KMT2D and KDM6A. This review assesses our current understanding of KS, which was originally named Niikawa–Kuroki syndrome, and aims to guide surveillance and medical care of affected individuals as well as identify gaps in knowledge and unmet patient needs. Ovid MEDLINE and EMBASE databases were searched from 1981 to 2021 to identify reports related to genotype and systems-based phenotype characterization of KS. A total of 2418 articles were retrieved, and 152 were included in this review, representing a total of 1369 individuals with KS. Genotype, phenotype, and the developmental and behavioral profile of KS are reviewed. There is a continuous clinical phenotype spectrum associated with KS with notable variability between affected individuals and an emerging genotype–phenotype correlation. The observed clinical variability may be attributable to differences in genotypes and/or unknown genetic and epigenetic factors. Clinical management is symptom oriented, fragmented, and lacks established clinical care standards. Additional research should focus on enhancing understanding of the burden of illness, the impact on quality of life, the adult phenotype, life expectancy and development of standard-of-care guidelines.Kabuki syndrome (KS) is a rare neuro-developmental disorder caused by variants in genes of histone modification, including  Kabuki syndrome (KS) (Niikawa–Kuroki syndrome) is a rare, congenital disorder that was first recognized as a distinct entity by two independent groups of clinicians in 1981 . KS has We now recognize that the clinical phenotype of KS follows a highly variable and continuous spectrum that additionally includes cardiac, renal, genitourinary and/or inner ear structural defects, dental anomalies, developmental delays, hypotonia, feeding difficulties, hearing loss, as well as involvement of the immune, neurologic, ophthalmologic, gastrointestinal, and endocrine systems ,5,7. AspUpdated international consensus diagnostic criteria were recently published . HistoriKMT2D are associated with autosomal dominant KS type 1  whereas heterozygous or hemizygous variants in KDM6A are associated with X-linked dominant KS type 2 .The identification of the molecular basis of KS ,10 allowKMT2D, whereas a much smaller number (6–10%) harbor pathogenic variants in the coding regions of KDM6A [RAP1A/RAP1B, HNRNPK and ZMZ1 [The vast majority of individuals (>80%) with a clinical diagnosis of KS harbor pathogenic variants in the coding regions of number 6–% harbor of KDM6A ,12,13. Pand ZMZ1 ,15,16. LKMT2D and KDM6A are, de novo, either associated with loss-of-function  resulting in haploinsufficiency or with missense variants, which may pose a challenge for variant classification [KMT2D present with a unique clinical phenotype that includes choanal atresia, athelia, thyroid abnormalities, abnormal pubertal development, and short stature [KMT2D variants may also be associated with isolated alobar holoprosencephaly [The majority of pathogenic variants in both fication ,10. Mosafication ,23,24,25 stature ,27. KMT2ncephaly ,29KMT2D encodes a lysine-specific methyltransferase, responsible for post-translational histone 3 lysine 4 (H3K4) mono-, di- and tri-methylation, which is exclusively associated with actively transcribed genes [KDM6A encodes an X-linked H3K27 demethylase that removes repressive epigenetic marks and interacts with KMT2D in regulating gene expression in the activating signal cointegrator-2 -containing complex (ASCOM) complex [ed genes . KDM6A e complex . TranscrThis review aims to appraise our current knowledge of Kabuki syndrome with specific attention to clinical presentation and associated genotypes to identify gaps in knowledge and unmet patient needs.A literature search was conducted using EMBASE and Ovid MEDLINE databases by two independent individuals (KB and MT). Search terms and inclusion and exclusion criteria are detailed in A total of 2418 citations matching the search terms were identified . After removal of duplicates and evaluation of abstracts for exclusion and inclusion criteria , 152 citA total of 1369 individuals with KS were identified in the published literature. When gender was reported (n = 582), 43% were male, and 57% were female. The average age at the time of publication was 9.97 years . Reported ethnicity (n = 475) was as follows: Scandinavian (18%), US-American (14%), British (14%), Italian (10%), Chinese (8%), Brazilian (5%), Korean (4%), Australian (4%), Turkish (4%), French (3%), German (3%), Japanese (3%), Canadian (2%), Czech (2%), Thai (2%), Welch (1%). Case reports from Taiwan, Spain, Ireland, Palestine, Iran, Morocco, Egypt, Columbia, and Ecuador were also identified.KMT2D were identified. The remaining 195 individuals carried a clinical diagnosis of KS without molecular confirmation. The majority of KMT2D variants were truncating (nonsense: n = 241 or frameshift: n = 137), followed by missense (n = 163), small deletions (n = 95), and splice site variants (n = 66). When recorded (n = 718), the relative frequency of variants correlated with the size of the exon and was the highest for exons 39 (20% of variants), 48 (15%), 31 (11%), 34 (7%), 11 (5%), and 10 (5%) of KMT2D. Five mosaic KMT2D cases were identified with reported levels of mosaicism of 68%, 40%, 32% and 37% in blood [A total of 1174 individuals with pathogenic or likely pathogenic variants in in blood . One stuKDM6A were identified. When described (n = 56), the majority of KDM6A variants were truncating , followed by splice site (n = 11), missense (n = 10), large deletions (n = 3), small insertions (n = 3), indel (n = 2), and small deletions (n = 2). In a recent study which evaluated 61 pathogenic KDM6A variants in patients with KS type 2, truncating variants were distributed across the entire gene whereas missense variants were primarily clustered in the TRP2, TRP3, TRP 7, and Jmj-C domains [KDM6A case was identified; the level of mosaicism was not reported [A total of 89 individuals with pathogenic or likely pathogenic variants in  domains . One mosreported KDM6A variants  and 5 inherited KMT2D variants  [In total, 19 cases of inherited pathogenic variants from affected parents were identified including 14 inherited herited) ,23,25,32herited) ,34,35,36herited) ,21. In therited) . Prenataherited) . In famiThe cardinal facial features associated with KS include long palpebral fissures; eversion of the lateral third of the lower eyelid; arched and broad eyebrows with a sparse lateral third; a broad/depressed nasal tip with short columella; and large, prominent, or cupped ears . DysmorpThe reported frequency of congenital heart defects in KS varies, ranging between 28 and 80%; most are diagnosed prenatally or at an early age ,39 and pImmune deficiency and autoimmune disorders manifest in 60–73% of KS patients. Hypogammaglobulinemia occurs in up to 58% and IgA deficiency in up to 80% ,48,49,50Autoimmune disorders typically manifest in late childhood between 4 and 13 years . The mosRare cases of premature death have been reported in severely immunologically impaired patients ,52,58,65Congenital central nervous system malformations and functional neurological abnormalities are common. Muscular hypotonia is highly prevalent (51–98%) and may lead to long-term sequelae including developmental delays and oromotor dysfunction . EmerginStructural central nervous system anomalies may have an ethnic association; most cases of cortical dysplasia were reported in Caucasian individuals, although reporting biases cannot be excluded . The repKMT2D are frequently implicated in tumorigenesis and depending on the biologic context, this methyltransferase may exert either tumor suppressive or promoting functions [KMT2D may be at an increased risk for developing cancer due to somatic second hit mutations [The incidence of cancer and whether individuals with KS have an increased predisposition for malignancy is unknown. Somatic mutations in the histone methyltransferase unctions ,81. It hutations ; neuroblutations ; low-grautations ; synoviautations ; aggressutations ; hepatocutations ; giant cutations ; spinal utations . Hematolutations ; acute lutations ; Burkittutations ,92. Notautations ,94. Piloutations ,96.KDM6A increased the release of insulin from pancreatic islet cells in murine models [Endocrine dysfunction is common in KS. Postnatal growth restriction occurs in 35–85% of individuals and is far more common than growth hormone deficiency ,98,99. Re models .Two cases of isolated central diabetes insipidus (DI) are described in the literature ,103. AltIn KS, the risk for diabetes mellitus type II may be as high as 20% in early adulthood . In suchIn total, 30–40% of individuals with KS have a genitourinary anomaly . Both reOcular abnormalities occur in 38–72% of KS patients ,114. In Feeding difficulty and reflux are highly prevalent in KS , but conThere is a paucity of information regarding fertility and reproductive health pertaining to KS. Familial transmission of KS both to and from affected males and females has been reported ,21,22,37There is a paucity of data concerning prenatal and perinatal complications of KS. One study reported abnormal second and third trimester ultrasounds and quad screens in 69% and 44% of cases, respectively . PolyhydPostnatal growth restriction (60–83%) and short stature (31–81%) ,97 are cThe natural history of KS is poorly characterized. In one Japanese study, childhood height ranged from −2.1 to −5 SD below the mean for healthy controls; 27% of children had height within the normal range; none were greater than +0.5 SD above the mean . KS1-speInterestingly, up to 50% of individuals with KS will become overweight or obese in late childhood or adolescence ,97,127. Developmental milestones are often delayed in children with KS. Cognitive impairment, hypotonia, skeletal anomalies, congenital heart defects, hearing loss, and gastrointestinal anomalies can cause delays in speech and language development, feeding and swallowing, walking, sitting upright, and toilet training ,132,133.In one study, the average age of acquisition of developmental milestones was as follows : sittingLanguage acquisition varies in KS ,135. AltKDM6A is similarly associated with more severe psychomotor developmental delays [KDM6A versus protein-altering variants may also display more severe intellectual disability [No clear pattern of psychomotor development has emerged, and data are limited to sporadic case reports ,138,139;l delays . In KS2,l delays . Patientsability .Intellectual disability (ID) is one of the cardinal features of KS, presenting in up to 90% of cases ,99. ID iSevere ID is rare and only described in a few cases ,76,146. A variable pattern of oromotor, speech, and language deficits reflects neurologic, orofacial, structural, hearing, and cognitive impairment.Receptive and expressive language delays present across multiple language sub-domains  ,119; howSome studies report improvements in speech, language, and oromotor function with age; however, others conclude the opposite, noting worsening language impairment over time ,152. RegAutistic-like behavior, hyperactivity, inattention, impulsivity, self-mutilation, sleep disturbances, multiple phobias, emotional dysregulation, impairment of adaptive skills, difficulty with communication and peer interactions, poor eye contact, and anxiety disorders are among the diverse behavioral phenotypes associated with KS ,154,155.Severe behavioral phenotypes  are rare, occurring only in patients with severe cognitive deficits . ChildreMore consistently, children with KS are socially outgoing, talkative, and affectionate, with good socialization skills and affable nature ,130. A sKMT2D and KDM6A in transcriptional regulation, growth, and development. Current literature lacks consistent clinical characterization of KS, posing challenges in drawing meaningful genotype–phenotype correlations. Limited long-term follow-up similarly restricts our understanding of the natural history of the disease. Unfortunately, clinical management is fragmented and remains largely symptom oriented.A broad and continuous spectrum of clinical phenotypes is associated with KS, with notable variability between affected individuals. Advancements in genetic sequencing improve our understanding of this disease and elucidate the role of This review holistically examines KS and details the natural history as described in current literature to guide the surveillance and expectant management of KS . Clinici


