Seed longevity is the most important trait related to the management of gene banks because it governs the regeneration cycle of seeds. Thus, seed longevity is a quantitative trait. Prior to the discovery of molecular markers, classical genetic studies have been performed to identify the genetic determinants of this trait. Post-2000 saw the use of DNA-based molecular markers and modern biotechnological tools, including RNA sequence (RNA-seq) analysis, to understand the genetic factors determining seed longevity. This review summarizes the most important and relevant genetic studies performed in Arabidopsis (24 reports), rice (25 reports), barley (4 reports), wheat (9 reports), maize (8 reports), soybean (10 reports), tobacco (2 reports), lettuce (1 report) and tomato (3 reports), in chronological order, after discussing some classical studies. The major genes identified and their probable roles, where available, are debated in each case. We conclude by providing information about many different collections of various crops available worldwide for advanced research on seed longevity. Finally, the use of new emerging technologies, including RNA-seq, in seed longevity research is emphasized by providing relevant examples. Sustainable agriculture depends on the judicial use of natural resources, including use of crop varieties that are resistant to pests and diseases and do not require pesticide spraying, thus providing environmental benefits in addition to fulfilling the energy requirements of mankind. At present, >7000 plants are cultivated for food, shelter, and other purposes , and appSeed longevity is defined as the maximum time period during which seeds can germinate ,6,7 and Seed quality can be reduced in parental plants owing to adverse environmental conditions, premature germination  and pathSeed deterioration during storage may involve many physical and chemical changes, including disrupted intracellular integrity, decreased enzyme activity, lipid peroxidation, and nonenzymatic reactions ,14. SeedSeed deterioration varies between different varieties of the same species. Even within a variety, the storage potential of individual lots varies, and within a seed lot, individual seeds have different storage potentials . Broken,A number of mechanisms of seed aging have been identified , includiCanna [Lotus [Albizia benth., Cassia L., Goodia, and Trifolium L. seeds can germinate after 100 years [Lactuca sativa L.), onions (Allium cepa L.), parsnips (Pastinaca sativa L.), and rye  [Festuca rubra subsp. commutata Gauidin) and annual ryegrass seeds have similar appearances and chemical compositions. However, ryegrass seeds were stored much better under comparable conditions [Genetic differences between species are also responsible for differential seed longevity; for example, seeds of Canna  and Lotua [Lotus  are thou00 years . Seeds oreal L.) . The infreal L.) ; howevernditions . Seed londitions .Very little is known about the genetic basis of differences in seed quality because this trait is strongly affected by environmental factors during seed formation, harvest, and storage and is probably controlled by many genes. Therefore, seed longevity is a composite trait because in genetic studies of longevity , geneticThis review focuses on the genetic determinants of seed longevity in a variety of plant species after describing classical genetic studies.Awareness of seed longevity dates back to ~2500 years ago (372 BC–287 BC) when Theophrastus discussed seed deterioration in his botanical writings ,36 SimilZea mays L.). Maize seeds have been declared more susceptible to aging [luteus 2 or luteus 4 genes, although the physiological basis of this difference remains obscure. Later, two unrelated studies [Classical genetic studies were first initiated and probably the best characterized in maize  to determine variation in seed longevity after various artificial ageing protocols. One or more QTLs were identified for various traits  after various treatments (controlled deterioration test (CDT), H2O2 treatment, and abscisic acid), with some QTLs for different co-locating traits. Using various Arabidopsis mutants, the same group of scientists concluded that abscisic acid insensitive3 (abi3), abscisic acid deficient1 (aba1), and aberrant test shape (ats) mutants have reduced longevity [Arabidopsis was demonstrated using the same mutation approach used by [A. thaliana T-DNA insertional line  1a-1) had extended seed longevity, whereas metabolite profiling of dry Atfahd1a-1 seeds showed low δ-tocopherol levels. This indicates that the role of vitamin E in extending seed longevity is complex. In another investigation using mutant technology in Arabidopsis, employing genotypes with altered expression of protein L-isoaspartyl methyltransferase (PIMT1), it was concluded that the PIMT repair enzyme system contributes to seed longevity in concert with other anti-aging pathways to improve seed longevity and vigor [Another detailed investigation was undertaken by Clerkx et al.  through ongevity . Consequongevity  in Arabi used by . The aut used by  found thnd vigor .Arabidopsis was made one year later with the simultaneous analysis of six RIL populations where five loci were discovered (germination ability after storage 1 (GAAS1) to GAAS5). Although GAAS loci co-located with dormancy loci (delay of germination (DOG)), a negative correlation between longevity and dormancy was observed [Subsequent experiments, based on RNA interference against the three seed-expressed dehydrins, viz. LEA14, XERO1, and RAB18 (responsive to abscisic acid 18), revealed that at least one of the three seed-specific dehydrins plays a role during long-term cold storage at low moisture content . Furtherobserved .Arabidopsis seeds [RSL1 (from the ring finger of Seed Longevity1). The role of GAs was further clarified in a similar study using the same mutant approach and it was concluded that GAs may act in seed coat reinforcement [Through mutant analysis and a forward genetics approach in a subsequent study, under both natural and accelerated aging treatments, a RING-type zinc finger putative ubiquitin ligase was identified as imparting a long life to is seeds  by enhanorcement .Arabidopsis mutants varying in different seed attributes were harnessed [To understand the influence of selective environmental influences on seed longevity, 12 arnessed  and temparnessed .Arabidopsis after CDT were made when tonoplast intrinsic proteins  were studied in abi3-6 mutants, where TIP2 transcript and protein levels were significantly reduced in the mutants. It was concluded that TIP3s may help extend seed longevity under the expressional control of ABI3 during seed maturation. Thus, TIP3s are members of the ABI3-mediated seed longevity pathway, together with small HSPs and late embryogenesis abundant (LEA) proteins [Arabidopsis. Aged dry seeds and after-ripened seed proteomes were markedly dissimilar and showed that antioxidant systems, including vitamin E, are essential for seed longevity. The abundance of seed storage proteins (SSPs) also indicated that they act as buffering agents to protect seeds against oxidative stress during storage [Further developments to understand seed longevity in proteins . Simulta storage .Arabidopsis [Arabidopsis, seeds of two mutants (atm and atr) were found to be highly resistant to aging. Therefore, ATAXIA TELANGIECTASIA MUTATED (ATM) and RAD3-RELATED (ATR) are important determinants of seed viability. Taken together, the physiological functions of sensor kinases, including ATM and ATR, in linking genome integrity to germination, which influence seed quality, are crucial for plant survival in the natural environment and sustainable crop production. The same year also witnessed the use of ectopic expression methodology to understand the role of 1-cys peroxiredoxin (1-Cys Prx aka PER1) in seed longevity after experimental ageing [PER1 protein from the sacred lotus (Nelumbo nucifera Gaertn.) NnPER1 is ectopically expressed in Arabidopsis and causes enhanced germination after aging. The main reason for this enhanced germination was the significantly lower levels of ROS release and lipid peroxidation.A role for genome integrity has been critically addressed in seed longevity in bidopsis . Using ml ageing . Seed-spArabidopsis during priming after CDT [Arabidopsis seeds. Using Arabidopsis mutants, they found that the SSPs during germination in aspg1-1 mutants were very impaired in both naturally aged and artificially aged (after CDT) seeds, which led them to conclude that ASPG1 is an important player in seed longevity, dormancy, and germination, and acts via SSPs degradation and regulation of GA signaling.The year 2017 saw the use of RNA sequencing technology (RNA-seq) to address seed longevity in fter CDT . In totafter CDT  and thisArabidopsis. Surprisingly, all traits were modified by cytonuclear reshuffling, with certain combinations providing favorable effects of novel cytonuclear combinations on longevity and other traits demonstrating the existence of suboptimal genetic combinations in natural populations for these traits. Furthermore, certain combinations exhibited a positive influence on longevity compared to natural combinations. In addition, to shed further light on the role of oxidative stress in seed longevity after subjecting seeds to CDT, another study [Arabidopsis seeds lacking functional NADP-MALIC ENZYME 1 (NADP-ME1) have reduced seed viability relative to the wild type. Furthermore, NADP-ME1 loss-of-function mutant seeds exhibited higher levels of protein carbonylation. NADP-ME1 catalyzes the oxidative decarboxylation of malate to pyruvate with the simultaneous production of CO2 and NADPH, whose expression is increased in imbibed aged seeds compared with non-aged seeds. Its activity during testa rupture promotes the normal germination of aged seeds. Hence, it was concluded that NADP-ME1 activity is required to protect seeds against oxidation during dry seed storage.Cytoplasmic genomes are an additional source of natural variation in seed longevity . Cytoplaer study  found thvps29) Arabidopsis mutant. Major changes were observed in the retromer mutant with respect to SSPs and synthesis of lipid reserves. These changes negatively altered vigor and longevity. It was concluded that retromers stimulate energy metabolism, including cell wall biogenesis, underlining the importance of retromer function in seed biology.In addition to developments in genetics and genomics, the role of proteins in seed longevity after CDT has also been addressed. For example,  investigArabidopsis were used to map seed longevity loci by employing a genome-wide analysis, and several multiple genomic regions associated with variation in seed longevity were detected after subjecting seeds to various experimental aging conditions, as well as natural aging [PSAD1, SSLEA, SSTPR, DHAR1, CYP86A8, MYB47, and SPCH) and five negative  seed longevity genes. In addition, the protective role of the seed coat during seed aging was strengthened by cytochrome P-450 hydroxylase, CYP86A8, and transcription factors, MYB47, KNAT7 and SEP3. The same group also reported the up-regulation of several peroxidase genes in an Arabidopsis mutant (cog1-2D) [prx2 prx25) and triple (prx2 prx25 prx71) mutants possessed reduced longevity because