Seed development, dormancy, and germination are key physiological events that are not only important for seed generation, survival, and dispersal, but also contribute to agricultural production. RNA-binding proteins (RBPs) directly interact with target mRNAs and fine-tune mRNA metabolism by governing post-transcriptional regulation, including RNA processing, intron splicing, nuclear export, trafficking, stability/decay, and translational control. Recent studies have functionally characterized increasing numbers of diverse RBPs and shown that they participate in seed development and performance, providing significant insight into the role of RBP–mRNA interactions in seed processes. In this review, we discuss recent research progress on newly defined RBPs that have crucial roles in RNA metabolism and affect seed development, dormancy, and germination. RNA-binding proteins (RBPs) centrally regulate all aspects of RNA metabolism from synthesis to decay . The funGRP1A and RZ-1A significantly increased during seed maturation and desiccation. Their encoded proteins potentially act as molecular chaperones to stabilize seed stored-mRNA and maintain its original integrity and translatability during seed dormancy and under various natural conditions [Transcriptome data have been used to identify more than 12,000 and 17,000 mRNAs in the mature dry seeds of Arabidopsis and rice, respectively ,12. Thesnditions ,15. Recenditions ,17. Our The RRM was the first conserved domain to be identified in RBPs and appears to be common to all eukaryotes . In addiDrosophila melanogaster and FBF (Fem-3 mRNA-binding factor) from Caenorhabditis elegans [1-3AU(A⁄U) motif in the 3′-untranslated region (UTR) of the target RNA, mediated by its eight sequential tandem PUF repeats. Each PUF repeat comprises approximately 36 amino acids and specifically recognizes one RNA nucleotide [The dsRBD is the second most widespread RNA recognition motif and usually comprises 65 to 70 amino acids that form an αβββα fold . How dsR elegans   in a T-DNA mutant screen [APUM5 was highly expressed in silique ends and was markedly upregulated after treatment with abscisic acid (ABA) and NaCl, suggesting that it may function in seeds in response to ABA and abiotic stress. Germination rate was hypersensitive to salt and osmotic stress in seeds that overexpressed APUM5, whereas APUM5-RNAi seeds showed increased tolerance of these stresses [apum24 mutant is deficient in seed setting, whereas the apum23 knock-out mutant develops normal embryos [apum23 null mutant and the apum24 knock-down mutant, including significantly delayed germination and abnormal development of leaves and roots [apum23 null mutant seeds germinated more slowly than wild-type seeds, regardless of whether or not they were stratified, suggesting the involvement of APUM23 in seed dormancy release and germination [APUM23 also caused hypersensitivity to salt stress, with delayed germination and seedling establishment, possibly resulting from changes in the expression of genes involved in ABA biosynthesis and signaling [PUF RBPs have been reported to affect RNA decay, translational repression, pre-ribosomal RNA processing, and chromosome stability . Some Art screen . APUM5 wstresses . APUM5 wstresses . APUM23 stresses . They ar embryos . Their c embryos ,41. Addind roots ,42. Notaignaling .APUM9, APUM10 and APUM11, may function together with REDUCED DORMANCY5 (RDO5) in the regulation of seed germination and dormancy, as the expression of all three genes was significantly up-regulated in the rdo5 mutant after imbibition. RNAi transgenic lines with simultaneous down-regulation of three APUMs exhibited enhanced dormancy compared with wild type. By contrast, the overexpression (OE) of APUM9 resulted in reduced seed dormancy. Together, these results suggest that APUM9 has a strong negative effect on seed dormancy [APUM9-OE plants compared with wild type. Five  were involved in the ABA signaling pathway. Moreover, APUM9 bound to the 3′-UTR of FBS1 and COL4, subsequently destabilizing their neighboring GFP reporter gene in an N. benthamiana transient expression system. It therefore appears likely that APUM9 directly targets ABA signaling genes for degradation, thereby promoting the transition from dormancy to germination [APUM9, APUM10, and APUM11 in an rdo5-1 background completely rescued the reduced dormancy phenotype of rdo5-1 [Xiang et al. (2014) reported that members of another PUF subgroup, including dormancy . Interesmination . These Af rdo5-1 . In the cpl1 null and dsRBD defective mutants exhibit strong tolerance to ABA and NaCl stress during seed germination, indicating the requirement for CPL1, and especially the dsRBD, in typical seed germination behavior [cpl1 mutant, the cpl2 mutant with a defect in the dsRBD is hypersensitive to high salinity and shows a lower seed germination rate, suggesting that CPL1 and CPL2 have distinct functions in seed behavior under stress [DsRBD-type RBPs are mainly involved in post-transcriptional regulatory events such as the editing, trafficking, and portioning of substrate mRNAs and the biogenesis of various small RNAs. Based on the phylogenetic analyses of dsRBD sequence, 16 dsRBD-type RBP have been identified . Six havbehavior . CPL1 inbehavior ,49,50. Cbehavior . By contr stress . Interesr stress . Moreover stress . CPL1 inr stress . These fr stress .DOG1), the major quantitative trait locus that specifically controls seed dormancy in Arabidopsis, is tightly controlled on different levels [DOG1 by directly binding its promoter region, while indirect regulation mode is adopted by histone methyltransferase KYP/SUVH4 [DOG1 mRNA. Alternative polyadenylation selectively occurs on the proximal transcription termination site (pTTS) and the distal TTS (dTTS) of the DOG1 sense transcript, resulting in the production of short DOG1 (shDOG1) and long DOG1 (lgDOG1), respectively [cpl1-8 and cpl1-9 mutants, defective in CPL1, exhibit priority polyadenylation of pTTS, increased expression of shDOG1, and enhanced seed dormancy [DOG1 target specificity during seed dormancy control has not been identified. More recently, the eceriferum (cer) mutant identified from a stem surface glossiness screen for wax-deficient mutants was shown to harbor a mutation in CER11, and CER11 was demonstrated to be CPL2. Notably, the seed coat of cer11/cpl2 showed reduced mucilage extrusion and deposition, as well as delayed columella secondary cell wall development [cer11 context, demonstrated by the fact that secretion of MUCILAGE-MODIFIED 2 (MUM2), a cell-wall modifying enzyme, is delayed in cer11/cpl2 mutant seeds [Recently, CPL1 and CPL2 were reported to affect seed dormancy. The transcript of DELAY OF GERMINATION1  mutants were identified from ABA sensitivity germination screens and are sensitive to salt and osmotic stresses during germination. HYL1 may affect the ABI5- or ABI3-mediated ABA signaling pathway to control seed germination and seedling establishment by repressing the activity of two mitogen activated protein kinases (MAPKs), SNP1 and MPK3, although the specific mechanism remains unclear [hyl1 and dcl1-11 mutants was hyposensitive to glucose, which may be explained by alterations in 38 glucose-related pri-miRNAs and their corresponding miRNAs. Notably, the hyl1 mutant showed lower expression of ABI3, ABI4, and ABI5 after glucose treatment, contradictory to the hyl1 ABA hypersensitive seed germination phenotype, suggesting an interaction between ABA, glucose, and miRNAs in the control of seed germination [DCL1 and HYL1 expression in lettuce and Arabidopsis seeds [DCL1 short integuments (sin1/dcl1-7), the hypomorphic mutant of HEN1 (hen1-8), and the null mutant of HYL1 (hyl1-2) are defective in asymmetric growth of the integuments, leading to abnormal ovule development and reduced female fertility [dcl1 and hyl1-2 mutants. Transcriptome analysis has identified many miRNAs specifically expressed in the embryo, but their molecular mechanisms of action remain largely unknown [Most dsRBD-type RBPs, such as DCLs, HYL1/HYL1 homologs and HENs, are localized to the nucleus and influence the biogenesis of various small RNAs ,66. In ae to ABA ,71. HEN1e to ABA . The hen unclear . Interesmination . HYL1 acmination ,76,77. Tis seeds . Mutatioertility ,80. Embr unknown .MIR156 subfamily or overexpression of MIR9678 increased seed dormancy and suppressed seed pre-harvest sprouting (PHS) [In crops, rice miR156 and wheat miR9678 have been reported to function in seed dormancy through the regulation of the GA and GA/ABA pathways, respectively. Dysfunctions in one ng (PHS) ,83. ThesCSPs are predominantly expressed in shoot apices and siliques at both the RNA and protein levels, but they have different roles in plant growth, seed development, and performance [CSP4 leads to reduced silique length and embryo lethality, whereas CSP2 knock-down plants show changes in vegetative development and flowering control [CSP1 confers sensitivity to dehydration or salt stress with a delayed germination phenotype. By contrast, CSP2-OE lines exhibit accelerated seed germination under salt stress conditions [CSPs are highly similar, their specific biological roles differ, and they deserve further study.GRPs are involved in alternative splicing or transcriptional regulation. Many GRPs function in various stress-related seed germination processes . The Araformance . The ove control ,87. Overnditions . CSP2 handitions . Intrigunditions . Thus, aGRP4 is induced by cold stress, markedly repressed by salinity, and slightly repressed by dehydration. Intriguingly, GRP4-OE seeds exhibit delayed germination under salt and dehydration stress, but no noticeable differences were observed under cold or freezing stress [GRP4, GRP7 is repressed by ABA, high salt, and mannitol treatment. Its knock-out mutant is hypersensitive to these stresses, displaying delayed seed germination and root development. Moreover, the grp7 mutant has significantly higher expression of RD29A and RAB18 than the wild type in response to stresses and ABA. It has therefore been proposed that GRP7 positively modulates gene expression in the ABA and stress signaling pathway [GRP7 knock-out mutant under NaCl or mannitol treatment, which suggested that GRP7 had a negative effect on abiotic stress response. This discrepancy may have arisen from differences between the two mutants or from differences in the stress treatments or plant growth conditions; more accurate experiments are needed to assess the effects of GRP7 on stress tolerance [Another two of the Arabidopsis eight GRPs, GRP4 and GRP7, also affect plant stress response. g stress . In cont