tissues and whole blood samples without ASFV propagation and enrichment through a cell culture. Moreover, we compared the genome of the Vietnamese strain to those of the China/2018/AnhuiXCGQ and related European p72 genotype II strains. Our results provide important insights into the evolution of ASFV following its transboundary spread and essential genetic information for developing vaccines, and diagnostic markers, and implementing effective control measures to prevent the spread of ASF.® ASFV qPCR kit , that amplifies a target sequence in the ASF viral B646L (p72) genome. A total of 10 mg of individual spleen tissues and 200 µL of whole blood obtained from deceased pigs were homogenized with a grinding process in phosphate-buffered saline (PBS) and lysed using the addition of Proteinase K, heated at 56 °C for up to 1 h, and centrifuged at 12,000× g for 10 min. A total of 200 μL of the lysate supernatant was used for viral DNA extraction with a QIAamp Viral DNA Mini Kit 250  [ASFV-infected spleen tissue samples were collected in 21 breeding sows from a pig farm in Hung Yen VN/HY/2019-ASFV1) and 24 fattening pigs in Quynh Phu (province) (VN/QP/2019-ASFV1) in northern Vietnam. The pigs displayed several distressing symptoms, including a sudden loss of appetite, high fever, anorexia, vomiting, lethargy, respiratory distress, coughing, instances of abortion, and disseminated cyanosis, and the first to show ultimately died 3 days later in the first outbreak in 2019 -ASFV1 an. A totalLibrary preparation was performed according to the TruSeq Nano DNA Library Prep Guide . In brief, 100 ng of 3 genomic DNA samples from each VN/HY/2019-ASFV1, VN/QP/2019-ASFV1, and VN/HY/2022-ASFV2 sample was sheared using an LE220 Focused-ultrasonicator  with a duty factor of 15% and peak incident power of 450 W at 200 cycles per burst for 50 s. Sheared DNA fragments were then end-repaired, size-selected to obtain DNA fragments of approximately 350 bp, and adenylated according to the manufacturer’s instructions. After ligating index adapters to the ends of the DNA fragments, DNA libraries were enriched using eight cycles of PCR, according to the manufacturer’s instructions. After size selection and PCR amplification, the quality of the library and band size were assessed using D1000 Screen Tapes on a Tapestation 2200 . Finally, libraries were quantified using the PicoGreen dsDNA quantitation assay  and determined using a Victor3 plate reader .Illumina Inc. uses a unique bridged amplification reaction on the surface of a flow cell. A flow cell containing the DNA libraries was prepared using the cBot fluidics station and then loaded onto the HiSeq™ 4000 platform (Illumina Inc.) for automated cycles of extension and imaging. The sequencing-by-synthesis cycle was repeated for a paired-end read length of 2 × 100 bp.The whole genomes of VN/HY/2019-ASFV1, VN/QP/2019-ASFV1, and VN/HY/2022-ASFV2 were sequenced using the Illumina HiSeq™ 4000 platform. First, sequence reads were assembled using SPAdes to generate a single-cell assembler . Each VNThe CLC Genomics Workbench 22.0.2 (QIAGEN) was used for a comparative genomic analysis. For comprehensive genome comparison, 25 ASFV strains were selectively isolated as the first outbreak and reported in several countries by 2019. First, the whole-genome average nucleotide identity (ANI) and alignment percentage (AP) were computed using the ANI 1.0 module . In addiIn 2019, during the first outbreak in Vietnam, the pigs of sows in Hung Yen and Quynh Phu province pig farms showed the first clinical signs of the disease, including high fever, anorexia, vomiting, lethargy, respiratory distress, coughing, abortion, and unwillingness to stand, and the first sow died 3 days later. ASFV was detected, using commercial real-time PCR targeting of the B646L (p72) gene, in 21 of the 33 dead pigs in Hung Yen in February 2019 and all 24 pigs in Quynh Phu (province) in May 2019. After the first outbreak in Vietnam, ASFV continued to occur and ASFV-positive whole blood samples were collected in Hung Yen from five dead pigs in November 2022. We collected three genomic DNA samples at various time points (2019–2022) and from different regions in Vietnam (Hung Yen and Quynh Phu provinces). We conducted whole-genome sequencing on the collected samples from Hung Yen and Quynh Phu in 2019 and Hung Yen in 2022, with cycle threshold (Ct) values of 26.94, 13.58, and 15.1 using real-time PCR, respectively.After whole-genome sequencing, we successfully analyzed the VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1 strains circulated in Hung Yen and Quynh Phu in 2019, respectively. Two contigs of each Vietnamese strain were obtained using de Devo SPAdes assembly software, and 196 ORFs were identified using the RAST server. The complete genome sequences of VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1 were 189,113 and 189,081 bp in length, respectively. Compared with the ASFV China/2018/AnhuiXCGQ genome, which represents the first ASFV outbreak in China in 2018, both sequences showed deletion of nucleotides, spanning from position 420 to 810 at the 5′ end. Compared with the VN/HY/2019-ASFV1 genomic sequence, that of VN/QP/2019-ASFV1 showed a deletion of 37 bp at the 3’ end. The pairwise comparison showed six variable nucleotide sites between VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1, including four deletion and two insertion sites. However, these two genome sequences showed 100% ANI, even with these deletion and insertion sites .We first reported the partial genome sequence of B646L (p72), EP402R (CD2v), and E183L (p54) in Vietnam in 2019. These sequences were classified into p72 genotype II and CD2v serotype 8, which are identical to the Georgia 2007/1 and China/2018/AnhuiXCGQ strains . The GenAs shown in Most of the p72 genotype I strains with variable MGFs, including MGF360, MGF110, and MGF505, exhibited more modifications and gene deletions than p72 genotype II, which includes VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1. Comparing the MGF360-1L, MGF110-14L, and KP177R genes of VN/HY-ASFV1 with the Estonia 2014 genomic sequence, the genomic sequence of VN/HY-ASFV1 shared 10.32–89.26% nucleotide and 40.62–83.2% protein identity with some of the genes missing from the Estonia 2014 strain . CompariIn comparing the sequences of MGF110-1L of VN/HY-ASFV1 and VN/QP-ASFV1 with the genome sequences of p72 genotype I, 64 mutations were identified, and three nucleotides were deleted at positions 316–318. Moreover, at the 3′ end, 498 nucleotides were deleted in p72 genotype II, including VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1, except for ASFV-SY18, ASFVL14, and Georgia 2007_1, which had a deletion of 170 nucleotides . The 5′ The complete genome sequence of VN/HY2022-ASFV2 encodes 203 ORFs and 189,607 bp in length, including approximately 500 additional nucleotides at the 3′ end, with 99.98% ANI compared to VN/HY/2019-ASF1 and VN/QP/2019-ASF1. The ORFs that differed between VN/HY2022-ASFV2 and VN/HY/2019-ASF1 were ASFV_Ch_ACD_00290 QDL88036.1, ASFV_Ch_ACD_00290 QED21566.1, F8221_gp208, EP402R, and H233R. ASFV_Ch_ACD_00290 QDL88036.1 was only encoded in VN/HY/2019-ASF1 and VN/QP/2019-ASF1. EP402R and H233R, which were divided into four and two ORFs, respectively, were only detected in VN/HY2022-ASFV2. By comparing VN/HY2022-ASFV2 with VN/HY/2019-ASF1 and VN/QP/2019-ASF1, we detected 16 ORFs  showing nucleotide mutation, insertion, and deletion sites, and prominently identified 19 nucleotides’ mutation in MGF_360-21R, 66 nucleotides’ insertion in I196L, 11 