of low seed coat permeability. Altered polyphenolics were concluded to be the likely reason for low permeability (and hence reduced seed longevity).In 2020, natural variations of 270 ecotypes of al aging . Furthercog1-2D)  with enhAtHB25 transcription factor regulates seed permeability and longevity in naturally aged seeds as well as in seeds subjected to CDT by increasing the accumulation of lipid polyesters in the seed coat [LACS2). Its importance in seed longevity was also demonstrated by transferring LACS2 into wheat and tomato, thus identifying AtHBD25 as a trans-species regulator of seed longevity. Finally, AtFAHD1 [Arabidopsis.More advancements took place in 2021, when it was demonstrated that the eed coat . They fu AtFAHD1  has alsoqLG-2), 4 (qLG-4) and 9 (qLG-9) were identified after accelerated aging treatment [qLS-9), 11 (qLS-11), and 12 (qLS-12) following AA treatments were subsequently identified the following year [In crop plants, seed longevity studies were first initiated in rice in 2002 when three QTLs in 98 backcross inbred lines (BILs) on chromosomes 2  reported by [qLG-9. Embryonic and/or endospermic factors were concluded to influence longevity. Consistent with previous reports, another three rice storability QTLs were detected on chromosomes 1, 3, and 9 [qLG-9 was fine-mapped in a 30 kb interval  [Os09g0369400) and an unknown protein (Os09g0369500)) were annotated in this region.Using chromosome segment substitution lines (CSSLs), the role of hull, seed coat, and embryo on the effect of rice storability QTLs  . FurtherArabidopsis when the potential of mutants was used to understand seed longevity. It was demonstrated that a rice aldehyde dehydrogenase (OsALDH7) plays an important role in maintaining seed viability after accelerated ageing treatments by detoxifying the aldehydes generated by lipid peroxidation [qMT-SGC5.1, qMT-SGC7.2, and qMT-SGC9.1 on chromosomes 5, 7, and 9, respectively, in one RIL population) and three QTLs ) were detected after various storage periods [qSS-2, qSS-3, qSS-4, qSS-6, qSS-9, and qSS-11, on chromosomes 2, 3, 4, 6, 9, and 11, respectively, for rice storability were identified in a set of 182 BILs after 32 and 48 months of storage at 40–60% relative humidity, where qSS-9 was the most stable [qSS-9 to gain an understanding of seed storability in rice and possibilities for its improvement. However, before the storability QTL on chromosome 9, Os03g0700400 was identified as a candidate gene for a seed lipoxygenase (sLOX3) QTL on chromosome 3, after application of a map-based cloning strategy. It was determined that sLOX3 negatively influences seed longevity, probably by facilitating the colonization of some seed pathogens [OsLOX2, was found to act like sLOX3 in rice seed longevity after accelerated aging [In 2008–2009, the rice community replicated research in xidation . Later,  periods . Similart stable  as it waathogens ,89. Lateed aging .qSSh-2-1 and qSSh-2-2) were repeatedly detected in both treatment conditions, whereas the remaining four  and seven QTLs  were detected only once in the natural and artificial aging treatments, respectively. The existence of several QTLs  was confirmed using CSSLs.Another related investigation used a set of 85 BILs to locate seed storability QTLs in rice under natural storage conditions and after AA . The autPIMT gene in rice (OsPIMT1) increases seed longevity during AA, probably via its involvement in the repair of detrimental isoAsp-containing proteins that accumulate in acceleratory aged embryos [With the development of more sophisticated technologies to develop rice mutants in 2015, it was demonstrated that the  embryos ., chromosome 2 (RM174), chromosome 4 (RM124), chromosome 7 , chromosome 8 , and chromosome 9 (RM160). Additionally, a clarification of the role of tocopherols in seed longevity after aging at high temperatures in rice [AKR1) enhanced seed longevity by detoxifying toxic compounds and glycation products [osa-miR164c and down-regulation of osa-miR168a were observed in aged seeds. Concomitant changes in the cytomembrane permeability of seeds and the expression of osa-miR164c target genes (OsPM27 and OsPSK5) and osa-miR168a target genes (OsAGO1 and OsPTR2) under aging conditions coincided with changes in seed vigor induced by osa-miR164c and osa-miR168a. Hence, miRNAs can be targeted for future research and improvement in longevity.Pioneer association mapping (AM), also known as genome-wide association study (GWAS), was initiated in 140 rice genotypes , and 10  in rice  was madeproducts . In 2019products . Up-reguqSL-2 and qSL-8, on chromosomes 2 and 8, respectively, where the latter was regarded as a novel QTL for longevity [3 bi-parental population with 45 annotated genes potentially relevant to seed longevity located in that area [SS1-1, qSS1-2, qSS2-1, qSS3-1, qSS5-1, qSS5-2, qSS7-1, qSS8-1, and qSS11-1) [qSS1-2 and qSS8-1 colocalized with the qSS1/OsGH3-2 and OsPIMT1 loci. Finally, bulked segregant analysis of two BILs through whole-genome sequencing (BSA-seq) was used to locate potential longevity QTLs when subjected to AA treatments [Another genetic study mapped novel loci linked to seed longevity in rice using 172 RILs after natural aging. It uncovered two QTLs, ongevity . The stuongevity , indicatongevity . Chromoshat area . AM was qSS11-1)  after aceatments . Two maiZeo1  [Aleurain (Ale) which is a barley vacuolar thiol protease whose expression is regulated by the plant hormones gibberellic acid and abscisic acid. On chromosome 2H, dehydration responsive element binding protein (DREB) was reported as a candidate gene, which, together with the ethylene-responsive element (ERE) binding factors, belong to the APETALA2/ethylene-responsive element-binding protein family that play an important role in the regulation of abiotic and biotic stress responses, respectively. DREB expression is activated by drought, cold or ethylene [Barperm1, have been proposed as a candidate gene [Barperm1, heat shock cognate 70 kDa protein 2, and APETALA2-like protein. Enoyl-ACP reductase, ethylene-responsive element-binding factor 1 (EREBP-1), and sucrose synthase have been reported at the site of chromosome 7H loci.Following the successful application of genetic studies in rice, investigations have started in other crops, including barley. Three DH mapping populations were employed by  viz. ‘Strtility)  was founethylene . On chroate gene . AM analate gene  and theyate gene . The mosArabidopsis under the control of constitutive promoters. Both NADP-ME MLOC_35785.1 and UDP-glycosyltransferase MLOC_11661.1 were able to restore the nadp-me1 seed longevity phenotype.More refined effort towards genetic understanding of seed longevity in barley was attempted by  through Further developments revealed the association of miRNAs with seed aging in barley in an investigation that involved distinctive seed lots that belonged to a single genotype (cv. ‘Damazy’) which varied in viability after over 45 years of storage in a dry state . The dryAegilops tauschii introgression lines following AA [Aegilops chromosomes 1D and 5D, indicating the existence of genetic variability in seed longevity in wheat. This was followed by a detailed investigation in 2012 of classical linkage mapping ) and GWAS was performed on 96 winter wheat accessions [Per2 (peroxidase) and Wip (wound-induced protein) genes [The first report addressing the genetic basis of longevity in bread wheat appeared in 2010 reporting a pilot investigation of seed longevity traits in wheat/owing AA . Overallcessions . In ITMIn) genes , whereasn) genes . The chrn) genes . The autn) genes , using tn) genes  and somen) genes , for absThe studies of ,115 wereankyrin (ANK)-3-like isoform X1 and 60S ribosomal protein L10a, pentatricopeptide repeat-containing protein, geraniol 8-hydroxylase-like, ETHYLENE-INSENSITIVE 2-like (EIN2) and RDM16-like isoform X2, and carotenoid 9,10-cleavage dioxygenase-like isoform X1 and aspartyl protease family protein 2-like. ANK proteins play a prominent role in plant immunity, development, and growth by being involved in protein–protein interactions [MAP kinase signaling pathways [EIN2 governs stable miRNA164 expression during aging [carotenoid cleavage deoxygenases that cleave carotenoids and apocarotenoids are produced by their actions, which play various roles in the growth and development of plants [Fusarium head blight resistance [In addition to additive QTLs, epistatic QTL networks of seed longevity loci were reported for the first time in ITMI/MP , which wractions , whereasractions . Geraniopathways . EIN2 gong aging . Likewisf plants . Membersf plants . Some ofsistance .rhomboid 19, disease resistance RPM1-like and its isoform X1, eukaryotic translation initiation factor 3 subunit M, subtilisin-like protease isoform X2, the ELMO CED-12 family, and Brahma 1.Thirty-eight diversity array technology (DArT) markers out of a total of eight hundred and forty were reported to be associated with seed longevity-related traits in the GWAS analyses , and a nAnother report in 2017  attemptestem rust resistance protein Rpg1 and NBS-LRR resistance-like protein [FAR1-related sequence 6-like protein w , which starch synthase 3, stem rust resistance proteinRpg1, NBS-LRR resistance-like protein, dolichyl-diphosphooligosaccharide-protein glycosyltransferase, glutaminyl-peptide cyclotransferase-like, and wheat alpha-Amy2/53. In a latter report [FAR1-related sequence 6-like protein, delta-1-pyrroline-5-carboxylate synthase (P5CS), and MICOScomplex subunit mic60 protein, which is essential for the integrity of cellular membranes and genomes [More reports emerged in 2018 and 2019 when loci for longevity were examined in-depth in an RIL population composed of 246 genotypes using the classical linkage mapping approach  and in ar report , 23 loci genomes .The most recent report addressing seed longevity in wheat from a genetic perspective appeared in 2020 when an analysis of 150 DH lines  was repopathogen-related protein 2, superoxide dismutase 4, catalase 3, opaque endosperm 2, metallothionein 1, and golden plant 2 have been reported as candidate genes for longevity. In addition, five novel candidate genes  were reported.Natural variability of maize seed viability at the beginning of 21st century using RILs has been examined ,132. TheEarly harvesting is preferred in maize, but it can result in poor quality and low seed vigor . To tackstress-responsive glyoxalase family gene and an aldehyde dehydrogenase gene. The functions of the remaining six candidate genes were associated with energy processes, including an ATP synthase F1 subunit alpha gene, glyceraldehyde 3-P dehydrogenase gene, V-type (H+)-ATPase domain gene, phosphoglucomutase gene, 3-phosphoglycerate kinase gene, and isocitrate lyase gene. Eight candidate genes were functionally associated with protein metabolism. Four candidate genes had functions related to general protein metabolism or translation, including an elongation factor 1-g2 gene, an elongation factor 1B-g gene, a calreticulin 1 gene, and an Asp aminotransferase gene. The other four candidate genes encoded HSPs, including a hypothetical ACD ScHsp26-like gene, HSP16.9, HSP17.2, and hsp20/alpha crystallin family protein gene. In addition, five candidate genes were associated with protein modification and signal