pathway . Howeverolerance . In addiolerance .RZ-1A-OE seeds showed delayed germination, whereas faster germination and earlier seedling onset were observed in the loss-of-function mutant after salt, dehydration, and freezing treatments. Furthermore, seed germination was affected by ABA or glucose, implying that RZ-1A negatively regulated seed germination through an ABA-dependent pathway [RZ-1C expression observed in embryos, endosperm of germinated seeds, and newly initiated leaves and root tips [Arabidopsis RZ-1A, RZ-1B, and RZ-1C are characterized by a zinc finger motif between the RRM domain and the C terminus. RZ-1B and RZ-1C fall into one phylogenetic clade, whereas RZ-1A belongs to a separate group, suggesting that they may have different biological functions ,96. RZ-1 pathway ,98. By cOsGRP1, OsGRP4, and OsGRP6 in the Arabidopsis grp7 knockout mutant rescued the cold-sensitive germination phenotype of the grp7 mutant [Triticum aestivum) harbor three RZ proteins, referred to as OsRZ1–OsRZ3 and TaRZ1–TaRZ3, that are homologous to Arabidopsis RZs. The OsRZs showed noticeable up-regulation only in response to cold stress, whereas the expression of the three TaRZs was affected differently by high salt, drought, and cold stress. Only OsRZ2 affected seed germination under cold stress; the three TaRZ proteins had no significant effect under cold stress but had different effects on germination under high salt and dehydration [The rice genome encodes at least six GRPs, designated OsGRP1–OsGRP6. The overexpression of 7 mutant . Both riydration ,101. Thegun1 mutant is more sensitive to ABA treatment than abi4, suggesting that the germination regulation of gun1 may be independent of ABI4. Furthermore, a later study showed that both ABA and sucrose delay the seed germination rate of the gun1 mutant [ahg11), slow growth 1 (slg1), and slow growth 2 (slo2) show enhanced ABA hypersensitivity and delayed germination, and appear to act by editing mitochondrially encoded NAD genes of complex I  [cis-splicing of nad2 intron3 and stabilization of nad1 exon2–exon3 transcripts, respectively [nad1, nad2, nad4, and nad5) [abo5, ppr19, and abo6 mutant seeds also show delayed germination. Disruptions in PENTATRICOPEPTIDE REPEAT PROTEIN FOR GERMINATION ON NaCl (PNG) lead to an ABA hypersensitive phenotype with delayed germination, but it is unclear how PNG changes the transcript abundance of nad1, which encodes a complex I subunit [ppr40 mutant exhibits hypersensitivity of germination to ABA. No obvious alterations in RNA processes or in the abundance of mETC complex subunits were identified, but the ppr40 mutant had a strongly reduced capacity for complex III electron transport and accumulated more reactive oxygen species (ROS) [ppr96 loss-of-function mutant exhibited an ABA insensitive phenotype with a faster germination rate. Future identification of the mitochondrial targets and molecular mechanisms of PPR96 in ABA tolerance will deepen our understanding of mitochondrial function, ABA, and seed germination [PPR proteins are encoded by the nuclear genome but predominantly targeted to the chloroplasts and/or mitochondria, where they have a significant role in organelle RNA metabolism, biogenesis, and function . Many PP1 mutant . These ror nad7) ,115,116.ectively ,118. Thend nad5) . Likewis subunit . Some PPes (ROS) . In contmination .aba3-1. Dysfunction in MEF11 led to increased seed dormancy. Remarkably, after one month of ripening and stratification, mef11 mutant seeds still germinated slightly more slowly than the wild type. Moreover, hypersensitivity to the germination inhibitor paclobutrazol (a gibberellin biosynthesis inhibitor) and abiotic stresses (NaCl and mannitol) was also observed in mef11 mutant seeds. These alterations in seed behavior may result from a loss of editing of the mitochondrial cytochrome c maturation FN2 (ccmFN2) gene, which encodes a cytochrome c-heme lyase subunit required for cytochrome c generation. Increased respiration and altered production of hydrogen peroxide (H2O2) and nitrogen monoxide (NO) were observed in mef11 mutant seeds, as well as in the mitochondrial function-deficient ppr mutants described above [Recently, the PPR protein MITOCHONDRIAL EDITING FACTOR11 (MEF11), also known as LOVASTATIN INSENSITIVE1 (LOI1), was isolated from a suppressor screen of the ABA biosynthesis-deficient mutant ed above ,124.Both ROS and NO function as positive signals that contribute to the transition from seed dormancy to germination, and they probably act through ABA signaling ,126. Addwsl) mutant exhibits chlorotic striations on leaves. The WSL gene encodes a chloroplast-localized PPR, and its mutant shows enhanced sensitivity to ABA, high salt, and sugar, with a delayed seed germination rate. Both mis-regulation of splicing in the chloroplast ribosomal protein subunit L2 (RPL2) transcript and decreased accumulation of plastid rRNAs and translation products were observed in the wsl mutant and may have caused its sensitivity to abiotic stresses [ccmC, COX2, COX2, NAD2, and NAD4). The ogr1 mutant has pleotropic phenotypes, including delayed seed germination under normal conditions, but its mechanisms of action have not been described in detail [ppr8522 and emp4 embryos successfully germinated with sucrose supplementation, suggesting that a metabolic defect affects germination in the two mutant embryos [In rice, the white stripe leaf (stresses . OGR1 isn detail . In maiz embryos ,106.Plant RBPs are a huge family with diverse biological roles. Nonetheless, only few of them have been functionally characterized with respect to seed biology, including seed development, dormancy, and germination regulation . Seed ge


Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) proteins (APOBEC3s) are deaminases that convert cytosines to uracils predominantly on a single-stranded DNA, and function as intrinsic restriction factors in the innate immune system to suppress replication of viruses (including retroviruses) and movement of retrotransposons. Enzymatic activity is supposed to be essential for the APOBEC3 antiviral function. However, it is not the only way that APOBEC3s exert their biological function. Since the discovery of human APOBEC3G as a restriction factor for HIV-1, the deaminase-independent mode of action has been observed. At present, it is apparent that both the deaminase-dependent and -independent pathways are tightly involved not only in combating viruses but also in human tumorigenesis. Although the deaminase-dependent pathway has been extensively characterized so far, understanding of the deaminase-independent pathway remains immature. Here, we review existing knowledge regarding the deaminase-independent antiretroviral functions of APOBEC3s and their molecular mechanisms. We also discuss the possible unidentified molecular mechanism for the deaminase-independent antiretroviral function mediated by mouse APOBEC3. Humans and mice have at least 11 and 5 proteins, respectively, which belong to the activation-induced cytidine deaminase and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (AID/APOBEC) family. These include AID, APOBEC1, APOBEC2, APOBEC3A, 3B, 3C, 3D, 3F, 3G, 3H, and APOBEC4 for humans and AID, APOBEC1, APOBEC2, APOBEC3, and APOBEC4 for mice ,7,8. OneIn this review, following a brief introduction to the AID/APOBEC family members and the deaminase-dependent APOBEC3 function, we focus on the deaminase-independent antiretroviral functions of APOBEC3s. We also discuss one more possible molecular mechanism for the deaminase-independent pathway that mA3 seems to exert on MuLV replication.AID/APOBEC family members are evolutionarily conserved cytidine deaminases that act on the preferred nucleotide contexts of DNA and RNA . They plOf the 11 members of the human AID/APOBEC family, AID is thought to be the oldest, and was firstly identified as an essential factor for the function of activated B cells in the germinal center . AID actAPOBEC1 was the first member of the AID/APOBEC family to be identified and acts in lipid metabolism via its RNA editing activity. Editing a cytosine within the apolipoprotein B pre-mRNA encoding apolipoprotein B100 by this enzyme generates a new stop codon in the mRNA, resulting in the translation of a shorter form named apolipoprotein B48 ,29. SeveThe physiological function of APOBEC2 is proposed to be related to muscle development in mammals since it is expressed in skeletal and cardiac muscle tissues . HoweverLike APOBEC2, APOBEC4 does not exhibit deaminase activity, although it has a conserved deaminase domain with some differences from that of other AID/APOBEC family members ,51. APOBAPOBEC3 genes on chromosome 22 in tandem arrangement, APOBEC3A (A3A), APOBEC3B (A3B), APOBEC3C (A3C), APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H). The number of APOBEC3 genes varies among mammals: horses, cows, and rodents , for example, have six, three, and one per haploid genome, respectively. Differences in the number of APOBEC3 genes in mammals as well as in the amino acid sequences of APOBEC3s are likely due to positive selection in APOBEC3 molecular evolution, which is attributed to the long-lasting host-virus arms races [APOBEC3 gene has expanded in simian primates beyond the well-known APOBEC3 locus (chromosome 22 for humans being one example) by a molecular mechanism called retrocopying [Humans have at least seven ms races ,60,61,62ms races . Thirteeocopying . In retrC (where C is the target base) which is hardly targeted by AID that favors the WRC sequence  [C element, and among these APOBEC3s, the more preferred sequence seems to be TC depending on bases flanking the target C and DNA structures such as open or stem structures. As for functions, APOBEC3s exert antiviral activity against not only retroviruses including HIV-1 and endogenous retroelements but also HBV, adeno-associated virus, foamy virus, Epstein–Barr virus (EBV), coronavirus, and some other viral species [Although all APOBEC3 members convert cytosines to uracils in a single-stranded DNA, the preferred target sequences are different to varying degrees among members ,68,69,70R = A/G) . Other h species ,83,84,85Among APOBEC3s, human APOBEC3G was the first identified restriction factor against HIV-1 infection. HIV-1, however, antagonizes it with viral infectivity factor (Vif), a key regulator targeting APOBEC3G for proteasome-mediated degradation . APOBEC325–31-C-X2–4-C, where X represents any amino acid (2+) is coordinated by the histidine and two cytidines in the Z-domain. A hydroxyl group (–OH) derived from a water molecule is also coordinated to Zn2+ as the fourth ligand, and