Among the post-translational modifications of proteins, ADP-ribosylation has been studied for over fifty years, and a large set of functions, including DNA repair, transcription, and cell signaling, have been assigned to this post-translational modification (PTM). This review presents an update on the function of a large set of enzyme writers, the readers that are recruited by the modified targets, and the erasers that reverse the modification to the original amino acid residue, removing the covalent bonds formed. In particular, the review provides details on the involvement of the enzymes performing monoADP-ribosylation/polyADP-ribosylation (MAR/PAR) cycling in cancers. Of note, there is potential for the application of the inhibitors developed for cancer also in the therapy of non-oncological diseases such as the protection against oxidative stress, the suppression of inflammatory responses, and the treatment of neurodegenerative diseases. This field of studies is not concluded, since novel enzymes are being discovered at a rapid pace. Post-translational modifications (PTM) of proteins require the activity of enzyme writers and erasers, and their presence is recognized by readers, protein modules, or structures that bind to specific chemical structures and organize protein–protein interactions. In PTM reactions, the amino acids that are modified may be available or may already be involved in other modifications. Therefore, different types of PTM may interfere with the same amino acids.The purpose of this review is to update the current knowledge on ADP-ribosylation reactions. There are several types of enzymes, broadly defined as polyADP-ribosylating (PARylating) and monoADP-ribosylating (MARylating) enzymes. In the following paragraph we introduce the polymer-forming writers, the poly(ADP-ribose) polymerases. Then, a short presentation introduces the diphtheria-toxin-like ADP-ribose transferases (ARTDs), the cholera-toxin-like ARTCs, and the MARylating sirtuins. Several ADP-ribosylating enzymes are linked to cancer development, and the review discusses the proposed applications of specific ART inhibitors for the containment of cancers and their therapeutic potential.Two main groups of enzyme writers of protein ADP-ribosylation are described. MARylating enzymes attach one unit of ADPR, while PARylating enzymes synthesize and attach long PAR polymers. Scaffolding functions are ascribed to certain mono(ADP-ribose) (MAR) and poly(ADP-ribose) (PAR) structures. Several types of proteins may read these domains and are recruited to MAR and PAR sites. Enzymes that erase the modification in part or completely are described in detail. The ADP-ribosylation of phosphorylated ends of RNA and DNA will not be discussed in the review.+ as a substrate to transfer the ADP-ribose (ADPR) moiety to target amino acids and release nicotinamide (NAM): when one unit of ADPR is attached, they are named mono-ADP-ribosyl-transferases (mARTs) and the reaction is called MARylation [The writers use NADRylation ,2 , leucine (L), threonine (T), valine (V), or tyrosine (Y) instead of glutamate ; all theAmong cholera-toxin-like mono(ADP-ribosyl) transferases (ARTCs), four genes are expressed in humans, but only ARTC1 and ARTC5 are active .+ to remove acyl and acetyl groups from lysine, forming 2′-O-acyl-ADP-ribose [In addition to these PARP-domain-containing enzymes, there are few sirtuins with MARylating activity. SIRT4, SIRT6, and SIRT7 perform a specific MARylation of selected targets and in this way regulate cell metabolism and transcription . SirtuinP-ribose ,16. AmonP-ribose  in mitocIn the pioneering years, PARP1 was principally thought to be involved in modification of acidic amino acids, with glutamate and aspartate as acceptors for auto-modification. Recently, it was shown that serine residues of target proteins such as histones are the most abundant acceptor sites for PARylation, especially after DNA damage. This is made possible by the interaction of PARP1 with histone PARylation factor 1 (HPF-1), that changes the tertiary structure of PARP1 and PARP2, granting them specificity for serines. In the absence of HPF1, PARP1 preferentially auto-modifies itself, becoming unavailable for HPF1 interaction ,19. Durin units [Archeglobus fulgidus, that has been applied to proteomic studies for the enrichment of ADP-ribosylated proteins [+ consumption, and cell death, allowing for an efficient DNA repair after DNA damage [There are several motifs for readers to recognize in MARylation and PARylation. The PAR-binding motif (PBM) is present in proteins involved in the DNA damage response (DDR), chromatin remodeling, or RNA processing. The PAR-binding zinc finger (PBZ) motif is present in proteins involved in DDR, such as the aprataxin PNK-like factor (APLF), and in the regulation of DNA damage-activated checkpoints, i.e., checkpoints with FHA and RING domains (CHFR). The WWE domain is present in two groups of proteins: E3 ubiquitin ligases such as RNF146 and, in a few cases, ARTDs . The RNF146/Iduna ubiquitin ligase associates with tankyrase, involved in PARylation-dependent ubiquitylation (PARdU) reactions: tankyrases PARylate their substrate proteins, and then RNF146 recognizes the PAR portion of PARylated proteins by its WWE domain and is allosterically activated to ubiquitylate them for proteasomal degradation ,28,29,30n units ,34. ADPRproteins ,36,37. AA damage . MARylatA damage . ALC1/CHA damage ,40. SincPAR glycohydrolase (PARG) cleaves ADPR/ADPR bonds. PARG is a macrodomain-containing enzyme that efficiently cleaves PAR chains through exo- and endo-glycosyl hydrolytic activity. However, PARG cannot remove the terminal ADPR linked to protein substrates. PARG has an important role in SSB and DSB DNA repair, which was shown using a cell-permeable PARG inhibitor  and funcO-acetyl ADPR (OAADPr) deacetylase activity. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) transforms the last ADPR group, into phosphoribose bound to the proteins leaving 5′AMP. Similarly, nucleoside diphosphates linked to some moiety X (NUDX/NUDT) hydrolases cleave ADP-ribose to produce 5′ AMP and ribose-5-phosphate (R5P) [+ salvage pathway [TARG1 performs hydrolase activity toward MAR/PAR as well as te (R5P) ,52,53. V pathway ,55. PAR- pathway . When ov pathway ,56. The Concerning PARP1, several post-translational modifications regulate its activity. A SUMOylation on K486 renders this lysine unavailable for acetylation, determining a competition between SUMOylation and acetylation modifications of PARP1 . Sirtuin+-binding pocket within the catalytic domain, increasing PARP1 activity, which leads to auto-PARylation. Furthermore, PARP1 and CDK2/cyclin E are recruited to chromatin, resulting in PARylated proteins and the opening of chromatin through the displacement of histone H1. The receptor tyrosine kinase c-Met associates with and phosphorylates PARP1 at Tyr907 (pTyr907 or pY907) [2O2 treatment. The C-Met-dependent PARP1 phosphorylation at Tyr907 increases PARP1 activity and results in a reduced affinity to PARP inhibitors, thereby rendering cancer cells resistant to PARP inhibitors. On the other hand, cancer cells treated with c-Met inhibitors become more sensitive to PARP inhibitors. EGFR is another kinase that phosphorylates PARP1 [The extracellular signal-regulated kinases 1/2 (ERK1/2) promote PARP1 activation through the direct phosphorylation of PARP1 at serine 372 and threonine 373 . The phor pY907) . The enzes PARP1 , and theVarious cellular functions are disrupted by the altered expression of ADPR writers, readers, and erasers. Several enzymes involved in ADP-ribosylation are linked to some form of cancer. Specific enzymes may cooperate in one or several forms of intrinsic hallmarks of cancer: uncontrolled proliferation, evasion of growth suppressors, cell death resistance, genome instability, reprogrammed energy metabolism, and escape from replicative senescence ,65,66,67BRCA1/2 mutations, in which lower-fidelity non-homologous end-joining (NHEJ) occurs, resulting in chromosomal aberration and in cell death [BRCA1 mutant MDA-MB-436 breast cancer cells [BRCA1, BRCA2, ATM, ATR, FANC, RAD51, BRIP1, or PALB2 mutations. Cancers with defective ATM protein or aberrations of the MRE11A-RAD50-NBN complex are included in BRCAness. ATR, RAD51, RAD54, DSS1, RPA1, NBN, CHK1, CHK2, FANCD2, FANCA, FANCC, FANCM, and BARD1 mutated tumors also respond to PARP inhibitors [Various cancers were shown to respond well to PARP1/2/3 inhibitors (PARPi). Olaparib, rucaparib, niraparib, veliparib (ABT-888), pamiparib, simmiparib, and talazoparib can be applied for the treatment of breast and ovarian cancers showing ll death ,68. Howell death . A noveler cells . PARP1/2er cells . BRCAneshibitors ,73.RNASEH2B deleted cancers, in HER negative cancers, in few cases—as the pretreatment with adjuvant taxanes or anthracycline [PTEN, as prostate cancers, can be sensitive to PARP inhibitors, due to the downregulation of RAD51. Since lysine methylase SMYD3, often overexpressed in tumors, regulates BRCA1/2 and DNA repair, the inhibition of SMYD3 with BCI-121 promotes cancer inhibition by PARP inhibitors such as olaparib [BRCA1 and BRCA2 mutated breast and ovarian cancers, and for therapy in platinum sensitive ovarian cancer patients with defective BRCA1/2 genes [BRCA1/2 and other mechanisms is common; these mechanisms include: PARP1 loss of function and the silencing of DNA damage response (DDR) proteins such as 53BP1 and REV7, that reactivate the HR pathway [PARG [EZH2, MUS81) [RNASEH2B mutant prostate cancers were found to be hypersensitive to PARP inhibitors [RAD51C-silenced high-grade serous ovarian cancers, RAD51 re-expression was associated with an acquired loss of RAD51 promoter methylation during PARP inhibitors therapy [ARH3 was shown as a mechanism to develop PARP1/2 inhibitor resistance [PTEN, overexpressing SAMHD1 with 53BP1 loss, PARP inhibitors were ineffective [BRCA2-deficient cells confers sensitivity to PARP inhibitors, while its depletion counterbalances the chemosensitivity of BRCA2-deficient cells, by promoting both HR and fork stability [PARP inhibitors were also tested in acycline . Cancersolaparib . Olapariolaparib ,72,73. T/2 genes . Olapari pathway ; selectiay [PARG ,76,77; l, MUS81) ; high ex, MUS81) . Ribonuchibitors . In RAD5 therapy ,43,75. T therapy . The lossistance . The appsistance . In cellffective . E2F7 istability . BRCA1-deficient TNBC cells [+-like PARP1 inhibitors, binding to sites different from the NAD+ binding site, have been studied for prostate cancer treatment [BRCA1-, BRCA2-, and PALB2-deficient cells [PARP3 deficiency or inhibition leads to the growth suppression of BC cells ,87. The BC cells . PARP3 sBC cells . Non-NADreatment . Severalreatment , while Areatment . A differeatment . Topoisont cells . Researcnt cells . Furthernt cells . A list High levels of