transduction. These included a ubiquitin E2 gene, calcium-dependent protein kinase, a gene containing a predicted RING-finger domain, a CAAX prenyl protease 1 gene, and a cucumisin-like serine protease. Two other candidate genes had functions related to cell growth and division, including a predicted cyclin-dependent kinase A gene and an MEK homolog1 gene.The first comprehensive use of SNPs for studying seed longevity also appeared in maize in 2014  in two u2 (IF2) population were convened in 2015-16 to examine genetic differences between homo-and heterozygous maize lines after AA and subsequent evaluation in field experiments [2 and RIL populations, respectively, with only one QTL (qGP5) common between both. In the IF2 population, a QTL (qGI10b) was detected on chromosome 10 in the same region as that reported in the same population [qGE3a) in the RIL population in this study was the same QTL previously reported [qVI4b and qGE3a detected in the RIL population were suggested to be ZmLOX1 and ZmPLD1.The RIL population used in  and an ieriments . Twenty-pulation , which dreported . In addi2:3 population and RIL population [ZmPIMT and ZmLOX11 genes in seeds, which implies that they are highly tissue-specific or even differentially expressed in mono- and dicots. In addition, several novel candidate genes have been suggested for longevity.Another report on QTL mapping for seed longevity in maize after AA appeared in 2018 in an Fpulation . The autpulation  were alspulation , which rZmDREB2A regulates the longevity of maize seeds following AA by stimulating the production of raffinose while simultaneously limiting auxin-mediated cell expansion [DREB2A mutant (zmdreb2a) lines, with decreased expression of GRETCHEN HAGEN3.2  deactivating enzyme) and increased IAA in their embryos, produced longer seedling shoots and roots than the null segregant (NS) controls. In contrast, zmdreb2a seeds with decreased expression of RAFFINOSE SYNTHASE (ZmRAFS) and less raffinose in their embryos, exhibited decreased seed aging tolerance compared to the NS controls. When they overexpressed ZmDREB2A in maize protoplasts, there was an increased expression of ZmGH3.2, ZmRAFS genes and that of a Rennila LUCIFERASE reporter (Rluc) gene, which was controlled by either the ZmGH3.2 or ZmRAFS promoter. They also provided evidence that ZmDREB2A directly binds to the DRE motif of the promoters of both ZmGH3.2 and ZmRAFS.In 2020, the use of maize mutants provided evidence that xpansion . The aut2:3 population and a population of RILs constructed from a cross between “Dong156” and “Dong237” [cQTL-7 on chromosome 7  linked to cQTL-7 and cQTL-10 could be used to select maize germplasms with a high degree of seed storability.Another attempt to map QTLs for seed longevity in maize was made in 2021 by utilizing an FDong237” . Genotypine with ), and cQ2:3 population composed of 153 lines from a cross of ‘Birsa soya-1′ × ‘JS 71-05’. Four  independent SSR markers were significantly associated with seed longevity at a distance of 158.63 cM, 75.4 cM, 105 cM and 25.51 cM on chromosomes A2, D1b, H and J, respectively [Satt434, Satt538, Satt281, and Satt598 on chromosomes H, A2, C2, and E, respectively) were associated with seed coat permeability. In addition, SSR Satt281 has been linked to electrolyte leaching on chromosome C2. The SSR Satt281 was also reported in a subsequent longevity analysis of 21 soybean genotypes [The first study to resolve the genetics of seed longevity after experimental ageing in soybean was initiated in 2008  in an F2ectively . The samectively , where fenotypes .Satt371, Satt453, and Satt618 were identified as candidate markers for linkage with seed storability and testa color. In the same year, a QTL mapping study of soybean for seed longevity in an F2:3 population derived from a soybean line  and a landrace cultivar from Myanmar  was published [2:4 seeds after AA, as well as under ambient conditions, revealed 13 markers from six linkage groups  in association with seed storability. QTL mapping detected three QTLs on linkage groups C1, F, and L, and the SSRs involved were Satt476 and Satt399, Satt269, Satt423, Satt523, and Satt143, respectively.To link seed coat color with longevity, Hosamani et al. used 33 genotypes of soybean that differed in storability and seed coat color with 53 SSR and 51 randomly amplified polymorphic DNA (RAPD) markers . Black sublished . The F2:ERF110, HSF6AB, NFXL1, and members of the DREB2 family, as well as transcription factors associated with auxin and developmental cell fate during flowering, organ growth, and differentiation. Transcriptional transition occurred concomitantly with seed chlorophyll loss and detachment from the mother plant, which was enriched with AP2/EREBP and WRKY transcription factors and genes associated with growth, germination, and post-transcriptional processes that prepared the seed for the dry quiescent state and subsequent germination.Another investigation to elucidate the genetic mechanism behind seed longevity was undertaken during detailed physiological and molecular characterization of late seed maturation . A two-fA comprehensive investigation to identify QTLs employing high-density genetic linkage maps of soybean for seed longevity in two RIL populations derived from “Zhengyanghuangdou” × “Meng 8206” and “Linhefenqingdou” × “Meng 8206” using both natural and AA protocols identified 34 QTLs on 11 chromosomes, all of which were novel . Twenty-3 progenies of the cross exhibiting brown, yellow, and black seed coat color have been undertaken [Satt162, Satt523, and Satt453, which are linked to seed coat color and seed permeability, exhibited a specific-size allelic fragment in soybean genotypes and crosses with better seed longevity, confirming the results of a previous report [Biochemical and molecular analyses of the parents exhibiting black (Birsa soya-1) and yellow seed coat color (EC 241780) and the 11 Fdertaken  to unders report ..The latest report on seed longevity in soybean appeared in 2021, where the investigators analyzed the QTLs for seed longevity in an RIL population under −20 °C conservation and AA conditions . Two majLactuca sativa cv. Salinas” × “Lactuca serriola accession UC96US23” identified multiple QTLs under CD and conventional storage conditions. However, they did not find a correlation between the results of CD and conventional storage, which led to the conclusion that CD conditions are not predictive of aging in conventional storage environments. QTLs for seed longevity were reported on chromosomes 1, 2, 3, 4, 6, 7, and 8 after various treatments, where chromosomes 1 and 4 were involved in multi-treatment QTLs. Four genes  were suggested as candidate genes on chromosome 4, including heat shock transcription factor 4, phosphoglycerate/biphosphoglycerate mutase protein family, CC-NBS-LRR and TIS-NBS-LRR class disease resistance protein, glycosyl transferase family 43, methionine adenosyltransferase 2, ubiquitin-conjugating enzyme 16, and pfkB-type carohydrate kinase protein family. The other locus on chromosome 4 carried AtPAP2 as a candidate gene, which produces phytochrome-associated protein 2 (AUX/IAA family). There were other possible candidate genes, including AMP-dependent synthetase and ligase, a zinc finger (GATA type) family protein, an ABC transporter family protein, and LOX. The two regions on chromosome 1 were reported to carry approximately nine candidate genes, viz. AtCPN60A, AtCKS1, AtMTN2, AtABA2, AtCYSA, AtEIN2, AtBGL2, RPT1A, and AtHSD5, which include RuBisCO subunit binding protein, cyclin-dependent kinase, methyladenosine nucleosidase, ABA2, cysteine-type endopeptiase inhibitor, EIN2, glucosidase, 26S proteasome AAA-ATPase subunit RPT1a, and hydroxysteroid dehydrogenase 5.Seed longevity has been thoroughly explored at the genetic level in one report . QTL mapPhytophthora nicotianae was detected in a previous study [One of the first systematic studies to approach seed longevity loci under ambient conditions in tobacco was initiated on a collection of 122 RILs derived from a cross between the cultivars “Florida 301” and “Hicks” . They weus study . A subseus study  where anSolanum lycopersicum cv Moneymaker” × Solanum pimpinellifolium) was performed [galactinol synthase gene (Solyc02g084980.2.1), which is a key enzyme of the RFO pathway. A reverse genetics approach using T-DNA knockout lines in genes encoding enzymes of the RFO pathway  and overexpression of the cucumber galactinol synthase 2 gene in Arabidopsis was undertaken. The analysis demonstrated that the galactinol synthase 2 mutant and galactinol synthase 1 galactinol synthase 2 double mutant contained the lowest seed galactinol content, which coincided with lower seed longevity. It was thus concluded that galactinol content of mature dry seeds can be used as a biomarker for seed longevity in Brassicaceae and tomatoes.To address the problem of reduced seed longevity after AA in tomato, QTL mapping in 50 RILs of tomato  collaborate to further our understanding of the processes of seed aging. Although the quest for seed immortality is a mission impossible , researcGenetic research, in particular, involves two versatile methods: linkage analysis and association mapping to dissect quantitative traits  includinArabidopsis) [Experimental examination of seed longevity is a time-consuming and labor-intensive process as it involves growing a population of plants for one complete cycle, harvesting the seeds and post-harvest treatments, and subsequent seed storage. The harvested/stored seeds are then treated experimentally, followed by germination tests  to obtain an estimate of longevity in that population. With the arrival of high throughput scoring of seed germination in at least model plants (idopsis) , new metQgene [QTL Cartographer [ICIM mapping [QTL.gCIMapping) [QTL. gCIMapping [ICIM mapping software (version 4.2.53) [Sophisticated methods of QTL detection have been developed and incorporated in various programs to facilitate breeders to capture hidden or otherwise uncaptured trait variation. Initially, in wheat, a single marker analysis was performed to map longevity loci in pioneer longevity studies . SimilarQgene , QTL Carographer  and ICIM mapping . More reMapping)  has beenMapping) ,111, QTLIMapping  might beIMapping ,130,153, 4.2.53)  in const 4.2.53) ,166. HenThe discussion above indicates that the most important steps to discover the genetic determinants of seed longevity are population development, genotyping, intensive phenotyping, and computational analysis. All of these steps are expensive. Consequently, scientists, breeders, seed technologists, physiologists, molecular biologists, and genebank curators must exchange the germplasm developed by them in their respective projects to accelerate genetic research on seed longevity. With the arrival of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) and its increasing popularity in the plant research community , substanIn conclusion, genetic advances have been successful in establishing genetic determinants of seed longevity in wheat, barley, rice, maize, and other crop plants, in addition to horticultural plants, including tomato and lettuce. New techniques, including CRISPR/Cas9, RNA sequencing, and MACE sequencing, remain to be applied in the seed science research.