it performs a nucleophilic attack on the C4 site of the cytosine ring in the initial stage of the process of deamination reaction [All APOBEC3s contain one or two cytidine deaminase domains (CDs), which are also called zinc-coordinating domains (Z-domains), that consist of H-X-E-Xino acid  [59,62,1reaction . During reaction ,104. Intreaction . The NTDreaction ,110,111.When APOBEC3s are aberrantly expressed at the wrong time and/or in excess amount in human cells, its deaminase activity can generate mutations in host genomic DNAs irrespective of pathogen infection. Previously, more than 20 mutational signatures were extracted by analyzing somatic mutation patterns in genomes derived from several human cancers. The one among these mutational signatures could be due to the deaminase activity of APOBEC family members, which probably act in collaboration with DNA replication and/or DNA repair systems . In part+ T cells that express physiological amounts of APOBEC3s, most likely APOBEC3G restricts HIV-1 replication via inhibition of even the initial step of reverse transcription in addition to massive cytosine deamination on viral DNA [As mentioned above, it is clear that human APOBEC3G inhibits HIV-1 (ΔVif) through a deaminase-dependent mechanism. However, it has also been reported that it represses HIV-1 replication in a deaminase-independent manner ,126,127.iral DNA . A cataliral DNA . HIV-1 riral DNA . APOBEC3iral DNA . Furtheriral DNA ,132.Human T cell leukemia virus type 1 (HTLV-1) is sensitive to the deaminase-independent antiviral function of APOBEC3G . Human AHBV is also sensitive to the antiviral function of some human APOBEC3s ,138,139.The mammalian genome comprises a large number of transposons such as retroelements. The retroelements include endogenous retroviruses (ERVs), which are long terminal repeat (LTR)-positive elements, and L1, which is the most common among non-LTR elements . L1 is tParamyxoviridae, through a deaminase-independent mechanism [Of note, APOBEC3G appears to be able to interfere with replication of some RNA viruses including measles virus (MV), a negative-strand RNA virus that belongs to echanism . MV RNA Coronaviridae together with SARS-CoV, MERS-CoV, HCov-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1, all of which are pathogenic to humans with severe syndromes or general common colds. Human APOBEC3C, 3F, and 3H inhibited replication of HCoV-NL63 without heavy hypermutation [Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 and has already spread all over the world ,158,159.mutation . Both demutation . HoweverThe parvovirus adeno-associated virus requires a helper virus such as adenovirus for efficient viral replication in the cell nucleus . This viReplication of mouse gammaretrovirus, Friend or moloney MuLV, and betaretrovirus, mouse mammary tumor virus (MMTV), is inhibited by mA3 with few or no G-to-A mutations ,165,166.As described above, the deaminase-independent antiviral function of APOBEC3s is widely involved in the restriction of many types of virus, strongly suggesting that this function is as necessary as the deaminase-dependent function for the complete host defense system.Several molecular mechanisms for the deaminase-independent antiretroviral function of APOBEC3s have been reported .One of the known deaminase-independent antiretroviral mechanisms exerted by APOBEC3s is the inhibition of reverse transcription process in which, for instance, APOBEC3G inhibits the negative-sense (-), single-stranded DNA synthesis ,168. AddWe and others previously showed that mA3 restricted MuLV in their natural hosts without inducing significant levels of G-to-A hypermutation ,163,164.Recently, it has been shown that the P50 protein, which is translated from an alternative spliced MuLV gag RNA, binds mA3 in cells and inhibits its incorporation into viral particles . Since nThe critical question here is to what extent does each deamination-independent antiretroviral activity play a role in overall APOBEC3-mediated antiretroviral activity including the deaminase-dependent pathway. This would depend on a type of virus, APOBEC3s, and cells infected with pathogens.A deaminase-independent function of APOBEC3B influences tumor development ,183. ForWe predict there being at least one more deaminase-independent mechanism by which mA3 inhibits MuLV replication. Previously, we observed that mA3 reduces MuLV viral particle production in the supernatant of cells infected with MuLV or transfected with an MuLV molecular clone despite comparable amounts of viral proteins generated in these cells and the mA3-negative control cells . Martin 6-methyladenosine (m6A) modifications were identified in cellular mRNAs [6A-modified RNA isolation (m6A-seq) has enabled the location of the modification on RNAs to be identified [6A modification on cellular transcripts influences a wide range of RNA biology including RNA splicing, RNA nuclear export, translation, and RNA degradation in the processing-body (P-body), which is a cytoplasmic compartment involving mRNA stability and storage [6A modification on viral RNAs affects the viral life cycle [6A modification of MuLV gRNAs promotes viral gene expression and replication [5C), is also detected on cellular and viral RNAs, and has critical roles in RNA metabolism [5C modification on HIV-1 gRNA affects the alternative splicing of viral RNAs and the efficiency of translation [5C modification also accumulates on MuLV gRNA, and this modification appears to be required for nuclear export of the gRNA [5C residue on cellular mRNA is recognized by the YBX1 protein, and the mRNA is stabilized [6A and m5C [1G), and 7-methylguanosine (m7G). It was recently reported that cytidines of HIV-1 RNAs are acetylated by NAT10, and this modification results in stabilization of viral RNAs [Over 100 types of RNA modification on both noncoding and messenger RNAs have been identified so far, and knowledge of their physiological roles has been accumulating . Of thesar mRNAs . The deventified . The m6A storage ,199,200. storage ,205,206.fe cycle ,216,217.lication . Anothertabolism ,220,221.the gRNA ,221. Theabilized ,223. Cou and m5C . These mral RNAs . Howeverral RNAs . Among tral RNAs . Thus, iAll human and mouse APOBEC3s interact with DNA and RNA. The enzymatic activity exerted on mammalian genomes and viral single-stranded DNA has been well characterized. In contrast, the importance of a deaminase-independent function on cellular and viral RNAs is apparent, but its role remains obscure. Further studies deciphering this role are required for an improved understanding of APOBEC3 function. These analyses may enable the identification of a virus replication process that can be targeted by novel antiretroviral drugs.


Backgrounds: Hepatocellular carcinoma (HCC) is a lethal malignancy worldwide that is difficult to diagnose during the early stages and its tumors are recurrent. Long non-coding RNAs (lncRNAs) have increasingly been associated with tumor biomarkers for diagnosis and prognosis. This study attempts to explore the potential clinical significance of lncRNA DUXAP8 and its co-expression related protein coding genes (PCGs) for HCC.Method: Data from a total of 370 HCC patients from The Cancer Genome Atlas were utilized for the analysis. DUXAP8 and its top 10 PCGs were explored for their diagnostic and prognostic implications for HCC. A risk score model and nomogram were constructed for prognosis prediction using prognosis-related genes and DUXAP8. Molecular mechanisms of DUXAP8 and its PCGs involved in HCC initiation and progression were investigated. Then, potential target drugs were identified using genome-wide DUXAP8-related differentially expressed genes in a Connectivity Map database.Results: The top 10 PCGs were identified as: RNF2, MAGEA1, GABRA3, MKRN3, FAM133A, MAGEA3, CNTNAP4, MAGEA6, MALRD1, and DGKI. Diagnostic analysis indicated that DUXAP8, MEGEA1, MKRN3, and DGKI show diagnostic implications . Prognostic analysis indicated that DUXAP8 and RNF2 had prognostic implications for HCC . The risk score model and nomogram showed an advantage for prognosis prediction. A total of 3 target drugs were determined: cinchonine, bumetanide and amiprilose and they may serve as potential therapeutic targets for HCC.Conclusion: Functioning as an oncogene, DUXAP8 is overexpressed in tumor tissue and may serve as both a diagnostic and prognosis biomarker for HCC. MEGEA1, MKRN3, and DGKI maybe potential diagnostic biomarkers and DGKI may also be potentially prognostic biomarkers for HCC. By 2018, it was estimated that nearly 841,800 new liver cancer cases and 781,631 deaths due to liver cancers were recorded across 20 world regions Long non-coding RNAs (lncRNAs) regulate normal cellular functions et al. reported that DUXAP8 expression was positively correlated with lymph node metastasis and clinical stage in esophageal cancer DUXAP8, a pseudogene derived from lncRNA, promotes growth of pancreatic carcinoma cells by epigenetically silencing CDKN1A and KLF2 https://cancergenome.nih.gov/). Criteria of patient selection as following: patients were pathologically diagnosed primary HCC, vital data including HBV infection status, alcohol history, vascular invasion and radical resection should be collected, all the patients have definite alive/die status and survival time. LncRNAs function together with their co-expression-related PCGs and co-expression coefficient. Therefore, evaluated to acquire its PCGs was conducted using R 3.5.0 (https://www.r-project.org/) HCC patient expression data was downloaded from The Cancer Genome Atlas database  Body distribution of DUXAP8 and its top 10 PCGs were obtained from the Gene Expression Profiling Interactive Analysis database  curves of DUXAP8 and the PCGs were depicted using data from tumor and non-tumor tissue expressions using Graphpad 7.0. An Area Under Curve (AUC) value ≥ 0.7 represented high diagnostic significance. The joint-effect analysis was performed using the diagnosis-related lncRNA and genes.In addition, prognostic significance was calculated and visualized using Kaplan-Meier plots. Multivariate analysis was performed using prognosis-related clinical factors. The joint-effect analysis was also performed using prognosis-related lncRNA and genes.1 x β1 of gene1 + expression of gene2 x β2 of gene2 +… + expression of genen x βn of genenA risk score model and nomogram were constructed for HCC prognosis prediction. Risk scores, patient survival status, DUXAP8 gene expression heat maps, Kaplan-Meier plot, and time-dependent ROC curves were entered into the model. The risk score was formulated as follows: risk score = expression