The global population is projected to experience a rapid increase in the future, which poses a challenge to global food sustainability. The “Green Revolution” beginning in the 1960s allowed grain yield to reach two billion tons in 2000 due to the introduction of semi-dwarfing genes in cereal crops. Semi-dwarfing genes reduce the gibberellin (GA) signal, leading to short plant stature, which improves the lodging resistance and harvest index under modern fertilization practices. Here, we reviewed the literature on the function of GA in plant growth and development, and the role of GA-related genes in controlling key agronomic traits that contribute to grain yield in cereal crops. We showed that: (1) GA is a significant phytohormone in regulating plant development and reproduction; (2) GA metabolism and GA signalling pathways are two key components in GA-regulated plant growth; (3) GA interacts with other phytohormones manipulating plant development and reproduction; and (4) targeting GA signalling pathways is an effective genetic solution to improve agronomic traits in cereal crops. We suggest that the modification of GA-related genes and the identification of novel alleles without a negative impact on yield and adaptation are significant in cereal crop breeding for plant architecture improvement. We observed that an increasing number of GA-related genes and their mutants have been functionally validated, but only a limited number of GA-related genes have been genetically modified through conventional breeding tools and are widely used in crop breeding successfully. New genome editing technologies, such as the CRISPR/Cas9 system, hold the promise of validating the effectiveness of GA-related genes in crop development and opening a new venue for efficient and accelerated crop breeding. The Earth’s population is expected to reach 9.7 billion by 2050 . To matcA major breakthrough in modern agriculture occurred in the 1960s, at the time of the “Green Revolution” , which esd-1, encoding a defective gibberellin 20-oxidase (OsGA20ox), results in lower bioactive GAs, leading to a semi-dwarf phenotype with improved lodging resistance and higher crop production [Sdw1/denso that contains gibberellin 20-oxidase2 (HvGA20ox), the orthologous of OsGA20ox2, and the mutant forms of REDUCED HEIGHT (TaRHT) with a DELLA protein in wheat show a similar phenotype to rice sd-1 [Since then, many genes responsible for the semi-dwarf phenotype have been identified in major cereal crops such as wheat, barley, and rice. In rice, oduction ,7. The bice sd-1 ,9,10.The above “Green Revolution” genes are still being deployed in the breeding of modern crop varieties, all of which are involved in gibberellic acid (GA) biosynthesis pathways or GA response ,9,11. GAent-kaurene is produced from a separate pool of trans-geranylgeranyl diphosphate (GGPP) via a GGPS (GGPP synthase) in proplastids [ent-kaurene from GGPP via ent-copaly1 is catalyzed by separating bifunctional enzymes encoded by the TPS genes ent-copaly1 diphosphate synthase (CPS) and ent-kaurene synthase (KS). CPS is a class II (proton-initiated) cyclase, and KS is a class I (initiated by phosphate ionisation) cyclase  and 2-oxoglutarate-dependent dioxygenases (2ODDs) [ent-kaurene oxidase) and KAO (ent-kaurenoic acid oxidase) are two mono-oxygenases, which are required to catalyse the formation of GA12, a common precursor for all GAs in plants [ent-kaurene to ent-kaurenoic acid by KO occurs in three steps via the intermediates ent-kaurenol and ent-kaurenal, depending on the mechanism for hydroxylations on C-19 [osko2-1, which presents a severe GA deficiency and a dwarf phenotype without flowering [OsKO2 (CYP701A6), is required for GA biosynthesis [The transformation of  (2ODDs) . KO  family promote the final conversion of GA12 and GA53 in parallel pathways into bioactive GA9 and GA20, respectively [GA12, a product of KAO, lies at a key point in the pathway in higher plants and is hydroxylated at C-13 and/or C-20 . GA12 caectively . This isectively . Previouectively . The C-2In the final step, to produce biologically active hormones, the conversions of GA9 and GA20 to GA4 and GA1, respectively, involve hydroxylations on C-3β catalysed by the ODD gibberellin 3-oxidase (GA3ox) . MoreoveSeveral mechanisms have been detected to regulate GA homeostasis by inactivating GAs , with 2βGAMT genes are expressed in developing seeds and encode enzymes that methylate the 6-carboxyl group of C19-GAs, which leads to GA inactivity. Lines with GAMT gene knockout show an increased content of GAs, confirming their roles in GA regulation. It has been reported that the product of a rice EUI (ELONGATED UPPERMOST INTERNODE) gene, a cytochrome P450 mono-oxygenase (CYP714D1), can convert GAs into their 16α, 17-epoxides [EUI in rice, which causes dwarfism and reduced GA4 content in the uppermost internode. This effectivity of epoxidation varies with the specific substrates of GAs, with GA12, GA9, and GA4 being more effective.Other mechanisms for GA inactivation mainly include epoxidation via GA methyl transferase 1 (GAMT1) and GA methyl transferase 2 (GAMT2). GAMT1 and GAMT2 are two members of the SABATH group of methyl transferases found in Arabidopsis and are specific for GAs. The epoxides . EpoxidaWUSCHEL-RELATED HOMEOBOX 14 (WOX14) promotes GA biosynthesis by stimulating GA3ox gene expression and represses GA2ox genes [GA3ox1 [SlbHLH95 downregulates the two GA biosynthesis genes SlGA20ox2 and SlKS5, leading to decreased GA biosynthesis [Furthermore, some transcription factors are involved in the upstream regulation of GA biosynthesis in plants. For example, in Arabidopsis, the overexpression of ox genes . The tra [GA3ox1 , while A [GA3ox1 ,44,45. Mynthesis .GA acts as an important phytohormone that plays a major role in most crop developmental processes, including seed germination, stem elongation, leaf expansion, and flowering, by increasing cell division and elongation  47,48,4,50.PHYTOCHROME-INTERACTING FACTORs (PIFs), GAMYB, SUPPRESSOR OF CONSTANS 1 (SOC1), and SQUAMOSA PROMOTER-BINDING PROTEIN-LIKEs (SPLs), and the downregulation of SPINDLY (SPY). DELLA-mediated PIFs are required for coordinating light and GA signals to regulate hypocotyl elongation [GAMYB is the downstream gene of DELLA and positively regulates GA-responsive genes that are involved in mediating flowering. For example, one of the GAMYB-like genes, MYB33, directly activates the floral meristem identity gene LEAFY (LFY) to promote flowering [microRNA172–FLOWERING LOCUS T (miR172-FT) module, leading to increased APETALA1 (AP1) and upregulated SOC1, which are two other pathways involved in GA signalling to activate LFY for promoting flowering [SPY encoding nucleocytoplasmic protein is another negative regulator of GA signalling by activating the GlcNAcylation of DELLA [SPY could partially rescue the non-germination and dwarf phenotype caused by GA-deficient mutants [Bioactive GAs activate GA signalling, which is one of the major pathways that mediates plant development ,51. GA songation . GAMYB ilowering . The uprlowering ,62,63,64of DELLA . The dow mutants ,67.GA not only affects phenology development via the endogenous and environmental exogenous genetic pathways but also interacts with phytohormone pathways, such as abscisic acid (ABA), jasmonic acid (JA), cytokinins (CK), auxin (IAA), and brassinosteroids (BR), to control plant phenology.RGA-like2 (RGL2), by binding to its promoter region. DELLA activates XERICO to increase ABA biosynthesis [OsAP2-39 (includes an APETALA 2 (AP2) domain) operates as a central regulator in the antagonistic network between ABA and GA that controls plant growth. OsAP2-39 upregulates OsNCED-1, which leads to increased ABA and the elongation of the uppermost internode (EUI), which are involved in the deactivation of GAs [In Arabidopsis, ABA antagonises GA in several developmental processes, especially the regulation of seed dormancy and germination. ABA is synthesised by 9-cis-epoxycarotenoid dioxygenase (NCED) and activates ABA-INSENSITIVE (ABI) to regulate the balance between ABA and GA. The interaction between ABI3 and ABI5 regulates ABA and GA metabolic genes by activating SOMNUS (SOM) to regulate seed germination at high temperatures . In addiynthesis . ABI4, aynthesis ,72. In aynthesis . In ricen of GAs .Fusarium graminearum [In addition, excessive GA activates ABA catabolism, leading to ABA reduction and, thus, accelerates flowering and spikelet initiation at the central part of the barley spike ,76,77. Tminearum ,79.OsJAZs, OsJAZ8 and OsJAZ9, interact with SLRs in rice to regulate the antagonistic mechanism of JA and GA [JA, normally induced by abiotic stress, antagonises the GA response through the interaction between the JASMONATE-ZIM domain (JAZ) and DELLA, which regulates the balance between plant defence and growth . When JAA and GA .GA2ox and promote the expression of ISOPENTENYL TRANSFERASE7 (IPT7), a CK biosynthesis gene [CK is another phytohormone that has an antagonistic effect on GA in a wide variety of developmental activities, including cell differentiation, meristem maintenance, as well as shoot and root elongation. KNOXs act as the key intermediate regulators between GA and CK levels in the shoot apical meristem (SAM), which activate sis gene ,84,85. Tsis gene ,87.Similar to Arabidopsis, five functional class I KNOX genes in rice have a similar function that positively regulates CK signalling by activating IPT . OsIPT2 AUXIN RESPONSE FACTOR (ARF) family, as well as AUXIN INDUCIBLE/INDOLE-3-ACETIC ACID INDUCIBLE (AUX/IAA), are involved in auxin response. These not only regulate GA metabolism enzymes, including GA20ox, GA3ox, and GA2ox, but also negatively regulate DELLA [OsGA3ox2 expression levels, leading to decreased cell numbers and cell elongation, thereby resulting in a shortened UI [Auxin regulates GA signalling and biosynthesis in mediating plant growth. The major bioactive auxin is indole-3-acetic acid (IAA), which is rapidly synthesised in the tissues with extended cell division . The trate DELLA ,93. Mostte DELLA . In ricetened UI . Anothertened UI . IAA postened UI .GA20ox1 and GA3ox1 [BR is considered to act synergistically with GA in mediating the numerous aspects of plant growth. BR induces the inactivation of BRASSINOSTEROID-INSENSITIVE 2 (BIN2), which allows BRASSINAZOLE-RESISTANT1/BRI1-EMS-SUPPRESSOR 1 (BZR1/BES1) to be rapidly dephosphorylated and activated by PHOSPHATASE 2A (PP2A) and translocated into the nucleus to activate PRE1 and repress IBH1. An antagonistic cascade formed by PRE1, IBH1, and HBI1 regulates plant growth . GAs crod GA3ox1 ,100,101.BR also promotes cell elongation by regulating GA metabolism genes in rice. For example, BR significantly induces D18/GA3ox2, resulting in increased levels of GA1 in rice seedlings. Compared to Arabidopsis, rice has a divergent pattern, wherein the responding pathway is highly dependent on tissue and endogenous hormone levels. For instance, the excessive application of bioactive BR induces GA2ox3 transcriptional activity to induce GA inactivation. In the feedback mechanism, leaf angles enlarged by the enhanced BR can be reduced by GA treatment . MoreoveSeed dormancy is an essential agronomic trait that allows the crop to germinate under favorable conditions but delays this process