TNKS1 and/or TNKS2 expression have been observed mainly in colon, lung, brain, breast, ovarian, and liver cancers . SeveralTNKS1 and TNKS2 mutations [Wnt-driven cancers such as CRC, HCC, and lung cancers may benefit from Tankyrase inhibitors ,101,102.utations , followeutations ,115,116.utations . K-756 autations ,113. Theutations . Tankyrases recruit specific motifs (RxxPDG “hexapeptides”) in their binding partners via an N-terminal region of ankyrin repeats. These ankyrin repeats form five domains termed ankyrin repeat clusters (ARCs), that bind the substrates . Tankyraγ-STAT1 signaling and of the TNF-α/IL-β proinflammatory pathway [ARTD9 PARP9/BAL1), ARTD7 (PARP15/BAL3), and ARTD8 (PARP14/BAL2) were originally identified as highly expressed in B-aggressive lymphomas (BAL) . The mac, ARTD7  may lead to a loss in ARTD10 activity . ARTD10 However, to complete a MARylation cycle, the writers and the eraser should be expressed at similar levels. MAR hydrolases cleave the modifications promoted by ARTD10. MacroD2, expressed in neuroblastomas, and MacroD1, overexpressed in various cancers, can counteract the tumor suppressive role of ARTD10 . The MARARTD11 MARylates ubiquitin E3 ligase β-TrCP, regulating the IFN-I-dependent antiviral activity . ARTD11 ARTD14/PARP7 is amplified and highly expressed in squamous cell carcinomas, in ovarian and lung tumors, and associated with poor survival ,139,140.ARTD15/PARP16 associates with the endoplasmic reticulum (ER) ,151,152.ARTD17/PARP6 is an enzyme involved in some cancers. It has been identified as a negative regulator of cell-cycle progression in HeLa cells: ARTD17 overexpression was reported to arrest cells in the S-phase; this activity is dependent on a functional catalytic domain. In colorectal cancer, ARTD17 promotes cancer growth . In addiConcerning cholera-toxin-like ARTs, ARTC1 MARylates the Grp78/BiP chaperone, required for protein quality control in endoplasmic reticulum ; this moA list of MARylating enzymes and their potential inhibitors applied to cancer treatment is presented in + binding pocket; the sirtuins are not inhibited by PARP inhibitors such as olaparib and similar compounds, PJ34, and XAV939, due to differences in their NAD+ binding site [SIRT1, SIRT2, SIRT3, and SIRT6 are blocked by a sirtuin inhibitor selisistat (Ex527) targeting the NADing site ,167.SIRT4 is localized in the outer mitochondrial membrane. SIRT4 MARylates GDH  and deac+-binding inhibitors block both deacetylation and MARylation reactions, it is not possible to discriminate between the two activities in SIRT6 studies [SIRT6 was found to be overexpressed in skin cancer and non-small cell lung cancer (NSCLC) ,169, ost studies . SIRT6-s studies . SIRT6 h studies .SIRT6 MARylates itself, as well as PARP1 ,185,186,SIRT6 functions upon DNA damage, and activates two DNA repair pathways, the nonhomologous end joining (NHEJ) and the base excision repair (BER). SIRT6 induces, by a protein–protein interaction, the formation of a multiprotein complex  ,189,190:+-binding site [A study on the structure of bacterial SIRT bound to the acetylated +2 arginine peptide showed that the arginine entered in the active site and reacted through a deacetylation reaction intermediate, yielding an ADP-ribosylated peptide . New coming site , effectiing site ,192.+-binding pocket and the catalytic site, together with the flanking residues E185 and N189. In SIRT6 and SIRT7, E185 and N189 are in the opposite direction, facing the surface of the cavity, and the two residues interact forming a loop. These residues are important for their role in the ADP-ribosylation reaction: E185 is the catalytic residue that starts the reaction, while N189 is the first acceptor of the ADP-ribosyl moiety [SIRT7 has been involved in genome integrity and non-homologous end-joining (NHEJ) DNA repair . SIRT7 hl moiety . In TablIn this review the most studied players of MARylation and PARylation have been reviewed. The main objective was to relate the proteins targeted by these post-translational modifications and their interactions with specific proteins toward a disruption of their roles in the maintenance of cell functions through MARylation/PARylation inhibitors. These disfunctions lead to cell proliferation, anomalies in growth control, loss of cell-to-cell contacts, and tumor development. Furthermore, worthy data have been produced through clinical trials on several types of PARP1/2/3 inhibitors, on tankyrase/PARP5a/b inhibitors, and on MARylation inhibitors targeting ARTD14/PARP7. The deregulated cell functions may derive from the MAR/PAR cycling control, stemming from the overexpression of eraser enzymes that have been associated with various cancers. Approaches using PARP1/2 inhibitors or broad PARP inhibitors have been proposed and tested not only in cancer treatment ,99,100, Similarly to MAR erasers, several RNA viruses express nsp3, a macrodomain protein with eraser activity that subverts the stress granule structure and re-establishes the translation and function of stalled mRNAs .Recently, new data have been produced on MacroD1, MacroD2, and TARG, uncovering their interactomes and their cell specificity ,199. AmoFurthermore, compounds able to inhibit and discriminate between ARH3 and ARH1 have been developed and could be tested to protect cells from the deregulated MAR/PAR hydrolytic activity or the inhibition of the nsp3 macrodomain of SARS-CoV2 ,200.The perspectives for ADP-ribosylation’s future studies lead in the direction of antiviral therapies and the development of more specific inhibitors of the MAR/PAR modifications and their control, for the amelioration of oncological and non-oncological diseases.Finally, a new enzyme with supposed PARylating activity has been associated with mitochondria: Neuralized-like protein 4 (NEURL4) possesses an H-Y-E-like ART domain in the C-terminal domain, in addition to six Neuralized Homology Repeat (NHR) domains, functioning as protein–protein interaction scaffolds ,202,203.Further studies analyzing 3D structures of MARylation/PARylation enzymes should help develop new inhibitors. It is envisaged that in the next year new therapies will be tested via the exploitation of PARylation and MARylation inhibition, the safety of the administration of new drugs, and the recovery to a healthy state.


Autism Spectrum Disorder (ASD) comprises a heterogeneous group of neurodevelopmental disorders with a strong heritable genetic component. At present, ASD is diagnosed solely by behavioral criteria. Advances in genomic analysis have contributed to numerous candidate genes for the risk of ASD, where rare mutations and s common variants contribute to its susceptibility. Moreover, studies show rare de novo variants, copy number variation and single nucleotide polymorphisms (SNPs) also impact neurodevelopment signaling. Exploration of rare and common variants involved in common dysregulated pathways can provide new diagnostic and therapeutic strategies for ASD. Contributions of current innovative molecular strategies to understand etiology of ASD will be explored which are focused on whole exome sequencing (WES), whole genome sequencing (WGS), microRNA, long non-coding RNAs and CRISPR/Cas9 models. Some promising areas of pharmacogenomic and endophenotype directed therapies as novel personalized treatment and prevention will be discussed. Autism is a heterogeneous disorder with a high heritability    and acco(MECP2)] . Mendeli(MECP2)]  through Twin and family studies point to genetic basis of ASD susceptibility and is a highly heterogeneous genetic disorder. There are over 100 ASD candidate genes, and CACNAIC, GABAA receptor subunit, FOXP2, HOXA1, HOXB1, HTR2A, MTHFR, RELN, RAY1/ST7, IMMP2L, SLC6A4, OXTR, UBE3A and WNT-2 ] ] . RecentlSHANK3]] . Rare CNSHANK3]] . A similSHANK3]] . This stR, SHANK  and foun for ASD .In conclusion, CNVs represents alterations of the normal number of gene copies and include deletions, insertions, duplications and complex multi-site variants. The above reviewed studies ,46,47,48CDH10) and cadherin 9 (CDH9), and with the most significant SNP being rs4307059 [SEMA5A (rs10513025) [MACROD2 (rs4141463) [MSNP1 (rs4307059) [Genome-wide association studies (GWAS) have identified potential contributions of common variants of small effect to pathogenesis of ASD ,49. GWASs4307059 . A larges4307059  looked a0513025) , MACROD24141463) ,52 and M4307059) ,53 and wPITX3 which encodes a transcription factor for neuronal differentiation and CUEDC2 involved in the ubiquitination-proteasomal degradation pathway [EXT1, ASTN2, ANO4, MACROD2, and HDAC4. More recently, a unique Danish population resource under iPSYCH project [PTBP2, CADPS, and KMT2E. Moreover, the identified ASD-candidates in this study showed the highest expression during fetal corticogenesis [KIZ and XRN2 (Chr 20), MFHAS1, XKR6, MSRA, and SOX7 (Chr 8). The other associated genes were KCNN2, KANSL1, MACROD2, WNT3, MAPT, CRHR1, NTM, MMP12, and BLK [XRN2, NKX2-4, PLK1S1, KCNN2, NKX2-2, CRHR1-IT1, C8orf74 and LOC644172. Both the analysis by MAGMA and PASCAL showed common loci to be XRN2, KIZ, and KCNN2 [A genome-wide significant (GWS) risk loci was detected at 10q24.32 and was associated with social skills  and this pathway . This st project , detecteogenesis . Gene-ba and BLK . Summary and BLK  employed and BLK , identifnd KCNN2 ,56. PASCGWAS approaches ,55,56,57Recent advances in the development of next-generation sequencing (NGS) technologies provide researchers with unprecedented possibilities for genetic analyses to unravel genetic causes of autism. Pathways of transcriptional regulation and chromatin remodeling are affected by causative mutations in neurodevelopmental disorders  , and theAMT, PEX7, SYNE1, VPS13B, PAH, POMGNT1). Genes implicated here also include ones known to regulate or be regulated by synaptic activity . A population-based approach on 933 ASD cases and 869 controls [Whole Exome Sequencing (WES) is an approach to selectively sequence the coding regions (exons) of a genome to uncover rare or common variants associated with a disorder or phenotype , as morecontrols , showed controls .WES analysis of the Simons Simplex Collection  identified new de novo likely gene-disrupting (LGD) mutations involved in this heterogeneous disorder , and moshttps://www.nimhgenetics.org/, accessed on 1 August 2021). The genes carrying VOIs were enriched for biological processes related to cell projection organization and neuron development. Missense variants in one gene, CEP41, associated significantly with ASD [CEP41 pathogenic alleles in zebrafish model, showed that variants in embryos induces axonal defects and also affects cranial neural crest (CNC) cell migration and exhibited deficits in social behavior. More recently, Kim and co-workers [FMR1 testing, chromosomal microarray (CMA) and/or WES, recommend WES as the first-tier approach in the