nucleotides’ deletion in H233R, and 9 and 3 nucleotides’ deletion in MGF_110-14L of VN/HY/2019-ASF1 and VN/QP/2019-ASF1, respectively.Based on the IGR between the I73R and I329L genes, ten nucleotide tandem repeats of GGAATATATA were considered molecular fingerprints or genetic markers that are highly variable, specific, and easy to amplify. The four IGR repeats were classified as IGR I (two copies), II (three copies), III (four copies), and IV (five copies), depending on the number of tandem repeats . The IGRSequence alignment was performed for the whole-genome sequences of the ASFV strains downloaded from NCBI using the CLC Genomics Workbench. The phylogenetic analysis using the maximum likelihood method revealed that all 27 ASFV strains could be divided into two p72 genotypes . VN/HY/2In this study, we analyzed the first outbreak cases of Vietnamese ASFV strains circulating among northern pig farms in Hung Yen in February and Quynh Phu, Thai Binh in May 2019, and later in Hung Yen in November 2022. Notably, we used ASFV-infected pig spleen tissue and whole blood samples without the need for sub-culturing the virus. When the first ASFV outbreak was reported in Vietnam, we reported that ASFV might have spread from the first ASFV strain named China/2018/AnhuiXCGQ, which was detected in China in August 2018, sharing the same p72 genotype II and CD2v serotype 8 . In the In the present study, the results of the multiple sequence alignment analysis for 28 ORFs, particularly EP153R, I196L, KP177R, and MGFs of VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1, showed distinct deletion regions compared to Estonia 2014 and the p72 genotype I strains. Mutations observed in EP153R, I196L, and KP177R are associated with increased virulence and transmissivity, potentially rendering the ASFV less susceptible to vaccines ,38,39. MIn addition, the IGRs located between the I73R and I329L of VN/HY/2019-ASFV1, VN/QP/2019-ASFV1, and VN/HY/2022-ASF2 were classified as IGR II in p72 genotype II, which differs from IGR I in p72 genotype I. IGR II was the most common variant circulating in Vietnam, and IGR I was detected only in Hanoi and northern Vietnam in 2019. Recently, we detected four IGR III variants in three different regions of northern Vietnam; however, IGR III variants are not common in Vietnam and have been previously reported in China. Therefore, IGR III variants detected from 2020 to 2021 in northern Vietnam, near the Chinese border, are assumed to be the result of the transmission of ASFV from China ,34.In the present study, the phylogenic analysis of 27 ASFV strains revealed that Vietnamese strains had a closer genetic relationship with Estonia 2014, ASFV-SY18ASFV/LT/1490, Pol/2015/Poldiaskie, and Russia/Odintsovo_02/14 than China/2018/AnhuiXCGQ in the p72 genotype II group. The Estonia 2014 strain first occurred in Estonia in 2014 and quickly spread to neighboring countries. ASF outbreaks occurred sequentially in Latvia, Lithuania, Poland, and Russia in 2015 and in Belarus, the Czech Republic, Slovakia, and Ukraine in 2016 . During In conclusion, the whole-genome analysis of ASFVs in Vietnam during the first outbreak in 2019 provides important insights into the origin and evolution of the virus in the country. The genetic analysis showed that the ASFV strains circulating in Vietnam were closely related to strains previously detected in Estonia, China, and Poland, suggesting that the virus had likely originated from one of these countries. Our data also showed that the ASFV strains in Vietnam are undergoing rapid evolution and acquiring mutations in genomes involved in virulence or transmission, highlighting the need for continued surveillance of ASFV in Vietnam and other countries to track the virus’s evolution and prevent further outbreaks.


While the interplay between aging, immunosenescence, and IAV is recognized as key in severe lower respiratory tract infections, the role of specific patient attributes in shaping innate immune reactions and inflammasome activity during IAV infection remains under-investigated. In this study, we utilized an ex vivo infections, and the ensuing immune responses were profiled using flow cytometry.Our cohort consisted of thirty adult patients who underwent video-assisted thoracoscopic surgery during 2018–2019. Post-surgical lung tissues were exposed to H3N2 IAV for P = 0.022) and macrophages (P = 0.003) in the IAV-exposed group relative to controls. Notably, interleukin-2 levels correlated with variations in TLR1 expression on epithelial cells and PD-L1 markers on macrophages. Age emerged as a modulating factor, dampening innate immune reactions, as evidenced by reduced interleukin-2 and interferon-γ concentrations (both adjusted P < 0.05). Intriguingly, a subset of participants with pronounced tumor necrosis factor-alpha post-mock infection (Cluster 1) showed attenuated cytokine responses in contrast to their counterparts in Cluster 2 and Cluster 3 . Individuals in Cluster 2, characterized by a low post-mock infection NP expression in macrophages, exhibited reduced variations in both NP and TLR1–3 expressions on these cells and a decreased variation in interleukin-2 secretion in comparison to their Cluster 3 counterparts, who were identified by their elevated NP macrophage expression .We observed pronounced IAV activity within lung cells, as indicated by marked NP upregulation in both epithelial cells (ex vivo H3N2 IAV exposure, reflecting real-world lower respiratory tract infections. While these findings provide a foundation for tailored therapeutic strategies, supplementary studies are requisite for thorough validation and refinement.Our work elucidates the multifaceted interplay of patient factors, innate immunity, and inflammasome responses in lung tissues subjected to  Among tVarious risk factors heighten the susceptibility to acute or severe lower respiratory tract infections. These risks encompass aging, existing health conditions, weakened immunity, smoking, incomplete vaccination, and socio-environmental factors such as overcrowding and malnutrition . Aging pFurthermore, the age-associated decline in immune function, known as immunosenescence, augments vulnerability to infections . As an eex vivo lung tissue model. We theorized that specific patient traits might impact the immune response during the early stages of ex vivo H3N2 IAV infection. Our exploration aims to clarify the intricate relationships between patient characteristics, immune responses, and H3N2 IAV infection, potentially guiding personalized influenza treatment strategies.In this study, we set out to: (1) assess the effects of H3N2 IAV on primary lung tissue cultures, emphasizing TLRs, the PD-1/PD-L1 pathway, and other immune biomarkers, and (2) determine the role of patient characteristics, especially age, in H3N2 IAV infection using an 22.1The study protocol was approved by the Institutional Review Board at the Chang Gung Medical Foundation, Taoyuan, Taiwan . The procedures were in line with the ethical principles of the Declaration of Helsinki 1975. Before enrollment, all participants provided written informed consent.We conducted a prospective case-control study. Twelve patients aged 65 years or older (case group) and 24 patients aged less than 65 years (control group) who underwent video-assisted thoracoscopic surgery (VATS) at Chang Gung Memorial Hospital, Linkou Main Branch, Taoyuan, Taiwan, between August 1, 2018, and December 31, 2019, were recruited. Both groups were matched for sex and body mass index (BMI). The study protocol is illustrated in The inclusion