when seeds are still located on the mother plant. GA plays a significant role in the transition of the seed from dormancy to germination. Several genes acting as GA response regulators that promote seed germination have been identified in cereal crops  106,107107.CYTOCHROME P450 genes encode gibberellin 13-oxidase, which plays a significant role in manipulating the development of seeds/siliques. cyp72a9 mutants exhibit accelerated germination due to an increased level of GA4 [RGL2, one of three RGA-like (RGL) genes involved in the DELLA subfamily, is the major negative regulator of seed germination [rgl2 alleles being more resistant to the inhibitory effect of paclobutrazol (PAC) on germination than rgl1-1, gai-t6, and rga-t2. Loss-of-function mutations in RGL2 completely restore germination, in the absence of exogenous GA, to ga1-3 seeds with a nongerminating phenotype [SPINDLY (SPY) is also involved in GA signalling to control seed germination, as SPY can partially regulate the GA signal transduction pathway that is independent of GA. AtSPY is considered the negative regulator of GA signalling, and SPY mutants promote seed germination by conferring resistance to PAC with an inhibitory effect on seed germination and rescue the non-germinating phenotype in ga1-2, an allele of ga1-3 [In Arabidopsis, l of GA4 . Besidesmination , with rghenotype . In addiof ga1-3 ,111.SPY homologues in cereal crops also have a similar function. For example, HvSPY can suppress the expression of α-amylase induced by GA and maintain seed dormancy [SLENDER1 (HvSLN1) is another repressor of α-amylase activity induced by GA in barley seeds, and the mutation in HvSLN1 exhibits non-dormancy with high α-amylase expression in the aleurone [LEA33, one of five Late Embryogenesis Abundant (LEA) genes, promotes seed germination via enhancing GA biosynthesis in rice embryos [TaGA20ox1 and TaGA3ox2 in wheat led to breaking seed dormancy through increasing GA content [The dormancy ,138. Howaleurone ,139,140. embryos . GA oxid content ,125.Thus, current studies are in agreement that DELLA may be the most direct regulator of GA-regulated agronomic traits and abiotic stress tolerance. Moreover, orthologous genes generally have the same biochemical function and exhibit a similar phenotype in cereal crops. The genes involved in GA metabolism and GA signalling pathways for regulating seed dormancy and germination are either DELLA-dependent or -independent GA responses. Meanwhile, several DELLA proteins that regulate seed germination in Arabidopsis are orthologous to GA response height-mediating factors in cereal crops such as RGL1/RGL2 . DELLA pGA is also a crucial factor that determines plant stature such as plant height and biomass. Plant height is an important agronomic trait that plays a central role in crop performance. Plant height is positively correlated with its biomass during the vegetative stage, while short plants possess higher resistance to lodging. Therefore, by modifying GA-related genes, plant height and yield components can be manipulated. RGA [GAI [RGL2, are considered negative regulators of the GA response and inhibit stem elongation [RGA/GAI in rice is OsSLR1, a DELLA protein, which is a repressor of GA signalling [OsSLR1 reduces GA responsiveness and exhibits dwarf phenotype in rice [In Arabidopsis, RGA  and GAI RGA [GAI , which songation . The fungnalling . The ove in rice ,141.HvSLN in barley and REDUCED HEIGHT (TaRHT) in wheat are found to be the orthologous genes of OsSLR. Both genes have an effect, similar to OsSLR, on plant height [t height ,133.OsGA20ox2 at the SD1 locus. The removal of this locus causes a semi-dwarf phenotype due to the low amount of active GA in the shoot [OsGA20ox3 exhibits a similar semi-dwarf phenotype, while the overexpression of OsGA20ox3 shows an elongation phenotype [OsGA2-oxidase 6 (OsGA2ox6) is related not only to the semi-dwarf phenotype but also more tillers, leading to a high yield [HvGA20ox2, located in semi-dwarf locus sdw1/denso, is homologous to OsGA20ox2 [The well-known “Green Revolution gene” in rice has been mapped as he shoot . Moreovehenotype . Similargh yield . In barlsGA20ox2  and has Rht24 in wheat was identified as a novel locus for height reduction, which encodes a GA2 oxidase [TaGA2oxA9 can not only reduce plant height without yield loss but also increase plant nitrogen use efficiency [A few years ago,  oxidase . Interesficiency .sdw1/denso alleles, sdw1.d with a 7 bp deletion in exon1, reduces the thousand-seed weight [However, GA-related semi-dwarfing genes can also have adverse effects on agronomic performance such as decreased coleoptile length ,144,145.d weight .GA20ox, GA3ox, and GA2ox, act as the main regulators in GA biosynthesis for environmental signals; of particular interest is the role of GA2ox genes in response to abiotic stress. In Arabidopsis, DWARF AND DELAYED FLOWERING 1 (DDF1) interacts with AtGA2-oxidase 7 (AtGA2ox7) to upregulate gene expression, which leads to GA deficiency and enhanced salt tolerance [OsGA2ox6 and OsGA2-oxidase 8 (OsGA2ox8) promotes the accumulation of osmoregulators such as proline, and, thus, plants can adapt to osmotic stress and drought [Genes encoding dioxygenases, including olerance . In rice drought ,120.SD1 activated by ETHYLENE-INSENSITIVE 3-like 1a (OsEIL1a), an ethylene-responsive transcription factor, promotes GA synthesis, particularly GA4, leading to stem elongation above the water surface to restore gas exchange between crop tissues and air [Submergence 1A (Sub1A) in rice acts as an ethylene response factor that confers submergence tolerance by impeding shoot elongation via stimulating the GA signalling repressors SLR1 and SLR1 Like-1 (SLRL1) [GA also plays a central role in mediating crop shoot elongation to improve crop survival under waterlogging conditions . Under w and air . Submerg (SLRL1) .Rht12, RhtB1b, and sdw1, can enhance heat and drought stress tolerance through an expanded growth period, increased tiller number, improved lodging resistance, and preventing head loss, thus, improving crop adaptability and grain quality [In wheat and barley, GA-related genes, such as  quality ,135,136.Biotic stress, including bacteria, fungi, viruses, nematodes, insects, and infections, is another major environmental factor that affects crop productivity .AtGA2ox7 in Arabidopsis is involved in enhancing plant resistance to pathogens [OsGA20ox3 RNAi lines show improved resistance to rice blast and pathogens X. oryzae pv. Oryzae and upregulated defence-related genes, while plants with an overexpression of OsGA20ox3 are more sensitive to pathogens [gibberellin-insensitive dwarf1 (Osgid1), and probenazole-inducible protein (PBZ1), regulated by GA signalling, are involved in resistance to rice blast fungus [athogens . In riceathogens . A GA-int fungus . Genes iArabidopsis thaliana, as well as the crop species rice, wheat, barley, maize, tomato, and grape [ZmGA20ox3 in maize [PROCERA encoding DELLA protein in tomato [MaGA20ox2 in banana [OsGA3ox1 was also verified as a significant gene in regulating starch accumulation in mature pollen grains [HvARE1 edited by gene editing led to increased nitrogen use efficiency in barley [CRISPR/Cas9 technology has shown great promise for functional gene research and crop improvement and has been used to validate gene functions in the model plant nd grape ,152,153.in maize , PROCERAn banana  were edin grains . Moreoven barley .SD1 in rice and sdw1/denso in barley, both encoding GA20-oxidase, has dramatically increased grain yield and led to paradigm-shifting agricultural practices termed a “Green Revolution”. Many alleles of sd1 and sdw1/denso with economic values were identified through fine-tuning plant phenology and yield potential. However, only a few of them have been wildly used in breeding programs on a global scale. In addition, the allelic effects on crop agronomic performance should be explored in diverse genetic backgrounds to validate the effectiveness of gene function and the adverse influence on other agronomic traits. Therefore, the modification of GA-related genes and the identification of novel alleles without a negative impact on yield and adaptation are significant in cereal crop breeding for plant architecture improvement.In the past half-century, the use of With the arrival of the era of genome editing technologies , the functional analysis of genes involved in GA signalling and the investigation of functional alleles of known GA-related genes can be conducted in a more efficient and less time-consuming manner, when compared with the conventional breeding. The use of genome editing technology, such as CRISPR/Cas9, to modify GA-related genes can alter plant morphology and performance and, thus, generate a wild range of germplasms for specific environmental conditions and agricultural practices.


Pancreatic neuroendocrine neoplasms (PanNENs) are rare neoplasms arising from islets of the Langerhans in the pancreas. They can be divided into two groups, based on peptide hormone secretion, functioning and nonfunctioning PanNENs. The first group is characterized by different secreted peptides causing specific syndromes and is further classified into subgroups: insulinoma, gastrinoma, glucagonoma, somatostatinoma, VIPoma and tumors producing serotonin and adrenocorticotrophic hormone. Conversely, the second group does not release peptides and is usually associated with a worse prognosis. Today, although the efforts to improve the therapeutic approaches, surgery remains the only curative treatment for patients with PanNENs. The development of high-throughput techniques has increased the molecular knowledge of PanNENs, thereby allowing us to understand better the molecular biology and potential therapeutic vulnerabilities of PanNENs. Although enormous advancements in therapeutic and molecular aspects of PanNENs have been achieved, there is poor knowledge about each subgroup of functioning PanNENs.Therefore, we believe that combining high-throughput platforms with new diagnostic tools will allow for the efficient characterization of the main differences among the subgroups of functioning PanNENs. In this narrative review, we summarize the current landscape regarding diagnosis, molecular profiling and treatment, and we discuss the future perspectives of functioning PanNENs. Neuroendocrine neoplasms (NENs) are a heterogenous group of rare tumors with different morphological features, immunophenotype, molecular profiling and clinical presentation that can virtually originate in every part of the body, including lung, small intestine and pancreas ,2.Pancreatic NENs (PanNENs) arising from the islets of Langerhans, account for 12% of all NENs and 1–3% of all types of pancreatic cancers .Around 15% of cases secrete hormones leading to clinical symptoms and thus are known as functioning PanNENs. Based on the secreted peptide, the functioning PanNENs can be divided into insulinoma (insulin), gastrinoma (gastrin), glucagonoma (glucagon), somatostatinoma (somatotastin) and VIPoma , along with other less common tumors producing serotonin and adrenocorticotrophic hormone (ACTH) ,7. HowevThe current WHO’s classification  divides 2; <3% Ki67 index), G2  and G3 (>20 both mitotic count/mm2 and % Ki67 index). While PanNECs are poorly differentiated tumors that are always characterized by the mitotic count and Ki67 index of >20 [PanNETs are well-differentiated tumors and are classified in G1 , von Hippel–Lindau disease (VHL), neurofibromatosis type 1 (NF1) and tuberous sclerosis complex (TSC) ,12. All MEN1 encodes for a protein, menin, ubiquitously expressed mainly in the nucleus. Menin plays crucial roles in the regulation of the expression of cyclin-dependent kinase inhibitors through the interaction with KMT2A/2D , inhibition of PI3K/mTOR, activation of DNA repair genes (BRCA1 and RAD51) and preservation of telomere stability [MEN1 mutations [VHL codifies for pVHL, a negative regulator of hypoxia-inducible factor (HIF) and the loss of pVHL induces an increase of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) [NF1 gene product, neurofibromin, is a negative regulator of rat sarcoma (Ras)/mitogen-activated protein kinases (MAPK) and PI3K/mTOR signals. Consequently, the loss of neurofibromin promotes the activation of both RAS/MAPK and PI3K/mTOR [TSC1 and TSC2 encode for hamartin and tuberin, respectively (negative regulators of PI3K/mTOR) [tability . Howeverutations . The VHLCDKN1B, MUTYH, CHEK2 and BRCA2 [APC, RAD50, RECQL4, FANCC, MAPKBP1 and PIF1 genes [Interestingly, Scarpa et al. found that in sporadic PanNETs, 17% of patients harbored germinal mutations in nd BRCA2 , while oF1 genes ,16. ThisMEN1, DAXX/ATRX, PTEN, TSC2) in 68 resected and sporadic PanNETs (mostly G1/G2), thereby highlighting the main genetic differences between PanNETs and PDAC [MUTYH, CHEK2, BRCA2), chromatin remodeling , telomere alteration  and PI3K/mTOR signaling pathway  [ARID2, CDKN2A, SETD2, and gene fusions, as EWSR1-BEND2/FLI, TSC1-TMEM71 and CHD7-BEND2 in PanNETs [Concerning the sporadic PanNETs, most of our information concerning signaling network derived from two large-scale reports: a whole-exome sequencing study  and a whand PDAC . Second, DEPDC5) . Althoug PanNETs ,18.MEN1, DAXX, DMD, SETD2, ATRX and CREBBP [Metastatic forms were also analyzed in order to identify molecular mechanisms underlying molecular progression. A whole-genome sequencing performed on metastasis derived from 20 advanced or metastatic PanNETs (G2/G3), and showed mainly somatic aberrations in d CREBBP . From a d CREBBP .TP53 and RB1), particularly to the liver, as also observed in other types of NETs (gastro-entero-pancreatic and lung NETs) [Another molecular mechanism of metastatization was identified in a recent study where it was observed that the immunohistochemical expression of P53 and RB1 was normal in NETs G1/G2, but abnormal in NETs G3. Noteworthy, these alterations in the protein expression have been found only in NET G3 metastasis and not in primary tumors ,23,24. Tng NETs) ,23,24.MEN1, ATRX/DAXX and, less frequently, TP53, CDKN2A, ARID1A, LRP1B and APC genes, without any KRAS mutation or Rb loss [ATM, VHL and IDH1 in 15 gastro-entero-pancreatic NET G3 [Few studies interrogated the genomic landscape of PanNETs G3. For a total of 44 PanNETs G3, three studies detected a few mutations affecting  Rb loss ,26,27. Ac NET G3 .KRAS, TP53, BRAF, RB1, APC, MYC, ARID1A, ATM, KDM5A, ESR1, CDKN2A, ARID1A and LRP1B [BRAF, MYC and ARID1A mutations, were more frequent in large-cell PanNEC [TP53, CDKN2A, ARID1A, LRP1B and APC) with PanNETs G3, and thus is more similar genetically and phenotypically with PDAC [KRAS mutation demonstrated that they are valid predictors of the response to platinum-based chemotherapy and indeed specifically detected in PanNEC [MEN1/ATRX/DAXX and RB1/TP53 to discriminate between PanNET G3 and PanNEC [RB1 mutations to discriminate PanNET3 G3 versus PanNEC [Unlike PanNETs G3, PanNEC often showed Rb loss and mutations in nd LRP1B ,27,29,30ith PDAC . These dith PDAC . Accordin PanNEC . The curd PanNEC ,32. HoweINS, IAPP, INSM1); the MLP subtypes are associated with a high rate of liver or lymph node metastasis and enriched for genes correlated with stroma, hypoxia and pancreatic progenitor-specific genes; the intermediate subtype is more similar to islet/insulinoma tumors but more aggressive with a high frequency of MEN1 and DAXX/ATRX mutations [n = 84). This study has found four different subgroups: proliferative, stromal/mesenchymal, alpha cell-like and PDX1-high [YAP1 and WWTR1 , thereby increasing the activation of epithelial-mesenchymal transition and angiogenesis [YAP1 and WWTR1. Alpha cell-like subgroup showed increased expression of transcription factor ARX and mitochondrial proteins , and is enriched by oxidative phosphorylation-related genes associated with frequent mutations in MEN1, DAXX or ATRX [MEN1, ATRX, DAXX) with a low incidence in proto-oncogenes. Strikingly, the PDX1-high subgroup showed high expression levels of PDX1 and is associated with high frequency in proto-oncogenes, including CTNNB1 (p.D32N), HRAS (p.Q61R), NRAS (p.Q61R), KRAS (p.L19F and p.Q22K) and RET (p.V292M) [In 2015, Sadanandam et al. identified three transcriptomic subgroups in human PanNETs: islet/insulinoma, metastasis-like primary (MLP) and intermediate subtype . The islutations . These tDX1-high . The proDX1-high . Accordiogenesis . The str or ATRX . This ma or ATRX  have demp.V292M) .SETD2, MEN1 and ATRX can influence histone deacetylases (HDACs) and DNA methylation [RASSF1, HIC1, APC, CDKN2A, MGMT, MLH1, TIMP3, BRCA1 and VHL, involved in key pathways  are hypermethylated in PanNENs [APC promoter hypermethylation in MEN1-related PanNETs [MEN1 mutations, while the β-like one is associated with MEN1/DAXX/ATRX wild-type. Instead, the intermediate subgroup presents mutations in MEN1 and/or DAXX/ATRX with an increase in copy-number variation (CNV) events, compared to the α- and β-like subgroups [ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup is associated with tumors harboring mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses. T3 subgroup includes PanNETs G1 tumors carrying mutations in MEN1 and recurrent loss of chromosome 11; this subgroup is associated with a better prognosis [The epigenetic regulation of DNA via methylation is strongly associated to tumor initiation and development in several human cancer types . The glohylation ,38,39. Ihylation . Previou PanNENs ,42,43. A PanNENs ,45. In a PanNENs . This st PanNETs . Based o PanNETs . The α-lubgroups . Moreoveubgroups . Indeed,ubgroups . As the ubgroups , anotherubgroups . The T1 rognosis . Using trognosis , confirmInterestingly, different candidate protein biomarkers, such as FASLG, which is negatively correlated with Ki67 and found in lower levels in PanNETs G3 , could bNOVA1, which is highly associated with cell proliferation, invasion, and migration. This study shed the light on the role of splicing machinery in carcinogenesis, thereby paving the way for the development of a new class of biomarkers in PanNETs [Finally, the analysis of spliceosomes in 20 PanNETs discovered the overexpression of  PanNETs .The information about the molecular landscape of sporadic PanNENs are summarized in MEN1 mutation develop insulinoma [DAXX and/or ATRX is associated with an increased risk of metastasis [It is the most common functioning PanNEN and accounts for about 4–20% of PanNENs with four cases per 1 million person-years ,54. Insusulinoma . The bessulinoma ,58. The sulinoma . For allsulinoma . Moreovetastasis . In a mutastasis .It accounts for 4–8% of all PanNENs  and is mGlucagonoma predominantly involves the tail of the pancreas with an incidence rate of about two cases per 20 million person-years and accounts for 1–2% of all PanNENs ,70. It cIt represents 4% of PanNENs with an estimated incidence of one case per 40 million person-years with a female predominance (2:1) . SomatosIt accounts for 0.6–1.5% of all PanNENs with an incidence of one case for 10 million person-year, yet it is observed in approximately 5% of MEN1 patients . VIPoma It accounts for 0.58–1.4% of all PanNENs and derives by uncontrolled proliferation of Kulchitsky cells or Enterochromaffin cells that express serotonin ,89. MostIt is an extremely rare tumor characterized by uncontrolled production of ACTH from bronchial carcinoid cells. This, in turn, can lead to the increase in glucocorticoid levels and, consequently, to the Cushing syndrome ,95. OccaGiven their low incidence, all the available information regarding genetic alterations of functioning PanNENs are derived from low-scale reports or case reports.YY1 (T372R) mutation is predominant (30%) [MEN1 mutations occur rarely (7%) [MEN1 is a tumor suppressor, and it interacts with several proteins, receptors, and transcriptional factors [DAXX, histone methyltransferases (MLL1-2), BRCA1/RAD5 1 [YY1 mutations and loss of chromosome 11, while the second one had nearly no YY1 mutations and gains of chromosomes 7, 3p, 5p and 13q [MAFA missense mutation drives the development of familial insulinomatosis, characterized by the synchronous and metachronous occurrence of insulinomas, multiple MEN1-associated insulinoma precursor lesions and the rare development of metastasis [MAFA regulates insulin expression and other genes involved in glucose-stimulated insulin secretion. This missense mutation promotes β cells proliferation [INS/IGF2 locus, CDNK1C, MEN1, KDM6A, MLL3/KMT2C, YY1, KDM5B, and SMARCC1 [According to recent studies on sporadic insulinoma, nt (30%) ,101,102 ely (7%) . As ment factors . Among m1/RAD5 1 . The YY11/RAD5 1 . The mTO1/RAD5 1 . The mTO1/RAD5 1 . Moreove and 13q ,109. In  and 13q . Several and 13q . Moreove and 13q . It has tastasis . MAFA referation . Based oferation : (1) betferation . Insulin SMARCC1 ,115.MEN1 mutations [HER-2/neu proto-oncogene or chromosome 9q, deletion of the p16/MTS1 tumor suppressor gene or deletion of chromosome 3p [CDKN2A gene is the most common epigenetic alteration observed in gastrinoma [In gastrinoma, a high rate (about 40%) of somatic utations ,116,117 utations  are repoosome 3p . Amplifiosome 3p . The proosome 3p . Moreoveosome 3p . Howeverstrinoma .MEN1 mutations, including MEN1 E179V [MEN1 mutations, one heterozygous mutation 928G>C (G310R) in exon 7 [DAXX [MEN1 or VHL [Regarding the molecular profile of glucagonoma, few studies have been performed. In these studies, it was observed that glucagonoma is characterized by a high rate of N1 E179V  and two n exon 7  and the n exon 7 . Recentl 7 [DAXX . It form 7 [DAXX . Moreove1 or VHL . This mu1 or VHL . To dateMEN1 mutations [HIF2A somatic mutations were found in somatostinoma, as well as in other rare neuroendocrine tumors affecting other organs, such as paragangliomas [HIF2A is associated with a new syndrome of paraganglioma and somatostatinoma associated with polycythemia [In addition to the high rate of utations , HIF2A sngliomas . In partcythemia ,132. Undcythemia . For thiMEN1 mutations [MSH2 and overexpression of CXCR4 in the hepatic metastasis [MSH2 promote defects in the mismatch repair system and, thus, tumor development [Our knowledge about the molecular landscape of VIPoma is related to the low number of case reports, most of them reported utations ,129. In tastasis . Mutatioelopment .Concerning serotonin-producing tumors, a recent study revealed that they are characterized by few pathogenic mutations and that TGF-β pathway activation signatures were associated with extracellular matrix remodeling . TGF-β rFinally, in a series of seven ACTH-producing tumors, the hypomethylation in pro-opiomelanocortin promoter was found leading to the ACTH abnormal releasing . No furtThe aforementioned mutations found in each subgroup of functioning PanNENs are summarized in the following table .The surgical resection remains the only possible treatment for patients with localized functioning PanNENs without widespread metastasis ,140,141.However, all patients with metastatic functioning PanNENs and without contraindication should have surgical exploration with the aim to remove the primary tumor and peritumoral lymph nodes .The first consideration in the management of patients with advanced and unresectable functioning PanNENs is hormonal control to improve survival and quality of life . The somRegarding therapies that directly control tumor growth, the systemic treatment is indicated when unresectable and multiple metastasis are present. The somatostatin analogues are considered the first choice for low grade advanced functioning PanNENs (<10% Ki67) with a positive expression of somatostatin receptor ,148,149.When patients progress under somatostatin analogues treatment or as a first-line treatment in patients with tumor-negative expression of