of geneThe nomogram was constructed using prognosis-related genes, DUXAP8, and clinical factors. Differential expressions and clinical factors indicate varied points. The 1, 3-, and 5- year overall survivals (OS) were included in the nomogram. OS prediction at 1, 3, and 5 year can be found accordingly from the points.GSEA was performed to assess molecular mechanisms of DUXAP8 and the PCGs. Gene ontologies (GOs), including biological processes , cellular components , molecular functions , and Kyoto Encyclopedia of Genes and Genomes  pathway sets were used to find the biological processes associated with DUXAP8 and its PCGs.https://string-db.org/) The co-expression matrix for DUXAP8 and its PCGs of Pearson correlation were constructed using R 3.5.0. Different colors indicate either positive or negative correlations. The GGI network was constructed and their co-expression relationship with other genes was determined using the geneMANIA plugin of Cytoscape software https://portals.broadinstitute.org/cmap/). Positively correlated drugs were considered as potential targets. The 2D and 3D structure of the target drugs were obtained from PubChem Compound (https://www.ncbi.nlm.nih.gov/pccompound/).Genome-wide differentially expressed genes (DEGs) in volcano plots and heatmaps were determined and plotted using the edgeR method https://david.ncifcrf.gov/) Enriched metabolic pathways and biological processes were analyzed using the Database for Annotation, Visualization and Integrated Discovery  and oncomine https://www.oncomine.org/resource/main.html) databases to confirm our results.For further confirming potential values of these PCGs in HCC, we further used HCCDB Survival and statistical analyses were performed using SPSS 16.0 . Median survival time, log-rank p-value, adjusted p-value, 95% confidence interval (CI) and hazard ratio (HR) were calculated using the Kaplan-Meier method and Cox proportional hazards regression model. A p-value of ≤ 0.05 was statistically significant.Supplementary Table 1. Co-expression-related PCGs were identified and are shown in Supplementary Table 2. The top 10 PCGs were; RNF2, MAGEA1, GABRA3, MKRN3, FAM133A, MAGEA3, CNTNAP4, MAGEA6, MALRD1, and DGKI (Table 1). Pearson correlation coefficient R was used to evaluate the correlation between these PCGs and DUXAP8. PCGs with an ∣R∣ value of ≥0.2 were visualized in the network .A total of 370 HCC patients were enrolled in this study. Clinical factors found to be linked with HCC were HBV infection, radical resection, and tumor stage OS . Detailed demographic characteristics are shown in RNF2 were highly expressed in liver in comparison to other PCGs . In comparison, DUXAP8 and RNF2 were highly expressed in kidney, lungs, and intestines relative to other PCGs. Except MALRD1, DUXAP8 and the other 9 PCGs were differentially expressed between tumor and non-tumor tissues . All PCGs and DUXAP8 were overexpressed in tumor tissues.Body maps of DUXAP8 and the top 10 PCGs showed that DUXAP8 and MEGEA1, MKRN3, and DGKI were found to be potential diagnostic markers . Other PCGs showed no diagnostic significance . The joint-effect analysis of DUXAP8, MEGEA1, MKRN3, and DGKI indicated that their joint effect provided better diagnostic significance than when applied individually , except for MAGEA1 and MKRN3. The AUC value of this combination was equal to that of MAGEA1 alone .In the diagnosis analysis, DUXAP8, DUXAP8, RNF2, and GABRA3 showed prognostic value in univariate analysis . However, only DUXAP8 and RNF2 showed prognostic value in multivariate analysis, after adjustment for tumor stage, HBV infection, and radical resection factors . Also, combination of DUXAP8 and RNF2 showed better prognostic value than individual effects .In the prognostic analysis, RNF2 . low DUXAP8 and RNF2 expressions were associated with low risk and hence better prognosis . Time-dependent ROC curves suggested that the risk score model could predict the 1 and 3 years survival rate . A nomogram was constructed using tumor stage, HBV infection status, radical resection status, and expressions of DUXAP8 and RNF2 . Low expressions of DUXAP8 and RNF2, tumor stage I, without HBV infection and radical resection, were correlated with low-risk scores, and hence better patient survival.A risk score model was constructed using risk scores, patient survival status, and expressions levels DUXAP8 and Figure ). KEGG pathway analysis revealed that DUXAP8 was associated with fatty acid metabolism, tryptophan metabolism, and the citrate cycle (TCA cycle) pathways . For RNF2, GO analysis revealed that it enriched in cell division, mitotic nuclear division, cell cycle checkpoint, sister chromatid segregation, chromatin modification and histone binding . KEGG pathways analysis showed that RNF2 was associated with oxidative phosphorylation, PPAR signaling pathway, and the TCA cycle pathways .GSEA analysis of GO terms for DUXAP8 revealed that it was enriched in mitotic nuclear division, cell cycle phase transition, positive regulation of cell cycle phase transition, meiotic cell cycle, cell division, centrosome, chromosome condensation, and histone binding . The GGI network indicated that CNTNAP4, MKRN3, DGKI, GABRA3, MAGEA6, and MAGEA1 were co-expressed with other genes . Many MAGE family members interacted with them. Proteins encoded by CNTNAP4, GABRA3, DGKI, MAGEA6, MAGEA3 and MAGEA1 genes show co-expressional and co-occurrence relationship .The co-expression matrix of DUXAP8 and PCGs revealed that most of them were positively correlated with each other. In the matrix, deep blue color indicated highly and positively correlated interactions . Detailed drug information is provided in Supplementary Table 3. The 2D and 3D structures of these three chemical compounds are in Figure 12. GO analysis was performed for the DEGs and results are shown in Figure 13. The DEGs were enriched in the synapse, extracellular region, postsynaptic membrane, ion channel complex, multicellular organismal development, and extracellular ligand-gated ion channel activity. The top 10 GO terms and KEGG pathways were: chemical synaptic transmission, ligand-gated ion channel activity, extracellular region, postsynaptic membrane, extracellular ligand-gated ion channel activity, neuroactive ligand-receptor interaction, and retrograde endocannabinoid signaling (Table 7). Detailed results of GO terms and KEGG pathways enrichment are shown in , respectively.DEGs were identified using edgeR and presented in heatmap and volcano plots as shown in MAGEA1  tissues indicating poor OS in PC patients making it a potential therapeutic target CDKN1A and KLF2, and by binding to EZH2 and LSD1DUXAP8, a 2107 bp RNA, was initially found to be overexpressed in gastric cancer (GC) tissues, its overexpression resulted in larger tumor size, advanced tumor stage, lymphatic metastasis, and poor prognosis of GC patients et al. found that DUXAP8 is upregulated in renal cell carcinoma (RCC) tissues and promotes the proliferation and invasion of RCC cells by downregulating microRNA-126 expression Zheng A genome-wide analysis revealed that DUXAP8 was highly expressed in esophageal cancer. GO enrichment analysis for DUXAP8 and its co-expressing PCGs showed that they were enriched in the cell cycle, cell division and DNA repair, suggesting an important role in the tumorigenesis and progression of esophageal cancer 19H22N2O) is a natural compound used as an antimalarial drug We found cinchonine, bumetanide, and amiprilose as the potential drugs targeting DUXAP8 in HCC. Cinchonine (COur study indicates that higher expression of DUXAP8 correlated with poor prognosis in HCC. We also found that DUXAP8 is involved in the mitotic nuclear division, cell division, regulation of cell cycle phase transition, histone binding, oxidative phosphorylation, and the TCA cycle pathways. Therefore, we infer that DUXAP8 functions as an oncogene in HCC. This conclusion is in agreement with previous reports in ESCC, PC, BC, GC, and RCC.Live cell imaging or readouts of active cellular processes have revealed that lncRNAs play various roles in cellular pathologies and can influence the efficacy of new therapeutic targets for cancer  SNF2 is overexpressed in tumor tissues leading to poor patient survival. Molecular mechanism results demonstrate that SNF2 is involved in cell division, mitotic nuclear division, cell cycle checkpoint, sister chromatid segregation, chromatin modification, histone binding, oxidative phosphorylation, PPAR signaling pathway, and the TCA cycle. Therefore, we speculate that SNF2 is an oncogene in HCC A recent study showed that downregulation of SNF2, a co-expression-related gene of DUXAP8, decreases cell growth and metastases of HCC cells. RNF2 promoted HCC cell proliferation by accelerating cell cycle progression SNF2 in several malignanciesSNF2 is an oncogene in HCC.We show thatThis study for the first time demonstrates the oncogenic role, diagnostic and prognostic value of DUXAP8 in HCC. This study has some limitations that need to be recognized. First, our findings concerning DUXAP8 and its PCGs need to be validated using a larger population. Second, well-designed functional trials are necessary to identify deeper mechanisms of DUXAP8 and its PCG in HCC. Thus, further clinical trials are required to assess the translational potential of DUXAP8.MEGEA1, MKRN3, and DGKI can be used to diagnosis HCC, while DUXAP8 and RNF2 can be used to predict the prognosis of HCC. In addition, we found that DUXAP8 and its PCGs demonstrated better diagnostic and prognostic performance when these factors were used jointly as opposed to single application. A risk score model and nomogram exhibited good prognostic prediction performance of DUXAP8 and its PCGs in HCC. Molecular analyses revealed that DUXAP8 and PCGs are involved in mitotic nuclear division, cell division, regulation of cell cycle phase transition, oxidative phosphorylation, and PPAR signaling pathway. Furthermore, three potential target drugs; cinchonine, bumetanide, and amiprilose, which were identified as candidate drugs targeting DUXAP8 in HCC. This study for the first time demonstrates the oncogenic role, diagnostic and prognostic value of DUXAP8 in HCC. Thus, further clinical trials are required to assess the translational potential of DUXAP8.This study investigated the functions of DUXAP8 and its PCGs in HCC. We found that DUXAP8 and its PCGs are differentially expressed in the liver and overexpressed in tumor tissues. Diagnostic and prognostic analysis indicated that DUXAP8, Supplementary figure.Click here for additional data file.Supplementary tables.Click here for additional data file.