Intrauterine adhesion (IUA) is an endometrial fibrotic disease with unclear pathogenesis. Increasing evidence suggested the important role of competitive endogenous RNA (ceRNA) in diseases. This study aimed to identify and verify the key long non-coding RNA (lncRNA) associated-ceRNAs in IUA. The lncRNA/mRNA expression file was obtained by transcriptome sequencing of IUA and normal samples. The microRNAs expression date was downloaded from the Gene Expression Omnibus database. Differential expressions of mRNAs, lncRNAs and miRNAs were analyzed using the DESeq2 (2010) R package. Protein interaction network was constructed to explore hub genes. TargetScan and miRanda databases were used to predicate the interaction. Enrichment analysis in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were performed to identify the biological functions of ceRNAs. Regression analysis of ceRNAs’ expression level was performed. There were 915 mRNAs and 418 lncRNAs differentially expressed. AURKA, CDC20, IL6, ASPM, CDCA8, BIRC5, UBE2C, H2AFX, RRM2 and CENPE were identified as hub genes. The ceRNAs network, including 28 lncRNAs, 28 miRNAs, and 299 mRNAs, was constructed. Regression analysis showed a good positive correlation between ceRNAs expression levels . The enriched functions include ion transmembrane transport, focal adhesion, cAMP signaling pathway and cGMP-PKG signaling pathway. The novel lncRNA-miRNA-mRNA network in IUA was excavated. Crucial lncRNAs such as ADIRF-AS1, LINC00632, DIO3OS, MBNL1-AS1, MIR1-1HG-AS1, AC100803.2 was involved in the development of IUA. cGMP-PKG signaling pathway and ion transport might be new directions for IUA pathogenesis research. It is often manifested as hypomenorrhea, amenorrhea, and secondary infertility, and can lead to miscarriage and placental implantation . At presThe pathogenesis of IUA is still unclear. In recent years, microarray analysis and transcriptome sequencing have been widely used to detect differentially expressed gene in tissues or cells and further study its biological functions. In the past, scholars have used this method to analyze the differential genes and pathways in the development of IUA ,4. Long Multiple ceRNAs may play important and complex roles in IUA. Thus, this study performed transcriptome sequencing on the endometrium of IUA patients and normal endometrium, and then conducted a comprehensive bioinformatics analysis to construct the lncRNA-miRNA-mRNA network and excavate the crucial ceRNAs in IUA, aiming to supply a theoretical basis for deeply investigating the pathogenesis of IUA and promote the development of molecular targeted therapy for IUA.2.2.1This study was conducted after being approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University. All participants signed consent forms. The endometrium of three severe intrauterine adhesion  was used to isolate total RNA from samples. The spectrophotometer  was used to assess RNA purity. RNA integrity was measured using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system .Samples with RNA integrity number >7.0 were selected for sequencing. Firstly, 3 μg RNA per sample was used to obtain the rRNA free residue. Briefly, rRNA Removal Kit  was used to remove ribosomal RNA from total RNA sample, then the products were purified by ethanol precipitation. Subsequently, sequencing libraries were established using rRNA free residue by Directional RNA Library Prep Kit for Illumina  following manufacturer’s instructions, and libraries quality was checked on the Agilent Bioanalyzer 2100 system. Then, TruSeq PE Cluster Kit v3-cBot-HS (Illumina) was used to generate the cluster on a cBot Cluster Generation System according to the manufacturer’s instructions. Lastly, the libraries were sequenced on an Illumina Hiseq 4000 platform and 150 bp paired-end.2.3Reads numbers mapped to each gene were counted using HTSeq v0.13.5. Fragments per kilobase per million of both lncRNAs and coding genes were calculated based on the length of genes and reads count, and used for differential expression analysis by the DESeq2 (2010) R package . Use ‘in2.4MiRanda database was used to screen miRNAs targeted by differentially expressed lncRNAs (DELs). The obtained miRNAs were further intersected with the differentially expressed miRNAs (DEMs) in GSE160634 for improving the accuracy. Then, TargetScan was used to predict the targeted genes of the intersecting miRNAs. Similarly, targeted genes were taken at intersection with the differentially expressed mRNA (DEGs) in sequencing file. According to the ceRNAs theory , this st2.5Several gene expression data of fibrotic disease were analyzed . Regression analysis and correlation coefficient calculation were implemented using SPSS Statistics v25.0 software package .2.6Gene Ontology (GO) enrichment analysis of DEGs was performed using the clusterProfiler R package (v3.12.0), aiming to explore the enriched biological processes of DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs was used KOBAS v3.0 software, aiming to obtain the enriched pathway of DEGs. The p-value calculated by Fisher’s exact test represented the enrichment degree. P-value <0.05 was considered significantly for both GO terms and KEGG pathways.2.7The protein interaction network was constructed to further explore the hub genes of IUA. We import the differential gene TOP500 into the String database, limit the research species to ‘Homo Sapiens’ to obtain the protein interaction relationship, set the connection score > 0.5, and finally export the differential gene protein interaction network data file. We use the network analyzer in Cytoscape software to visualize and construct a PPI network diagram, and use cytoHubba to get the top 10 hub gene based on degree.3.In this study, we hypothesized that multiple ceRNAs may play important and complex roles in IUA. First, we performed differential expression analysis and constructed ceRNA network according to interaction, and then regression analysis validated good positive correlation between lnRNA-mRNA expression levels. Enrichment analysis identified critical functions of ceRNA were transmembrane transport, cAMP signaling pathway and cGMP-PKG signaling pathway.3.1There were 915 DEMs and 418 DELs identified. Among them, 353 mRNAs and 249 lncRNAs were overexpressed, while 562 mRNAs and 169 lncRNAs were under-expressed (Supplementary Table 2). In GSE160634 dataset, 183 miRNAs were differentially expressed, in which 14 miRNAs were over-expressed and 169 miRNAs were under-expressed.3.2The GO analysis of 915 DEGs identified that many genes were significantly enriched in the DNA packaging (GO:0006323 p = 1.11E-10), extracellular matrix organization (GO:0030198 p = 3.46E-08), acute inflammatory response (GO:0002526 p = 8.96E-07), positive regulation of leukocyte cell–cell adhesion (GO:1903039 p = 2.89E-05), muscle system process (GO:0003012 p = 1.25E-07), and regulation of T cell activation (GO:0050863 p = 2.17E-06) . KEGG pa3.3The PPI network was established using STRING and Cytoscape consisting of 420 nodes and 1802 edges. The top 10 genes according to degree in cytoHubba were AURKA, CDC20, IL6, ASPM, CDCA8, BIRC5, UBE2C, H2AFX, RRM2 and CENPE, which were identified as hub genes .Figure 3.4The final integrated lncRNA-miRNA-mRNA interaction film (Supplementary Table 3) was imported into Cytoscape, then ceRNAs network was constructed . It inclGO and KEGG enrichment analyses were performed. The significantly enriched biological processes included regulation of blood circulation, muscle organ development, multicellular organismal signaling, regulation of ion transmembrane transport, and actin-mediated cell contraction. The significantly enriched KEGG pathways included hypertrophic cardiomyopathy (HCM), vascular smooth muscle contraction, cGMP-PKG signaling pathway, focal adhesion, and cAMP signaling pathway. 3.5The ceRNAs network constructed in the present study revealed the interaction of lncRNAs and mRNA in IUA. To confirm this finding, regression analysis was performed using several gene expression datasets of fibrotic disease including liver fibrosis, heart fibrosis, and skin fibrosis. The results showed a very good positive correlation between lnRNA-mRNA expression levels . For exa4.Intrauterine adhesion is a fibrotic endometrial disease that seriously threatens women’s reproductive health, and the pathogenesis is still unclear. Recent microarray analysis has identified some lncRNAs, miRNAs, and mRNAs associated with IUA. Previous study confirmed that miRNAs mediating the interactions of lncRNAs and mRNAs were important to the development of many diseases. Thus, deeply exploring lncRNA-miRNA-mRNA ceRNAs network will improve the understanding of the occurrence and development of IUA.This study identified a total of 915 DEGs. Among the DEGs, AURKA, CDC20, IL6, ASPM, CDCA8, BIRC5, UBE2C, H2AFX, RRM2 and CENPE were identified as hub genes. IL6 is a recognized inflammatory factor associated to fibrosis , and itsLncRNAs were involved in various biological functions of fibrotic diseases . This stThe ceRNAs network of lncRNA–miRNA–mRNA was established. Among the miRNAs in ceRNAs network, miRNA-326 could target block the 3ʹUTR of transforming growth factor-beta1 (TGF-β1) RNA, further suppressing the activation of the TGF-β1/Smads signaling pathway in endometrial stromal cells from patients with IUA . The miRAlthough this study provides important molecular information about the occurrence and development of IUA, there are some limitations. First of all, the number of samples used for transcriptome sequencing is relatively small. Second, this study only verified the correlation of ceRNA in the GEO data set, luciferase reporter assays and co-immunoprecipitation analysis are still needed for further verification. In the future, we will conduct deep mechanism studies on the critical lncRNAs in the ceRNA network, especially lncRNAs associated with ion transport, aiming to provide new direction for the pathogenesis research of IUA.5.This study established a novel lncRNA-miRNA-mRNA network in IUA based on the ceRNAs mechanism. Some potential genes such as MYL3, MAPK10, POPDC3, PLN, HLF, SLC6A9, lncRNAs such as ADIRF-AS1, LINC00632, DIO3OS, MBNL1-AS1, and miRNAs such as miRNA-326, miRNA-155-5p, miRNA-874-3p, miRNA-503-5p, miRNA-149-5p that might be involved in the occurrence and development of IUA were excavated. cGMP-PKG signaling pathway and ion transport might be new directions for exploring the pathogenesis of IUA.Click here for additional data file.