somatostatin receptor, chemotherapy  or targeIn addition, for advanced functioning PanNENs with high levels of somatostatin receptors, the peptide receptor radionuclide therapy might be a sustainable option. Indeed, NETTER-1 trial has provided encouraging evidence for the treatment in advanced PanNENs, but this approach should be further validated in other trials ,158.To date, two clinical trials are recruiting for patients with functioning PanNENs, a phase III trial investigating the effects of cabozantinib, a tyrosine-kinase inhibitor (NCT03375320), and one study evaluating the possibility and the safety of radiofrequency ablation of the tumor under ultrasonography guidance (NCT03834701).Immunotherapy is positively changing the treatment of some solid tumors, including lung cancer and melanoma , suggestOverall, immunotherapy remains a promising therapeutic approach for the treatment of advanced PanNENs ,166,167.In addition, novel immunotherapeutic approaches using chimeric antigen receptor (CART)-cells demonstrated promising positive results in vitro and in vivo in PanNETs .The functioning PanNENs are a heterogeneous group of tumors with different morphogenic and clinical features ,2. Due tMen1 inactivation in pancreatic islet cells (hTS/Men1/−−). This mouse model allowed us to identify a crucial dualism between these two proteins in exacerbating PanNETs progression [The basic and preclinical research allowed us to better characterize the main pathways underlying the functioning PanNENs. Among all mice models , it is wgression .To date, a study incorporating human tumor tissues, in vivo models and large organoid biobank ,181 coulMoreover, the development of high-throughput techniques has clearly accelerated the research on PanNENs, allowing us to identify the common genetic and epigenetic alterations and paving the way for the introduction of targeted therapy, even if for a small number of patients ,2.Recently, a novel multi-gene liquid biopsy, based on real-time PCR, the NETest, has been demonstrated to be a reliable and accurate tool for the diagnosis, grading, staging, progression, and therapy responses of PanNENs ,185,186.In order to improve the clinical management of functioning PanNENs, a set of important initiatives should be taken in a five-year perspective plan. Liquid biopsy may be considered as a promising surrogate for tissue biopsy, allowing for the longitudinal monitoring of the disease in a non-invasive manner. This approach could significantly help towards early diagnosis, assessment of treatment efficacy, as well as detection of early onset of resistance, thus allowing adjustment of the treatment . AlthougHER-2 amplification in gastrinoma that could be the subject of targeted therapy  [Today, targeted therapy in functioning PanNENs is limited only to very few agents . However, it is worth stating that this targeted approach is not chosen based on the molecular profiling of the patients. Interestingly, genetic profiling of different functioning PanNENs has revealed different actionable alterations, such as tuzumab) . ConsequHowever, there are different limitations in the studies on functional PanNENs. They are rare (about 15% of PanNENs) and heterogenous types of tumors, which make it difficult to recapitulate their features in vitro and in vivo. Most preclinical models  develop Indeed, the main studies deciphering the molecular, transcriptomic, and epigenetic profiles of PanNENs  have beeFurthermore, the previous successful clinical trials ,155,156 To solve this gap of information in the future, we highly encourage the establishment of international multi-institution consortiums in order to share knowledge and increase the available samples for the research. This will translate into the recruitment of more patients for conducting clinical trials for each subgroup of each primary tumor site to decipher selectively the molecular features .


Leptospira spp., there are five sphingomyelinases exclusively expressed by pathogenic leptospires, in which Sph2 is expressed during natural infections, cytotoxic, and implicated in the leptospirosis hemorrhagic complications. Considering this and the lack of information about associations between Sph2 and leptospirosis severity, we use a combination of immunoinformatics approaches to identify its B-cell epitopes, evaluate their reactivity against samples from leptospirosis patients, and investigate the role of antibodies anti-Sph2 in protection against severe leptospirosis. Two B-cell epitopes, Sph2(176-191) and Sph2(446-459), were predicted in Sph2 from L. interrogans serovar Lai, presenting different levels of identity when compared with other pathogenic leptospires. These epitopes were recognized by about 40% of studied patients with a prevalence of IgG antibodies against both Sph2(176-191) and Sph2(446-459). Remarkably, just individuals with low reactivity to Sph2(176-191) presented clinical complications, while high responders had only mild symptoms. Therefore, we identified two B-cell linear epitopes, recognized by antibodies of patients with leptospirosis, that could be further explored in the development of multi-epitope vaccines against leptospirosis.Sphingomyelin is a major constituent of eukaryotic cell membranes, and if degraded by bacteria sphingomyelinases may contribute to the pathogenesis of infection. Among  Leptospirosis is a tropical and neglected emerging zoonotic disease that afflicts humans and other animals . It is cLeptospira, which can infect humans and almost all mammals, as well as reptiles and amphibians  and other bacteria including Listeria ivanovii (SmcLUniprot ID: Q9RLV9), Bacillus cereus , Staphylococcus aureus (Uniprot ID: A0A7U4AUV1), and Pseudomonas spp. strain TK4 (Uniprot ID: Q93HR5) were conducted by MAFFT [Sequence alignment and identification of conserved patterns among sphingomyelinases from by MAFFT .Sequences predicted as antigenic linear B-cell epitopes were synthesized using fluorenylmethoxycarbonyl (F-moc) solid-phase chemistry ,42 , resulting in 51 leptospirosis patients (MAT positive), reactive in MAT, and 36 negative controls (MAT negative). The study was reviewed and approved by the Oswaldo Cruz Foundation Ethical Committee and the National Ethical Committee of Brazil (number CAAE: 31405820.8.0000.5262).Samples of confirmed leptospirosis cases and the control group were screened for the presence of naturally acquired antibodies against the synthetic peptides via ELISA as previously described .Briefly, MaxiSorp 96-well plates  were coated with 100 μg/mL of a peptide. After overnight incubation at 4 °C, plates were washed with phosphate-buffered saline (PBS) and blocked with PBS-containing 5% non-fat dry milk (PBS-M) for 1 h at 37 °C. Individual serum samples diluted 1:100 on PBS-M were added in duplicate wells, and the plates were incubated at 37 °C for 1.5 h. After three washes with PBS-Tween20 (0.05%), bound antibodies were detected with peroxidase-conjugated goat anti-human IgG  or goat anti-human IgM , diluted at 1:1000 (in PBS-M), and incubated for 1 h at 37 °C, followed by TMB . The reaction was stopped by the addition of HCl (1N), and the absorbance was read at 450 nm using an xMark™ microplate absorbance spectrophotometer . The results for total IgG and IgM were expressed as the reactivity index (RI)—the ratio between the mean optical density (OD) of tested samples and the mean OD of 44 control group samples plus 2.5 standard deviations (SD). Subjects were considered IgG responders to a particular antigen if the RI was higher than 1.p-value < 0.05 was considered significant.The obtained data were analyzed using GraphPad Prism 8.0 . First, to determine if a variable was normally distributed, the one-sample Kolmogorov–Smirnoff test was used. Differences in frequencies of IgG and IgM responders to synthetic peptides were evaluated using Fisher’s exact test, while the reactivity indices against synthetic peptides between responders to each epitope were compared using the Mann–Whitney test. A two-sided L. interrogans serovar Lai Sph2. In this way, considering that each amino acid residue was predicted as inserted in a linear B-cell epitope by at least five algorithms, we identified two sequences as continuous B-cell epitopes in the studied protein: GHDERAKRISKSDYVK (Sph2(176-191)) and TPTKSGHKKKYDQV (Sph2(446-459)). As shown in In this study, we used a combination of nine algorithms to predict linear B-cell epitopes from (176-191) and Sph2(446-459), were predicted as antigenic, presenting Vaxijen scores of 1.173 and 1.763, respectively.The protein Sph2 was considered a protective antigen, obtaining a score of 10.458 in the VaxiJen algorithm. Regarding predicted B-cell linear epitopes, both sequences, Sph2Leptospira spp. sphingomyelinases. Moreover, when compared with sphingomyelinases of other bacteria, Sph2 presented identities ranging from 42% to 49%  and Sph2(446-459) were considered non-conserved among microorganisms not belonging to the genus Leptospira, because they presented an E-value greater than one on BlastP. Interestingly, compared with Sph2 from other pathogenic leptospires and other sphingomyelinases, we observed a different profile of conservation between both epitopes. Sph2(176-191) presented more than 70% of identity with pathogenic leptospires, highlighting 100% of identity in 20% of studied pathogenic leptospires . This epitope also presented 100% of identity when compared with L. interrogans serovar Lai Sph4, and presented a mean identity of 42.2% when compared with sphingomyelinases from other bacteria. On the other hand, Sph2(446-459) was highly conserved among all studied leptospires SPh2, presenting more than 70% of identity. Interestingly, the epitope Sph2(446-459) was also highly conserved in other Leptospira interrogans serovar Lai sphingomyelinases, presenting identities that ranged from 71.4% to 100%, but presented low conservation when compared with other studied bacteria sphingomyelinases, presenting identities less than 37.5%  and five in the central metal-binding site . Considering this, when we compared aligned amino acids in Sph2 from L. interrogans serovar Lai with other pathogenic leptospires, the histidine at position 293 (H293) was the unique amino acid conserved in all studied leptospires. Moreover, the number of modified amino acids in the catalytic or metal-binding sites ranged from one to six among the studied Sph2. In this context, while Sph2 from L. interrogans serovar Bataviae; L. alstonii serovars Pingchang and Sichuan; L. borgpetersenii serovars Pomona and Hardjo-bovis; L. noguchii serovars Panama and Autumnalis; and L. santarosai serovar Arenal presented no modifications in amino acid residues in the catalytic or metal-binding sites, L. interrogans serovar Australis presented six (67%) different residues. Additionally, there were eight surface-exposed amino acids described as being associated with the interaction with the host cell membrane . Interestingly, these amino acids seemed to be less conserved among pathogenic leptospires, only one out of the three known serovars  presented no changed amino acids in the alignment, while the other studied proteins presented from three to six changed amino acids. Additionally, when compared with sphingomyelinases from other bacteria, Sph2 presented differences only in amino acids involved with the interaction with host cells, while all amino acids of the catalytic site and metal-binding sites remained unaltered  was inserted in Sphingomyelinase C domain (Sph2(155-440)). Finally, aiming to allow the visualization of predicted epitopes, catalytic and metal-binding sites, and amino acids associated with the interaction with the host cell membrane in the Sph2 3D structure, we highlight these structures in the Alphafold predicted model (ID: AF-P59116-F1)  .Leptospira spp. (MAT positive), presenting antibody titers on MAT ranging from 1:800 to 1:12800 (mean: 1:2839 ± 2407), and were grouped as reactive against Leptospira spp. (RL), while 36 patients were non-reactive against Leptospira spp. (NRL).The studied population was composed of 87 Brazilian febrile suspected leptospirosis cases based on their contact with rodents, floods, or other risk factors. Among them, 51 patients were reactive to p = 0.134). Moreover, in both groups, RL and NRL, about 11.5% of deaths cases were reported. As shown in The studied population presented was 35.7 (±17.5) mean years old, and clinical and epidemiological data were obtained between 1 and 74 days after the beginning of symptoms (mean 12.5 ± 12). Our studied group was mostly composed of men (85%), but we did not observe statistical differences in the frequency of men in RL (90%) and NRL (78%) (p = 0.0159), calf pain , renal insufficiency , and pulmonary hemorrhage  were statistically more frequent in RL patients.Regarding reported symptoms, fever (84%), myalgia (77%), and headache (63%) were the most prevalent symptoms among studied patients. However, comparing the frequencies of symptoms among groups, only jaundice , while the samples of 14 individuals (27%) cross-reacted with two serovars, one individual sample (2%) cross-reacted with serovars Australis, Hebdomadis, and Autumnalis, and another (2%) recognized the serovars Copenhageni, Canicola, Icterohaemorrhagiae, and Tarassovi  and Sph2(446-459) in plasma samples from 51 confirmed leptospirosis patients (RL: MAT reactive to Leptospira spp.) and 36 non-confirmed cases (NRL: non-reactive to Leptospira spp.). First, using the defined cut-off, the predicted epitopes were specifically recognized by serum samples of the patients from the RL group, as the NRL samples were non-responders against the predicted epitopes ((176-191) and Sph2(446-459) were specifically recognized by 33.3% (n = 17) and 19.6% of RL individuals, respectively, with a prevalence of IgG immune response to both epitopes. As shown in (176-191) and Sph2(446-459), while only one (2%) and two (3.9%) patients presented IgM antibodies, respectively ((446-459), while the other two IgM responders also presented IgG antibodies. Moreover, among 20 individuals that presented antibodies against at least one Sph2 epitope, ten individuals only presented antibodies against Sph2(176-191), three against Sph2(446-459), and seven individuals presented antibodies against both epitopes. Regarding the magnitude response, there were no statistical differences between IgG reactivity indexes against each peptide, which ranged from 1.05 to 3.23 (median: 1.27) against Sph2(176-191) and ranged from 1.07 to 1.81 (median: 1.19) against Sph2(446-459)  c.(176-191) in patients reactive to serovars: Tarassovi (n = 6), Copenhageni (n = 3), Canicola (n = 1), Copenhageni/ Icterohaemorrhagiae (n = 3), Copenhageni/Tarassovi (n = 2), Tarassovi/Icterohaemorrhagiae (n = 1), and Copenhageni/Hebdomadis (n = 1). Furthermore, specific antibodies against Sph2(446-459) were detected in patients reactive to serovars: Tarassovi (n = 4), Copenhageni (n = 2), Grippotyphosa (n = 1), Copenhageni/Icterohaemorrhagiae (n = 2), and Copenhageni/Tarassovi (n = 1).Regarding the detection of antibodies in patients infected by different serovars, we detected specific antibodies against Sph2(176-191) and Sph2(446-459). In this context, considering that pulmonary hemorrhage was a symptom exclusively observed among RL patients, we highlight that its frequency was statistically higher among responders to Sph2(176-191) (29.4%) than among non-responders . However, this difference was not observed among responders (20%) and non-responders (12.2%) to Sph2(446-459) (p = 0.6116).Based on the observed differences in the frequencies of symptoms between RL and NRL patients, we further explored the frequencies of cases presenting jaundice, renal insufficiency, pulmonary hemorrhage, calf pain, and deaths by comparing the frequencies of these symptoms between individuals whose sera recognized or not the epitopes Sph2(176-191) (23.5%) and Sph2(446-459) (30%) were not statistically different when compared with non-responders to Sph2(176-191)  and Sph2(446-459) , and neither when compared with the observed frequency of death cases in the NRL group . Regarding other symptoms, we did not observe statistical differences between responders and non-responders to synthetic peptides. Remarkably, the frequency of renal insufficiency cases among non-responders to Sph2(446-459) (41.5%) was statistically higher than the frequency observed in NRL , while the frequency among responders to Sph2(446-459) (30%) was statistically similar to both non-responders (p = 0.7208) and NRL (p = 0.3442). Additionally, responders and non-responders to Sph2(176-191) presented similar frequencies of renal insufficiency , both higher than the NRL group . Additionally, we observed higher frequencies of jaundices among responders to Sph2(176-191)  and non-responders to Sph2(446-459)  than among NRL (11.1%), and observed higher frequencies of calf pain among responders to Sph2(176-191) (76.5%) than NRL . Remarkably, clinical complications, such as pulmonary hemorrhage, were only observed in individuals with low or no-reactivity against Sph2(176-191), while individuals with high reactivity indexes to this epitope presented only mild symptoms.As shown in Leptospira spp. [Leptospira, sphingomyelinases play an important role in their survival in the mammalian host by mediating the lysis and release of essential nutrients from the host cells [L. interrogans serovar Lai, Sph2 and SphH are proven to have cytotoxic properties and are expressed during infection [L. interrogans serovar Lai Sph2 and to explore their association with the clinical data of naturally infected patients.Sphingomyelin (SM) is a major constituent of eukaryotic cell membranes and the ability to degrade this phospholipid by bacteria may consequently contribute to the pathogenesis of infection. Sphingomyelinases are a group of hemolysins, present in both eukaryotes and prokaryotes, which are related to phospholipid metabolism in the former and that frequently act as toxins in the latter , that arira spp. ,44. In pst cells , but arest cells ,46. Amonnfection ,47. HoweL. interrogans serovar Lai Sph2. This approach had been used and improved by our group in recent years to predict epitopes in viruses [(176-191)) and TPTKSGHKKKYDQV (Sph2(446-459)), which did not present similar epitopes described in BLASTP, in the Immune Epitope Database http://www.iedb.org/home_v3.php (accessed on 20 June 2020) (data not shown), and also did not present similar sequences described in humans and mice, or in other databases from PeptideAtlas http://www.peptideatlas.org/map/ (accessed on 20 June 2020). Therefore, predicted epitopes were considered non-conserved among hosts and other bacteria, suggesting them as antibody targets specific against Leptospira spp. In this context, when compared with other sphingomyelinases from L. interrogans serovar Lai, we observed that Sph2 is highly similar to Sph4, presenting more than 99% of identity, while other sphingomyelinases presented identities from 53% to 66.4% (L. interrogans serovar Lai and serovar Bataviae (identity of 90%), Sph2 from serovar Lai was more similar to L. noguchii serovars Panama and Autumnalis (identities of 89.6%) than to other serovars of L. interrogans , corroborating studies that recognized the great numbers of serovars as an obstacle that has hampered the development of a universal vaccine for leptospirosis [First, we used a combination of algorithms to predict linear B-cell epitopes in  viruses ,26, bact viruses , and pro viruses ,48,49, rto 66.4% . Moreovespirosis .L. interrogans serovar Lai and Sph2 from other pathogenic leptospires. Remarkably, the epitope Sph2(446-459) was highly conserved among Sph2 from pathogenic leptospires and among sphingomyelinase from L. interrogans serovar Lai, presenting more than 71.4% of identity when compared with studied sequences. However, Sph2(176-191) was highly conserved in only three studied Sph2  and in Sph4, presenting 100% of identity with these proteins, while presenting less than 56.3% of identity when compared with other studied proteins. From our point of view, the high conservation of Sph2(446-459) among pathogenic leptospires suggests that this epitope may be inserted in a region under low selective pressure by the host immune response. In line with this assumption, according to the prediction of algorithms InterPro family, TIGRFAMs, and nSMase (data not shown), this epitope is located out of the Sph2 Sphingomyelinase C domain (Sph2(155-440)), supporting the hypothesis of low selective pressure given that all amino acids involved in Sph2 activities are located on the Sphingomyelinase C domain [Based on these data and aiming for the future constructions of multi-epitope vaccines, we explored the conservation of predicted epitopes among other sphingomyelinases from C domain , a commoC domain .L. interrogans serovar Lai presents all amino acids described in the catalytic and metal-binding sites highly conserved (100% of identity) when compared with sphingomyelinases from other bacteria, such as Listeria ivanovii and Bacillus cerus, which are considered membrane-damaging virulence factors that induce hemolysis, a reduction in phagocytosis [L. interrogans serovar Lai [L. interrogans serovar Copenhageni Fiocruz L1-130 to lyse sheep erythrocytes [(176-191) was closely located to the catalytic site, metal-binding site, and the region related to interaction with the host membrane. This finding suggests that antibodies against Sph2(176-191) could hamper the functionality of Sph2 by blocking the interaction with the host, or even the ligation to cofactor or substrate. However, studies aiming to identify the active sites of Sph2 from Leptospira spp. and to evaluate their activity and the role of antibodies against their epitopes remain a lack in the literature.In the same way, we evaluated the variability of amino acids in the catalytic site, metal-binding site, and the region of interaction with host membranes . First, ocytosis , and escocytosis . Additioocytosis , which iovar Lai , and prohrocytes . Based o(176-191) and Sph2(446-459) in blood samples from 51 Brazilian patients with leptospirosis, which presented reactivity (or cross-reactivity) to 14 Leptospira spp. serovars, with the prevalence of Tarassovi and Copenhageni. Interestingly, this observation is in disagreement with the recent revision of Browne and collaborators, which reported the serovars Icterohaemorrhagiae, Canicola, and Pomona as the most prevalent in human leptospirosis in the Americas from 1930 to 2017, with Pomona and Canicola being the most prevalent in Brazil [Until now, antibodies against Sph2 were detected in the blood of mares following leptospiral abortion, but not in horses immunized with bacterins , and in n Brazil . From ouLeptospira spp. were able to recognize at least one of these epitopes, with a prevalence of IgG antibodies among responders to both Sph2(176-191) and Sph2(446-459). Both epitopes Sph2(176-191) and Sph2(446-459) were recognized by patients’ samples reactive to at least one of the most prevalent serovars , while Sph2(176-191) was also recognized by patients’ samples reactive to serovars Canicola and Hebdomadis, and Sph2(446-459) was also detected in samples reactive to serovar Grippotyphosa. Remarkably, among the serovars detected in our studied population, only samples reactive to serovars Wolffi (n = 1), Pomona (n = 1), Wolffi/Sejroe (n = 1), and Australis/Hebdomadis/Autumnalis (n = 1) presented no reactivity to at least one of predicted epitopes. However, considering the low level of identity shared by L. interrogans serovar Lai Sph2(176-191) and other pathogenic species, we cannot discard a higher frequency of responder and reactivity index investigating the epitopes from other important serovars. Therefore, the