Acute myeloid leukemia is mainly characterized by a complex and dynamic genomic instability. Next-generation sequencing has significantly improved the ability of diagnostic research to molecularly characterize and stratify patients. This detailed outcome allowed the discovery of new therapeutic targets and predictive biomarkers, which led to develop novel compounds , nowadays commonly used for the treatment of adult relapsed or refractory AML. In this review we summarize the most relevant mutations affecting tumor suppressor genes that contribute to the onset and progression of AML pathology. Epigenetic modifications , DNA repair dysregulation , cell cycle inhibition and deficiency in differentiation  as a consequence of somatic mutations come out as key elements in acute myeloid leukemia and may contribute to relapse and resistance to therapies. Moreover, spliceosomal machinery mutations identified in the last years, even if in a small cohort of acute myeloid leukemia patients, suggested a new opportunity to exploit therapeutically. Targeting these cellular markers will be the main challenge in the near future in an attempt to eradicate leukemia stem cells. TE. TETET2 rozygous . Both hoce in OS , althougce in OS . The fre 12%–34% . They oc 12%–34% . TET2 muutations ,53. Cleaoversial . RecentlWilms tumor 1 (WT1) is a tumor suppressor gene responsible for the development of familiar Wilms’ tumor, from which it takes its name . Th. Thadditrrations ,96. Othe mutants . Furtherhibitors . Lastly,hibitors .In this second section, we describe other frequently mutated AML tumor suppressors whose function is not involved in epigenetic mechanisms.nucleophosmin (NPM1), located on 5q35, encodes a nucleus–cytoplasm shuttling protein [NPM1 (NPM1c) support the importance of NPM1c as a cooperative event in leukemogenesis, but not to initiate leukemia [NPM1 mutations on prognosis has been extensively examined over the last decade. They can be found in 25%–30% of AML patients, and their frequency rises in adult AML (near 30%–40%), especially CN-AML [FLT3-ITD mutations, correlating with good response to conventional therapy and high complete remission rates, EFS and/or OS [FLT3 (fms-related tyrosine kinase 3) mutations. Indeed, co-occurrence of NPM1 and FLT3, either when the latter harbors the more common ITD or the less frequent D835 point mutations, significantly improves the response and the survival outcomes over that of an isolated FLT3 mutation, thus defining a highly favorable prognostic AML group [NPM1 mutations is generally used for monitoring MRD [NPM1 and FLT3 mutations is consolidated [DNMT3A, IDH1, IDH2 and TET2 mutations are identified as concomitant to NPM1 mutations [NPM1 may be involved in chromosomal translocations with ALK ), which represents the anaplastic large-cell lymphoma molecular landmark [RAR-α ), causing a subtype of acute promyelocitic leukemia (APL) [The gene  protein . In 2005 protein . Functio protein , DNA rep protein , ribosom protein . NPM1 is protein ,105, thu protein . They le protein ,107. As  protein . Further protein . Mouse mleukemia . The impy CN-AML ,110. Paty CN-AML  in the and/or OS . Risk asML group . Furtherring MRD ,115. Intolidated , with a olidated . Moreoveutations ,117, conlandmark  and withia (APL) .CEBPA, an intronless gene on chromosome 19q31.1, encodes for a zinc finger transcription factor [CEBPA or with a mutation in the CEBPA basic region displayed a complete block of myeloid differentiation and increased levels of hematopoietic stem cells [CEBPA gene occur in 5%–20% of de novo AML [CEBPA disrupt the DNA-binding and homodimerization domains, while out-of-frame insertions or deletions in the N-terminal result in abolishing the translation of full length CEBPA, leading to overexpression of the shorter p30 isoform [CEBPA mutations, therefore the 2016 WHO classification of myeloid neoplasms defined it as a distinct diagnostic entity [CEBPA are also associated with mutations in TET2, the most frequently co-mutated gene (34%), followed by GATA2 (21%), WT1 (13%), DNMT3A (9%) and ASXL1 (9%) [CEBPA and granulocyte colony-stimulating factor receptor (CSF3R) mutations has been reported [CEBPA biallelic mutations feature a CSF3R mutation; in these patients the co-occurrence induces a worsening outcome [n factor , which pn factor . In partn factor . Recentln factor . Moreoveem cells . The posem cells . The secem cells . The ratem cells . Mutationovo AML , in both isoform .The pecu isoform . Resultsc entity . MultiplXL1 (9%) ,132,133.reported ,135. Not outcome ,137.Tumor protein 53 (TP53) is a tumor suppressor gene located on chromosome 17p13.1. It regulates cell cycle arrest, apoptosis, senescence and DNA repair. It has been initially described as “the guardian of the genome” referring to its role in preserving genome stability through the prevention of mutations [+ ions and arginine amino acid residues; and a C-terminal homo-oligomerization domain (OD) [TP53 mutations, including hematological malignancies, where it has been observed mutated in 5%–20% of AML patients [TP53 mutations or deletions increases dramatically (approximately 70%) [TP53 mutations are associated with resistance to chemotherapy, poor OS and poor disease-free survival (DFS) [TP53 mutations occur in the region encoding the DNA-binding domain. Notably, six mutational hot-spots residues were identified R175, G245, R248, R249, R273 and R282, with R273 and R248 being the most frequently mutated [+/-TP53 clones [NPM1, FLT3, MDM2 and ARF) [MDM2 and MDMX overexpression [FLT3-ITD mutations, which also promote TP53 inactivation. Additional aberration including SIRT1 overexpression with subsequent TP53 deacetylation, CRM1 overexpression and nuclear export of TP53, destruction of TP53 regulator PML in PML-RARα positive AMLs and NPM1 mutations via dysregulation of ARF-MDM2-TP53 axis [utations . The encain (OD) . More thpatients . Howeverely 70%) ,141. TP5al (DFS) ,143. Theal (DFS) ,145. TP5al (DFS) . Thus, tal (DFS) ,143. The mutated ,147. Mut- clones . They arand ARF) ,149 but and ARF) ,149. Finpression , miRNA oP53 axis ,152,153 P53 axis . FinallyP53 axis .In this last section we will discuss other mutations, with high relevance in leukemogenesis but that occur less frequently in the AML mutational landscape. enhancer of zeste 2 (EZH2) gene encodes for a catalytic component of the polycomb repressive complex 2 (PRC2), and its role in epigenetic regulation is enacted through di- and trimethylation of lysine 27 of histone H3, thus leading to transcriptional repression. In normal hematopoiesis, EZH2 is involved in maintaining multipotency and self-renewal of HSCs [EZH2 gene is located on this chromosome arm and these patients are often resistant to chemotherapy and characterized by a poor prognosis [SRSF2 and U2AF1 (near 10% of AML patients) decreases EZH2 transcript [U2AF1 and inactivating EZH2 mutations are mutually exclusive [The  of HSCs ,155. Low of HSCs , but morrognosis ,156, Finanscript . For thixclusive . SF3B1, U2AF1 and SRSF2, are well established mutated genes in MDS, with different frequency (45–85%) depending on their subtypes [RUNX1, ASXL1, IDH2 and TET2 mutations, and they found an association with poor prognosis in term of shorter DFS and OS.Splicing factors (SFs), notably subtypes ,159,160.subtypes . They idmiRNA play a pivotal role in sustaining AML by dysregulating several processes such as proliferation, apoptosis, quiescence and disease progression ,163. It GATA2 gene encodes for a transcription factor involved in the regulation of hematopoietic stem cell activity and self-renewal [GATA2 mutations are identified in MDS [GATA2 gene mutations are also found in de novo AML [GATA2 mutations are often associated with CEBPA biallelic mutations and, with lower incidence, to NPM1 and FLT3 mutations [CEBPA biallelic and GATA2 mutations show a more favorable prognosis and better OS than those with CEBPA biallelic mutations alone [GATA2 mutations frequently occur in Emberger syndrome, in monocytopenia and mycobacterial infection (MonoMAC) and in secondary AML [The -renewal  and in m-renewal . GATA2 md in MDS  in blastd in MDS . In addinovo AML ,176, beinovo AML . Interesutations . Furtherns alone . Curiousdary AML ,180,181.NPM1 and FLT3, but also the choice of treatment, since some of them become therapeutic targets   . In addi


Background: The present study aims to evaluate the diagnostic roles of various immunohistochemical (IHC) markers in thymic tumors, including thymic carcinoma (TC) and thymoma (TM). Methods: Eligible studies were obtained by searching the PubMed databases and screening the searched articles. Thirty-eight articles were used in the present meta-analysis and included 636 TCs and 1861 TMs. Besides, for IHC markers with statistical significance, a diagnostic test accuracy review was performed. Results: The comparison of various IHC expressions between TC and TM was performed for 32 IHC markers. Among these IHC markers, there were significant differences between TC and TM for beta-5t, B-cell lymphoma 2 (Bcl-2), calretinin, CD1a, CD5, carcinoembryonic antigen (CEA), cytokeratin19 (CK19), CD117, glucose transporter 1 (Glut-1), insulin-like growth factor 1 receptor (IGF-1R), mesothelin, MOC31, mucin1 (MUC1), p21, and terminal deoxynucleotidyl transferase (TdT). Markers with higher expressions in TCs were Bcl-2, calretinin, CD5, CEA, CD117, Glut-1, IGF-1R, mesothelin, MOC31, MUC1, and p21. Among these markers, there were no significant differences between TC and TM type B3 in immunohistochemistries for Bcl-2 and CK19. On the other hand, β-catenin and CD205 showed a considerable difference in IHC expressions between TC and TM type B3, but not between TC and overall TM. In diagnostic test accuracy review, MUC1 and beta-5t were the most useful markers for TC and TM, respectively. Conclusions: Taken together, our results showed that the expression rates for various IHC markers significantly differed between TC and TM. The IHC panel can be useful for differentiation from limited biopsied specimens in daily practice. Thymic epithelial tumors (TETs), which originate from thymic epithelial cells, include thymoma (TM), thymic carcinoma (TC), and thymic neuroendocrine tumors ,2. TheseAlthough various IHC expressions were reported, single specific markers for each TET are not available. In our study, we evaluated the IHC expression patterns of TCs and TMs, alongside the performance of a diagnostic test accuracy review for various IHC markers.Relevant articles were obtained by searching the PubMed database through 31 January 2020. For searching, the keywords used were as follows: “thymic carcinoma or thymoma” and “immunohistochemistry”. The titles and abstracts of all searched articles were screened for inclusion and exclusion. Included articles had the information for the immunohistochemistry of the TC and TM. However, case reports, non-original articles, or those written in English were excluded from the present study. The PRISMA checklist shown in the Data associated with various IHC expressions of TC and TM were extracted from each of the eligible studies ,42,43,44Q and 2I statistics and expressed as p-values. Additionally, sensitivity analysis was conducted to assess the heterogeneity of eligible studies and the impact of each study on the combined effects. Due to the use of various evaluation criteria and tumor types in the eligible studies, a random-effect model rather than a fixed-effect model was determined to be more suitable for this meta-analysis. The Begg’s funnel plot and Egger’s test were performed to assess publication bias, with fail-safe N and trim-fill tests additionally used to confirm the degree of publication bias if found. The results were considered statistically significant at p < 0.05. The diagnostic test accuracy review of various IHC markers was performed using R software ver. 3.6.3 . We calculated the pooled sensitivity, specificity and diagnostic odds ratio (OR) according to individual data collected from each eligible study. By plotting the sensitivity and 1-specificity of each study, the summary receiver operating characteristic curve (SROC) was able to be constructed with curve fitting performed via linear regression. Due to all of the data being heterogeneous, accuracy data were pooled by fitting the SROC and measuring the area under the curve (AUC).A meta-analysis was performed using the Comprehensive Meta-Analysis software 2.0 package . The expression rates of various IHC markers were investigated by dividing them into TC and TM markers. Comparisons of IHC expressions between TC and TM type B3 were also performed. Heterogeneity between the studies was checked using n = 105), non-human studies (n = 81), and a language other than English (n = 34) (n = 636) and TM (n = 1861).In this study, 934 relevant articles were searched for on the PubMed database and reviewed for a meta-analysis. Of these, 409 articles had no or lack of sufficient information for a meta-analysis. In addition, 267 were excluded due to non-original articles. Among the remaining articles, 220 reports were excluded for the following reasons: articles in other diseases ((n = 34) . Finally(n = 34) . These sFirst, the significant differences in IHC expressions between TC and TM were investigated. There were significant differences in immunohistochemistry for beta-5t, B-cell lymphoma 2 (Bcl-2), calretinin, CD1a, CD5, carcinoembryonic antigen (CEA), cytokeratin19 (CK19), CD117, Glut-1, IGF-1R, mesothelin, MOC31, MUC1, p21, and TdT . Among tThe diagnostic test accuracy reviews were performed for candidates of IHC markers, which were showed the statistical differences between TC and TM type B3. Five positive markers, including CD5, CD117, Glut-1, and IGF-1R, MUC1, and four negative markers, including beta-5t, CD1a, CD205, and TdT, were included in the present analysis . Among tAlthough the prognosis of each subtype of TETs is not clear, the prognosis of Type B3 TMs is clearly different from other subtypes of TMs and TC ,45,46; hTETs can be differentiated by histological characteristics ; howeverVarious tumors can occur in the anterior mediastinum, including TETs, germ cell tumors, and metastatic tumors. Differentiating between TET subtypes can be useful when deciding on treatments and predicting prognoses. Due to the similar histological findings of TC and TM type B3, diagnoses from small biopsied specimens are challenging in daily practice. However, single and specific IHC markers for each tumor are not yet defined. Specific markers have high expression in the target and low expression in the reference. According to our results, positive markers may be suitable for CD5, CD117, MUC1, and Glut-1 in differentiating TC from TM type B3. On the other hand, beta-5t, CD1a, and TdT are considered to be negative markers. In this study, we initially evaluated the statistical differences between TC and TM using odds ratios. In addition, we analyzed the expression rates of each IHC marker with statistical significance between TC and TM. These results should be considered before applying diagnostic markers in daily practice. Diagnostic implications, regardless of the statistical significance, are limited when IHC expression rates are high in both compared subgroups. Therefore, our results showed that the estimated expression rates were useful for the selection of an IHC panel.Kim et al. suggested that an IHC panel using EZH2, CD117, and CD205 was useful for differentiation between TC and TM type B3 . In the The direct invasion and metastasis of primary lung cancers in the anterior mediastinum can be detected . Based oThis study has a limitation in that we performed the diagnostic test accuracy review for individual IHC markers, but it was difficult to conduct the diagnostic test accuracy review for a combination of markers due to limited eligible study information. Therefore, the recommended IHC panels could not clarify the diagnostic role of the differentiation of thymic masses.In conclusion, our results showed that significant differences in IHC expression between TC and TM identified positive markers, including CD5, CD117, Glut-1, IGF-1R, and MUC1, and negative markers, including beta-5t, CD1a, CD205, and TdT against TC. An IHC panel including positive and negative markers, as well as CD205, could be useful to differentiate between thymic masses in daily practice.