The maintenance of animal health and preventing disease is an important point for sustainable aquaculture. In this sense, medicinal herbs can help farmers to achieve this goal. Although many review articles have discussed the effects of these additives on growth performance, immune and antioxidant responses, as well as resistance to diseases in fish, limited knowledge is available on how herbs affect these parameters at the molecular level. Given that studies at the molecular level can provide an accurate picture of the mode of action of feed additives, in this paper, the results of studies focusing on the effect of herbs on the expression of different genes in fish are discussed.The crucial need for safe and healthy aquatic animals obligates researchers in aquaculture to investigate alternative and beneficial additives. Medicinal herbals and their extracts are compromised with diverse effects on the performances of aquatic animals. These compounds can affect growth performance and stimulate the immune system when used in fish diet. In addition, the use of medicinal herbs and their extracts can reduce oxidative stress induced by several stressors during fish culture. Correspondingly, aquatic animals could gain increased resistance against infectious pathogens and environmental stressors. Nevertheless, the exact mode of action where these additives can affect aquatic animals’ performances is still not well documented. Understanding the mechanistic role of herbal supplements and their derivatives is a vital tool to develop further the strategies and application of these additives for feasible and sustainable aquaculture. Gene-related studies have clarified the detailed information on the herbal supplements’ mode of action when administered orally in aquafeed. Several review articles have presented the potential roles of medicinal herbs on the performances of aquatic animals. However, this review article discusses the outputs of studies conducted on aquatic animals fed dietary, medicinal herbs, focusing on the gene expression related to growth and immune performances. Furthermore, a particular focus is directed to the expected influence of herbal supplements on the reproduction of aquatic animals. The provision of food for human beings is one of the main challenges facing humanity, which has attracted the attention of different countries to increase the number of aquaculture products in their food basket. Besides, high-quality proteins derived from aquaculture products have made the aquaculture industry, with an annual growth of 8%, the highest activity in the food industry , and in The aquaculture industry has applied different strategies to eliminate or reduce mortality from various diseases. One of the strategies is to use herbal extracts to improve the immune state. Among such strategies, these is the enhancement of the immune status of the cultured aquatic organism with immunostimulants. In aquaculture, herbal extracts, phytochemicals, and plant secondary metabolites are important feed additives that activate the specific or nonspecific immune system. In the literature, there have been many studies on the effects of Phyto-additives on cellular, humoral, and adaptive immune responses in fish ,3,4,5. IGH) and insulin-like growth factor, I (IGF-I) play a central role in regulating growth. Associated with reproduction, the presence of various genes including luteinizing hormone β (lhβ), follicle-stimulating hormone β (fshβ) , is a natural phytogenic compound that is found in cherries, grapes, and citruses such as lemon and orange . It has loticus) . Such reGHRL) in zebrafish (Danio rerio). GHRL is known as an appetite stimulant gene that is involved in GH secretion, food intake, and energy homeostasis.Apple cider vinegar mainly contains organic acids, minerals, some vitamins, and flavonoids. Ahmadifar et al.  found thIt seems that the mode of action of phytochemicals and their derivatives involves up-regulating the expression of growth-related genes, which activate a series of functions and eventually improve fish growth.Non-specific immunity is characterized by recognition of non-self molecules  to identHerbal bioactive components can act as immunostimulants and influence several immune-related pathways . An immuSalvia officinalis) and Spirulina platensis (Arthrospira platensis) on levels of immune markers, including lysozyme, nitric oxide, and immunoglobulin M (IgM), as well as increased resistance against Pseudomonas aeruginosa in Nile tilapia -8 and IL-1β) in gills, liver, and intestine of teleost species and increased activity of antioxidant enzymes  after supplementation with menthol oil [Thymus vulgaris essential oils up-regulated transcription of immune-related genes. Although the efficacy of the intervention was different at various concentrations, the dose of 1 mL/kg diet increased gene expression of complement component 3 (C3) and the cluster of differentiation 4 (CD4). A higher dose of 2 mL/kg enhanced lysozyme transcription levels. These findings were not consistent at the final time point: both concentrations increased gene expression of C3 and CD4, but the 2 mL/kg concentration reduced lysozyme and IL-1β gene expressions [cyp11b, hif-1α, casp-3, and il-1β gene expression and increases in steroidogenic acute regulatory protein (StAR) gene expression and serum lysozyme activity [Salvia officinalis and Lippia citriodora elevated the expression of genes involved in humoral immunity and inflammation, as well as leucocyte cell surface markers and antioxidant enzymes in gilthead seabream. This treatment did not exhibit any significant effect in non-induced pancreatic cells, and the significant findings were recorded after challenge with lipopolysaccharide. Another component extracted from essential oils is citral, which has been shown to exert anti-inflammatory and antibacterial effects due to its monoterpenoid structure [Several herbal compounds have been shown to modulate immune responses and treat infections. Plants from the Lamiaceae family (mints) have been studied in several recent experiments ,68,73,74thol oil . Becausethol oil  found thressions . In addiressions . The finactivity . The essactivity  a combintructure ,76. Moretructure . Mori ettructure .IL-1β, IL-8, transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), IgM, and IgT [By the same token, polyphenols have been described as anti-inflammatory , anti-mi and IgT . The fin and IgT .Glycyrrhiza uralensis, a species from the Fabaceae plant family, in immune response and resistance against Flavobacterium columnare in yellow catfish. Supplementation with the extracts increased gene expression of toll-like receptor (TLR) 2, TLR3, TLR5, TLR9, Myd88, and p65NFκB, and subsequently increased IL-1β and IL-8 gene expressions. The significance level was demonstrated only at the post-infection time point in head kidney specimens, not at the other time points. This indicates that the timing of supplementation matters to a significant extent for the supplement to act as an effective medication [Spatholobus suberectus, Phellodendron amurense, and Eclipta prostrata showed an immunoregulatory role via modulation of TLR5, IL-8, and mitogen-activated protein kinase (MAPK) pathways [On this subject, a study by Wang et al.  suggestedication ,82. ThesRadix bupleuri), Bangkal (Nauclea subdita), Buton (Eleutherine bulbosa), Stinging nettle (Urtica dioica), and contain phytochemicals such as flavonoids, triterpenoids, sterols, essential oils, lignans, tannins, alkaloids, and phenolics. These phytochemicals can improve immune dysregulation consequences of hyperlipidemia, increase levels of lymphocytes, phagocytosis, and defense against A. hydrophila infection, and act as antioxidant, anti-inflammatory, and anti-viral components. In addition to these, the herbs also up-regulated gene expression levels of MHC2, IKKα, TGF-β1, TNF-α, IL-1β, IL-6, and IL-8 .Chaihu (Trigonella foenum graecum), which has been shown to have anti-inflammatory, anthelmintic, and immunostimulant properties in several fish species including common carp [IL-1β and TNF-α, reduced levels of aspartate aminotransferase (AST), and alanine aminotransferase (ALT), and increased resistance against A. hydrophila after administration of 3% fenugreek seed powder in Nile tilapia [In addition to the aforementioned herbs, another health-promoting plant is fenugreek  at a dose of 10% per feed elevated phagocytosis and leukocyte count and showed activity against Streptococcus agalactiae; however, it did not alter hematocrit and leukocrit levels in Nile tilapia at any dose examined [Carica papaya) is a tropical plant from Central America and southern Mexico that contains unlike groups of bioactive agents, including saponins, flavonoids, carotenoids, and proteolytic enzymes. In vitro and in vivo research has demonstrated antibiotic, anti-inflammatory, and immunomodulatory effects of this tropical plant [Telfairia occidentalis) is a rich dietary source of phytochemicals, especially tannins. This ancient medicinal herb has been shown to exert antimicrobial effects via growth inhibition of common microorganisms and an immunostimulant role [Supplementation with a natural antibiotic called snogga wood  R. Br., Eomecon chionantha Hance, and Chelidonium majus are the main sources of SG [TNF-α, IL-1β, TLR/-4, TLR-7, and TLR-8. Interestingly, higher inclusion of cottonseed and rapeseed meal was negatively associated with immune-related gene expression in the intestinal mucosal barrier and survival after bacterial challenges [Plant alkaloids, such as sanguinarine, (SG) exert toxic effects in high doses by inhibition of Na⁺/K⁺-ATPase in membranes of animal cells  and showes of SG . SG has es of SG . Adminisallenges .Myrtaceae family. An example is Aiton (Rhodomyrtus tomentosa), which has been reported to be an anti-inflammatory agent in the treatment of various diseases [Traditional herbal medicine from southeast Asia is the diseases ,99,100. diseases .Moringaceae family has been considered to treat several animal diseases as these plants are rich in various phytochemicals including carotenoids, flavonoids, and xanthins, as well as vitamins such as vitamins C and A [The  C and A . Differe C and A . A study C and A . The aut C and A .E. piscicida and A. hydrophila [SOD and TNF-α gene expressions and suppression of HSP70 in Nile tilapia [In addition to the abovementioned components, blue-green algae or spirulina can be a potential feed supplement for the health and welfare of marine species. Pectin, the main ingredient of the cell wall in spirulina, showed an immunomodulatory effect in zebrafish via up-regulation of pro-inflammatory cytokines, chemokines, lysozyme, and mucin as a medication against drophila . The com tilapia .lhβ) and follicle-stimulating hormone β (fshβ)), in turn, regulate gonadal function, that is, gametogenesis and hormone production. Recent studies performed in zebrafish demonstrated that GnRH isoforms locally produced in the gonads could also directly act on testis and ovary and regulate gametogenesis in vitro in an autocrine/paracrine manner [cyp19a) or reducing the bio-conversion of testosterone to estradiol [VTG) by binding to the estrogen receptor instead of estradiol [erα, erβ1, and erβ2 [arα and arβ [cyp19a and cyp19b). cyp19a plays a role in female gender determination and differentiation, while cyp19b shows high expression in both sexes in adult fish [star, cyp11a1, cyp17a1, 3β-hsd, 11β-hsd2, 17β-hsd3, and ftz, androgen receptor (ar), sex-determining region Y-box 9a (sox9a), and double-sex and mab-3 related transcription factor 1 (dmrt1) have been reported in fish. Other than the referred reproduction-related genes affected by herbal extracts and PSMs in fish species, there are considerable numbers of genes affected in higher vertebrates. It is also acknowledged that the integrated regulation of reproduction can be altered through changes in apoptotic mechanisms, at different levels of the HPG axis. Here, we summarized the effects of a number of herbal extracts and PSMs on reproduction-related genes at different levels of the reproductive axis  levels. In vertebrates, hypothalamic gonadotropin-releasing hormone (GnRH) stimulates the synthesis and secretion of gonadotropins from pituitary cells . Gonadote manner . In addistradiol ,106. Besstradiol . Changesstradiol ,108. VTG and arβ , have beive axis  in diffecyp19a1b and vtg1 gene expressions increased, while a decrease in the expression level of the cyp19a1a gene was only found in medaka. In a different study, genistein was injected intraperitoneally to Dicentrarchus labrax  fish at a dose of 5 mg/kg, and after 24 h, scale and liver vtg2 and chgl gene expressions increased. At the end of the 5th day, similar results were found only in the liver tissue [Cyprinus carpio fish model it was found that ovary cyp19a1a, and liver vtgb2, and erβ gene expressions decreased after feeding female Cyprinus carpio fish with 0.01, 0.03, 0.06, and 0.09 g/kg genistein supplements for 60 days [esrrb and cyp1a [erβ. Moreover, only testicular BRDT gene expression changed in the daidzein exposure [Oncorhynchus mykiss juveniles showed that injection of 5 μg/g body weights of genistein and daidzein along with 50 μg/g body weight genistein to fish, for 24 h, liver vtg, and era1 gene expressions increases [vtg1 gene expression and decreased 17s-hsd3, cyp11b, and 11β-hsd2 gene expression in gonads [In addition to the aforementioned compounds, genistein, and daidzein, two natural Phyto-estrogens found in plants, affect reproductive processes depending on the dosage used, fish species, and age . Schiller tissue . In a ve 60 days . Moreovend cyp1a . Adult mnd cyp1a . Resultsexposure . Apart fncreases . Equol, n gonads .Eurycoma longifolia plant extract, is a quassinoid that increases the reproductive processes of male animals. Studies report that eurycomanone increases testosterone production in rat testicular Leydig cell-rich interstitial cells by blocking aromatase and phosphodiesterase enzymes [E. longifolia extract increases female fertility index, fecundity index, and the pup litter size [Clarias maggot fish. Brain fshβ and lhβ expressions and testis cyp11a1, star, cyp17a1, 3β-hsd, 17β-hsd, cyp19a1, ftz, ar, sox9a, and dmrt1 increased depending on time, dosage, or mode of application [Clarias magur fish 3 times in 21 days, and brain fshβ, lhβ ve cyp19a2 and ovary ftz, star, cyp19a1, 3β-hsd, 17β-hsd, and cyp17a1 gene expressions increased depending on dosage or mode of application [Eurycomanone, found in  enzymes  and 25 mter size . Bahat alication . In anotlication .Findings from all of these different studies demonstrated interactions between herbal extracts and PSMs with the regulation of different levels of the HPG axis. The changes observed in reproductive-related gene expression appear to be variable, depending on the species, mode, and duration of administration of herbal extracts and PSMs. However, herbal extracts and PSMs can influence reproduction either directly or indirectly by affecting the hormones of the HPG axis and/or by influencing apoptotic or steroidogenic pathways. The availability of sufficient steroidogenic enzymes is particularly important to support ovarian and testicular development and function, and the observed changes in gene expression of these enzymes would likely have important effects on the reproduction.This review summarizes some selected findings regarding the beneficial effects of herbal bioactive compounds on immune-related markers in fish species. The secondary metabolites of plants can stimulate immunity via innate and adaptive immune responses and can trigger immune cell activity, enhance phagocytosis, and enhance the secretion of inflammatory markers to resist various pathogens. Since they exhibit promising results in most investigations and have low toxicity and stressor levels, herbal bioactive compounds can be recommended as a prophylactic and therapeutic measure in the aquaculture industry. This review also reveals the direct and indirect actions of herbal extracts and phytochemicals on the reproductive axis (mainly steroidogenic pathways) in vertebrates, suggesting a potential pathway for future research.


N6-methyladenosine (m6A) modification plays an essential role in diverse key biological processes and may take part in the development and progression of hepatocellular carcinoma (HCC). Here, we systematically analyzed the expression profiles and prognostic values of 13 widely reported m6A modification-related genes in HCC.  The mRNA expression of 13 m6A modification-related genes and clinical parameters of HCC patients were downloaded from TCGA, ICGC, GSE109211, and GSE78220. Univariate and LASSO analyses were used to develop risk signature. Time-dependent ROC was performed to assess the predictive accuracy and sensitivity of risk signature.  FTO, YTHDC1, YTHDC2, ALKBH5, KIAA1429, HNRNPC, METTL3, RBM15, YTHDF2, YTHDF1, and WTAP were significantly overexpressed in HCC patients. YTHDF1, HNRNPC, RBM15, METTL3, and YTHDF2 were independent prognostic factors for OS and DFS in HCC patients. Next, a risk signature was also developed and validated with five m6A modification-related genes in TCGA and ICGC HCC cohort. It could effectively stratify HCC patients into high-risk patients with shorter OS and DFS and low-risk patients with longer OS and DFS and showed good predictive efficiency in predicting OS and DFS. Moreover, significantly higher proportions of macrophages M0 cells, neutrophils, and Tregs were found to be enriched in HCC patients with high risk scores, while significantly higher proportions of memory CD4 T cells, gamma delta T cells, and naive B cells were found to be enriched in HCC patients with low scores. Finally, significantly lower risk scores were found at sorafenib treatment responders and anti-PD-1 immunotherapy responders compared to that in nonresponders, and anti-PD-1 immunotherapy-treated patients with lower risk scores had better OS than patients with higher risk scores.  A risk signature developed with the expression of 5 m6A-related genes could improve the prediction of prognosis of HCC and correlated with sorafenib treatment and anti-PD-1 immunotherapy response. Hepatocellular carcinoma (HCC) is a common type of cancer and represents the leading cause of cancer-related death worldwide. HCC is still a serious burden to public health . There wZC3H13, WTAP, KIAA1429, METTL3, METTL14, RBM15, YTHDC1, YTHDC2, YTHDF1, YTHDF2, HNRNPC, ALKBH5, and FTO, are the most prominent [YTHDF1 is found to be related with poorer survival of HCC patients, and KIAA1429 and METTL3 are found to regulate migration and invasion of HCC, indicating an important role of m6A modification-related genes playing in HCC [RNA modification, discovered in the 1970s, has recently been recognized as a third layer of epigenetics that could modify a plethora of native cellular RNAs . N6-methrominent . These mrominent –19, theyrominent –23. For g in HCC –26.METTL3 and YTHDF1 [Recently, Zhou et al. explored the expression pattern and prognostic values of m6A modification-related genes of HCC patients, but they mainly focused on the role of d YTHDF1 . In the All the data analyzed in the present study were received from TCGA, ICGC, and GEO dataset, and written consents were already obtained before our study.https://cancergenome.nih.gov/). Meanwhile, corresponding clinical-pathological data, including gender, age, histologic grade, tumor T stage, N stage, M stage (M), TNM stage, overall survival (OS) time, and disease-free survival (DFS) time, were also downloaded. It was of note that 9 of 374 HCC patients were excluded because of absence of corresponding clinical-pathological data, and basic characteristics of 365 HCC patients were summarized in https://dcc.icgc.org/projects/LIRI-JP). The mRNA expression of 67 sorafenib-treated HCC patients of GSE109211 was downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/), and there were 21 sorafenib treatment responders and 46 nonresponders in GSE109211. Moreover, the mRNA expression of 27 melanoma patients with anti-PD-1 checkpoint inhibition therapy of GSE78220 was also downloaded from the GEO database. Four patients achieved complete response, 10 patients achieved partial response, and 13 patients achieved no response.mRNA expression of TCGA HCC cohorts, which included 374 HCC cases and 50 normal controls, was got from GDC Data Portal  is an online tool designed for estimating the abundances of 22 kinds of tumor-infiltrating immune cells with transcriptomic data [CIBERSORT  was used for statistical analysis. Wilcox test was performed to compare difference of m6A modification-related genes between HCC and healthy controls. Correlation of the 13 m6A modification-related genes with each other was compared by Spearman correlation analysis. One-way ANOVA was carried out to compare difference of m6A modification-related genes among different histologic grades and TNM stages. Chi-square analysis was carried out to analyze distribution of clinical-pathologic parameters between high-risk HCC patients and low-risk HCC patients. Univariate and multivariate Cox regression analyses were carried out to analyze the prognostic value of m6A modification-related genes and risk signature. Kaplan-Meier analysis with log-rank test was carried out to analyze difference of OS or DFS between patients of different clusters or with risk scores. Time-dependent ROC was carried out to analyze the predictive accuracy and sensitivity of risk signature. Additional statistical analyses were performed with STAMP . P < 0.0FTO, YTHDC1, YTHDC2, ALKBH5, KIAA1429, HNRNPC, METTL3, RBM15, YTHDF2, YTHDF1, and WTAP was found in the tissues of HCC patients compared to normal tissues . Interestingly, we also found that the expression of most of the 13 m6A modification-related genes seemed to be lower than those of other 32 kinds of tumors. Besides, most of the 13 m6A modification-related genes were positively correlated with each other  + (0.02∗YTHDF1) + (0.11∗METTL3) + (0.04∗KIAA1429) − (0.1∗ZC3H13). We calculated the risk score for every HCC case and assigned them into high-risk group and low-risk group on the basis of the median risk score. The expression of YTHDF2, YTHDF1, METTL3, and KIAA1429 tended to be higher in patients with high risk score; the