diagnosis of ASD patients [To identify genes with private gene disrupting and missense variants of interest (VOI), Patowary and colleagues  looked awith ASD  and over-workers  performepatients .WES studies have extended our in-depth knowledge on the rare de novo SNVs and CNVs impacting protein coding genes leading to dysregulation of signaling pathways controlling nervous system development, neuronal activity, synaptic homeostasis, immune response, chromatin modification, transcription and translation ,10,11,12whole genome for SNVs, indels, SV and CNVs in coding and non-coding regions. Non-coding regions cover almost 98% of the human genome [Whole Genome Sequencing (WGS), on the other hand, examines the n genome  and thisPTPN11, CACNA1G, TRIP12, PTK7, SUPT16H and SCN3A indicating the importance of particularly severe de novo missense mutations. Moreover, the well-established increase in de novo substitutions with paternal age was observed with strong correlation between the number of de novo SNVs and indels and increase in paternal age; and also for noncoding de novo SNVs and indels [To understand the genetic etiology of ASD, Turner and colleagues , analyzed indels .SCN2A.An analysis of whole-genome sequences on 5205 individuals, identified 18 new candidate genes for autism , includihttps://david.ncifcrf.gov/, accessed on 2 August 2021) [KMT2E, PHF3, KDM5B, KMT2C, CHD8, ILF2, FOXP2, FOXP1, MED13L, MECP2, HOXA1, ADNP, HOXB1, ARID2) were annotated as transcription regulators under biological processes (BP) category with 2.4-fold more than expected with a p-value = 0.004. Similarly, DNA binding, a molecular functions (MF) category, was 2.2-fold more than expected with 11 genes  and a p-value = 0.02. Also, a total of 21 genes  were found to be associated with nucleus cellular component (CC) with 1.9-fold (p = 0.002)   was used= 0.002) .Non-coding RNAs (ncRNAs) regulate gene expression at the transcriptional and post-transcriptional level and are important to control epigenetic pathways essential for targeting of histone modifying complexes, chromatin remodeling and DNA methylation. This section will focus more on information on ncRNA especially microRNAs (miRs or miRNAs) and long noncoding RNA (LncRNA), These non-coding RNAs exhibit tissue-specific, cell-specific expression and are important in the development and functioning of the brain.http://www.mirbase.org/, accessed on 17 July 2021 [miRNAs or miRs are approximately 18–25 nucleotides, noncoding transcripts that control messenger RNA (mRNA) and protein levels by interacting with the 3′ untranslated region (UTR) of specific mRNAs . miRs cauly 2021 . In addiuly 2021 . These mOver thirty publications were reviewed on miRs and ASD that were published in the past 10–15 years; predominately in the previous 5 years. The focus was on human, not animal or computational, miR manuscripts. Based on the author’s choice, only 15 papers  78,79,8,90,91,92This collection of papers examined miRs differentially expressed in post-mortem brain tissue, lymphocyte and lymphoblastoid cells lines, saliva, serum, and whole blood. Of these 15 manuscripts, specimens were derived from both children and adults.As for miR technologies used in these miR biomarker discovery papers, whole genome sequencing (WGS), miR arrays, and quantitative RT-PCR were used, and most were validated by a secondary means to suggest a final, smaller number of differentiated miRs expression profiles. In some cases, the same miRs were found to be expressed in different tissue sources, as well as similar expression patterns were observed (up or down regulation). A few examples of this are the miR-146a, miR-664-3p, miR-151a-3p, and miR-27a-3p . In two As a single miRNA can target approximately hundreds to thousands of mRNA transcripts and a single mRNA transcript could be targeted by multiple miRNAs , so studhttp://www.noncode.org/, accessed on 2 August 2021) [Long noncoding RNAs (lncRNAs) are the type of non-protein coding transcripts, which are more than 200 nucleotides in length ,96. LncRst 2021) . LncRNAsst 2021)  and can Human LncRNA microarray analysis on postmortem brain samples displayed over 200 differentially expressed LncRNAs in ASD . The salMSNP1AS is antisense to and can bind moesin (MSN) transcript, and overexpression of MSNP1AS causes a decrease in moesin protein that regulates neuronal architecture [LOC389023, positioned in chromosome 2q14.1, within the DPP10 gene [NEAT1, PANDA, and TUG1), only NEAT1 and TUG1 showed significant upregulation in ASD cases [Another LncRNA MSNP1AS was highly overexpressed in the postmortem cerebral cortex of individuals with ASD . This stitecture . DeWitt itecture  showed MP10 gene , can regP10 gene ,107. PerP10 gene , which aP10 gene . In anotEpigenetic mechanisms can regulate cellular processes including differentiation, apoptosis, and metabolism; and clinical implications of dysregulated ncRNAs (miRNA and LncRNA) in ASD could have potential for personalized therapies as diagnostic biomarkers or indicators of prognosis.CADPS2 has an important role in BDNF secretion, as it regulates the exocytosis of synaptic and dense-core vesicles in neurons. [TAOK2 gene has been reported by WGS and WES approach, and functional analysis show these mutations differentially impact kinase activity, dendrite growth, and spine/synapse development [The generation of a relevant disease specific knockout model for autistic traits/behaviors has been difficult because of the genetically heterogeneous nature of ASD. A number of rodent models  for rare variations (de novo and CNV) detected in ASD have been used to understand the etiology of ASD ,110,111.neurons. . A condineurons. , showed elopment .Human in-vitro models using induced pluripotent stem cell (iPSC)-derived neurons and astrocytes are particularly valuable for ASD studies and there are a number of protocols in use to differentiate iPSC into neurons ,114. JinCHD8 gene are associated with ASD [CHD8 in a control iPSC line [CHD8+/− iPSC model showed that CHD8 regulates multiple genes implicated in ASD pathogenesis. Identified genes influence brain volume and are involved in cell communication, extracellular matrix and neurogenesis that are critical for brain development [CHD8+/− and isogenic control (CHD8+/+) cerebral organoids [DLX gene family, which encodes a transcription factor involved in GABAergic interneuron differentiation. Additionally, genes expressed in CHD8 mutant and wild-type organoids were enriched for pathways involved in neurogenesis, neuronal differentiation, forebrain development, Wnt/β-catenin signaling and axon guidance [AFF2/FMR2, ANOS1, ASTN2, ATRX, CACNA1C, CHD8, DLGAP2, KCNQ2, SCN2A and TENM1) on neuronal function [AFF2/FMR2, ASTN2, ATRX, KCNQ2 and SCN2A with significantly reduced spontaneous excitatory postsynaptic current frequencies in iPSC-derived excitatory neurons. The results from KO studies show that ten ASD-risk genes of varying function have similar transcriptional rewiring and electrophysiological phenotypes in human iPSC-derived glutamatergic neurons [Mutations in the with ASD , and WESwith ASD . CRISPR/PSC line  and CHD8elopment . Furtherrganoids  and showguidance . The devguidance . Notch sguidance . NOTCH2Nguidance . CRISPR/function . Associa neurons .The above outlined studies with CRISPR/Cas9 system, show this RNA-based genome-editing tool has the potential to engineer pre-clinical models to understand the molecular and cellular pathways in ASD, as it allows site-specific genome editing to generate isogenic cell lines from iPSCs. Future, gene editing strategies will help us understand better, the events at the molecular level of synaptic dysfunction, calcium signaling, chromatin remodeling and transcriptional regulation.Gottesman and Gould  define eThe most reliable endophenotypes in ASD fall in following clinical subgroups: hormonal, biochemical, immunological, morphological, neurophysiological/neuroanatomical, neuropsychological, and behavioral . StudiesCYP2D6 and CYP3A4. SNPs in CYP2D6, and CYP3A4 genes that encode enzymes responsible for risperidone and aripiprazole metabolism contribute substantially to interindividual variability in dose requirement. The genetic polymorphisms of CYP2D6 are contributing to predicting dosing for risperidone and aripiprazole [CYP2D6 and CYP3A4 genotypes in ASD vulnerable populations can inform clinicians to dosing and treatment options for risperidone and aripiprazole. The other agents commonly used include selective serotonin inhibitors, mood stabilizing antiepileptic agents such as lamotrigine, stimulants as well as alpha-receptor antagonist, and anxiolytic agents.Advances in the field of pharmacogenomics (PGx) will help realize the objective of personalized medicine in ASD. The majority of PGx studies to date have focused on commonly utilized medication classes for ASD such as antipsychotics, antidepressants and stimulants ,133. ASDiprazole ,135 and iprazole . Pre-empNon-core symptoms found in patients include irritability, aggressive behaviors, repetitive behaviors and mood related conditions. The complexity in identifying comorbid psychiatric disorders is often limited by the limitations in communications. This conditions like most of the rest of psychiatry are merely behavioral diagnoses and do not predict a molecular cause. The use of pharmacogenomics testing in the general population is established. While it also does not suggest a molecular cause PGx testing provides supportive data to assist with treatment decisions. While there are a few PGx studies with ASD patients in general they follow the more established findings seen in neurotypical psychiatric patients. Additionally, most of the studies that have been done suffer from low numbers of subjects and limited genetic data. Some focus primarily on pharmacokinetic data while others have limited pharmacodynamics data relating to receptors and transporters. Our adult psychiatry clinic (UAMS) uses commercial laboratories with limited data to perform PGx assays. It results in helping identify treatment refractory cases as well as in patients with medication intolerability.ASD is highly heritable and heterogenous group of neurodevelopmental disorder characterized by alterations in social interaction, communication, and repetitive behaviors. The above listed studies show genetic heterogeneity in ASD. Large cohort studies using WGS, miRNA, LncRNA has the potential for biomarker development. Carefully designed studies incorporating PGx and endophenotype stratified patient population will help further define the unique genetic underpinnings involved in cellular and molecular pathways of ASD and help in personalized therapies. Finally, the introduction of genome editing technology, CRISPR/Cas9 will help us better understand the impact of the mutations in the neural circuitry which is important in molecular dysregulation of ASD.