Heat shock proteins (HSPs) constitute a large family of conserved proteins acting as molecular chaperones that play a key role in intracellular protein homeostasis, regulation of apoptosis, and protection from various stress factors . Apart from their intracellular localization, members of different HSP families such as small HSPs, HSP40, HSP60, HSP70 and HSP90 have been found to be localized on the plasma membrane of malignantly transformed cells. In the current article, the role of membrane-associated molecular chaperones in normal and tumor cells is comprehensively reviewed with implications of these proteins as plausible targets for cancer therapy and diagnostics. Heat shock protein (HSP) families consist of constitutive and stress-inducible members such as HSPB , DNAJ HSP40), HSPA HSP70), HSPC HSP90), HSPH HSP110) and their related chaperokines HSPD/E HSP60/HSP10) and CCT (TRiC) 10 and th,3, their0, HSPA  inducible form of Hsp70 (HSPA1A)  and (2) the ER-localized glucose-regulated protein 78 (GRP78) (HSPA5)  have bee123 the radiolabeled chaperone accumulated inside the tumor . S. S109]. O-linked carbohydrate moiety . P. P161]. Listeria monocytogenes exhibited high levels of mHsp60 [L. monocytogenes infections (104–106 CFU/mL) increased the expression of mHsp60 and, as a result, enhances cell adhesion, but not invasion [Hsp60 plasma membrane localization was shown for Daudi cells . Applicaf mHsp60 . Intriguf mHsp60 . Furtherinvasion . Subsequ2+-dependent manner to phosphatidylserine (PS) with high affinity (DK = 1.5 × 10−5 M) [Calreticulin, another ER-residing stress protein present on tumor cell membranes, was shown to induce migratory capacity in melanoma cells . Applica 10−5 M) ,178. Fur 10−5 M) ,180. ThuHsp40 (Hdj1) was shown to be associated with Hsp70 on the surface of CX+ colon and Colo+ pancreatic carcinoma cells . PreviouLarge parts of sHSPs  are embedded into lipid bilayers (a) or they can be present on the cell surface (b) ,166,167.Apart from their important intracellular functions as molecular chaperones in regulating intracellular proteostasis, many HSPs have been shown to be expressed on the surface of various types of solid and hematological malignancies. An increased expression density of mHSPs is associated with tumor progression, resistance to anti-tumor therapies , and an enhanced invasive and metastatic potential. Certain membrane-bound HSPs, including mHsp60, mHsp70 and their endoplasmatic analogues mGRP78, mGRP96, and membrane calreticulin, were demonstrated to induce innate and adaptive anti-cancer immunity. Exposure of the intracellular endoplasmatic chaperones on the plasma membrane of tumor and, in some cases, damaged normal cells, followed by subsequent activation of immunity represents a possible mechanism of immunologic surveillance for elimination of malignantly transformed or damaged cells. Some membrane-bound chaperones  could also play a role in tumor cell association with extracellular matrices, thus influencing cell motility and invasion. Exposure of HSPs on the membrane is directly related to the composition of the lipid bilayer. Presumably, pharmacological modulation and/or modification of membrane lipid structures and microdomains could be further exploited in regards to chaperone expression, which in turn could be used for cancer therapies ,187,188.


Large indoor gatherings pose a high risk for transmission of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), and have the potential to be super-spreading events  test results from nasopharyngeal swab specimens. Both persons reported attending a wedding reception on August 7, and both experienced onset of fever, cough, and sore throat on August 11. Three more persons who reported attending the same reception received positive SARS-CoV-2 test results the next day, prompting initiation of an outbreak investigation by MeCDC on August 14.MeCDC used the Council of State and Territorial Epidemiologists’ COVID-19 case definitions . MeCDC developed transmission chains using MicrobeTrace The reception, attended by 55 persons, was held on August 7 in a rural Maine town located in county A. The town had a total population of approximately 4,500 persons and no previously reported COVID-19 cases. COVID-19 incidence in county A before the reception was 97 cases per 100,000 persons. The bride, groom, and groom’s family (seven persons) traveled from California to Maine on August 6. In compliance with the governor of Maine’s executive order,The reception was held at a lodging establishment in county A that had an attached restaurant and four dining areas, including the event room, breakfast room, bar, and an open deck. Guests were seated indoors in the event room, which had 10 tables, with 4–6 guests seated around each table. The total number of wedding guests (55) exceeded Maine’s 50-person limit for indoor gathering in a shared space. Facility staff members had conducted temperature checks for all guests at the facility entrance; these were reported as normal. Although the facility had signs posted at the entrance instructing visitors to wear masks, guests did not comply with this requirement nor maintain a physical distance of ≥6 feet, and staff members did not enforce these measures; all staff members wore masks. The facility did not collect contact information from guests. A member of the wedding party informed MeCDC of the total number of guests but did not provide their contact information. MeCDC investigators linked cases to the event by backward tracing,https://stacks.cdc.gov/view/cdc/96482). Fifty-one (89.5%) patients were symptomatic, and 51 (89.5%) cases were confirmed  reported cough onset on August 10 and attended an in-person school meeting the same day. Two school staff members subsequently received diagnoses of COVID-19 on August 14 and 17. Local schools delayed reopening by 2 weeks while all exposed staff members completed isolation or quarantine.https://stacks.cdc.gov/view/cdc/96482) on August 8 and 9. Patient B1 reported fever, chills, cough, myalgia, runny nose, and headache on August 11, but nevertheless worked on August 11 and 12; patient B1 was tested for SARS-CoV-2 on August 13 and received a positive result on August 18.After the reception, one of the guests (patient A2) had a close interaction with patient B1 (A2’s parent), a health care worker at long-term care facility (LTCF) A . During August 15–19, patient A3 worked daily 8-hour shifts in two separate correctional facility housing units while symptomatic. On August 19, four staff members, including patient A3, received confirmation of COVID-19 diagnoses, which led MeCDC to initiate an investigation at this facility.One wedding guest (patient A3) was a correctional facility staff member and reported onset of cough, myalgia, runny nose, sore throat, and a new onset loss of taste sensation on August 14 . Investigation revealed noncompliance with CDC’s recommended mitigation measures.To mitigate transmission, persons should avoid large gatherings, practice physical distancing, wear masks, stay home when ill, and self-quarantine after exposure to a person with confirmed SARS-CoV-2 infection.