criteria for participation were as follows: (1) age above 20 years, (2) undergoing VATS due to suspected lung cancer nodules, and (3) willingness to participate and provide written informed consent. Participants were excluded from the study if they met any of the following criteria: (1) recent respiratory tract viral infection within the last 2 weeks, (2) known history of chronic viral infections such as human immunodeficiency virus, hepatitis B virus, or hepatitis C virus, or (3) known history of interstitial lung disease, severe chronic obstructive lung disease, pulmonary fibrosis, or other lung infectious diseases. Data collected for each participant included age, sex, BMI, history of lung cancer, medical comorbidities , and smoking history.2.23, which was distant from the resection margin and any gross pathology, was carefully dissected from the lobe. The dissected tissue was then briefly stored in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 1% penicillin-streptomycin  and 1% gentamicin  during transport to the laboratory, with a maximum transport time of 30 minutes   was used for washing the tissue three times, followed by a single wash with unsupplemented RPMI 1640 medium. Subsequently, the tissue was incubated overnight at 37°C in a 5% CO2 atmosphere using RPMI 1640 medium supplemented with 1% penicillin-streptomycin and 1% gentamicin.Upon arrival at the laboratory, the tissue was further processed. It was cut into 1-mm2.3ex vivo infection of resected lung tissue with seasonal H3N2 IAV strain A/Texas/50/2012 and mock infection were carried out following established protocols  and type IIA elastase (10 U/mL), both sourced from Sigma Chemical Co., St. Louis, MO, USA, operating at a temperature of 37°C for a predetermined duration of 15 minutes, as previously detailed in reference . Subsequ2.4Cells that exhibited immunoreactivity for the IAV nucleoprotein (NP) were determined to be virally infected . An incrFor the identification of NP-positive cells, samples were resuspended in BD Pharmingen stain buffer  supplemented with heat-inactivated fetal bovine serum (FBS) proteins (incubated at 56°C for 30 minutes). Intracellular staining for viral NP used the BD Cytofix/Cytoperm fixation/permeabilization kit , following manufacturer instructions by Becton Dickinson Biosciences. Post-fixation and permeabilization, samples were incubated for 30 minutes in stain buffer (FBS) on ice, away from light, to block Fc receptors, minimizing nonspecific antibody binding common in lung macrophages, as detailed in reference . During To create a baseline, an unstained singlet population was obtained from digested tissue, excluding dead cells using Fixable Viability Stain 700 . The resulting lung tissue cell sample was analyzed for intracellular IAV NP expression using the FACSAria IIu Cell Sorter. FACS data were then processed using FACSDiva software v5.0.3. The flow cytometric analysis methodology aligned with standard procedures as outlined in earlier literature.2.5-/EpCAM+ biomarkers, whereas macrophages were defined by their CD45+/HLA-DR+ patterns . Subsequently, the samples were subjected to a 30-minute incubation on ice in the absence of light, with the introduction of fluorescently labeled antibodies or their respective isotype controls. All antibodies utilized in this process were procured from Becton Dickinson Biosciences, San Jose, CA, USA.During the flow cytometric analysis, the categorization of epithelial cells hinged on the presence of CD45patterns . T cellsSpecific targeting of CD45, EpCAM, HLA-DR, CD3, CD4, and CD8 molecules was accomplished using the following fluorescently labeled antibodies , samples were incubated with appropriate fluorescently labeled antibodies or isotype controls, following the manufacturer’s protocols. For PD-1 detection, BV421 mouse anti-human CD279 (PD-1) antibody  was used, and for PD-L1 detection, APC Mouse anti-human CD274 (PD-L1) antibody  was used -2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) in the culture supernatants. Each capture bead  had distinct fluorescence and was coated with a specific capture antibody. Upon incubation with standards or specimens, sandwich complexes formed and were identified using flow cytometry as outlined in earlier literature .2.9ex vivo H7N9 IAV infection  for continuous and skewed variables, and as numbers (proportions) for categorical variables. Percentage (%) difference was calculated using the formula: /variable for mock infection group × 100. In this calculation, value of mock infection group served as reference. To ensure that there were no gaps in the assay results and to avoid inaccuracies in estimating concentrations, samples with undetectable values were treated as follows: they were plotted as either 0.1 pg/mL or one-half of the lower limit of quantitation for that particular cytokine, whichever value was greater .U test was employed to assess differences between two groups , while the related-samples Wilcoxon signed-rank test was used to assess within-group differences  when appropriate. For independent categorical variables, such as age groups, Fisher’s exact test or chi-square test was used to analyze differences between the subgroups when appropriate. To examine relationships between variables of interest, the Spearman correlation test was utilized. In comparing differences in outcome variables by age group (age ≥ 65 years vs. age < 65 years), generalized estimating equations were applied with adjustments for potential confounding factors .In the analysis of continuous variables, the Mann-Whitney post hoc analysis, the researchers evaluated the profiles of TLRs, PD-1/PD-L1 pathways, viral expressions, and cytokines. To identify the regulation patterns of these variables, unsupervised cluster analysis was conducted based on the % differences of the 18 variables of interest. The hierarchical cluster analysis algorithm was used to determine the number of clusters and assess their robustness. Subsequently, between-group comparisons were performed using the independent-samples Kruskal-Wallis test to examine if there were significant differences among the identified clusters. Given the multiple tests conducted, to control for the increased risk of type I error, the significant P-values obtained were adjusted using the Bonferroni correction method.In the P-value of < 0.05 was considered statistically significant.All statistical calculations are performed with G*Power 3.1.9.2 , SPSS , and GraphPad Prism for Windows  software packages. A two-sided 33.12. 3.2During the early phase of H3N2 IAV infections, a distinct upsurge in NP expression was observed in both epithelial cells and macrophages of the H3N2-infected cohort, relative to the mock-infected group Table 2.