Coronavirus (COVID-19) has severely impacted lifestyles worldwide. Responses to COVID-19 have intentionally restricted the factors that encourage regular and frequent PA; opportunity, capability and motivation. However, the effects of these restrictions are likely to have differed by gender and age and different intensities of PA. This rapid review builds on previous evidence by synthesising the global impact of COVID-19 on adult PA through specific intensities and types of PA and evaluating this by gender and age.A rapid systematic search of seven electronic databases  was performed from December 2019 to January 2021. Studies investigating adult change in PA, exercise or sedentary behaviour due to COVID-19 were included.From an initial database search identifying 3,863 articles, 66 remained for synthesis after applying eligibility criteria. Results demonstrate decreases among all intensities and types of PA—walking (6 out of 7 papers), moderate-only (5 out of 6 papers), vigorous-only (5 out of 6 papers) and MVPA (4 out of 5 papers); as well as overall PA (14–72% participants reported a decrease). Reflecting that COVID-19 responses were designed to have universal effects, they also achieved whole-society decreases in PA behaviour, accented in older age groups.There is a universal need to address the low levels of PA post-COVID-19. The consequences of decreased PA across all intensities has powerful, potentially recoverable, impacts. Universal declines have implications for public health officials and PA advocates for post-COVID-19 initiatives to promote PA. As a highly transmissible disease, coronavirus COVID-19) required physical distancing protocols and/or self-isolation 9 require. In respCapability, reflecting having the knowledge and ability to be physically active, declines as engagement reduces. COVID-19 restrictions undermined the usual PA prompts in daily life, such as remembering to be physically active when routines have changed ,11, therOpportunities for PA also changed with quarantines and lockdowns. Closure of facilities left fewer places to be active, reducing opportunities to be active and to socially interact with others. Women are typically more reliant on social support to engage in PA , leavingMotivation for being physically active involves both reflective  and automatic (e.g. emotion and habit) processes . RestricA recent systematic review identified, as a result of COVID-19, the majority of studies report a decrease in PA and an increase in sedentary behaviour across several populations . AlthougA rapid review was performed following the Cochrane rapid review methods recommendations . This reThe study selection process was performed in-line with rapid review guidelines . StudiesData relating to participant characteristics , data collection method (e.g. questionnaire), data collection period (i.e. when questionnaire was launched and length of data collection) and PA, exercise or sedentary behaviour outcomes were extracted. One researcher (AC) extracted all data; another researcher (SB) verified the extracted data and amended as necessary. Amendments were shared with the first researcher and any disagreements were resolved through discussion, until agreement was reached. Where data were only presented in chart or graph format, WebPlotDigitizer v4.2, an online data extraction tool, was used to ascertain numerical values. WebPlotDigitizer report high validity and reliability and has been suggested for use in systematic reviews when numerical values aren’t provided within a manuscript ,23. For Within PA, there are several key concepts used; for clarity, definitions have been provided. PA is defined as any bodily movement produced by skeletal muscles that results in energy expenditure . This inThe methodological quality of the included studies was assessed using the modified assessment scale –28 of DoThrough the original database search, 3,863 articles were identified. After applying eligibility criteria, 66 articles remained for synthesis . A flowcA total of 44 studies reported changes in PA as a result of the COVID-19 pandemic .The majority of studies (n = 25) ,34,38–56Across all 25 studies, the percentage of participants who reported a decrease in PA ranged from 14–72%, whereas no change in PA ranged from 11–77%, and those reporting an increase ranged from 3–46%.Ten studies –68 reporTen studies ,66,69–73Four studies reported PA by age ,61,70,73Seventeen studies ,68,74–82Six studies ,66,68,74Five studies reported results by moderate-to-vigorous PA (MVPA) –79,81,82Seven studies investigated change in time spent in walking behaviour ,74,80,82Three studies reported change in step counts ,37,77. AThree studies reported changes by gender ,74,79. CTwo studies reported changes by age ,79. CastTwelve studies ,76,83–89Seven studies ,84,87,88Three studies reported the amount of training pre and during the COVID-19 pandemic. Two studies ,83 reporOne study  reportedTwo studies report exercise behaviour by gender ,88. Hu eNo studies investigated differences across age groups.Eighteen studies ,84,90,91Eight studies ,74,78,91Seven studies reported categories of change  in sedentary behaviour ,72,84,90Three studies ,62,82 reOnly two studies reported change in sedentary behaviour by gender ,74. YamaOnly one study , reporteThis rapid review summarises the impact of COVID-19 by intensity-specific PA and sedentary behaviour in healthy adults, differentiated by age and gender. Based on 66 studies, this review identified similar decreases across all intensities of activity. Additionally, while most evidence suggests a similar generalised decrease in PA by gender through COVID-19, studies investigating change in PA by age were inconclusive.Seen through a COM-B lens , this reResults discovered a general decrease in PA, seen across all intensities of activity: walking (6 out of 7 papers), moderate-only (5 out of 6 papers), vigorous-only (5 out of 6 papers) and MVPA (4 out of 5 papers)—in conjunction with increased time spent sedentary (7 out of 8 papers). COVID-19 restrictions de-activated elements of COM-B, while also imposing powerful social and environmental barriers that reduced PA. Although the impacts were predictable, an avoidable, downward spiral ensued. Lost opportunity and capability resulted from the closure of gyms and leisure centres  and disrSurprisingly, this review suggests a similar decrease in PA behaviour (7 out of 10 papers) for both genders. Restrictions were expected to impact women more than men  due to wOverall, when amalgamating the results by all types and intensities of activity the results for the impact of COVID-19 on activity by age are inconclusive, likely due to the lack of homogeneity amongst the studies. However, although different age ranges were applied, when solely assessing PA, evidence suggests older age groups reduced their PA more than younger age groups ,70,73. OThe limitations of a rapid review must be acknowledged. First, there was no search of grey literature or scan of references lists of included studies, and only papers published in English were included. Therefore, it could have resulted in missed articles that fit inclusion criteria. Secondly, as per rapid review guidelines , only onThis review presents a synthesis of the literature assessing the change in PA and sedentary behaviour among healthy adults from pre to during COVID-19 by gender and age. Overall, there were similar decreases in all intensities and types of activity by gender. Results remain inconclusive on the impact by age when amalgamating all types and intensities of activity, however, evidence could suggest older age groups reduced their PA more than younger age groups. These results have implications for public health officials and PA advocates for post-COVID-19 initiatives to promote PA.S1 PRISMA checklist(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Camellia sinensis produces an abundance of specialized metabolites, which makes it an excellent model for digging into the genetic regulation of plant-specific metabolite biosynthesis. The most abundant health-promoting metabolites in tea are galloylated catechins, and the most bioactive of the galloylated catechins, epigallocatechin gallate (EGCG), is specifically relative abundant in C. sinensis. However, the transcriptional regulation of galloylated catechin biosynthesis remains elusive. This study mined the R2R3-MYB TFs associated with galloylated catechin biosynthesis in C. sinensis. A total of 118 R2R3-MYB proteins, classified into 38 subgroups, were identified. R2R3-MYB subgroups specific to or expanded in C. sinensis were hypothesized to be essential to evolutionary diversification of tea-specialized metabolites. Notably, nine of these R2R3-MYB genes were expressed preferentially in apical buds (ABs) and young leaves, exactly where galloylated catechins accumulate. Three putative R2R3-MYB genes displayed strong correlation with key galloylated catechin biosynthesis genes, suggesting a role in regulating biosynthesis of epicatechin gallate (ECG) and EGCG. Overall, this study paves the way to reveal the transcriptional regulation of galloylated catechins in C. sinensis.The R2R3-MYB transcription factor (TF) family regulates metabolism of phenylpropanoids in various plant lineages. Species-expanded or specific MYB TFs may regulate species-specific metabolite biosynthesis including phenylpropanoid-derived bioactive products.  Camellia sinensis, along with coffee and cocoa, is one of the world’s three major non-alcoholic beverages. Worldwide, approximately two billion cups of tea are consumed daily (1). Tea was used first as a food in ancient China, then it served as a medicine to prevent and cure common diseases before developing into a popular beverage (Tea from ed daily . Tea probeverage . Nowadaybeverage .C. sinensis) is rich in characteristic metabolites, such as polyphenols, amino acids, caffeine, and terpenes, that significantly contribute to its pleasant flavors and industrial and medical value , anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR), and serine carboxypeptidase-like acyltransferases (SCPLs)  . HoweverVitis spp.) and maize (Zea mays) genomes, and differential expression of duplicated genes resulted in control of anthocyanin biosynthesis in different tissues  comprise the largest family of TFs in advanced plants . In addi tissues . Some splineages . In CapsC. sinensis, some R2R3-MYB TFs have a demonstrated role in regulation of phenylpropanoid biosynthesis in C. sinensis, such as CsAN1, CsMYB5a, CsMYB6A, and CsMYB75 that regulate anthocyanin pigments and CsMYB5e and CsMYB5b that involve in proanthocyanidin biosynthesis in C. sinensis tissues , and abscisic acid (ABA) treatments and a key CsR2R3-MYB gene regulated some main anthocyanin components. However, the C. sinensis specific and expanded R2R3-MYB TFs that are potential candidate regulators of galloylated catechins biosynthesis have still not been identified.In  tissues , 2020. RC. sinensis, this study focused on the specifically expanded CsR2R3-MYB genes and the high content of EGCG in C. sinensis. Differentiating from previous works, we adapted a more general genome-wide analysis method of the R2R3-MYB superfamily according to Arabidopsis thaliana, which greatly help to find out the specifically expanded CsR2R3-MYB genes and the high content of EGCG in C. sinensis , first leaves, second leaves, mature leaves, old leaves, stems, and roots of three plants were sampled as one biological replicates in spring of 2021. Three biological replicates of different tissues were immediately frozen in liquid nitrogen and stored at −80°C.The “Lingtoudancong” variety of Camellia sinensis MYB protein sequences were retrieved from the Tea Plant Information Archive database.2 In total, 222 MYBs and MYB-related genes were predicted in the ‘‘Shuchazao’’ genome, and they were all submitted to Pfam website3 to search for the two consecutive and conserved repeats of the MYB domain. However, only 118 of the MYBs TFs fit this characteristic, which were different from previous studies  was the same as described in the literature  model; maximum number of motifs: 20; minimum motif width: 6; maximum motif width: 50; minimum number of sites per motif: 2; and maximum number of sites per motif: 118  of the TBtools software. The Myb-binding sites were predicted in the 2000-bp upstream regions of the catechin biosynthesis downstream genes with PlantCARE online website8.Functional motifs and conserved domains were identified with The MEME Suite tooltif: 118 . The seqR2R3-MYB TFs and catechin biosynthesis genes from different tissues of C. sinensis were used to generate a heatmap with TBtools software using the normalized method.The RNA-seq data were downloaded from TPIA for transcript abundance analyses. The expression levels of the candidate Actin (TEA019484.1) was used as the housekeeping gene  according the manufacturer’s instructions. First-strand cDNA was synthesized using a HiScript III RT 1st Strand cDNA Synthesis kit  in a reaction volume of 20 μL. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in a Bio-Rad CFX384 Touch™ system. Each 10 μL reaction mixture was comprised of 4.4 μL qPCR SYBR Green Master Mix , 4.4 μL double distilled water, 0.2 μL of each primer (10 μmol/μL) and 1 μL of cDNA template. The reaction program was as follows: 95°C for 5 min; then 39 cycles at 95°C for 5 s and 60°C for 30 s. A melting-curve analysis was carried out at 95°C for 5 s, which was followed by a temperature increase from 60 to 95°C. All qRT-PCR reactions were carried out in independent triplicate and ing gene . The relns ± SDs . The qRTn = 3).Reference standards of C, EC, EGC, ECG, and EGCG were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. . ABs, first leaves, second leaves, mature leaves, old leaves, stems and roots of “Lingtoudancong” were ground into fine powders and freeze-dried. Approximately 0.2 g of each sample powder was extracted with 8 mL of 70% methyl alcohol (diluted with ultrapure water). After ultrasonic extraction for 30 min, the supernatant was collected by centrifugation. One milliliter of the liquid supernatant was filtered through a 0.22 μm Millipore membrane. The extracts of each sample were independently injected into three XSelect HSS C18 SB columns  for three independent replicates. The catechin monomers were separated using 0.1% aqueous formic acid (A) and 100% acetonitrile (B) as mobile phases on a Waters Alliance Series HPLC system . Detection was performed at 280 nm. The data were presented as the mean ± SD  were aligned with those of A. thaliana (126), and their evolutionary history was inferred by constructing a neighbor-joining phylogenetic tree , Tea Expanded Subgroup B (TESB), and Tea Expanded Subgroup C (TESC). Remarkably, SMYB5 and S5 subgroups were comprised of more tea plant R2R3-MYB members than A. thaliana members. For example, subgroup S5 had three C. sinensis members , while A. thaliana contributed only one member, AtMYB123/TT2. Similarly, the SMYB5 subgroup had four members from C. sinensis , but just one member (AtMYB5) from A. thaliana. In Arabidopsis, the members of SMYB5 and S5 subgroups are involved in the phenylpropanoid pathway. AtMYB123 controls the biosynthesis of proanthocyanidins (PAs) in the seed coat and AtMYB5 was partially redundant with AtMYB123 , TESB (4), TESC (4), S5 (3) and SMYB5 (4)] attracted our attention and were selected for further analysis as potential candidates involved metabolic processes specific to C. sinensis.Overall, the phylogenetic analysis results highlighted five special subgroups TESA, TESB, TESC, S5, and SMYB5, which expanded in or were exclusively present in R2R3-MYB genes of the five tea specifically expanded subgroups TESA, TESB, TESC, S5, and SMYB5, the homologous R2R3-MYB genes from kiwifruit, coffee, cacao, and grape were used to construct phylogenetic tree (R2R3-MYB TFs presented a trend of expansion in C. sinensis (21) similar to coffee (20), but greater than cocoa (11), kiwifruit (11), and grape (18). We surmised that the C. sinensis expanded R2R3-MYB function may have occurred through divergent evolution during speciation. For example, subgroup TESA was expanded in tea (6) relative to coffee (3), cocoa (5), kiwifruit (5), and grape (1). In the TESB subgroup, coffee (4), grape (4), and kiwifruit (3) had comparable numbers of members with C. sinensis (4), but only two homologous genes were found in cocoa. Subgroup TESC contained homologous genes in all species except for kiwifruit. Remarkably, no isogenous genes of TESA, TESB, or TESC subgroups were present in A. thaliana, indicating that these R2R3-MYBs evolved only in tea plant and probably have novel functions.To evaluate the 21 tic tree . The numR2R3-MYB genes serve, we searched for the functional characteristics of the selected R2R3-MYB genes from four close relative species. Only a few homologous genes  have been experimentally verified. In the TESA subgroup, GSVIVT01026868001 played an inhibitory role in flower development  mainly regulated plant epidermal cell fate  analyses were performed among the whole CsR2R3-MYB genes  and CsMYB117b (TEA002233.1), suggesting they are pseudogenes in C. sinensis. The genes were classified into seven expression clusters, based on their distinct transcript patterns in various tissues and organs  because they were preferentially expressed in ABs and young leaves.Catechins are the major type of polyphenols in the tea plant, comprising up to 70% of the polyphenols deriving from the phenylpropanoid pathway . Figure R2R3-MYB TFs and the catechin biosynthesis genes were investigated in different tissues. The results clearly showed that the genes in the catechin biosynthesis pathway display tissue-specific expression patterns. The genes downstream of catechin biosynthesis  were highly expressed in ABs and young leaves, whereas the upstream genes  were highly expressed in root and flower  have several Myb-binding domains  that were not tender parts-specific indeed showed a low correlation or negatively correlated to catechin content. Comparatively, the expression level of the nine candidate R2R3-MYB TFs was positively correlated to the transcript abundance patterns of the catechin biosynthesis pathway downstream genes  and correlated with the contents of EGCG and ECG, with inter-gene-to-metabolite Pearson’s correlation coefficients over 0.55. CsMYB30, CsMYB34, CsMYB37, and CsMYB42 exhibited good performance compared with the others, each having a correlation coefficient exceeding 0.75, indicating an extremely strong correlation. Notably, CsMYB34 had the strongest correlation with the catechin biosynthesis pathway downstream genes (>0.9), especially with CsSCPL1A7 (0.99). Besides that, the coefficients between CsMYB34 and EGCG and ECG contents were 0.89 and 0.86, respectively.To identify the relationship between transcript abundance and catechin contents, a comprehensive gene-to-metabolite correlation analysis was conducted. As shown in R2R3-MYB TFs and catechins, HPLC and qRT-PCR assays were carried out. Different tissues of the tea plant were tested for the contents of different catechin monomers via HPLC   grouped in cluster A and were highly expressed in roots, consistent with the absence of catechins in roots  had distinctly high expression in old leaves, and were not AB- or young leaf-specific. Remarkably, CsMYB30, CsMYB32, CsMYB34, and CsMYB37, which clustered with critical genes downstream of catechins biosynthesis (cluster D) were highly expressed in ABs and first leaves, where EGCG and ECG accumulated  . The genin roots . CsMYB22umulated , prelimiCsMYB30, CsMYB32, CsMYB34, CsMYB37, key genes of the catechin biosynthesis pathway, and the accumulation of catechins was conducted  as well as ECG and EGCG contents (>0.7). Particularly, CsMYB37 showed the highest correlation level with the highest inter-correlation coefficients within the four major functional genes CsSCPL1A7, CsANR, CsLAR, CsDFR and the contents of EGCG and ECG reaching to 0.79, 0.78, 0.88, 0.85, 0.87 and 0.8, respectively. However, CsMYB30, CsMYB34, and CsMYB37 were less related to the other downstream catechin biosynthesis genes  according to their lower Pearson’s correlation coefficients. Consistent with the in silico results in CsMYB32, the galloylated catechin contents and the biosynthesis genes was weaker than the other three R2R3-MYBs. In more detail, it had a relatively low correlation coefficient with ECG (0.52) and EGCG (0.67), and a relatively low correlation coefficient with genes CsSCPL1A, CsANR, CsLAR, and CsDFR .For further confirmation, gene-to-metabolite correlation analysis of onducted . The resCsMYB30, CsMYB34, and CsMYB37 might be the key TFs regulating galloylated catechins biosynthesis for the abundance of EGCG in C. sinensis.The results provide convincing clues that C. sinensis is an advantageous model system to dig into plant-specific metabolite biosynthesis and genetic regulation with rich and differential metabolite profiles. Its metabolites, such as flavonoids, caffeine, and volatile terpenes, accumulate in abundance and share characteristics with the same metabolites in other plant lineages .Plants are rich in metabolites that allow them to adapt to the environment and resist biotic and abiotic stress . These mC. sinensis   and UDP-glucose: galloyl-1-O-b-D-glucosyltransferase (UGGT) are recruited to catalyze the last two reactions in this bioprocess  . Two keyoprocess . The bioR2R3-MYB TFs confer tissue-specific or development stage-specific patterns for metabolites in the same biosynthesis pathway; often, multiple paralogs coexist in one species  among the tea-specific R2R3-MYB TFs.Based on the consideration of the massive accumulation of galloylated catechins (ECG and EGCG) in tea plant and tea-specific s family , we firsCsR2R3-MYB TFs, backed by multiple sequence alignment among C. sinensis and A. thaliana, suggests that most of the members in this family are conserved. Most R2R3-MYBs share similar functions to the homologous counterparts studied in A. thaliana. Some of the R2R3-MYB TFs that clustered in TESA, TESB, and TESC subgroups evolved exclusively in C. sinensis, but have isogenous genes in Actinidia chinensis, V. vinifera, Theobroma cacao, and Coffea canephora genomes. Thus, we speculated that TESA, TESB, and TESC are either obtained in C. sinensis or lost in A. thaliana lineages after divergence from their most recent common ancestor during two whole-genome duplication (WGD) events. In addition, members of SMYB5 and S5 subgroups, regulating flavonoids biosynthesis in A. thaliana, are greatly expanded in C. sinensis, which suggests that they might be either functionally redundant genes or genes that undertake some novel functions in the tea plant.The comprehensive and comparative phylogenetic analysis of CsR2R3-MYB TFs that were preferentially expressed in these tender tissues along with the major catechin-biosynthesis genes were the most promising candidates putatively regulating the biosynthesis of galloylated catechins. Consistent with previous results  in the phenylpropanoid pathway that are mainly relevant to the generation of condensed polymer PAs, was in fruits, flowers and roots. However, CsMYB42 was preferentially expressed in tender tissues and had a strong correlation with catechin biosynthesis genes and the contents of ECG and EGCG in the “Shuchazao” variety , CsMYB34 (S5 subgroup), and CsMYB37 (SMYB5 subgroup) were confirmed as the potential R2R3-MYB TFs relevant to the extensive accumulation of ECG and EGCG in C. sinensis, however, further investigation is needed. This study has laid a theoretical framework and valuable foundation for the future work, as we have provided a preliminarily evidence for illuminating that the high accumulation of galloylated catechins in C. sinensis are likely to be caused by the expanded R2R3-MYB TFs. Nevertheless, it is still necessary to further exploration and validate these results.Considering that galloylated catechins highly accumulate in ABs and young leaves, we speculated that the  results , our stu variety , while i variety . Thus, dR2R3-MYB gene members, classified into 38 subgroups, were identified in the C. sinensis genome. Notably, five subgroups  containing 21 R2R3-MYB TFs were identified to be remarkably expanded in or completely unique to C. sinensis. Furthermore, gene structure predictions, expression profile validation, and correlation analyses were subsequently conducted to screen out candidate R2R3-MYB TFs regulating galloylated catechin biosynthesis. CsMYB30, CsMYB34, and CsMYB37 were specifically expanded and their expression level were strongly correlated with galloylated catechin contents, suggesting the probable function in galloylated catechin biosynthesis. The present study firstly revealed the C. sinensis specific and expanded R2R3-MYB TFs that are potential regulators of galloylated catechins biosynthesis, which underpinned a basic understanding of the uniquely massive accumulation of galloylated catechins in C. sinensis.A total of 118 The original contributions presented in the study are included in the article/JL performed the qRT-PCR, analyzed and interpreted the data, made the data charts, and wrote the manuscript. SL conceived the project and supervised the research. PC carried out the HPLC test. JC, ST, and WY collected the materials for the experiments and provided useful suggestions. FC reviewed and edited the manuscript. PZ funded the research and reviewed the manuscript. BS designed the project, supervised the research, interpreted the data, and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