Acidithiobacillus ferridurans JAGS, determined using PacBio single-molecule real-time (SMRT) sequencing. The circular genome of JAGS  contains 3,001 protein-coding sequences, 46 tRNAs, and 6 rRNAs. Predicted genes indicate the potential to fix CO2 and N2 and to utilize Fe2+, S0, and H2 as energy sources.We report a complete genome sequence of  Acidithiobacillus ferridurans JAGS, determined using PacBio single-molecule real-time (SMRT) sequencing. The circular genome of JAGS  contains 3,001 protein-coding sequences, 46 tRNAs, and 6 rRNAs. Predicted genes indicate the potential to fix CO2 and N2 and to utilize Fe2+, S0, and H2 as energy sources.We report a complete genome sequence of  Acidithiobacillus is of importance because of its adaptation to extremely acidic and heavy metal-rich environments  sample collected in September 2010 from Clarabelle Mill, near Sudbury, Ontario, Canada. Govindarajan  and then was cultured in 9K liquid medium  of the predicted 16S rRNA gene of strain JAGS was performed. The average nucleotide identity based on BLAST (ANIb) was calculated using JSpeciesWS , 2,833 proteins, 46 tRNAs, and 6 rRNAs. The functional annotation of the CDSs indicated the potential to use ferrous iron, reduced sulfur compounds, and hydrogen as energy resources and to fix COCP044411, BioProject accession number PRJNA573091, BioSample accession number SAMN12799170, and SRA accession number SRR10222673.The complete genome sequence has been deposited in GenBank under accession number


Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite the progress of new treatments, the risk of recurrence, morbidity, and death remains significant and the long-term adverse effects in survivors are substantial. The fraction of cancer stem-like cells (CSCs) because of their self-renewal ability and multi-lineage differentiation potential is critical for tumor initiation, growth, and resistance to therapies. For the development of new CSC-targeted therapies, further in-depth studies are needed using enriched and stable MB-CSCs populations. This work, aimed at identifying the amount of CSCs in three available human cell lines , describes different approaches based on the expression of stemness markers. First, we explored potential differences in gene and protein expression patterns of specific stem cell markers. Then, in order to identify and discriminate undifferentiated from differentiated cells, MB cells were characterized using a physical characterization method based on a high-frequency dielectrophoresis approach. Finally, we compared their tumorigenic potential in vivo, through engrafting in nude mice. Concordantly, our findings identified the D283 human cell line as an ideal model of CSCs, providing important evidence on the use of a commercial human MB cell line for the development of new strategic CSC-targeting therapies. Around 300,000 children and adolescents worldwide are diagnosed with cancer each year ,2,3,4. AIt has been hypothesized that only a small subset of tumor cells can initiate and support tumor growth. These rare stem cells, called cancer stem cells (CSCs), possess tumorigenic ability with marked capacity for proliferation, self-renewal, and differentiation potential ,17,18. IThrough a multidisciplinary approach, here we discriminated undifferentiated cell populations in three different MB cell lines , as representative of different molecular subtypes . Potentip = 0.0158), together with clear-cut reduced expression levels of SOX2 , Nestin  and Ki67 . Remarkably, they showed an increased level of CD44 differentiation surface marker, , reduced expression of CD44  and GFAP relative to DAOY cells . Phenotypic characterization carried out in this study showed a huge heterogeneity for stemness/differentiation-related markers, especially between D283 and DAOY cells and, importantly, that their expression levels were not influenced by oxygen culture conditions (p < 0.0001). In addition to CD133, we analyzed CD15, which reported a significantly higher percentage of CD15-positive cells in D283 cells (52.5%) than D341 (23.3%) and DAOY , strongly highlighting a cancer stem-like phenotype of D283 cells.DAOY cells generated an extremely low rate of medullospheres (MS), characterized by large and regular shapes A–C. The p = 2 × 10−5). On the contrary, DAOY-MS showed very similar crossover frequencies compared with their parental counterpart. The box plots graphic representation illustrates the median together with the min/max crossover frequency for each cell population  identify CSCs with distinct levels of differentiation [CD44 and CD24 have been widely analyzed in combination with other stem cell markers to isolate CSCs from a solid tumor ,40,41. Wntiation . Remarkantiation ,46. Thisntiation ,48,49,50ntiation . SimilarAlthough the physiological response to external stimuli, given by culture media enrichment, remains an important tool to increase the percentage of CD133+ cells, the novelty of our study consists in the identification of a commercial ready-to-use cell line for improving the study of CSC biology of a tumor with a high risk of mortality. Although the functional role and signaling mechanisms of CD133 are not well understood, the high expression of CD133 is associated with resistance of CSCs to chemotherapeutic agents ,53,54 anFurthermore, D283 cells showed a significantly higher level of CD133+/CD15+, making them more useful to study signals transduction pathways generated from both membrane receptors and to develop new therapeutic approaches compared to DAOY cells, expressing in basal conditions low levels of double-positive cells ,58.Importantly, we exploited a novel method to discriminate the cell differentiation status using real-time measurements in a microfluidic lab-on-chip (LOC) platform implemented in CMOS technology ,59,60. T2 incubators unless they were exposed to hypoxia in an H35 hypoxic chamber  in an atmosphere of 2% oxygen, 5% carbon dioxide, and balanced nitrogen.Human MB cell lines  were obtained from American Type Culture Collection . Cell lines were routinely maintained in complete growth medium Eagle’s Minimum Essential Medium (MEM) with 2 mM glutamine and 100 U penicillin/0.1 mg/mL streptomycin. The DAOY and D283 medium were supplemented with 10% fetal bovine serum, while the D341 line was maintained in complete growth medium MEM with 20% fetal bovine serum. Cells were cultured in standard COTo evaluate the capacity to form MS, cells were plated at clonal density (1–2 cells/mm2) into ultra-low attachment 24-well plates in selective medium . After 7 days in culture, MS size and number were evaluated.To assess the initiating cell frequency of MB cell lines, cells were cultured in standard conditions until ready for passaging and then seeded serial dilutions of cells ranging from 1 to 250 cells/well in ultra-low attachment 96 well plates . Cells were then cultured in selective medium  for one additional week. The proportion of wells in which sphere formation was not observed was measured and initiating cell frequency (F) calculated according to the Poisson distribution.RNA isolation from cells was performed with RNeasy Mini Kit . After quantification, 2μg of total RNA was reverse transcribed with a high-capacity cDNA Reverse Transcription Kit , and qPCR was carried out as previously described  OligonucCells were lysed as previously described  and immu6 cells; Miltenyi Biotec, Bergisch Gladbach, DE); Alexa Fluor 647 mouse anti-Nestin, Alexa Fluor 488 mouse anti-Ki-67, PerCP-Cy5.5 mouse anti-Sox1, PerCP-Cy5.5 mouse anti-Sox2, FITC mouse anti-CD44, Alexa Fluor 488 mouse anti-β-Tubulin Class III , Alexa Fluor 647 mouse anti-GFAP and PE-Cy5 mouse anti-CD24  and mouse anti-CD15-FITC clone MMA BD Biosciences, cat n° 332778), all used at 4µl/106 cells. Briefly, MBs cell lines were harvested, fixed in BD Cytofix Fixation Buffer, and permeabilized with BD Phosflow Perm Buffer III  according to the manufacturer’s instructions of Stemflow human Neural lineage kit. Samples were stored in Perm Buffer at −20 °C for at least 30 min. After recovering, cells were stained with the above-described antibodies for 30 minutes. Samples were analyzed by a CytoFLEX flow cytometer . Data are presented as the percentage of positive cells in the live-gated cell population determined by physical parameters.MB cells were characterized for their expression of stemness/differentiation markers through the BD Stemflow™ Human Neural Lineage Analysis Kit , although with some modifications . In partThe HF–DEP crossover frequency is defined as the value able to move cells from negative to a positive DEP state ,63. In thttps://biit.cs.ut.ee/clustvis) [Integration of stem cell properties displayed by MB cell lines was generated by applying PCA to the following parameters obtained from 3 independent experimental replicates: Percentage of CD133, CD15, Sox2, Ki67, Nestin, CD24, βIII-tubulin, Sox1, CD44, and GFAP positive cells, cross-over frequency (MHz), number and area of neurospheres, neurosphere forming ability (%) and initiating cell frequency (%). The graph was generated by unit variance scaling of data and SVD calculation of components through the ClusVis web tool  suspended in 200 µL of Matrigel (BD Biosciences) were injected subcutaneously into mice (double or single flank). Inoculated animals were monitored daily and tumors measured with a caliper twice a week. Tumor dimension was estimated using the formula: Tumor volume = length x width2/2 and considered positive when tumor mass was ≥ 400 mm3. This animal study was performed according to the European Community Council Directive 2010/63/EU, approved by the local Ethical Committee for Animal Experiments of the ENEA, and authorized by the Italian Ministry of Health (n° 80/2017-PR).Female nu/nu CD1 mice (n = 14) of 6–8 weeks of age, were purchased from Charles River Laboratories Italia , and housed in sterilized filter-topped cages kept in laminar flow isolators, fed with autoclaved food and water ad libitum, and maintained in 12 h light/dark cycle. Aliquots of 6 × 10p) was calculated by two-tailed Student’s t-test. The crossover frequency data were analyzed using a one-way ANOVA test. Final tumor incidence was determined using Fisher’s exact test. All analyses were carried out using GraphPad Software, San Diego, CA, USA.All quantitative data were presented as mean ± SD and statistical significance (Altogether, our results provide evidence that D283 cells, characterized by an intrinsic ubiquitous expression of CD133, are endowed with stem-like features, are extremely tumorigenic, and characterized by peculiar dielectric characteristics. These features are valuable for designing biologically relevant experimental models in clinically oriented studies, making this cell line instrumental for the study of CSCs biology and for the development of more effective therapies against MB.


The present study aimed to contribute to the catalog of genetic mutations involved in the carcinogenic processes of uterine sarcomas (USs) and carcinosarcomas (UCSs), which may assist in the accurate diagnosis of, and selection of treatment regimens for, these conditions.We performed gene-targeted next-generation sequencing (NGS) of 409 cancer-related genes in 15 US , 5 UCS, and 3 uterine leiomyoma (ULM) samples. Quality, frequency, and functional filters were applied to select putative somatic variants.TP53 (48%), ATM (22%), and PIK3CA (17%) were the most frequently mutated genes. With respect to specific tumor subtypes, ESS showed mutations in the PDE4DIP, IGTA10, and DST genes, UCS exhibited mutations in ERBB4, and ULMS showed exclusive alterations in NOTCH2 and HER2. Mutations in the KMT2A gene were observed exclusively in ULM and ULMS. In silico pathway analyses demonstrated that many genes mutated in ULMS and ESS have functions associated with the cellular response to hypoxia and cellular response to peptide hormone stimulus. In UCS and ADS, genes with most alterations have functions associated with phosphatidylinositol kinase activity and glycerophospholipid metabolic process.Among the 23 samples evaluated in this study, 42 loss-of-function (LOF) mutations and 111 missense mutations were detected, with a total of 153 mutations. Among them, 66 mutations were observed in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. This preliminary study observed pathogenic mutations in US and UCS samples. Further studies with a larger cohort and functional analyses will foster the development of a precision medicine-based approach for the treatment of US and UCS. Sarcomas are rare heterogeneous tumors that affect the female genital tract and originate from tissues such as muscle, fat, bones, and fibrous tissue. Uterine sarcomas (USs) are the most commonly occurring gynecological sarcomas, representing 90% of the total cases . Based oJAZF1-SUZ12 gene fusion (YWHAE-NUTM2 gene rearrangement (previously termed YWHAE-FAM22). Recently, molecular alterations in ZC3H7B-BCOR, BCOR-ITD, EPC1-BCOR, JAZF1-BCORL1, and BRD8-PHF1 have been identified. This histological subtype demonstrates more aggressive clinical behavior and worse prognosis , it is a valuable contribution to the understanding of the pathogenesis of such tumors.Several genetic alterations have been associated with USs and UCSs, with few alterations being associated with specific histological subtypes. For instance, ESSs can be divided into two types: low-grade ESS (LG-ESS) and high-grade ESS (HG-ESS), both characterized by recurrent chromosomal translocations. In LG-ESS, the most common translocation, t 7; 17] p15; q21), is observed in almost 50% of the cases and results in the ; q21, ise fusion , reveale; 17 p15;rognosis . Howeverrognosis  demonstrBRCA1 and MED12 as well as the loss of expression of the proteasome β1i subunit LMP2 have been associated with ULMS development , CDKN2A, epidermal growth factor receptor (EGFR), HER2, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), platelet-derived growth factor receptor (PDGFR), and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PI3KCA) during the development of UCS. In addition, mutations in TP53, PTEN, protein phosphatase 2 scaffold subunit alpha (PPP2R1A), F-box, and WD repeat domain containing 7 (FBXW7) have already been identified, which may contribute to the development of therapeutic alternatives including the use of the inhibitors of PARP, EZH2, cell-cycle, and PI3K pathway  human samples including 14 ULMS, 12 ESS, 2 ADS, 12 UCS, and 3 ULM-non-cancerous tumor (as reference samples). All samples were obtained via surgical procedures performed between 2000 and 2012 at the Institute of Cancer of Sao Paulo (ICESP) and Clinics Hospital of the Faculty of Medicine, University of Sao Paulo (HCFMUSP). Tissues were stored at the molecular and structural gynecology laboratory (LIM-58) of the University of Sao Paulo Medical School (FMUSP).This study was performed in accordance with the Declaration of Helsinki and was approved by the Research Ethics Committee of the FMUSP with protocol number 477/15. Patients' medical records were revised and the following data were recorded: age at diagnosis, postmenopausal bleeding, adjuvant treatment, presence of metastasis or recurrence, and status.