+/CD4+ T cells. Nevertheless, these initial relationships lost their statistical significance post the application of the Bonferroni correction. In contrast, correlations concerning % differences in NP and TLR1–3 expressions in macrophages retained their significance post-adjustment.3.3There was a significant increase in in the secretion of cytokines IL-2, IL-6, IL-10, TNF-α, and IFN-γ in the H3N2 virus-infected group 24 hours after infection Table 2.3.4Initial assessment indicated only a handful of variables exhibiting age-dependent variations Table 4.3.5n = 21), Cluster 2 (n = 5), and Cluster 3 (n = 4), detailed in Unsupervised clustering led to the classification of participants into three discernible clusters: Cluster 1 , Cluster 2 (low NP expression in macrophages and low secreted TNF-α level), and Cluster 3 (high NP expression in macrophages and intermediate secreted TNF-α level), we were able to discern specific molecular and cytokine response trends that shed light on how different patient groups may react to IAV exposure. In this context, Cluster 2 served as our reference subgroup.Our detailed exploration into the innate immune responses and inflammasome activation post-Cluster 2, identified by its muted NP expression in macrophages during mock infections, likely represents individuals with limited previous exposure to IAVs. Their pronounced upswing in secreted cytokines TNF-α and IFN-γ upon H3N2 IAV exposure aligns with prior research , paintinIn contrast, the pronounced NP expression in Cluster 3 during mock infections could signal a history of exposure to IAVs, possibly without overt clinical manifestations. This silent sensitization seems to have equipped them with a faster and more intense immune response upon subsequent H3N2 challenges . The pro+ T cell defense against IAV, especially in the older demographic (Cluster 1, distinguished by its pronounced TNF-α secretion, unveils another facet of the immune response. With a demographic skew toward older individuals and those with a history of lung cancer, the heightened TNF-α secretion possibly hints at a backdrop of chronic inflammation, or even a predisposition toward malignant processes . This elographic . Taking ex vivo model, but several caveats exist. First, we didn’t use a western blot for IAV NP in this study, but we had validated infections through quantitative real-time PCR of IAV RNA in our previous study (ex vivo model offers direct insights into human tissue responses, it inevitably lacks the systemic interplay present in in vivo conditions. This limitation means that while our findings provide foundational knowledge, in vivo studies will be essential for translating these observations into clinical practice. Fourth, our primary focus on CD3+ and CD8+ T cells may have missed the nuanced responses from other specialized T cell subsets. Including these subsets could help elucidate the full spectrum of T cell responses to IAV infection. Fifth, the relatively limited sample size and short incubation period may restrict the scope and generalizability of our findings. Future investigations employing larger sample sizes and prolonged incubation periods can offer more robust and detailed insights.This study sheds light on the immune response and inflammasome activation during IAV infection using a human us study . Elevateus study . Second,us study . In lighus study  and our us study , we straus study . We inteex vivo H3N2 IAV infections, underscoring the central role of NP-expressing macrophages in the formative stages of the immune response. Aging, histories of lung cancer, and previous encounters with IAV emerge as pivotal factors that shape and modulate these responses. The molecular markers we’ve identified, notably NP expression in macrophages and TNF-α secretion during mock infections, offer a valuable schema to categorize patients based on the nature and magnitude of their immune reactions and subsequent inflammasome activations. Our ex vivo model stands out in its capability to mirror the myriad ways host factors mold innate immune responses to IAVs. This understanding, though preliminary, offers a bedrock upon which we can design more tailored and effective preventive and therapeutic interventions against H3N2 IAV and potentially other influenza viruses. To solidify our conclusions, studies with larger samples and a broader spectrum of immune cells are essential. This will enrich our understanding and improve strategies to manage IAV infections.In conclusion, our study has illuminated the nuanced responses of human lung tissue to The original contributions presented in the study are included in the article/The studies involving humans were approved by the Institutional Review Board at the Chang Gung Medical Foundation, Taoyuan, Taiwan. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.C-GH: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Resources, Software, Validation, Writing – original draft. Y-CW: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing – original draft. M-JH: Data curation, Formal Analysis, Investigation, Methodology, Writing – review & editing. Y-JL: Data curation, Investigation, Methodology, Writing – review & editing. T-HH: Data curation, Investigation, Methodology, Writing – review & editing. P-WH: Data curation, Formal Analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft. S-LY: Data curation, Investigation, Methodology, Resources, Writing – review & editing. K-CT: Supervision, Writing – review & editing. S-RS: Supervision, Validation, Writing – review & editing. L-AL: Conceptualization, Data curation, Formal Analysis, Visualization, Writing – original draft.


Yeast cells are equipped with different nutrient signaling pathways that enable them to sense the availability of various nutrients and adjust metabolism and growth accordingly. These pathways are part of an intricate network since most of them are cross-regulated and subject to feedback regulation at different levels. In yeast, a central role is played by Sch9, a protein kinase that functions as a proximal effector of the conserved growth-regulatory TORC1 complex to mediate information on the availability of free amino acids. However, recent studies established that Sch9 is more than a TORC1-effector as its activity is tuned by several other kinases. This allows Sch9 to function as an integrator that aligns different input signals to achieve accuracy in metabolic responses and stress-related molecular adaptations. In this review, we highlight the latest findings on the structure and regulation of Sch9, as well as its role as a nutrient-responsive hub that impacts on growth and longevity of yeast cells. Given that most key players impinging on Sch9 are well-conserved, we also discuss how studies on Sch9 can be instrumental to further elucidate mechanisms underpinning healthy aging in mammalians. Saccharomyces cerevisiae, several signal transduction pathways are involved in nutrient sensing, regulation of cell growth, and cell cycle progression. Among them, the TORC1 signaling pathway plays a key role in nitrogen sensing and signaling [SCH9  has been initially identified as a multicopy suppressor of the growth defect caused by a temperature-sensitive cdc25 allele [570 in the activation loop of Sch9 [723 and Ser726, both originally considered TORC1 target residues [288 by SNF1 when cells are starved for carbon [TPK1, TPK2, and TPK3 genes [Proper coordination between nutrient availability and cell growth is needed in all organisms to guarantee a successful adaptation to environmental changes. In the budding yeast ignaling ,3,4,5. Rignaling . The TORignaling . By phosignaling , TORC1 rignaling ,12,13,145 allele , hinting5 allele ,16,17, f5 allele . Full ac5 allele ,20,21 or5 allele  by phyto5 allele  for a de of Sch9 ,19,24,25 of Sch9 , and theresidues . In contr carbon ,30. In lr carbon , which iK3 genes . InteresK3 genes . In addiK3 genes ,35. MoreK3 genes ,37,38.In this review, we recapitulate the functions of the protein kinase Sch9, which serves as a hub that integrates inputs from different pathways, to modulate growth-related adaptations to nutrient changes, including stress response, autophagy, and longevity. We will additionally focus on the regulation of Sch9 by other signaling pathways and via lipid binding. Furthermore, a specific section will be devoted to exploring how regulatory events might affect Sch9 structure and