real cross-reactivity of these epitopes needs to be explored more in serological and epidemiological studies, since the absence of reactivity could be related to the limited number of studied patients and the expression of Sph2, which is markedly upregulated by changes in osmolarity and temperature [In this context, thinking about reactivity and cross-reactivity to predicted antibodies, first, we confirmed that the predicted epitopes were naturally immunogenic, since about 40% of serum samples from patients naturally infected by perature , determiL. interrogans serovar Copenhageni did not present protection when challenged with a virulent strain of L. interrogans serovar Pomona; however, the authors propose that this can be related to a lack of correct folding of the recombinant proteins [(176-191) ((176-191) presented only mild symptoms. Based on this data and on the position of Sph2(176-191) close to the catalytic site and the region of interaction of Sph2 with the host membrane, we conjecture that this epitope should be better explored as a target of protective antibodies against leptospirosis; however, more studies are necessary to prove this hypothesis. On the other hand, we also hypothesized that different levels of Sph2 expression related to Leptospira species and host factors can also be related to reactivity against Sph2 epitopes and could be associated with leptospirosis hemorrhagic symptoms, reinforcing the necessity of more studies to prove Sph2 and its epitopes as vaccine candidates.Moreover, the protective potential of antibodies against Sph2 is still unknown. Regarding its potential as a vaccine antigen, hamsters immunized with the recombinant Sph2 from proteins . Unfortu176-191) . We beliLeptospira elicit a serovar-dependent immunity [Leptospira antigens experimentally validated remains scarce, and justifies our study, since the identification of protective epitopes is the key to the design of an effective and universal multi-epitope vaccine for human leptospirosis.Up to now, the main vaccines proposed against immunity . Though,immunity . Moreoveimmunity , supportimmunity ,59,60,61L. interrogans serovar Lai and to assess their natural immunogenicity in human leptospirosis. However, further studies using patients from different regions and additional Leptospira spp. serovars are necessary to investigate the real protective role of antibodies against epitopes Sph2(176-191) and Sph2(446-459) in leptospirosis. Moreover, studies in animal models are essential to prove their potential as a vaccine antigen, singly and combined with other identified epitopes.In brief, we used immunoinformatics to predict linear B-cell epitopes in Sph2 from L. interrogans serovar Lai. Our data corroborate the association of Sph2 with hemorrhagic complications from leptospirosis. Moreover, based on its location and associations with clinical data and specific immune response, we suggest that Sph2(176-191) can be further explored in multi-epitope vaccines to prove its protective potential in animal models.Our study was the first to identify antibody targets in Sphingomyelinase 2 from


The group B coxsackieviruses (CVBs) exist in six serotypes (CVB1 to CVB6). Disease associations have been reported for most serotypes, and multiple serotypes can cause similar diseases. For example, CVB1, CVB3, and CVB5 are generally implicated in the causation of myocarditis, whereas CVB1 and CVB4 could accelerate the development of type 1 diabetes (T1D). Yet, no vaccines against these viruses are currently available. In this review, we have analyzed the attributes of experimentally tested vaccines and discussed their merits and demerits or limitations, as well as their impact in preventing infections, most importantly myocarditis and T1D. Picornaviridae family, and Homo sapiens are the natural hosts of enteroviruses, which are known to cause a wide range of diseases [Enteroviruses belong to the diseases ,2. In thdiseases ,4. Whilediseases , coxsackdiseases ,7,8,9,10diseases ,12. Whildiseases , CVBs coCVB was isolated for the first time at the Hudson River town of Coxsackie, New York, United States, in 1947 . Six serCVBs, positive-sense single-stranded RNA (ssRNA) viruses, are generally considered lytic viruses, but they can persist in defective forms, as demonstrated with CVB3 . They arCVBs are typically transmitted through the fecal–oral route. Infection of target cells requires interaction with two main receptors: the decay accelerating factor (DAF) and the coxsackievirus and adenovirus receptor (CAR) ,38; howepro, 2B, 2C, 3A, 3B, 3Cpro, and 3Dpol. While the structural proteins, also called capsid proteins, contribute to the assembly of the virus capsid, non-structural proteins are essential for viral replication and modulation of host responses [Generally, the CAR acts as an internalization receptor for viral entry into the cytoplasm. After uncoating, the positive-sense ssRNA genome is translated, leading to the production of a single polyprotein followed by proteolytic cleavage, which generates structural viral proteins (VPs) 1 to 4 and nonstructural proteins namely, 2Aesponses ,54,55,56esponses ,54,57,58Pathologically, both viral and host factors contribute to tissue damage in the affected organs ,59,60. BOf the many types of heart disease, myocarditis is one predominant cause of heart failure in pediatric populations and young adolescents ,62,63,64Likewise, diabetes has become a major health concern with a global prevalence estimated at 537 million in 2021 with a projected increase to 643 million by 2030 (occurring in ~9% of adults) , and theSince CVB infections are considered to be favored candidates in the occurrence of myocarditis/DCM and T1D, their prevention through vaccination can possibly lessen the incidence of both diseases. During the past two centuries, various vaccine platforms have been established, such as live-attenuated vaccines, inactivated vaccines, subunit vaccines, viral and bacterial vector vaccines, and nucleic acid vaccines, among others ,94. ManyThe major advantage of MLVs is their ability to induce both humoral and cell-mediated immune (CMI) responses, especially cytotoxic T lymphocytes (CTLs), which are critical in eliminating established infections. MLVs for CVBs have been derived by either serially passaging the viruses in various cell lines or introducing mutations in the structural proteins, namely VP1 or VP2 or stem-loops II and V. As indicated in Likewise, the CVB3-RD strain, which was attenuated after serial passaging of the CVB3 Nancy strain in human rhabdomyosarcoma cells, had a mutation in the puff VP2 region (T151S), rendering the virus non-cardio virulent ,103. SimSeveral clinical isolates of CVB3 collected over the years were tested to determine the major nucleotide determinants of cardio virulence, which led to the identification of nucleotide 234 in the 5′NTR as the primary determinant of cardiac virulence of CVB3 ,111. FurSimilar observations were noted with the temperature-sensitive mutants of CVB3 that replicate at 34 °C or 39.5 °C as vaccine candidates . These mAttempts have been made to create a multivalent vaccine by creating an attenuated CVB3 encoding the L1 loop of the adenovirus (Ad2) hexon protein and the protease 2A protein, which induced nAbs against both CVB and Ads, but protective effects of the vaccine were not investigated . LikewisTaken together, numerous approaches have been adopted to investigate the use of MLVs with mixed successes. Despite their ability to induce antibody and CMI responses, attenuated viruses can potentially revert to their virulence. Conversely, should the vaccine viruses retain mutations for a period of at least ~one month in vivo, protective immune responses would have been generated by then. Likewise, MLVs for CVB infections should be free of the autoimmune responses described above. These limitations can be overcome by developing inactivated/killed vaccines. SOCS-1) transgenic mice models, preventing CVB3-induced myocarditis and pancreatitis with no mortalities, as well as preventing CVB1- and CVB4-induced T1D. The immunogenicity of the hexavalent vaccine was also recapitulated in non-human primates. The three-dose vaccine regimen eliciting virus-reactive Abs may have contributed to the protective response. Although it was unclear whether T cell responses were generated in vaccinated animals, induction of IgG responses in vaccine recipients supports the possibility that the hexavalent vaccine might have induced virus-reactive T cells, as their help is critical for isotype switching. Inactivated vaccines have been traditionally prepared by killing the whole pathogen using formaldehyde, ultraviolet rays, X-rays, γ rays, β-Propiolactone (BPL), hydrogen peroxide, and zinc-finger reactive compound treatments, among others . AlthougdnaB gene of Rhodothermus marinus (Rma), termed Rma DnaB intein, which created a split functional N- and C-terminal intein to cyclize the VP1 protein of CVB3 in Escherichia coli (E. coli) [+ T cell responses [E. coli, termed FimH, revealed M-cell targeting and toll-like receptor 4-agonistic properties that also acted as an adjuvant was tested as a vaccine candidate [+ CD8+ T cells in draining lymph nodes with enhanced CTL responses [Ssp DnaX mini-intein spontaneous C-cleavage system in E. coli. Oral administration of these inclusion bodies induced mucosal immune responses and the vaccine recipients were protected from CVB3 myocarditis with a survival rate of 60% [Recombinant vaccines are routinely developed against infectious diseases, but only a few have been developed against CVB3. The VP1 protein has been used as a target to develop CVB3 recombinant vaccines. One study engineered a split intein from the E. coli) . This vaesponses  , C3d3, Shiga toxin B subunit (STxB), or mouse beta-defensin-2 (mBD2) DNA sequences and inserted into the pcDNA3 vector ,149,150.cis-acting replication element within the 2C protein of CVB3, designated MET-2C lenti. This vaccine induced a reduction in viral titers, myocardial lesions, and pro-inflammatory cytokines, but with a survival rate of only 50% [The RNA vaccine approach to creating CVB3 vaccines has been adapted to address three expectations: (a) the mRNA encoding the vaccine expresses a full-length mutant CVB3 polyprotein; (b) it would undergo self-cleaving to generate mature viral proteins for antigen processing and presentation; and (c) it would not give rise to infectious virus particles. One study created a plasmid pH3IH1 encoding CVB3 with a mutated cleavage site between viral 2A and 3B proteins to avoid infectious particle generation, leading to deletions at the beginning of the 5′NTR region  Table 1Table 1. only 50%  , and hence are non-infectious and do not revert to a virulent form by reversion, recombination, or re-assortment. VLPs are highly immunogenic and are expected to stimulate B and T cell immune responses ,173,174.Overall, multiple approaches have been used to develop CVB vaccines . A promiClinicalTrials.gov Identifier: NCT04690426). Any positive outcomes of this trial should boost efforts for developing similar vaccines that may lead to widespread acceptance.In contrast to other infectious diseases, vaccines for CVB infections have received little attention for many reasons. First, diseases caused by CVB are not severe enough to determine the immediate impact of CVB vaccines. However, from the perspective of myocarditis/DCM and T1D, the development of CVB vaccines may have merit. Still, their use is unlikely to have a dramatic effect on the occurrence of both diseases, since many non-CVBs and non-viral agents can trigger myocarditis and T1D . ArguablThe lessons learned from the use of vaccines against other RNA viruses, such as SARS-CoV-2, indicate that immune responses generated against vaccines may become irrelevant for escape mutants that may involve the phenomenon of original antigenic sin ,187,188.