expression of ZC3H13 seemed to be higher in patients with low risk score  were further used to examine the applicability of the signature. Risk score for every patient was computed. Similarly, the signature could effectively stratify high-risk HCC patients with poorer OS and low-risk patients with better OS  proved promising clinical efficacy, and previous study proved that combination treatment with anti-PD-1 antibodies and sorafenib exhibited a more potent antitumor effect, but only a small number of patients could achieve durable responses , 32, so YTHDF1 was significantly upregulated in HCC and positively correlated with pathology stage [KIAA1429 was higher in HCC and HCC cell lines, and KIAA1429 could regulate the progression of HCC by regulating ID2 m6A modification [METTL3 was significantly upregulated in HCC. Knockdown of METTL3 was also found to suppress the tumorigenicity and progression of HCC through YTHDF2-dependent posttranscriptional silencing of SOCS2 [YTHDF2 was significantly related to malignancy of HCC, and miR-145 could inhibit the tumorigenicity of HCC by decreasing YTHDF2 [m6A modifications are mainly controlled by methyltransferases and binding proteins and . Studiesgy stage . Cheng efication . Chen etof SOCS2 . Moreoveg YTHDF2 . CollectYTHDF1 and METTL3 expression was found to be related to poorer OS of HCC patients [THDF1, HNRNPC, RBM15, METTL3, and YTHDF2 were independent prognostic factors for OS and DFS in HCC patients. Next, a risk signature based on the expression of five genes could differentiate HCC patients into high-risk patients with poorer OS and DFS and low-risk patients with better OS and DFS. Interestingly, this risk signature together showed better predictive efficiency in predicting OS and DFS than TNM stage or any single gene estimation alone. Therefore, this risk signature might be an advantageous method for individualized therapeutic strategies in HCC patients. In addition, we also found that the risk signature was significantly associated with tumor-infiltrating immune cells, which might influence prognosis of patients with different risk scores. Significantly higher proportions of macrophages M0 cells, neutrophils, and Treg cells were found to be enriched in HCC patients with high risk scores. Previous studies showed that macrophages could be recruited to tumor tissues and become proangiogenic cells, which were significantly associated with microvessel density and poor OS and DFS of HCC [Whether expressions of m6A modification-related genes could be considered as prognostic biomarker is one of the trending research topics in m6A modification research . Upregulpatients , 25, 27.S of HCC , 35; ZhoS of HCC . These rS of HCC , which mAs an oral multikinase inhibitor, sorafenib is one of the standard care therapies for advanced stage HCC patients approved by FDA. It can prolong the survival time of HCC patients by inhibiting cell proliferation and angiogenesis and promoting cell apoptosis through inhibiting a variety of intracellular and cell surface kinases , vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR) , 39. HowThough the risk signature exhibited good performance for the prognosis of HCC, several limitations should be addressed. First of all, although the prognostic value of the risk signature has been validated in external cohort, independent cohorts consist of more HCC patients were required to further verify the model. Secondly, we did not explore the potential biological functions and pathways of risk signature. The experiment in vitro and in vivo should be carried out to uncover the relevant mechanisms. Finally, previously, Huang et al. suggested that the significant expression of m6A modification-related genes was found in circulating tumor cells (CTCs) . FurtherTHDF1, HNRNPC, RBM15, METTL3, and YTHDF2 were independent prognostic factors for OS and DFS in HCC patients. A risk signature developed with the expression of YTHDF2, YTHDF1, METTL3, KIAA1429, and ZC3H1 could improve the prediction of prognosis and correlate with sorafenib treatment and anti-PD-1 immunotherapy response.In conclusion,


This study was designed to understand the pivotal anti-cerebral ischemia/reperfusion injury (CIRI) targets and pathways of calycosin through network pharmacology and molecular docking analyses. In this study, bioinformatics tools were employed to characterize and identify the pharmacological functions and mechanisms of calycosin for CIRI management. The network pharmacology data identified potential, merged CIRI-associated targets of calycosin including tumor protein p53 (TP53), protein kinase B (AKT1), vascular endothelial growth factor A (VEGFA), interleukin 6, tumor necrosis factor (TNF), and mitogen-activated protein kinase 1 (MAPK1). Molecular docking analysis indicated the binding efficacy of calycosin with three of the targets, namely TP53, AKT1, and VEGFA. The biological processes of calycosin for the treatment of CIRI are mainly involved in the improvement of endothelial cell proliferation and growth, inflammatory development, and cellular metabolism. In addition, the anti-CIRI actions of calycosin were primarily through suppression of the toll-like receptor, PI3K-AKT, TNF, MAPK, and VEGF signaling pathways. Taken together, the current bioinformatic findings revealed pivotal targets, biological functions, and pharmacological mechanisms of calycosin for the treatment of CIRI. In conclusion, calycosin, a functional phytoestrogen, can be potentially used for the treatment of CIRI in future clinical trials. Some coin vitro . Guo et in vitro . Howeverin vitro –14. Our in vitro –18.In the present study, we used bioinformatics tools including network pharmacology and molecular docking to uncover the pharmacological functions and therapeutic mechanisms of calycosin as an anti-CIRI agent. A flow chart was created to summarize the main findings of calycosin action for the treatment of CIRI, obtained via bioinformatics analysis .All calycosin-putative genes were identified through a series of traditional Chinese medicine systems pharmacology (TCMSP), Swiss Target Prediction, and SuperPred databases. Meanwhile, other CIRI-associated pathogenic genes were collected using the DisGeNET and Genecards databases. Calycosin and CIRI-related genes were merged using the UniProt database. After re-determination using the Funrich software, the calycosin-anti-CIRI targets were screened and specified as described in previous reports –20.All merged genes of calycosin-anti-CIRI targets were used for re-analysis to create a target-functional network via the STRING database following an associated algorithm . Using CThe pharmacological functions and signaling pathways of all pivotal targets of calycosin-anti-CIRI were identified and uncovered using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database. Subsequently, pivotal targets were determined using Cytoscape software to create a network diagram of anti-CIRI targets of calycosin. After determining the log P value using the OmicShare tool, the advanced bubble diagrams used for biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG)-based molecular pathways were detailed and visualized, as reported previously –24.In brief, pivotal targets including tumor protein p53 (TP53), protein kinase B (AKT1), and vascular endothelial growth factor A (VEGFA) were screened and used for calycosin-associated molecular docking. The structure of these target proteins was retrieved from protein-data-bank (PDB) database with PDB IDs 2MWO, 3O96, and 5T89, respectively. Calycosin was docked to these proteins at their respective binding sites where their ligands are bound. The three-dimensional structure of calycosin prior to docking was demonstrated using ChemBio3D Draw setting of Chem Bio Office 2010 software. The software AutoDock Vina was used for all dockings and the molecular docked structures of calycosin with the target proteins of TP53, AKT1, and VEGFA were viewed using MGLTools in Autodock Vina software. The accuracy of the docking parameters was determined and identified using the root mean square deviation (RMSD) of the ligand molecules. The setting with an RMSD <4 Å is denoted as the threshold to conform to ligand molecules –26.After analyses of databases, 407 anti-diseased targets of calycosin and 198 diseased targets of CIRI were identified. In addition, a total of 51 merged targets of calycosin and CIRI are shown in a Venn diagram. The correlation network connected with these merged targets is shown in In the following analysis, the sub-network clusters obtained by the MCODE algorithm in Cytoscape software are shown in Based on the topological data and degree value to screen pivotal targets, the parameters of mean and large degrees of freedom in calycosin against anti-IRI target proteins were standardized. As a result, six core CIRI-associated targets of calycosin were screened and identified, including TP53, AKT1, VEGFA, interleukin 6 (IL6), tumor necrosis factor (TNF), and mitogen-activated protein kinase 1 (MAPK1) .We revealed the pharmacological mechanisms of calycosin for management of CIRI using enrichment analysis with pivotal targets. An advanced bubble chart showed top 20 biological processes of calycosin that can possibly be involved for treatment of CIRI, including its metabolic and apoptotic functions. The detailed mechanisms of calycocin action were determined from the enrichment analysis findings that revealed top 20 anti-CIRI pathways including modulation of cell proliferation and inflammation, and improvement of intracellular microenvironment . More daThe RMSD value of MLY, the original bound ligand in the TP53 protein (2MWO) was 2.675 Å. The ligand was bound at the target site through hydrogen bonding with the amino acid residues of the protein including ASP-1521 (2.7 Å), LYS-372 (2.6 Å), GLN-375 (1.8 Å), and LYS-373 (2.3 Å). In the calycosin-docked protein, calycosin was found to make hydrogen bonds with amino acid residues of ASP-1521 (2.5 Å), LYS-373 (2.4 Å), and SER-371 (2.5 Å) . In AKT1Calycosin formed hydrogen bonds with the amino acid residues of VAL-271 (3.2 Å), ASN-54 (2.9 Å), LYS-268 (2.4 Å), SER-205 (2.4 Å), TYR-272 (3.3 Å), and ASP-292 (2.3 Å) . In the Our current findings via bioinformatics analyses using network pharmacology and molecular docking revealed pivotal targets, biological functions, and molecular pathways of calycosin involved in CIRI management. We identified a total of six pivotal CIRI-associated targets of calycosin, including TP53, AKT1, VEGFA, IL6, TNF, and MAPK1. Moreover, molecular docking analysis demonstrated efficient binding of calycosin with thee of the targets, namely TP53, AKT1, and VEGFA. These findings strongly implicate an anti-CIRI action of calycosin. TP53, a well-reported anti-oncogene, can suppress intracellular DNA injury or genomic aberrations that are responsible for cell cycle arrest and cell growth . When muIn conclusion, the pivotal targets, biological functions, and molecular mechanisms of calycosin related to the treatment of CIRI are revealed through bioinformatics tools using network pharmacology and molecular docking. Furthermore, pharmacological targets, including TP53, AKT1, and VEGFA, have been identified before any experimental validation.Supplementary Tables