function.Although the experimental structure of Sch9 remains unresolved, the release of the Alphafold2 predicted protein structure database ,40 proviThe regions outlined in The N-terminus of Sch9 (amino acids: 1–183) is involved in membrane binding, and its structure is poorly predicted, indicating a predominantly disordered nature. In contrast, the N-terminus of AKT1 is well-folded and forms the PH (pleckstrin homology) domain a,c.2+ on the loops between the β-sheets, which subsequently generates a binding surface that interacts with negatively charged lipids on the top of the C2 domain [2+ using the following amino acids: D201 (between β1 and β2), D353 (between β5 and β6); and D386/E387 (between β7 and β8). This mechanism could potentially stabilize the membrane tethering of Sch9 or enable Sch9 to bind to membranes with varying lipid compositions, although the membrane binding capacity of this particular C2 domain is so far questionable [288, which is phosphorylated by SNF1 to promote the inactivation of Sch9 [The first well-folded domain of Sch9 is a C2 domain of type II topology, which is characterized by the positioning of the N- and C-termini at the bottom of the β-barrel. Some C2 domains have been shown to coordinate Ca2 domain . In conttionable   with the membrane lipid phosphatidylinositol 3,5-bisphosphate P2) [1–183 is predicted to be largely disordered, unlike many other known phosphoinositide-binding domains that have defined configurations [1–183 interacts with PIP2.Sch9 localizes throughout the cytoplasm, with a significant enrichment on the cytoplasmic surface of the vacuolar membrane ,11. The P2) ,17,57. Aurations ,59. It i1–183 fragment, suggesting rather a negative role [1–183 alone. While the full-length Sch9 exclusively localizes to the cytoplasm with enrichment at the vacuolar membrane, Sch91–183 alone is additionally found at signaling endosomes [2 is enriched on both vacuoles and signaling endosomes, this observation suggests that the non-N-terminal region of Sch9 biases the binding preference toward the vacuolar membrane. The underlying mechanism is again unknown but may involve the interaction of the 184–824 residues with other lipids or proteins residing uniquely on the vacuolar membrane. Alternatively, different physical properties of the two organelles, for example, the membrane curvature , may account for the altered preference. Of note, both Sch9 and AKT1 contain a motif upstream of the activation loop that closely resembles the proposed consensus sequence for the PIP2 binding [Other domains of Sch9 also influence its membrane targeting. As mentioned in the previous section, Sch9 has a C2 domain (184–402 residues), a typical lipid-binding motif . Surprisive role . The undndosomes . Since P binding . However2. Indeed, evidence exists that PIP2 responds to various cellular stresses and nutrient availability. Osmotic stress, for instance, stimulates PIP2 production in a manner dependent on the cyclin-dependent kinase Pho85 [2 by direct phosphorylation of the catalytic phosphatidylinositol-3-phosphate 5-kinase subunit Fab1 and its regulatory subunit Vac7 [2 production on signaling endosomes [2 is delivered to the vacuolar membrane, which is required to recruit TORC1 and its substrate Sch9 [2 acts as an important signaling lipid that links nutrient/stress-responsive pathways, such as Pho85 and TORC1 pathways, to downstream effectors, including Sch9 [2 has been hampered by the lack of visualization tools. The use of GFP-fused Sch91–183 as a PIP2 biosensor may therefore be helpful in advancing the research in this area [Membrane targeting of Sch9 is dynamically regulated in response to environmental changes. For example, glucose starvation and oxidative stress cause Sch9 to detach from the vacuolar membrane ,18,61. Tse Pho85 ,63. Mechndosomes . Upon fuate Sch9 ,26,57. Iate Sch9 . These fhis area .2 and that influences the activation of Sch9 is the V-ATPase, the vacuolar proton pump, whose assembly and activity are dependent on glucose availability [+ symporters and driven by the H+-ATPase Pma1, influences the TORC1 activation [Another vacuolar membrane client that requires PIPlability ,65. Initlability ,67. The lability ,69, but lability . Intereslability . How Ccrlability ,71. In alability . This retivation . Since Stivation , it like2-binding, the Sch9 function is also fine-tuned by the long chain base (LCB) phytosphingosine (PHS), an intermediate of the sphingolipid metabolic pathway [570 P pathway ,74. Here pathway ,23,75. Tway [570 a, an evechondria . This Ischondria ,78.2+ homeostasis [sch9Δ mutant cells display enhanced levels of the long-chain bases PHS and dihydrosphingosine (DHS) and their phosphorylated derivatives, decreased levels of several (phyto)ceramide species, and altered ratios of complex sphingolipids, a profile that is believed to contribute to the increased chronological lifespan of the mutant cells [pho85Δ cells are characterized by reduced LCB levels and markedly increased LCBP levels [2C levels [G53Rsnf1 mutant was shown to rescue the nitrogen starvation-induced cell death of a strain lacking Csg2, an enzyme required for mannosylation of IPC to produce MIPC [Sphingolipids are important components of membranes that, beyond their structural role, also fulfill additional specific functions in several fundamental cellular processes. For instance, the dynamic balance between the different sphingolipid metabolites, especially LCBs, their phosphorylated derivatives (LCBPs), ceramides, and complex sphingolipids, have been shown to accompany stress responses, mitochondrial functioning and oxidative phosphorylation, cell wall synthesis and repair, autophagy, endocytosis, and actin cytoskeleton dynamics, thereby affecting the growth and longevity of yeast cells ,79,80. Ieostasis ,83 and ant cells . Howevernt cells ,85. Besint cells . In addint cells . Hence, nt cells ,51. Thusnt cells . ConsistP levels . An exacC levels . This wouce MIPC . Given tAs TORC1 and Sch9 are central players in the nutrient-controlled signaling network of yeast, it is not surprising that they have a crucial role in controlling growth, stress responses, and longevity . For ins0) [288, thereby contributing to the inhibition of the TORC1 signaling pathway [0 program response to phosphate availability. In parallel to Rim15, TORC1 and PKA signaling similarly maintain inactive Yak1 in the cytoplasm by tethering this kinase to the cytoplasmic 14-3-3 anchor proteins Bmh1/2 [1 and to ensure proper entry into the stationary phase [MCK1 partially suppress the growth defects of sch9Δ cells under fermentative and respiratory conditions [TORC1 and Sch9 are intimately linked to the metabolic reprogramming during the diauxic shift transition and the proper entry of yeast cells into the non-dividing quiescent state (G0) . Here, t0) . In orde0) ,101. In 0) , we perf0) , but tha pathway ,35,103.  