Among various complications of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), renal complications, namely COVID-19-associated kidney injuries, are related to the mortality of COVID-19.In this retrospective cross-sectional study, we measured the sphingolipids and glycerophospholipids, which have been shown to possess potent biological properties, using liquid chromatography-mass spectrometry in 272 urine samples collected longitudinally from 91 COVID-19 subjects and 95 control subjects without infectious diseases, to elucidate the pathogenesis of COVID-19-associated kidney injuries.l-phosphate, phosphatidylcholine, lysophosphatidic acid, phosphatidylserine, lysophosphatidylserine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylglycerol, lysophosphatidylglycerol, phosphatidylinositol, and lysophosphatidylinositol increased, especially in patients with severe COVID-19 during the later phase, suggesting that their modulations might result from kidney injuries accompanying severe COVID-19.The urinary levels of C18:0, C18:1, C22:0, and C24:0 ceramides, sphingosine, dihydrosphingosine, phosphatidylcholine, lysophosphatidylcholine, lysophosphatidic acid, and phosphatidylglycerol decreased, while those of phosphatidylserine, lysophosphatidylserine, phosphatidylethanolamine, and lysophosphatidylethanolamine increased in patients with mild COVID-19, especially during the early phase (day 1–3), suggesting that these modulations might reflect the direct effects of infection with SARS-CoV-2. Generally, the urinary levels of sphingomyelin, ceramides, sphingosine, dihydrosphingosine, dihydrosphingosine Considering the biological properties of sphingolipids and glycerophospholipids, an understanding of their urinary modulations in COVID-19 will help us to understand the mechanisms causing COVID-19-associated kidney injuries as well as general acute kidney injuries and may prompt researchers to develop laboratory tests for predicting maximum severity and/or novel reagents to suppress the renal complications of COVID-19.The online version contains supplementary material available at 10.1186/s12929-022-00880-5. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with various complications. Among them, renal complications, especially acute kidney injury (AKI), are associated with critical conditions. These renal complications are known as COVID-19-associated kidney injuries. A high incidence of AKI has been reported, especially among critically ill patients, and patients with COVID-19-associated kidney injuries reportedly have a higher risk of in-hospital death , 14, 40.Regarding the etiology of COVID-19-associated kidney injuries, the mechanisms have not been fully elucidated at present, and both direct and indirect mechanisms have been proposed . ACE2 anN-acetyl-β-d-glucosaminidase [NAG], α1-microglobulin [α1-MG], neutrophil gelatinase-associated lipocalin [NGAL], and liver type fatty acid-binding protein [L-FABP]) and urine sediment findings are correlated with both the severity of COVID-19 and COVID-19-associated kidney injuries [Since COVID-19-associated kidney injuries are important in terms of clinical outcomes and the underlying mechanisms have yet to be fully elucidated, as described above, investigating biomarkers of COVID-19-associated kidney injuries will be important to understand the pathogenesis of such complications. Urine samples rapidly and accurately reflect renal conditions. Actually, recent studies have revealed that urinary chemical biomarkers  and ceramides have been well studied. S1P possesses potent anti-apoptotic and pro-survival properties , 33. A sAmong glycerophospholipids, lysophosphatidic acids (LPA) and lysophosphatidylcholine (LPC) have been well studied in the fields of nephrology. LPA is produced from LPC by autotaxin, and six kinds of LPA receptors have been identified . The rolAlthough several lipidomics analyses using serum or plasma samples from patients with COVID-19 have been performed , only onWe collected the residual urinary samples after routine clinical testing from 91 subjects who had been diagnosed as having COVID-19 using an RT-PCR assay between September 2020 and April 2021. The sampling times were classified into the following eight periods: day 1–3, day 4–6, day 7–9, day 10–12, day 13–15, day 16–18, day 19–24, and day 25–40 after symptom onset. Since the timing of RT-PCR testing varied largely among the patients, we used the day after symptom onset as the initial measurement in our investigation of lipid modulation. None of the subjects enrolled in the present study had been vaccinated at the time of sampling.The subjects were classified into three groups according to the maximum severity of COVID-19: maximum severity group 1 (did not require oxygen supplementation), maximum severity group 2 , and maximum severity group 3 . As a control, we collected 95 urine samples from volunteers without infectious diseases.The current study was performed in accordance with the ethical guidelines established by the Declaration of Helsinki. Written informed consent for sample analysis was obtained from some of the patients. For the remaining participants from whom written informed consent could not be obtained , informed consent was obtained in the form of an opt-out on our institution’s website, as follows. Patients were informed of the study through the website, and those who were unwilling to be enrolled were excluded. The study design was approved by The University of Tokyo Medical Research Center Ethics Committee (2602 and 2020206NI).We measured the levels of the lipid mediators listed below using four independent LC–MS/MS methods and the LC8060 system, consisting of a quantum ultra-triple quadrupole mass spectrometer . We simultaneously measured six ceramide species , Sph, and dhSph, as previously described . We alsoTo measure the urinary clinical markers, we used the reagents as described previously . Renal thttps://www.metaboanalyst.ca/). To examine differences in the time courses of urinary lipids among the control subjects and maximum severity groups 1, 2, and 3, we evaluated significant differences using the Kruskal–Wallis test, followed by the Steel–Dwass test as a post-hoc test. To examine differences in the urinary lipid levels longitudinally between specific time points in a specific maximum severity group, we used the paired Wilcoxon signed-rank test. To examine differences between the control subjects and the COVID-19 subjects, we performed non-parametric Volcano plot analyses. For the correlation studies, a Kendall rank correlation was performed to examine the correlations of lipids and clinical data with the maximum severity of COVID-19, using age, sex, and the presence of diabetes, hypertension, and current smoking as covariates of interest. To construct machine learning models, we used SPSS modeler ver. 18:2  and performed CHAID analyses, SVM analyses, and neural network analyses. The Spearman rank correlation was performed to examine correlations between lipids and clinical data. The independent effects of the clinical properties and the results of urinary laboratory tests on urinary lipid levels were investigated with a stepwise multiple regression analysis, using urinary lipid levels as objective variables and clinical information, maximum severity, eGFR, CRP, d-Dimer, urinary chemical markers, urinary sediment findings, SG, pH, and urinary sodium levels as possible explanatory factors. To examine differences between the subjects treated with antiviral reagents and those without, we used the Mann–Whitney U test. The graphs shown in the figures were prepared using Graphpad Prism 9  or MetaboAnalyst. P values of less than 0.05 were deemed as denoting statistical significance in all the analyses.The data were analyzed using SPSS  or MetaboAnalyst  and the total PS levels . Therefore, we think that these correlations were thought to have had a minimal impact on the interpretation of the dynamic modulations of the monitored lipid levels, as described below.The characteristics of all the subjects and the numbers of samples analyzed at each specific time point are described in Additional file The time courses for the urinalysis results and other clinical parameters are shown in Additional file Figure Figure p values at each time period were selected; their log2(FC) and p values are shown in Fig. To investigate time-course-dependent lipid modulations in greater detail, we next created separate volcano plots for each sampling point, as shown in Additional file p values of the lipids and clinical parameters with the 20 lowest p values at any specific time points. Among sphingolipids, SM (except for 36:3 SM) and Sph were positively correlated with maximum severity on day 10–12. On day 19–40, C18:0 Cer, C22:0 Cer, and C24:0 Cer were positively correlated with maximum severity. Regarding LPA, the 18:1 LPA, 20:3 LPA, and 16:1 LPA levels on day 10–12 and day 13–15 were negatively correlated with maximum severity. The 14:0 LPC and 16:1 LPC levels on day 7–9 and the 20:4 LPC levels on day 19–40 were positively correlated with maximum severity. Many PC species had strong positive correlations with maximum severity, while the 32:3 PC, 40:2 PC, 42:0 PC, 44:0 PC, 44:1 PC, and 44:2 PC levels during the middle phase (day 10–18) had rather strong negative correlations with maximum severity. Many PE species were negatively correlated with maximum severity, while 34:4 PE, 34:5 PE, and 38:6 PE at some sampling points were positively correlated with maximum severity. Several PG and PI species, especially on day 10–15, were negatively correlated with maximum severity. Among LPG species, 14:0 LPG was positively correlated with maximum severity. Meanwhile, LPS and PS species were, in general, negatively correlated with maximum severity. Additional file Next, we performed correlation analyses with the maximum severity of COVID-19, using age, sex, the presence of diabetes and hypertension, and current smoking as covariates of interest. Figure In addition to performing simple correlation studies, we investigated which lipids and clinical parameters were strongly correlated with the maximum severity of COVID-19 using machine learning techniques. Figure In the CHAID analyses, the PC-LPA-LPC axis throughout the time course, except day 16–18, had a high importance for determining maximum severity in the constructed models. The PE-LPE axis on day 7–15, PS on day 10–12, C16:0 Cer on day 1–6, SM on day 13–15, and 14:0 LPG on day 7–9 had some importance. In the SVM and neural network analyses, although the importance of each parameter was relatively low, the PC-LPA-LPC axis throughout the time course, sphingolipids during the early phase, and PS and the PE-LPE axis during the middle phase had importance for determining maximum severity in the constructed models.d-Dimer levels. The lipids which had a negative correlation with the urinary SG levels and a positive correlation with sodium levels are deemed to increase in the pathogenesis the renal factors or decrease in the pathogenesis of prerenal kidney injuries. As shown in Fig. Next, we investigated the correlations of the monitored lipids with clinical parameters. Figure The urinary TP levels were positively correlated with ceramides, Sph, LPC, PS, LPS, PE, LPE, and LPG in the early phase (day 1–6 and/or day 7–9) and in the late phase (day 16–18 and/or day 19–40). They consistently had positive correlations with the urinary C16:0 Cer and LPC levels. Regarding the urinary chemical biomarkers, generally, urinary sphingolipids except SM had positive correlations with urinary chemical biomarkers. Especially, the C16:0 and C18:1 ceramides had positive correlations almost throughout the monitored periods. The urinary glycerophospholipids, except PC, LPA, and LPI, generally had positive correlations with the urinary chemical markers Fig. C. IntereRegarding the urinary sediment findings, the urinary SM and dhS1P levels were negatively correlated with RTE, while the urinary ceramides levels were positively correlated with RTE and GraC, except the negative correlations observed in day 16–18. Among glycerophospholipids, the urinary PC, LPA, PG, LPG, PI levels had negative correlations with RTE in day 7–15. The urinary LPC levels had positive correlations with the urinary sediment findings in many time points. The urinary PS, LPS, PE, and LPE also had positive correlations with the urinary sediment findings in the early phase (day 1–9)  in maximum severity group 1 Fig. . ConsideRegarding the PC-LPC-LPA axis, the urinary total PC levels increased from the early phase (day 4–6) only in maximum severity group 3, while the total LPA levels increased during the later phase Fig. . However1, a producing enzyme for LPS, in the serum of COVID-19 patients [Regarding the PS-LPS axis, the urinary PS and LPS levels increased rapidly, especially in patients with severe COVID-19 Fig. . Althougpatients . Since Lpatients , 46, LPSpatients .The urinary levels of PE and LPE increased in COVID-19 beginning at an early phase. The upregulation of the PE-LPE axis might be characteristic of COVID-19, as shown in the volcano plots Fig. . PE is aRegarding the PG-LPG axis and the PI-LPI axis, the roles of PG in AKI are unknown, while PG reportedly suppresses toll-like receptor-mediated inflammation , suggestIntroduction [To the best of our knowledge, the modulations of sphingolipids and glycerophospholipids in the urine of AKI have not been well studied, while urinary levels of ceramides, SM, LPA, LPC, and PC have been demonstrated to be higher in chronic kidney diseases, especially diabetic nephropathy, as described in the oduction , 67, 69.Results, we could not completely match the backgrounds of both the COVID-19 and control groups, since some characteristics such as age differed largely among the maximum severity groups. In addition, since the eGFR levels were not obviously modulated in the present study (Additional file Since this was an observational study, the main limitation is that the possible involvement of these lipid modulations in the pathogenesis of COVID-19-associated kidney injuries remains unknown. However, the accuracy of the models predicting maximum severity that were constructed using machine learning methods was generally high, especially during the early phase of COVID-19 (Fig. In summary, analyses of urine samples collected from COVID-19 subjects showed that decreases in the urinary levels of C18:0, C18:1, C22:0, and C24:0 ceramides, Sph, dhSph, PC, LPC, LPA, and PG and increases in those of PS, LPS, PE, and LPE, especially during the early phase, might be derived from the direct effects of SARS-CoV-2 infection, while increases in the urinary levels of SM, ceramides, Sph, dhSph, dhS1P, PC, LPA, PS, LPS, PE, LPE, PG, LPG, PI, and LPI, especially during the later phase, might result from kidney injuries accompanying severe COVID-19. We believe that these results may prompt researchers to perform further investigations to develop laboratory testing methods based on sphingolipid and glycerophospholipid modulations for predicting the maximum severity of COVID-19 and/or novel reagents to suppress the renal complications of COVID-19.Additional file 1. Supplementary tables and figures.