In KTRs, high urinary carnosinase-1 concentrations were associated with increased risk of undetectable urinary carnosine . During median follow-up for 5.3 [4.5–6.0] years, 84 (12%) KTRs developed graft failure. In Cox regression analyses, high urinary carnosinase-1 excretions were associated with increased risk of graft failure  independent of potential confounders. Since urinary carnosine is depleted and urinary carnosinase-1 imparts a higher risk for graft failure in KTRs, future studies determining the potential of carnosine supplementation in these patients are warranted.Carnosine affords protection against oxidative and carbonyl stress, yet high concentrations of the carnosinase-1 enzyme may limit this. We recently reported that high urinary carnosinase-1 is associated with kidney function decline and albuminuria in patients with chronic kidney disease. We prospectively investigated whether urinary carnosinase-1 is associated with a high risk for development of late graft failure in kidney transplant recipients (KTRs). Carnosine and carnosinase-1 were measured in 24 h urine in a longitudinal cohort of 703 stable KTRs and 257 healthy controls. Cox regression was used to analyze the prospective data. Urinary carnosine excretions were significantly decreased in KTRs (26.5 [IQR 21.4–33.3] µmol/24 h versus 34.8 [IQR 25.6–46.8] µmol/24 h;  Kidney transplantation is the treatment of choice in patients with end stage renal disease. Compared to treatment by dialysis, it reduces mortality, is cost effective, and increases quality-adjusted life years ,2,3,4. HCNDP1 gene, encoding serum carnosinase-1 (CN1), is associated with lower risk of developing diabetic nephropathy in patients with type 2 diabetes  years after transplantation, and 257 healthy kidney donors . The characteristics of KTRs and healthy donors are depicted in p = 0.42). Similarly, there were no differences in median urinary CN1 concentrations and median urinary CN1/creatinine ratios between the studied groups. KTRs with proteinuria had higher urinary CN1 excretions in comparison to KTRs without proteinuria and to healthy subjects (data not shown). Of note, urinary CN1 was below the detection limit in 95 (14%) of KTRs compared to 33 (13%) healthy donors  compared to the healthy group. Likewise, urinary carnosine concentration and urinary carnosine/creatinine ratios were lower in KTRs . In line with this, the proportion of subjects with urinary carnosine below the detection limit was higher in the KTRs compared to healthy controls .Interestingly, urinary carnosine excretion was significantly lower in KTRs . Accordingly, serum creatinine levels  and the proportion of subjects with proteinuria  were also significantly higher in KTRs. Additionally, systolic and diastolic blood pressures were higher in KTRs. Furthermore, KTRs had higher levels of triglycerides, N-terminal pro-brain natriuretic peptide (NT-proBNP), and high-sensitivity C-reactive protein (hs-CRP) . In the KTR group, there were more subjects with diabetes and HbA1c levels were also higher (p < 0.001). The diet of the KTRs was characterized by lower energy intake and lower FFQ-derived protein intake compared to healthy controls (p < 0.001). KTRs had also lower creatinine excretion per 24 h (p < 0.001)  .p = 0.007). This inverse association was independent of age and sex, but lost significance after further adjustment for eGFR . No significant associations were found between urinary carnosine and urinary CN1 in the healthy group . Of note, in KTRs, urinary carnosine excretions were associated with meat intake .The low excretions per 24h and per mmol creatinine and low concentrations of urinary carnosine in KTRs prompted us to explore the association between urinary CN1 and urinary carnosine . In KTRsUnadjusted and multivariable-adjusted logistic regression analyses were performed to investigate the association of urinary carnosine with urinary CN1 concentrations in KTRs with adjustment for potential confounders. Urinary CN1 concentrations were log2-transformed for analyses. Model 1: crude association, Model 2: adjusted for sex and age, Model 3: adjusted as for Model 2 and for eGFR. Odds ratios and 95% confidence intervals were calculated with urinary carnosine as a dichotomous variable according to its detection cut-off in urine. Abbreviations: OR: odds ratio, CI: confidence interval, CN1: carnosinase-1, eGFR: estimated glomerular filtration rate, KTRs: kidney transplant recipients.p < 0.05) (The regression coefficients for the association of baseline characteristics and urinary CN1 excretions in KTRs are shown in  < 0.05) , Model 1p > 0.05).The adjustment for age, sex, and eGFR revealed an association of urinary CN1 excretion with HDL cholesterol and urinary creatinine excretion. In contrast, the association of urinary CN1 excretion with triglycerides, serum creatinine, graft rejection, animal protein intake, LDH, use of diuretics, and antihypertensive medication was no longer significant after adjustments . The associations of urinary CN1 excretion with NT-proBNP, proteinuria, sodium, and uL-FABP excretion weakened, yet remained significant after adjustment for age, sex, and eGFR  compared to KTRs with preserved renal grafts. This was also the case for urinary CN1 concentrations  and urinary CN1/creatinine ratios . In line with this, lower urinary carnosine excretions were found in KTRs experiencing graft failure  in comparison to KTRs with preserved renal grafts. Similar differences were found for urinary carnosine concentrations and urinary carnosine/creatinine ratios .During median follow-up of 5.3 [4.5–6.0] years, 84 (12%) KTRs developed graft failure. Of these, 62 (74%) developed chronic rejection, and eight (10%) had recurrence of the primary disease. KTRs who experienced graft failure had higher median urinary CN1 excretions . Dietary intake was adjusted for energy intake through the residual method. Abbreviations: BMI: body mass index, NT-proBNP: N-terminal pro-brain natriuretic peptide, CVA: cerebrovascular accident, TIA: transient ischemic attack, HDL: high-density lipoprotein, LDL: low-density lipoprotein, HLA: human leukocyte antigen, eGFR: estimated glomerular filtration rate, LDH: lactate dehydrogenase, RAAS: renin–angiotensin–aldosterone system, hsCRP: high-sensitivity C-reactive protein, uMDA: urinary malondialdehyde, uL-FABP: urinary liver-type fatty acid-binding protein.p > 0.05). Cox regression analyses revealed an association of log2 urinary CN1 excretions , log2 urinary CN1 concentrations , and log2 urinary CN1/creatinine ratio  with graft failure (Model 1). These associations remained materially unchanged after adjustment for age, sex, and BMI (Model 2). After cumulative adjustment for eGFR and proteinuria, the associations of urinary CN1 excretion , urinary CN1 concentration , and urinary CN1/creatinine ratio  with graft failure somewhat weakened, though they remained significant (Model 3). Moreover, these associations remained independent of additional adjustment for cardiovascular risk factors (Model 4), for transplantation-related parameters (Model 5), for transplantation complications (Model 6), and for urinary parameters (Model 7) (p > 0.05).Prospective analyses of the association between urinary CN1 excretions, CN1 concentrations, and CN1/creatinine ratios with death-censored graft failure are shown in Model 7) . No signCox regression analyses were performed to assess the associations of log2-transformed urinary CN1 excretion, urinary CN1 concentration, and urinary CN1 creatinine ratio with death-censored graft failure (number of events = 84) in KTRs (n = 703). Model 1: crude associations. Multivariable Model 2: adjusted for basic confounders . Multivariable Model 3 as for Model 2 with additional adjustment for eGFR and proteinuria. Subsequently, additive adjustments were performed based on variables already adjusted for in Model 3. Model 4: further adjustment for cardiovascular risk factors . Model 5 involved further adjustment for transplantation-related factors . Model 6: further adjusted for post-transplantation complications  and Model 7 involved further adjustment for urinary parameters . Abbreviations: CN1: carnosinase-1, HR: hazard ratio, CI: confidence interval, eGFR: estimated glomerular filtration rate, HDL: high-density lipoprotein.2-transformed urinary CN1 excretion, urinary CN1 concentration, and urinary CN1/creatinine ratio as continuous variables with graft failure are shown in The associations of logThe key findings of the present study are as follows: urinary carnosine levels are depleted in KTRs in comparison to healthy controls. Secondly, urinary CN1 concentrations are inversely associated with urinary carnosine in the KTR population, although this inverse association appears to be mediated by renal function. Thirdly, high urinary CN1 is independently and positively associated with risk of late graft failure in KTRs. These observations hold true for the analyses of urinary excretions, concentrations, and creatinine/ratios.Although primarily synthesized in the liver, CN1 is also expressed in the kidney ,8,41. Lip < 0.001)  . MoreoveCarnosine is predominantly present in skeletal muscle. As a dietary source, it is abundantly present in meat, with the highest concentrations being found in beef, mutton, pork, and to a lesser extent in poultry ,52. It iWe have previously shown in cross-sectional studies that urinary CN1 levels are positively associated with albuminuria and inversely with kidney function ,24. In aDespite the high impact of modern immunosuppressive and overall supportive therapy, patients undergoing kidney transplantation have limited long-term graft survival . One of Carnosine is generally considered as a bioactive food component with potential health benefits. Several physiological roles have been ascribed to carnosine, including high intracellular pH buffer capacity and protection against oxidative and carbonyl stress ,60,61. AAlthough our study has several key strengths, e.g., large sample size and a comprehensive collection of parameters that enables adjustment for multiple potential confounders, this study suffers from the usual inherent limitations of observational studies, which do not allow for establishing causative links or underlying mechanisms. Furthermore, we did not investigate the association of urinary carnosine with key inflammatory markers such as IL-6 and TNF-α and this could be assessed in future studies. In addition, the population of this study consisted of Caucasian subjects, which calls for caution in case of extrapolation to other ethnicities.In conclusion, high urinary CN1 levels are associated with low graft survival in KTRs, who in turn have low urinary carnosine levels. An adverse impact of high CN1 activity seems very likely, since CN1 regulates carnosine and other histidine-containing dipeptides directly involved in tissue protection. As a result, low carnosine might render the kidney vulnerable to oxidative and carbonyl stress. Urinary CN1 could potentially be a marker of the overall inflammatory state of the kidney.