pathway ,105,106. pathway . Moreove pathway ,108. It  pathway  as it ra pathway ,104,110. pathway ,113,114. pathway ,116. Hen pathway , the CDKs Bmh1/2 ,118. Oncs Bmh1/2 ,120. Herry phase . Yak1 alry phase . Moreovery phase . Besidesry phase ,124. Mckry phase ,126. Givry phase , the repry phase , and thery phase ,130,131 nditions . In linenditions .Finally, SNF1 also has a profound impact on stress responses. Indeed, Msn2/4 and Gis1 were originally retrieved as multicopy suppressors of SNF1 defects ,134, andFor optimal growth, the timely degradation of proteins to maintain proteostasis is essential. Different studies indicate that protein degradation is also used by yeast cells to adjust metabolic programming and fine-tune stress responses. For instance, TORC1 signaling is known to control the multivesicular body (MVB) pathway-driven degradation of plasma membrane proteins and lipids ,141,142.ATG genes by relieving repression mediated by the histone demethylase protein Rph1 and the Ume6-Sin3-Rpd3 histone deacetylase complex [ATG genes, as shown, for instance, for ATG8 [ATG39 [ATG genes via the transcription factors Gln3, Gat1, and Gcn4 [As the master regulator controlling cell growth and metabolic activity, TORC1 plays a central role in the regulation of autophagy. Under nutrient-rich conditions, TORC1 is active, and general autophagy is inhibited. This inhibition is accomplished by the TORC1 pool localized at perivacuolar signaling endosomes and involves the phosphorylation of Atg13 to prevent its association with Atg1 and, thereby, the induction of macroautophagy ,155,156. complex ,165,166.for ATG8  and ATG38 [ATG39 . Importa2 production and by priming Sch9 for its subsequent activation by TORC1 [In contrast to SNF1, which inhibits both TORC1 and Sch9 once activated under nutrient-limiting conditions ,102,169,by TORC1 . In addiby TORC1  and antaby TORC1 ,116. Henby TORC1 ,171. HowSch9, as part of the TORC1 pathway, is a prime determinant of cellular aging. Loss of Sch9 function increases the survival of stationary non-dividing cells, i.e., chronological lifespan (CLS) , and enhad libitum diet, is a potent modulator of longevity in several species. Although the longevity mechanisms of CR are not completely uncovered, it is clear that its benefits are related to alterations in the metabolic rate and the accumulation of reactive oxygen species (ROS). Initial studies have implicated inhibition of the TORC1-Sch9 axis as the longevity pathway through which CR modulates lifespan. In RLS, CR-induced longevity is mediated by reduced signaling through TORC1, Sch9, and PKA, which results in the downregulation of ribosome biogenesis. This proposed model for CR effects during RLS is independent of sirtuin 2 (Sir2) but likely links the signaling network from nutrients to ribosome assembly and protein synthesis [SCH9, suggesting that inhibition of the TORC1-Sch9 axis represents only one of the mechanisms through which CR modulates the lifespan [HSP26, HSP12, and SOD2 [SOD2 abolishes lifespan extension in sch9Δ mutant [Caloric restriction (CR), characterized by a 10–30% reduction in calories compared to an ynthesis . Regardilifespan . Downreglifespan ,181. CR lifespan . In the lifespan , which band SOD2 . ConsistΔ mutant .TOR1 and SCH9 results in the increased respiratory capacity associated with a higher ratio of mitochondrial respiratory-chain enzymes per mass during active growth [In natural scenarios, yeast and other organisms experience periods of nutritional stress, and an appropriate and efficient metabolic adaptation is, therefore, essential to ensure cell survival. Sch9 is a key player in this metabolic adaptation and in the assembled response to nutrient availability. Sch9 receives many different inputs and executes its function accordingly. As introduced above, one of the most important upstream kinases is the TORC1 kinase that targets Sch9 under favorable growth conditions ,185, bute growth ,188. Alte growth ,189.tor1Δsch9Δ cells is, therefore, associated with increased ROS levels and increased longevity by a hormesis-like phenomenon [The elevated respiratory capacity of enomenon . The so-enomenon ,191,192.11 of histone H3 under stress, and the loss of pThr11 prolongs CLS by altering the stress response at an early stage of the CLS [The TORC1-independent function of Sch9 on longevity seems to be mainly related to the activation of specific gene promoters and transcription factors. For example, as mentioned above, Sch9 regulates oxidative stress response by indirectly acting on the transcription factor Gis1 . Sch9 ca the CLS . TherefoSCH9 also significantly decreases the overall mutation frequency and DNA damage during CLS [SOD2 expression and, consequently, reduced superoxide-induced DNA damage found in sch9Δ cells further contribute to the observed reduced genomic instability.Deletion of the ring CLS , resultiring CLS ,195. TheSIC1 results in S phase entry and reduction in CLS by increased superoxide generation [1 arrest [SCH9, but not CR, protects against the premature aging phenotype of yeast cells lacking the RecQ helicase Sgs1 (WRN and BLM homolog) by inhibiting error-prone recombination and preventing DNA damage and dedifferentiation [As previously referred, Sch9 acts on cell cycle regulation by promoting an efficient G1 arrest . By inhineration ,199, and1 arrest . Importantiation . It appentiation .Yeast aging is likely a suitable natural scenario to understand the role of Sch9 in the interplay between different stress responses and nutritional status, which can constitute, per se, a particular form of stress. Because of their differential participation in yeast longevity, the two main yeast aging models can provide valuable information regarding the signal transduction mediated by Sch9 in response to multiple inputs.The investigation of the topology, regulation, and crosstalk of signaling pathways has proven invaluable in the dissection of the molecular mechanisms underlying pathophysiological processes ,201,202.560, Thr568, Thr574, Thr575, Ser709, Thr710, Ser711, and Thr721 residues, in addition to the more deeply studied Thr723 and Ser726 residues [In spite of the extensive studies which started to unravel the complex regulation and function of Sch9 and that are reviewed in depth above, recent findings hint at currently overlooked regulatory features. For instance, Sch9 was found to be phosphorylated by the CDK Bur1 at the Serresidues . Furtherresidues ,206, but3 in budding yeast [Sch9 does not have a readily identifiable orthologue in mammals. However, it shares functional similarities with the mammalian AGC kinase family member AKT , which exists in three different isoforms, AKT1, AKT2, and AKT3 , in whatng yeast  and the ng yeast .Thus, it is important to consider the limitations and differences between yeast and mammalian biology, but utilizing Sch9 as a model can still contribute to our understanding of the broader principles and mechanisms involved in the regulation of pathophysiological processes. Specifically, investigation of the post-translational modifications and their impact on the structure and function of Sch9 may translate to equivalent findings regarding the biochemical regulation of AKT and related protein kinases. Moreover, as examined in depth in this review, Sch9 could serve as a case study for lipid-dependent regulation of signaling effectors, as well as a molecular tool to investigate the subcellular distribution of specific lipid species . Finally