FTL and FTH1 expressions and (i) patient survival, using univariate, multivariate, Kaplan-Meier and Receiver Operator Characteristic analysis; and (ii) tumor-infiltrating immune cell subsets, using the bioinformatics tools Estimation of Stomal and Immune cells in Malignant Tumor tissues, Microenvironment Cell Population-counter, Tumor Immune Estimation Resource, and Tumor Immunology Miner. We found that FTL and FTH1 are upregulated and downregulated, respectively, in most of the human cancers analyzed. Tumor FTL levels were associated with prognosis in patients with lower grade glioma (LGG), whereas FTH1 levels were associated with prognosis in patients with liver hepatocellular carcinoma, HNSC, LGG, and kidney renal papillary cell carcinoma. In many cancers, FTL and FTH1 levels was significantly positively correlated with tumor infiltration by tumor-associated macrophages and T regulatory cells. These results suggest an important role for FTL and FTH1 in regulating tumor immunity to solid cancers.Ferritin is the most important iron storage form and is known to influence tumor immunity. We previously showed that expression of ferritin light chain (FTL) and ferritin heavy chain (FTH1) subunits is increased in head and neck squamous cell carcinoma (HNSC). Here, we analyzed solid tumor datasets from The Cancer Genome Atlas and Genotype-Tissue Expression databases to investigate correlations between  Ferritin, the most important iron storage form in humans, is an essential nutrient that plays crucial roles in diverse cellular physiopathological processes, including regulation of the tumor microenvironment and immunometabolism , 2. FerrFTL and FTH1 expression levels might correlate with tumor infiltration by diverse immune cell subsets. The Pan-Cancer Atlas, which is an essential resource for the development of precision medicine, seeks to reclassify cancers on the basis of their molecular similarities rather than their cellular origins. Along these lines, a comprehensive analysis of correlations between ferritin subunit levels and immune components in solid tumors could provide us with a better understanding of how iron metabolism influences tumor immunity.Increasing evidence supports a role for iron metabolism in supporting immunity to various cancers , 5, 6. IFTL and FTH1 in various cancer RNA-sequencing datasets, and we performed a comprehensive analysis of correlations between FTL and FTH1 levels and tumor-infiltrating immune cells, with the goal of uncovering a potential role for FTL and FTH1 in tumor immunology. To this end, we analyzed datasets from The Cancer Genome Atlas (TCGA), a landmark cancer genomics database started in 2006 [FTL and FTH1 levels and tumor infiltration by various immune cell subsets. These tools included Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (Estimate) [In the present study, we analyzed the expressions of  in 2006 –12, and stimate) , Microenstimate) , Tumor Istimate)  and Tumostimate) .FTL and FTH1 are differentially expressed in human cancers, we examined expression datasets from TCGA and GTEx databases. The results showed that FTL was upregulated in 21 of the 27 cancers analyzed (77.8%), downregulated in three cancers , and not significantly changed in three cancers  compared with normal tissues (FTH1 was downregulated in 22 of the 27 cancers (81.5%), upregulated in four cancers , and unchanged in only one cancer   .FTL and FTH1 levels and patient prognosis were first examined separately by univariate survival analysis. FTL expression was significantly associated with OS in 4 cancer types (−7), UVM , LIHC , and BLCA . FTH1 expression was significantly associated with OS in eight cancer types (−1), KIRP , LIHC , CESC , HNSC , LAML , LGG , and KICH . We then selected cancers in which FTL and FTH1 levels were associated with OS at P < 0.001, and performed Kaplan–Meier analysis of OS in patients stratified into high and low expression groups by ROC curve analysis. The results suggested that high FTL , KIRC , UCEC , and GBM . FTH1 expression was significantly associated with PFI in eight cancer types (−3), KIRP , CESC , HNSC , LGG , DLBC , KICH , and UVM . As was observed for OS, poor PFI was significantly associated with high FTL , PD-L1 in 10/32 (31.3%), CTLA4 in 13/32 (40.6%), TIM-3 in 26/32 (81.3%), LAG3 in 13/32 (40.6%), and LAIR1 in 26/32 (81.3%). FTH1 levels (PD-1 in 4/32 (12.5%) cancers, PD-L1 in 9/32 (28.1%), CTLA4 in 7/32 (21.9%), TIM-3 in 20/32 (62.5%), LAG3 in 2/32 (6.3%), and LAIR1 in 19/32 (59.4%). Interestingly, TIM-3 was positively related to FTL and FTH1 in the most cancers, so does LAIR1  of the correlation between pression  and sevepression  was mostFTL expression and CD4+ T cells  compared with normal tissues. FTH1 possesses ferroxidase activity and converts Fe2+ into Fe3+, its combination with FTL can efficiently reduce the toxicity of Fe2+ [FTH1 is thought to be a bifunctional molecule. Flavia et al. [FTH1 can enhance P53 expression and reduce the proliferation of non-small cell lung cancer. However, Salatino et al. [FTH1 is critical for proper functioning of the antioxidant system in ovarian cancer cells, suggesting that inhibition of FTH1 may improve cisplatin-induced cytotoxicity. In addition, the subcellular localization of FTH1 can affect its functions. Liu showed that a high cytoplasmic FTH1 level correlates with a favorable prognosis, whereas nuclear FTH1 is an adverse indicator, in triple negative breast cancer patients [FTH1. Clearly, FTH1 has multiple complex functions in cancer cells, and a broader understanding of its roles is urgently needed.In contrast to us study . Among t of Fe2+ , 29. In a et al.  found thpatients . It is sFTL and FTH1 correlated positively with levels of the immune checkpoint proteins TIM-3 and LAIR1, both of which can suppress tumor immunity [TIM-3 is a negative regulator of cytotoxic and helper T cells and can promote the formation of a suppressive tumor microenvironment through the TIM-3–galectin-9 pathway [LAIR1 is widely expressed in immune cells where it negatively regulates cell activation through interaction with several ligands, especially collagen [TIM-3 and LAIR1 levels and FTL and FTH1 levels. Moreover, few studies have assessed the influence of TIM-3 and LAIR1 on iron metabolism. We speculate that FTL and FTH1 may influence TIM-3 and LAIR levels by affecting immune cell infiltration into the tumor.Advances in our understanding of anti-tumor immunity have made targeted immunotherapies the most promising treatment methods for many cancers , 34. In immunity –38. TIM- pathway , 39. LAIcollagen . HoweverFTL and FTH1 expressions in solid tumors and infiltration of several other pivotal immune cells. Intriguingly, FTL and FTH1 levels are positively correlated with most of immune cells in some tumors, especially in LGG. However, both of them are positively correlated with the poor prognosis of LGG. Iron is essential for cell metabolism and proliferation [FTL and FTH1 on different immune cells should be various. In other words, the increasing level of immunosuppressive cells, like M2 and Treg, could completely offset the effect of anti-tumor immune cells. However, this needs to be confirmed in a large number of future studies.In addition to T cells, we investigated potential correlations between feration , howeverferation . We consFTL and FTH1 expressions and infiltration of both cell types in most cancers. Mounting evidence suggests that TAMs exhibit high plasticity and can switch between the anti-tumoral M1 subtype and the pro-tumoral M2 subtype, the latter of which is most abundant in solid tumors [FTL is mainly stored in M2-like TAMs in node-negative breast cancer [FTL and FTH1 levels and TAMs may be due to the high demand for ferritin in M2 TAMs. However, whether FTL or FTH1 inhibition can induce M2-to-M1 repolarization will need to be investigated in future studies.Moreover, our analyses revealed positive associations between d tumors , 42. Rict cancer . Cronin FTL and FTH1 levels are positively correlated with Tregs infiltration in most cancers [FTH1 could activate Tregs to produce interleukin-10 and suppress the immune response in vitro [FTL and FTH1 expressions and Treg infiltration in most solid tumors through analysis of mRNA and clinicopathological data from TCGA. While a robust source of iron is necessary for metabolic and redox reactions to support the proliferation and effector functions of T cells [FTL or FTH1 therapy could influence Treg infiltration by decreasing ferritin expression, which holds promise as a new mechanism of iron-based therapy. Whether such therapy could also enhance the activity of immunotherapies is another question that should be addressed in future studies.Finally, the present study shows that both  cancers . A simil cancers . Gray shin vitro . As note T cells , excessi T cells . The preFTL and FTH1 mRNA is increased and decreased, respectively, in most of the 27 solid cancer types examined. Furthermore, both ferritin subunits may have important physiological functions in TAMs and Tregs, and thus may play key roles in tumor immunity. Our results suggest that therapies targeting iron and ferritin homeostasis could be developed as potential treatments to synergize with specific immunotherapies to enhance anti-tumor responses.In summary, we have shown that the expressions of https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga/, OV: ovarian serous cystadenocarcinoma, LUAD: lung adenocarcinoma, LUSC: lung squamous cell carcinoma, PRAD: prostate adenocarcinoma; UCEC: uterine corpus endometrial carcinoma, BLCA: bladder urothelial carcinoma, TGCT: testicular germ cell tumor, ESCA: esophageal carcinoma, PAAD: pancreatic adenocarcinoma, KIRP: kidney renal papillary cell carcinoma, LIHC: liver hepatocellular carcinoma, CESC: cervical squamous cell carcinoma and endocervical adenocarcinoma, SARC: sarcoma, BRCA: breast invasive carcinoma; MESO: mesothelioma, COAD: colon adenocarcinoma, STAD: stomach adenocarcinoma, SKCM: skin cutaneous melanoma, CHOL: cholangiocarcinoma, KIRC: kidney renal clear cell carcinoma, THCA: thyroid carcinoma, THYM: thymoma, HNSC, READ: rectum adenocarcinoma, LGG, DLBC: lymphoid neoplasm diffuse large B-cell lymphoma, KICH: kidney chromophobe, UCS: uterine carcinosarcoma, ACC: adrenocortical carcinoma, PCPG: pheochromocytoma and paraganglioma, UVM: uveal melanoma, GBM: glioblastoma multiforme). Gene expression datasets for the corresponding normal tissues were downloaded from the Genotype-Tissue Expression (GTEx) program . Patients’ clinical information were organized in The expression levels of genes in various types of cancers were downloaded from TCGA  curve analysis, with the median expression level as the cutoff value. A multivariate Cox proportional hazard model was performed to evaluate the independent prognostic variables.Overall survival (OS) and progression-free interval (PFI) data were obtained from TCGA. Univariate survival analysis was used to analyze correlations between PD-1/PDCD1), programmed cell death-1 ligand 1 (PD-L1/CD274), cytotoxic T lymphocyte-associated protein 4 (CTLA4), T-cell immunoglobulin domain and mucin domain-3 (TIM-3/HAVCR2), lymphocyte-activation gene 3 (LAG3), and leukocyte-associated immunoglobulin-like receptor 1 (LAIR1).Expression datasets for 47 immune-related markers were also obtained from TCGA. The markers included pivotal immune checkpoint proteins such as programmed death-1 , RStudio (version 1.2.5033), and R Estimate package (version 1.0.13). The MCP-counter data were analyzed using R MCP-counter package (version 1.2.0).https://cistrome.shinyapps.io/timer/) is a deconvolution tool to calculate the abundance of tumor-infiltrating immune cell subsets, including B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells, in various human cancers. TIMINER (https://icbi.i-med.ac.at/software/timiner/timiner.shtml) is a computational framework that is based on the pre-ranked Gene Set Enrichment Analysis approach and was used here to define gene sets from 28 immune cell subtypes.TIMER (FTL and FTH1 expressions and tumor-infiltrating cells or immune-related markers were analyzed using Pearson’s Correlation Coefficient. P < 0.05 was considered statistically significant.Differences in FTL and FTH1 expressions between tumor and normal tissues were compared using Student’s t-test. Survival data were analyzed by univariate and Kaplan–Meier analysis of patient populations stratified by ROC curve analysis. Associations between Supplementary Figure 1Supplementary Tables