N-acylethanolamine (NAE) bioactive metabolite levels, and mitochondrial function in lean and obese Zucker rats in a model designed to study obesity and IR from overnutrition. We provided diets with 7% fat (w/w), with either a low PUFA/SFA ratio of 0.48, predominantly comprising palmitic acid (PA), (diet-PA), or the standard AIN-93G diet with a high PUFA/SFA ratio of 3.66  over eight weeks. In obese rats on diet-PA versus diet-C, there were reductions in plasma triglycerides, cholesterol, glucose, insulin concentrations and improved muscle mitochondrial function, inflammatory markers and increased muscle N-oleoylethanolamine (OEA), a bioactive lipid that modulates lipid metabolism and metabolic flexibility. Elevated palmitic acid levels were found exclusively in obese rats, regardless of their diet, implying an endogenous production through de novo lipogenesis rather than from a dietary origin. In conclusion, a reduced dietary PUFA/SFA ratio positively influenced glucose and lipid metabolism without affecting long-term PA tissue concentrations. This likely occurs due to an increase in OEA biosynthesis, improving metabolic flexibility in obese rats. Our results hint at a pivotal role for balanced dietary PA in countering the effects of overnutrition-induced obesity.We investigated the influence of varying dietary polyunsaturated fatty acid (PUFA)/saturated fatty acids (SFA) ratios on insulin resistance (IR), fatty acid metabolism,  Total ln-3 HUFA score = (20:5n3 + 22:6n3 + 22:5n3)/(20:5n3 + 22:6n3 + 22:5n-3 + 20:3n6 + 20:4n6 + 22:4n6 + 22:5n6 + 20:3n9) × 100Deuterated NAE and EC were added as internal standards to the samples before extraction for quantification by isotope dilution, aliquots of the lipid fraction were used for their quantification. NAE and EC quantification was carried out by an Agilent 1100 HPLC system  equipped with a mass spectrometry (MS) Agilent Technologies QQQ triple quadrupole 6420 with electrospray ionization (ESI) source , using a positive mode (ESI+), as described in .Plasma concentrations of triglycerides and cholesterol, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by a colorimetric enzymatic method using commercial kits . Blood glucose levels were determined using a rat-calibrated glucose monitor , and the plasma insulin levels were determined by ELISA . Basal fasting values of plasma glucose and insulin were used to calculate the homoeostatic model assessment (HOMA) index as [Glucose (mg/dL) × Insulin (mU/L)]/405 . Specifi2, 1 mM EDTA, 5 mM EGTA, and 0.1% (w/v) FA-free bovine serum albumin (BSA). Tissue fragments were homogenized with the above medium  in a Potter Elvehjem homogenizer  set at 500 rpm (4 strokes = min) and filtered through sterile gauze. The hepatic homogenate was then centrifuged at 1000× g for 10 min, and the resulting supernatant was again centrifuged at 3000× g for 10 min. The mitochondrial pellet was washed twice and finally resuspended in a medium containing 80 mM LiCl, 50 mM HEPES, 5 mM Tris-PO4, 1 mM EGTA and 0.1% (w/v) FA-free BSA, pH 7.0 [g, 10 min, 4 °C). The resulting supernatant was discarded, and the pellet was resuspended and centrifuged at 500× g for 10 min. The supernatant was centrifuged  and the pellet, containing the mitochondrial fraction, was washed once and resuspended in a suspension medium [Liver and limb leg muscle aliquots were finely minced and washed in a medium containing 100 mM KCl, 50 mM Tris-HCl, pH 7.5, 5 mM MgCl, pH 7.0 . The musn medium . The pron medium , using B2PO4, 1 mM EGTA and 0.1% (w/v) FA-free BSA to oxidize their endogenous substrates for a few minutes. Substrates were then added at the following concentrations: 10 mM succinate plus 3.75 mM rotenone; 40 µM palmitoyl-L-carnitine plus 2.5 mM malate. State 4 oxygen consumption was obtained in the absence of ADP, and State 3 oxygen consumption was measured in the presence of 0.3 mM ADP. The respiratory control ratio (RCR) was calculated as the ratio between states 3 and 4, according to Estabrook (1967) [Oxygen consumption in isolated mitochondria was measured with high-resolution respirometry Hansatech oxygraph  at the temperature of 30 °C. Isolated mitochondria were incubated in a medium (pH 7.0) containing 80 mM KCl, 50 mM HEPES, 5 mM KHk (1967) . In addik (1967) ,46.2O2) release was assayed by following the linear increase in fluorescence  due to the oxidation of homovanillic acid in the presence of horseradish peroxidase [2PO4, pH 7.8, 20 mM cytochrome c, 5 mM xanthine, and 0.01 U of xanthine oxidase. The SOD activity was monitored spectrophotometrically (550 nm) at 25 °C, by tracking the decrease in the reduction rate of cytochrome c by superoxide radicals, generated by the xanthine–xanthine oxidase system. One unit of SOD activity is defined as the concentration of the enzyme that inhibits cytochrome c reduction by 50% in the presence of xanthine + xanthine oxidase [The rate of mitochondrial hydrogen peroxide . All statistical analyses were conducted using GraphPad Prism 8.0.1 .Quantitative data are presented as mean ± SEM. Statistical significance among groups was assessed by the one-way ANOVA test followed by Tukey’s correction for multiple comparisons, and statistical significances were indicated as follows: * $, £ Our results showed higher food intake after eight weeks of dietary treatment in obese rats compared to lean rats A, and acTo evaluate the impact of different dietary PUFA/SFA ratios, data of each group were calculated as a percentage of Lean-C. Liver weight, and accordingly hepatic, and muscle total lipids, were increased in obese compared to lean rats, irrespective of the diet A,B. We fTo evaluate the impact of different dietary PUFA/SFA ratios on tissue FA profile, data of each group were calculated as a percentage of Lean-C. Values of FA are shown in In the liver, the FA characteristic of an enhanced de novo lipogenesis, i.e., myristic acid (14:0), 16:0, 16:1n7 and 18:1n9, increased in obese rats irrespective of the diets, albeit for the latter a slight increase was also observed in Lean-PA, with a concomitant reduction in PUFA, as shown in In adipose tissue , only 16As with the liver tissue, in adipose tissue, 18:3n3 and 18:2n6 were significantly decreased in relation to their concentrations in the diets, while their metabolites, long chain PUFAn3 and n6 , were found to have increased in obese rats, although to a lesser extent for 22:5n6 and 22:4n6, or even reduced , in obese rats fed diet-PA, with respect to their lean counterparts. The decrease in long chain PUFAn6 and n3 in rats fed diet-PA reflected the lower concentration in the diet of their precursors, 18:3n3, 18:2n6.In the muscle , 16:1n7 In plasma, similarly to the other tissues, 16:1n7 was increased in obese rats irrespective of the diets. 18:2n6 and 18:3n3 were significantly decreased in obese rats, and diet-PA further reduced their content, reflecting their dietary concentration. The n3HUFA score and PUFA were reduced in diet-PA-fed rats .N-palmitoleoylethanolamine (POEA) was increased in the muscles and liver of obese rats, in adipose tissue in lean and obese rats, and in plasma only in the lean rats  levels in the muscle and, as a trend, in the liver  showed a similar pattern to OEA in adipose tissue, plasma and muscle, with the last two showing no statistically significant differences, and remained unaffected in the liver  levels was induced by diet-PA in the muscle and, as a trend, in the adipose tissues D, and liTo assess whether the different diets influence the biosynthesis of specific FA-derived ligands of PPAR-α (OEA and PEA) or EC receptor (2-AG) and, thereby, the balance between these two systems, we also evaluated the ratio between OEA/2-AG. Diet-PA increased the OEA/2-AG ratio in muscle and non-significantly also in the liver, and in adipose tissue only in Lean-PA compared to Lean-C and Ob-PA E.N-arachidonoylethanolamine (AEA))  and EC receptors (2-AG and e (AEA)) . InteresThe analysis of plasma parameters related to lipid and glucose metabolism showed a marked increase in cholesterolemia and triglyceridemia, glycemia, insulinemia and HOMA-IR (homeostasis model assessment-estimated insulin resistance) in obese rats fed both diets compared to their lean counterparts A–E. InteA similar trend was observed for liver function  and inflammation  and monocyte chemoattractant protein-1 (MCP1)) parameters. However, for MCP1 the reduction