Meanwhile, LETM1 has been associated with the regulation of fundamental cellular processes, such as development, cellular respiration and metabolism, and apoptosis. This mini-review summarizes the diversity of cellular functions impacted by LETM1 and highlights the multiple roles of LETM1 in health and disease.Nearly 2 decades since its discovery as one of the genes responsible for the Wolf-Hirschhorn Syndrome (WHS), the primary function of the leucine-zipper EF-hand containing transmembrane 1 (LETM1) protein in the inner mitochondrial membrane (IMM) or the mechanism by which it regulates mitochondrial Ca Dependie brucei . A TXXR e brucei . Electroe brucei . The N-te brucei .+/H+ exchanger (KHE) as originally surmised or a Ca2+/H+ exchanger (CHE), as subsequent studies have shown m, loss of K+ acetate-induced swelling, and dissolution of cristae structure and mitochondrial morphology  came from studies in mitochondria expressing MDM38rphology . These e. brucei . The K+ chondria .T. brucei, LETM1 orthologs of higher organisms possess EF-hands, which may act as Ca2+ sensors and implicate LETM1 in Ca2+ regulation. Accordingly, LETM1 was identified as a putative CHE (LETM1-CHE) in a genome-wide RNAi screening in D. melanogaster cells, where mitochondrial Ca2+ influx/efflux was coupled to H+ extrusion/influx, respectively. LETM1-knockdown (LETM1-KD) decoupled the Ca2+/H+ antiport while LETM1 overexpression increased pH-induced Ca2+ transport 5-fold  following Ca2+ depletion in the sarco/endo-plasmic reticulum (SR/ER) and/or failure of Ca2+ sequestration through the sarco/endo-plasmic reticular Ca2+ ATPase , ultimately affecting NCLX-mediated Ca2+ efflux  and to nucleoprotein complexes called mitochondrial nucleoids, which are mitochondrial DNA (mDNA) complexed with regulatory proteins . Consequ+ transport using ionophores suggested that the absence of a LETM1-CHE/KHE activity may underlie the distorted morphology, lack of respiratory supercomplex formation, and decreased ATP production  is a progressive neurodegenerative disorder, associated with mitochondrial dysfunction . LETM1 ieostasis . The stuactivity . Future D. melanogaster exhibit motor defects, which correlates with reduced release of neurotransmitters , LETM1 expression is implicated in mitochondrial dysfunction. For example, a common mouse model used to study ALS, the end-stage superoxide dismutase SOD1(G93A) transgenic mice, exhibited a 2-fold increase in LETM1 expression in motor neurons of the hypoglossal nucleus (hMNs). This correlated with a decrease in mitochondrial Caeostasis .2+ dysregulation suggests that the loss of a LETM1-CHE activity and resulting Ca2+ dysregulation may underlie pathophysiology. Interestingly, however, LETM1-KD in the epilepsy model also decreased the expression of mitochondrial cytochrome b, a component of complex III , presumably encoded by LETM1, increased with matrix Ca2+ load and that CHE may be able to sense matrix Ca2+ buffering (2+ handling in the absence of NCLX. Future studies will need to address these knowledge gaps as to how the LETM1-CHE function in in vitro conditions is modulated in the presence or absence of one or more EF-hands in the LETM1 protein or factors such as the presence of other ionic exchangers.The diverse impact of LETM1 deficiency, as outlined above, suggests that LETM1 expression and function is critical for short-term dynamic processes such as cellular metabolism as well as long-term processes important for cell survival and development. Although the primary function of LETM1 as CHE or KHE is still being debated, it is evident that loss of LETM1 function adversely impacts mitochondrial Ca in vivo . Convers in vivo , do not uffering . The stuin vitro experiments that insertion of LETM1 complexes into membrane bilayers alone led to the formation of membrane invaginations (LETM1 interacts with proteins, such as CTMP, BCS1L, and MRPL36, and regulates processes like Akt signaling and protein import and assembly. Changes in LETM1 expression also affect these processes, implicating it in the pathophysiology of several disease phenotypes, including metabolic, neurodegenerative, and neoplastic diseases . The lininations . Four reinations , which iinations . These rinations , this suinations , this asAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tc1/mariner and pogo superfamily, as the most widely distributed transposons in nature, are widely reported in insects. However, in bees, the research on transposons has never been systematically reported. Therefore, this study focuses on annotating the transposons of the Tc1/mariner and pogo superfamily of bees that were sequenced, revealing the distribution, diversity, potential activities, evolutionary patterns, and structural characteristics of transposons in different bees.In the class of insects, bees are advanced evolutionary groups that play a pivotal role in ecosystem balance, agriculture, and food security. Transposons are the driving factors of genome evolution and speciation, which shape the genomes of almost all organisms. Among them, the pogo and Tc1/mariner transposons in the genome of each species, and performed a phylogenetic analysis and determined their overall distribution. The pogo and Tc1/mariner families showed high diversity and low abundance in the 37 species, and their proportion was significantly higher in solitary bees than in social bees. DD34D/mariner was found to be distributed in almost all species and was found in Apis mellifera, Apis mellifera carnica, Apis mellifera caucasia, and Apis mellifera mellifera, and Euglossa dilemma may still be active. Using horizontal transfer analysis, we found that DD29-30D/Tigger may have experienced horizontal transfer (HT) events. The current study displayed the evolution profiles  of the pogo and Tc1/mariner transposons across 37 species of Apoidea. Our data revealed their contributions to the genomic variations across these species and facilitated in understanding of the genome evolution of this lineage.Bees (Apoidea), the largest and most crucial radiation of pollinators, play a vital role in the ecosystem balance. Transposons are widely distributed in nature and are important drivers of species diversity. However, transposons are rarely reported in important pollinators such as bees. Here, we surveyed 37 bee genomesin Apoidea, annotated the  Bees (Hymenoptera: Apoidea), which originated in the early to mid-cretaceous era , are theAntarctic midge [migratory locust [Transposons, also called jumping genes, were once considered junk sequences but were later confirmed to play an important role in genome evolution and size ,12,13,14ic midge  to as muy locust . Transpoy locust ,18,19.Tc1/mariner, pogo, hAT, PiggyBac, CACTA, Helitron, and PIF-Harbinger. The Tc1/mariner superfamily is a member of the cut-and-paste group that was first discovered in Drosophila mauritiana (transposon mariner) and Caenorhabditis elegans . Generally, Tc1/mariner transposon usually has a single open reading frame (ORF) of about 340 amino acids (aa), flanked by two terminal inverted repeats (TIRs) and dinucleotide target site duplications (TSDs) of TA, and it may represent the most widely distributed transposon superfamily in nature [Tc1/mariner families, and have been suggested as major reservoir hosts for DNA transposons and RNA viruses [Transposons are usually divided into two types according to the structural organization and mechanism: Class I represents RNA transposons and Class II DNA transposons. DNA transposons have been widely reported and can be divided into three main types: cut-and-paste, peel-and-paste, and self-synthesizing transposons. Widely reported DNA transposons include: n nature ,21,22. T viruses ,31,32,33Tc1/mariner is classified into DD34E/Tc1 [mariner [TR [IC [maT [TRT [GT [VS [pogo transposon was first found in flies [pogo-like elements were detected, such as Tigger [Fot, Tan1, Pot1, Pot2, Flipper, and Aft1-transposons were found in the genome of fungi [Lemi1 was found in the genome of plants [pogo-like elements were found in the teleost genome [pogo and Tc1/mariner are two distinct superfamilies [Based on the phylogeny of the DDE-conserved catalytic motif, D34E/Tc1 ,36,37,38[mariner ,40,41,42iner [TR , DD36E/I [TR [IC , DD37D/m[IC [maT ,45, DD37maT [TRT , DD39D/G[TRT [GT , and DD4 [GT [VS . The pogin flies , then mos Tigger  was founof fungi ,52,53,54f plants ; and pogt genome . A previfamilies , but repmariner element has been reported in studies of the annotation of honeybees and bumblebees [pogo and Tc1/mariner transposons in the genome of each species; and determined their phylogenetic positions, classification, overall distribution, and structural characteristics. We also investigated the evolutionary patterns of DD29-36D/Tigger and DD34D/mariner transposons. Our data reveal the evolutionary landscape of pogo and Tc1/mariner transposons in Apoidea and will add to the understanding of their contributions to the evolution of the Apoidea genomes.The mblebees ,59. Howepogo and Tc1/mariner DNA transposons in 37 sequenced genomes of Apoidea (taxid:34735), we selected several representative transposase sequences from the well-defined families of pogo  and Tc1/mariner  superfamilies and subjected them to a TblastN search against the Apoidea genomes deposited in a whole-genome shotgun database  with default parameters (1e-10). The information of all genomes in this study is listed in To survey the distribution and diversity of pogo and Tc1/mariner transposases separately using MAFFT (v.7.310). The phylogenetic trees were inferred based on the conserved DDE domain using the maximum-likelihood method in the IQ-TREE program. According to the Bayesian information criterion, the best-suited aa substitution model for these data was the LG + G4 model, which was selected by ModelFinder. The reliability of the maximum-likelihood trees was estimated using the ultrafast bootstrap approach with 1000 replicates [www.timetree.org, accessed on 12 September 2021).For phylogenetic analysis, the conserved DDE domains of the identified transposases were aligned to the representative families of the plicates ,43. Infohttp://bioinf.cs.ucl.ac.uk/psipred, accessed on 1 March 2021). Putative nuclear localization signal (NLS) motifs were predicted using PSORT . The protein domains were identified using the profile hidden Markov models on the hmmscan online web server .Protein secondary structure predictions were performed using the PSIPRED program (EF1-a), heat shock cognate 70 (Hsc70-4), tubulin beta-3 (tub3), and 29 Ribosomal proteins, were used for the comparison with transposon distance to test the HT hypothesis. The three genes  were used as internal controls by other studies including HT analysis of transposons [EF1-a, Hsc70-4, tub3, and 29 universally conserved genes in the US National Center for Biotechnology Information (NCBI) database were not included in these calculations. Multiple alignments of EF1-a, Hsc70-4, tub3, 29 universally conserved genes, and all transposons in Apoidea were created using MAFFT, and the pairwise distances were then calculated using MEGA software  based on two aligned files. The species divergence times were estimated using the online Timetree program . The bar chart was created by Graphpad 8, and then statistical differences were analyzed using a one-factor ANOVA test in SPSS (25.0). Significant differences were assessed at p < 0.05. The genomic copies of DD34D/mariner were annotated by using RepeatMasker  with representative genome of each species, and then calculated the Kimura divergence parameter distance by using the calcDivergenceFromAlign.pl package from RepeatMasker. The Kimura two-parameter distance reflects the transpositional activity of DD34D/mariner on a relative time scale per genome [The pairwise genetic distance between transpsons and the conserved host genes was applied to determine whether these species have experienced HT events. The coding sequences of 32 host genes, including elongation factor1 alpha , we executed a Tblastn search using the NCBI whole-genome shotgun database  using 15 representative transposase sequences from different families of the pogo  and Tc1/mariner  superfamilies. We identified 164 unique elements in 37 species of Apoidea  were classified as members of the pogo superfamily and seven families  classified as members of the Tc1/mariner superfamily were identified in 37 species of Apoidea  of Apoidea, in which most species contained seven to eight transposon families. Most species of Apidae contained only two to four transposon families, particularly in the Apis genus, and most species exhibited only DD34D/mariner and DD41D/VS families. The exception was Habropoda laboriosa, which had one pogo family (Tigger), but variations of DDE domains  and six Tc1/mariner families . This species exhibited the highest diversity of pogo and Tc1/mariner transposons in Apoidea. Four transposon families  were detected in all bumblebees (five species), Melipona quadrifasciata and Frieseomelittavaria, whereas the five species of stingless bee  exhibited only the DD29-36D/Tigger and DD34D/mariner transposon families. The DD29-36D/Tigger, DD34D/mariner, DD34E/Tc1, and DD36E/IC families were detected in Eufriesea mexicana, and DD29-36D/Tigger, DD34D/mariner, DD34E/Tc1, and DD41D/VS families in Euglossa dilemma. Moreover, families of DD29-36D/Tigger, DD34D/mariner, and DD34E/Tc1 were found in Ceratina australensis and Ceratina calcarata, and Ceratina calcarata also includes a DD41D/VS family  and Dufourea novaeangliae , which represented the highest diversity across Apoidea