observed in Ob-PA with respect to Ob-C did not reach significance F–J.2O2 release compared to Lean-C and Ob-PA, while the latter group of animals showed a reduction in H2O2 release also compared to Lean-C animals  with varying proportions of 16:0, 18:1n9, 18:2n6, and 18:3n3, revealed that food intake did not vary among animals fed different diets. However, greater weight gain and higher final body weight were observed in rats fed diets with a low PUFA/SFA ratio (0.28 and 0.81) rich in 16:0 and containing low proportions of 18:1n9 . The conIn our study, there was a substantial increase in BMI in both groups of obese rats compared to lean rats, by 29% at the start of the study, and 57% in Ob-C and 36% in Ob-PA after eight weeks, with no discernible differences induced by diet composition. The BMI/Food Intake ratio, which can be considered an index of dietary energy substrate accumulation, predominantly fat, did not change after four weeks of dietary treatment. Yet, after eight weeks, lean and obese phenotypes were distinctly evident, irrespective of dietary treatment.Body energy reserves are controlled by complex systems that regulate food intake, energy expenditure, and substrate partitioning. Therefore, an increase in the BMI/Food Intake ratio may signify excessive fat deposition leading to disrupted homeostasis in the obese phenotype, with the accumulation of ectopic fat in the liver, muscles, and plasma, and reduced metabolic flexibility, i.e., the capacity to efficiently utilize and store energy substrates based on fuel availability. This accumulation of energy substrates may be associated with an impairment related to their disposal, likely due to a metabolic inflexibility in obese rats, and may exacerbate glucose and lipid metabolism and promote inflammation. Metabolic inflexibility, which hinders the effective utilization of energy substrates for body growth, might account for the slightly shorter body and tail lengths observed in obese rats, regardless of diet.Excessive dietary intake of SFA is usually associated with increased obesity-related hepatic inflammatory plasma markers such as ALT and AST ,50,51, aTherefore, our data corroborate that obesity triggers systemic inflammation, lipid and glucose metabolic impairment, IR, de novo lipogenesis, steatosis, and liver damage. However, unexpectedly, we found that diet-PA improved insulin sensitivity, reduced inflammation, and mitigated liver damage in obese rats. These findings suggest that the harmful effects previously attributed to dietary PA may be due to the extremely high fat content rather than to PA itself.We then explored possible mechanisms through which diet-PA exerted these positive effects in obese rats. The benefits do not seem to be directly associated with tissue PA concentrations. As already evidenced by others , we did Abdominal obesity predisposes to hepatic steatosis, via both the increased free fatty acid delivery to the liver and the hyperinsulinemia-induced increase in hepatic de novo lipogenesis . The susWe assessed the impact of diet-PA on the tissue concentration of FA-related bioactive metabolites, such as EC and NAE, as these can modulate glucose and lipid metabolism through the PPAR-α and EC systems ,18,19, pDiet-PA led to an increase in OEA levels in muscle, liver (trending), and adipose tissue in lean rats. OEA acts as a bioactive signal for the regulation of feeding and energy homeostasis, promoting satiety , reducinConversely, we found that different dietary concentrations of 18:2n6 substantially influenced its tissue levels and related desaturation and elongation metabolites. We thus investigated whether 18:2n6 tissue levels impact the concentrations of the EC AEA and 2-AG, both derivatives of 20:4n6, which may profoundly affect lipid and glucose metabolism. In human studies, 2-AG has been shown to positively correlate with decreased high-density lipoprotein cholesterol, and increased TG levels and IR ,69. It hWe analyzed the OEA/2-AG ratio to provide an indication of the balance between the PPAR-α system and the EC system (supported by 2-AG). This balance may regulate the homeostatic control of energy metabolism and body composition. This ratio was elevated in rats fed diet-PA in muscle tissue, and also trended higher in liver and adipose tissue, suggesting the dominance of PPAR-α activity.The PA-derived lipid mediator PEA exhibited a distribution pattern in the muscle similar to that of OEA, although it did not reach statistical significance. PEA is known to play an essential role in controlling the genesis of inflammation  that may2O2 release and no variation in SOD activity.In obese rats on diet-PA, we observed a beneficial impact on the restoration of mitochondrial respiratory activity, as evidenced by the heightened oxygen consumption rate with the FADH-linked (succinate) substrate. Notably, diet-PA had a pronounced effect on obese animals when we examined the mitochondrial FA oxidation rate using palmitoyl-carnitine as a substrate. Moreover, this increase in hepatic mitochondrial respiratory activity in Ob-PA rats did not result in an elevation in ROS production, unlike what was found in obese animals on diet-C. In fact, diet-C-fed obese rats displayed the highest HFurther, given the established role of skeletal muscle in metabolic flexibility, due to its association with mitochondrial dysfunction and IR , we deciTherefore, our data strongly suggest that the increase in muscle and liver of OEA, PEA and POEA, PPAR-α ligands, and the reduction in EC 2-AG, by enhancing the ratio between PPAR-α/EC system, might promote mitochondrial function and thereby improve glucose and lipid metabolism in dysmetabolic conditions such as obesity.However, despite the significant results observed in circulating molecules related to IR and lipid impairment, and markers of inflammation and mitochondrial functions, we did not observe a reduction in weight gain or improvement in the altered depots of body energy substrate. We theorize that significant changes in body composition might require a longer dietary treatment, suggesting that changes in metabolic flexibility could precede possible changes in body fat deposition and distribution.A significant improvement in glucose and lipid metabolism in obese conditions has been demonstrated to be greatly influenced by long-chain PUFAn3 20:5n3 and 22:6n3, particularly in the phospholipid form, and, more specifically, by the balance between n3/n6 ,76. IndeVery low levels of 18:3n3 in diet-PA led to a systemic reduction in the n3HUFA score, which is considered a biomarker of n3 FA intake and tissue status, in all tissues in both lean and obese rats . TherefoOur study unveils novel perspectives on PA, as it shows that long-term dietary PA did not significantly influence its tissue concentration, implying potential physiological mechanisms to limit an excess of tissue PA concentrations. Moreover, in obesity, regardless of the diet, the increase in PA and its metabolites sustained by enhanced de novo lipogenesis might contribute to the detrimental metabolic effects of obesity. A lower PUFA/SFA ratio, reducing dietary 18:2n6 levels balanced by 16:0 and 18:1, can decrease EC, 2-AG, and increase PPAR-α ligands in muscle, improving metabolic flexibility, mitochondrial function, and fat and glucose metabolism, without affecting weight and growth.Of particular interest, the ratio of PUFA/SFA in human milk is 0.4 , mirroriOverall, these findings propose that balanced dietary PA may play a key role in reversing metabolic derangement caused by overnutrition-induced obesity.