species   , which soneybees .Dufourea novaeangliae, Nomia melanderi, Lasioglossum albipes, and Megalopta genalis belong to the Halictidae family, but they had obvious irregular distribution of transposons, indicating putative HT events of these transposons across species within this lineage. By contrast, two very close species, Ceratina calcarata and Ceratina australensis, also had different distribution of transposons. Moreover, some relatively distantly related species on the phylogenetic tree were closer than closely-related species, for example, the DD29-30D/Tigger family in Dufourea novaeangliae and Megalopta genalis, which belong to Halictidae, but Dufourea novaeangliae was closer to the Osmia lignaria in the Megachilidae family on the clade than to Megalopta genalis, which is a more closely-related species of Nomia melanderi. Considering these findings, we speculate that some transposons might have been exposed to several episodes of HT.HT is an important form of asexual transmission in nature, and there is increasing evidence that transposons are important participants in HT. Generally, HT events will follow three conditions: (1) high sequence similarity of TEs from divergent taxa; (2) incongruence between TE and host phylogeny; (3) a patchy TE distribution within a group of taxa . In our pogo and Tc1/mariner in the bee genome, clusters that are inconsistent with the host phylogeny and have an identity of each cluster of the phylogenetic tree close to or greater than 80%, which may experience HT events, are selected. We detected the presence of HT signs in the four clusters . Then, the pairwise genetic distances between the four host-conserved genes  and these transposons were calculated and compared. we found that the average distance is smaller for DD29-30D/Tigger, DD33D/Tigger, DD34D/mariner-cluster 1, and DD34D/mariner-cluster 2 than for the four host genes  for DD29-30D/Tigger than for almost all host genes, except for EF1-a, RPL2, RPL18, RPL23, RPS8, and RPS9  across these species  domain at the N-terminus and a catalytic domain at the C-terminus. The N-terminal of Fot-like transposase comprises HTH and DNA-binding domain regions, and the C-terminal is a catalytic domain. The Tc1/mariner transposase contains an HTH domain at the N-terminus and a catalytic domain at the C-terminus. The DD29-36D/Tigger transposons invaded almost all species except the Apis genus, among which 26 species contain 58 transposons, representing a high diversity of transposons. The length of all transposons ranged from 907 bp to 4213 bp, and the length of the TIR was 10–33 bp. The overall copy number content was very low, but most transposons included an intact copy. Generally speaking, the last aa of the DD/D catalytic domain of the pogo transposon was ‘D’, but we found an exception in this study. DD33D and DD33E are similar in structure and have a very close phylogenetic relationship. Considering their phylogenetic location and all species that appear in stingless bees, DD33E may have been caused by the DD33D mutation at some stage. DD34D/mariner was found to be the most widely distributed transposon in this study. Almost all bees except Lepidotrigona ventralis hoosana have the DD34D/mariner transposon and it appears to represent a very young transposon family in Apoidea. We found that all DD34D/mariner transposons were around 1200 bp in length and that 24 transposons contained complete transposase. However, among the six Apis genomes studied , we could identify only complete ORFs but could not determine the boundary. DD34E/Tc1 is considered to be the origin of many Tc1/mariner transposons and was found to be a very widely distributed transposon in this analysis. All DD34E/Tc1 transposons were 952–1805 bp in length, and the TIRs were 24–221 bp in length. We also found six Fot-like transposons, namely DD35-36D/Fot in Colletes gigas, Lasioglossum albipes, Megachile rotundata, Osmia bicornis bicornis, Osmia lignaria, and Dufourea novaeangliae. Six transposons were found in DD36E/IC, but they all lost the function of encoding complete transposase. The first five base changes seem to be typical characteristics of some subfamilies. All DD37D/maT started with CAGGG, and DD38E/IT started with CACTA and CACTG. Lepidotrigona ventralis hoosana had an incomplete DD39D/GT transposase with a total length of 2381 bp, which retains the typical characteristics of DD39D/GT transposon beginning with CTCCC. In addition, the recently well-defined DD41D/VS family was also found in this study. Eleven of the 24 transposons had complete ORFs and were 1024–2258 bp in length. All of the bumblebee catalytic domains changed from DD41D to DD40D.Previous studies have shown that the Apis dorsata from other honeybees [Apis mellifera intermissa contains 5.16% TEs with low diversity, most of which comprise simple repeat sequences [Apis mellifera genome, the percentage of TEs is close to 8% [mariner transposons are the most common element within this class [Apis cerana accounted for 9.15% and simple repeats accounted for most TEs [Apis cerana genome and 0.57% (1.34 Mb) of the Apis mellifera genome. The TEs are significantly smaller in Apis cerana than in Apis mellifera [Bombus terrestris and Bombus impatiens, respectively, and most of them were retrotransposons, of which Gypsy was the most common element. The main types of DNA transposons are TIRs of mariner, which account for 1.6% and 2.7%, and their abundance is higher than that of Apis cerana and Apis mellifera [Transposons can generate new genes through genome rearrangement and overexpression, which play a role in the behavioral diversification of oneybees . Given toneybees ,14. The equences . In the se to 8% . DNA trais class . A recenmost TEs . The DNAellifera . In a stellifera . In addiellifera ,75,76,77Tc1/mariner and two families of pogo, which suggests that the genome of some bees represents high diversity of Tc1/mariner and pogo transposons. In our study, the genomes of 37 bee genomes were rich in pogo and Tc1/mariner transposons, and included nine different transposon families: DD29-36D/Tigger, DD35-36D/Fot, DD34D/mariner, DD34E/Tc1, DD36E/IC, DD37D/maT, DD38E/IT, DD39D/GT, and DD41D/VS. Among all transposon families discovered to date, DD34D/mariner is the most widely distributed and appears in almost all bee genomes. In addition, DD34D/mariner is also widely distributed in nature and has been reported in all species [As expected, in our analysis, the transposon diversity was also higher for bumblebees than honeybees. We detected at least seven families of  species ,80,81,82Tc1/mariner and pogo transposons do not display significant amplification in these lineages. This is consistent with previous findings that most Tc1/mariner is detected with low genomic coverage [mariner in the genomes of Apis mellifera, Apis mellifera carnica, Apis mellifera caucasia, Apis mellifera mellifera, and Euglossa dilemma. The evolutionary dynamics analysis showed that the DD34D/mariner elements of these five genomes are very young, and we speculate that these elements may remain active in the genomes of some species. The European honeybee is an important economic animal introduced into China in the last century, where there is a considerable amount of breeding. Active transposon elements may play an important role in the genome evolution and subspecies differentiation of European honeybees, and their existence raises the possibility of molecular operations on honeybees. In addition, the number of newly discovered families of the pogo and Tc1/mariner superfamily continues to increase as more genomic sequencing data are obtained, and this trend indicates that the diversity of the pogo and Tc1/mariner superfamilies may be much larger than the currently known families.It is generally believed that the copy number is related to transposon activity and that a genome with more intact transposon copies, which include the full-length transposase (no frameshift mutation) and the flanking TIRs with very high identity, usually reflects a recent invasion activity, suggesting that some copies may be still active . We examcoverage ,71. In aTc1/mariner and pogo transposons in 37 species, for a total of 164 transposons. Interestingly, we found that the genomes of some solitary bees were rich in DNA transposons. For example, Habropoda laboriosa exhibited DD29-36D/Tigger, DD34D/mariner, DD37D/MaT, DD41D/VS, DD34E/Tc1, DD36E/IC and DD38E/IT transposons; Dufourea novaeangliae exhibited DD29-36D/Tigger, DD34D/mariner, DD35-36D/Fot, DD34E/Tc1, DD36E/IC, DD37D/MaT, DD38E/IT and DD41D/VS transposons; and Colletes gigas exhibited DD29-36D/Tigger, DD35-36D/Fot, DD34D/mariner, DD34E/Tc1, DD36E/IC, DD37D/maT, DD38E/IT, and DD41D/VS transposons. Social bees exhibited significantly fewer transposons than solitary bees. For example, the Tetragonula genus exhibited only two transposon subfamilies, DD29-36D/Tigger and DD34D/mariner. In Apis genus, there are also two families of transposons (DD34D/mariner and DD41D/VS) that appear in the genome of honeybees. This seems to be related to the living environment. Solitary bees act alone and their long-term migration and living environment are more complicated and unstable, whereas social bees live in a group involving social activities and their living environment is more stable [Bombus genus exhibited four families, DD29-36D/Tigger, DD34D/mariner, DD41D/VS, and DD34E/Tc1. Interestingly, Bombus genus are a group that once transformed from solitary bees to social bees, which seems to further verify that the content of transposon may be related to the living environment.In this analysis, we detected a total of nine families of e stable . On furtApis mellifera, Apis mellifera carnica, Apis mellifera caucasia, and Apis mellifera mellifera exhibited a complete DD34D/mariner element (full-length and complete ORF). However, only the complete ORF of DD34D/mariner was exhibited by Apis cerana, Apis cerana cerana, Apis cerana japonica, Apis dorsata, Apis florea, and Apis mellifera intermissa, and the boundary could not be determined. Compared with its close relative, the European honeybee, the Asian honeybee seems to have a stronger selection of transposons in the genome. It is possible that the drones that develop from unfertilized eggs and carry haploid chromosomes experience strong selective pressure on some active elements [VS, we only found that Apis mellifera, Apis mellifera carnica, Apis mellifera caucasia, Apis mellifera mellifera, and Apis dorsata have the complete full-length transposon sequence, but no complete ORF was found. Moreover, Apis cerana, Apis cerana cerana, and Apis cerana japonica did not exhibit any DD41D/VS transposon elements. It seems that DD41D/VS may be an ancient invasion and that, during long-term genome evolution, European honeybees gradually lost their complete copy and Asian honeybees completely lost the entire transposon. Finally, some studies have noted that a host contains an endogenous enzyme system that can silence the transposon or some virus expression, such as RNA interference [We also found another interesting phenomenon. elements . In our rference . HoweverTransposons are parasitic DNAs whose only role is to replicate and propagate. When a transposon invades a host, the germline genome must be colonized to ensure that it remains in the population. This then increases the copy number , and theTigger. To find the HT origin of the DD29-30D/Tigger transposon, we performed Blast searching of this transposase in Dufourea novaeangliae in the NCBI nucleotide collection (nr/nt) database. The complete DDD spacing (DD30D) catalytic domains were detected in Nosema bombycis, Strongyloides stercoralis, and Ctenocephalides felis. Interestingly, these three creatures have parasitic abilities: Nosema bombycis is a parasite of silkworm, Strongyloides stercoralis is parasitic in humans, and the host of Ctenocephalides felis is the cat. These species may serve as potential vectors for Tigger transposons that have invaded the bee genome at a certain time; if so, this strongly suggests the existence of a host–parasitic relationship in bees. These findings suggest that the host–parasitic mode may also be the primary mode of HT of Tigger transposon.In this study, although most transposons did not show obvious HT signs, the potential HT traces were detected for DD29-30D/pogo and Tc1/mariner family transposons in 37 sequenced Apoidea genomes. In general, pogo and Tc1/mariner show high diversity in most species, except for a few honeybees, but have a low abundance. In addition, only DD34D/mariner has a high copy number in several species, appears as a complete structure, and may have potential activity. Finally, our results also show that DD29-30D/Tigger may experience HT events, and we speculate that they may have invaded their common ancestor before some species formed.Our research provides, for the first time, information about the distribution of