® according to the manufacturer’s instructions. DNA concentration, purity, and integrity were assessed by spectrophotometry  and fluorometry (Qubit - Thermo Fisher Scientific), respectively.Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit obtained from QIAGENTM Library kit 2.0 protocols, with 10 ng of input DNA per pool, totaling 40 ng of DNA from each sample. The FuPa reagent was used to partially digest primer sequences and phosphorylate the amplicons. Next, sequencing adaptors and barcodes were ligated to the amplicon by the enzyme Ligase using the Ion Xpress™ Barcode Adapters kit (Thermo Fisher Scientific), which were then purified using magnetic beads . Subsequently, emulsion PCR was performed using the Ion PI™ Hi-Q™ OT2 200 Kit (Thermo Fisher Scientific), followed by sequencing with Ion PITM Hi-QTM sequencing 200 and Ion PITM Chip.Sequencing libraries were prepared using the Ion Torrent Ampliseq Comprehensive Cancer Panel - Catalog number: 4477685 (Thermo Fisher Scientific), which contains ∼16,000 primer pairs multiplexed into 4 pools. This commercial panel was designed to assess the mutational profile of 409 cancer driver genes and drug targets along with signaling cascades, apoptosis genes, DNA repair genes, transcription regulators, inflammatory response genes, and growth factor genes . Prior tThe results were analyzed using the Torrent Suite v5.0.5 software (Thermo Fisher Scientific). Sequence variants (SNVs and indels) were identified using the Torrent Variant Caller (Ion Torrent - Thermo Fisher Scientific) and compared to the GRCh37 / hg19 genome version. VCF files were analyzed using VarSeq v1.8 software (GoldenHelix) for variant annotation and prioritization. The variants were filtered based on the quality and frequency criteria: coverage (>100), genotype quality score cutoff (GQS>50), variant base in at least 5% of reads, variant base present in at least 2 reads in each direction, homopolymer-length error<5, absence of genetic variants in population databases  or minor allele frequency (MAF)≤0.01%.Subsequently, variants were selected based on their effect on protein expression, with the following being considered: 1) variants described in the COSMIC database; 2) loss-of-function variants - Frameshift variants-nucleotide insertions/deletions, gain/loss of stop codons, splice site alterations); or 3) missense variants  predicted as possibly pathogenic in at least three of six prediction programs used  and occurring in oncogenes or tumor suppressor genes in OncoMD database. Variants not previously described in the COSMIC database were visually inspected using the integrative genomics viewer (IGV) program to exclude sequencing artifacts.Construction of genetic interaction networks was performed using Cytoscape platform version 3.7.0, which uses data from protein and genetic interactions, pathways, co-expression, co-localization, and protein domain similarity.Initially, 40 US and UCS  and 3 ULM samples were selected from the pathology department files; however, only 23  remained until the end of NGS analyses. Some losses occurred while performing multiplex PCR reactions (AmpliSeq™), during which we observed a high degree of fragmented DNA and many genetic artifacts in several samples. These issues are expected since tissue processing for paraffin inclusion and long storage time causes damage to the DNA structure (integrity). The clinical and pathological features of 40 patients with US and UCS who were enrolled in this study are summarized in Among the 23 samples deemed suitable for the evaluation of sequencing data, homogeneity average was 73.2%, median base coverage was 1257X, and horizontal coverage was 84.3% corresponding to 100X. Based on the NGS data, we selected point mutations with possible impacts on the function of the protein encoded by the altered gene  and small insertions and deletions (indels). Total variants detected in each sample and filtered variants for the selection of somatic alterations of interest are presented in An average of 1700 alterations were identified per sample (ranging from 746 to 3521), with an average of 1606 single nucleotide variants (SNVs) (ranging from 678 to 3406), 40 insertions (ranging from 23 to 77), and 55 deletions (ranging from 25 to 114). To select relevant somatic variants, a first filter was applied focusing on the quality and frequencies of these alterations. A second filter, focusing on variant functions and effects, was used to select the alterations that would be most relevant in alterations of gene functions. Collectively, in 23 samples that were evaluated, 42 LOF mutations and 111 missense mutations were detected, with a total of 153 filtered mutations, among which 66 were found in the COSMIC database .TP53 (11/23 - 48%), ATM (5/23 - 22%), and PIK3CA (4/23 - 17%) were the most frequently mutated genes.Among the 409 genes included in the panel, mutations were detected in 94 distinct genes, with 30 genes demonstrating mutations in more than one sample and 64 genes showing mutations in a single sample. ATM, TP53, and KMT2D) among the ULMS, ESS, and UCS samples. Nineteen genes were shared between 2 types of tumors, and 68 genes were mutated in a single type. Among them, 6 genes were mutated in more than one sample of the same histological subtype, namely, PDE4DIP (3 ESS samples), ITGA10, and DST (2 ESS samples), NOTCH2, and HER2 (2 ULMS samples), and ERBB4 (2 UCS samples). Quantitatively, this analysis shows similarities in the mutational profiles of ULMS and ESS, with 6 mutated genes in common (6.7%) between both subtypes. In the genes JAK3, APC, ATRX, CREBBP, MYB, and SYNE1, most of the mutations were characterized as missense mutations; however, in the SYNE1 gene, the two mutations observed in ULMS and ESS samples were determined as LOF mutations . In addition, mutations in the TRRAP, DNMT3A, EPHA7, KAT6B, and PRKDC genes indicate that UCS and ADS may exhibit molecular similarities.The Venn diagram  shows thATM and CREBBP), and ULM119 (benign tissue) with 2 mutations (MET and PDGFB). http://www.cbioportal.org/). ATM mutations were observed in 22% of the samples, with 3 missense-type mutations (2 ULMS and 1 ESS) and 2 LOF-type mutations (1 ESS and 1 UCS). PIK3CA appeared to be the third most mutated gene (17%) present in 3 UCS samples, with most of the mutations determined as the missense-type. APC, MTOR, DICER1, TRRAP, KMT2D, TSC2, PDE4DIP, and JAK3 showed a 13% mutational frequency. LOF mutations in PDE4DIP was found exclusively/specificaly in the ESS samples. NF1, CREBBP, and MYB demonstrated a 9% mutational frequency. Missense mutations in CREBBP and MYB were associated with ULMS and ESS (4 mutations in ULMS and 2 in ESS).Based on the data described in MTOR, PDK1, MDM2, TP53, CREBBP, NOTCH1, and HIF1A) and peptide hormone stimulus . Since uterine sarcomas are histologically classified into two primary subtypes, we used the same classification to study the association of the mutated genes with pure sarcomas (ULMS - ESS) and mixed tumors (UCS - ADS). PI4K2A, PIK3CA, PIK3CB, ATM, PI4KB, PIK3CG, PIK3C2B, PI4KA, PIK3C2A, PIK3C3, PIK3C2G, and PIK3CD) and glycerophospholipid metabolic process . Although UCS is no longer classified as uterine sarcoma but as metaplastic carcinoma, we included this tumor group in the analysis shown in Collectively, our results indicate that despite the molecular heterogeneity demonstrated by USs and UCSs, they share similarities in their mutational profiles. In addition, genetic interaction networks indicate that alterations in functions associated with hypoxia, response to peptide hormone stimulus in ULMSs and ESSs, and phosphatidylinositol kinase activity and glycerophospholipid metabolic process in UCS and ADS can influence the carcinogenic process of these tumors. Considering that NGS technology can provide a reliable molecular portrait of neoplasms quickly and cost-effectively , these rPIK3CA, DICER1, AR, and NF    . The exp(LG-ESS) . These f(LG-ESS) . HoweverNOTCH1 was the unique gene that shared mutations in the UCS and ULMS. Similarly, mutations in the DAXX gene have also been observed in the cases of ESS/ADS and ULMS/ADS, which share mutations in TSC2. Thus, our results suggest that besides exhibiting a similar tumor microenvironment, USs and UCSs also share genetic alterations. This observation is relevant to the understanding of the onset and evolution of these tumors. Furthermore, ULMS cases originating from ULMs have been reported; however, this hypothesis has not been proven yet , ITGA10 (c.2104G>A), and DST (c.16429C>T) genes. The variant PDE4DIP c.214C>T is described in the COSMIC database . The variant ERBB4 c.2513G>A is described in the COSMIC database  and is described in diffuse large B cell lymphoma and pancreatic ductal adenocarcinoma (PDAC). The NOTCH2 c.7223T>A variant is also pathogenic (score 0.85) and has already been described in meningioma, a primary non-malignant CNS tumor , ATM (2 ULMS/2 ESS/1 UCS), and KMT2D (1 UCS/1 ULMS/1 ESS) genes could be indicators of similarities in neoplastic progression. As specific signature genes, ESS exhibited mutations in the PDE4DIP, IGTA10, and DST genes. UCS showed mutations in the ERBB4 gene, and ULMS demonstrated exclusive alterations in the NOTCH2 and HER2 genes. Mutations in the KMT2A gene were observed exclusively in ULM and ULMS samples, and therefore, are potentially involved in the malignant transformation process. According to the Cytoscape platform, many genes that were mutated in the ULMS and ESS samples exhibit functions associated with the cellular response to hypoxia and peptide hormone stimulus. In UCS and ADS, most altered genes exhibit functions associated with phosphatidylinositol kinase activity and glycerophospholipid metabolic process. More studies should be conducted with a larger number of samples and functional analyses. However, the current screening contributes to the characterization of the complex genetic profile of USs and USCs.Using a platform to profile mutations in a panel of 409 genes, we identified that Da Costa LT and Dos Anjos LG were responsible for study conceptualization, literature organization and paper elaboration. Kagohara LT collaborated in analyses of data, manuscripts and reviews. Torrezan GT and De Paula CAA contributed to the study execution. Baracat EC and Carraro DM provided intellectual support. Carvalho KC analyzed the literature, critically reviewed the manuscript, supervised the research and developed the original idea.


TP53, HER2/ERBB2) were associated with molecular subtypes . The main finding was an increased tumor mutational burden (TMB) in IBC translating in the presence of many actionable genetic alterations (AGAs) with low frequency. Recently, a larger and more homogeneous series was reported [TP53, NOTCH2, MYH9, BRCA2, ERBB4, POLE, FGFR3, ROS1, NOTCH4, LAMA2, EGFR, BRCA1, TP53BP1, ESR1, THBS1, CASP8, and NOTCH1. The analysis was not corrected for multiple tests.Inflammatory breast cancer (IBC) is the most aggressive clinical form of breast cancer. Despite therapeutic progresses, ~50% of patients die from metastatic relapse. Since the last two decades, efforts have been made to better characterize IBC molecular biology and identify new therapeutic targets. High-throughput molecular analyses have been applied to clinical samples , mainly reported . In a moBased on these observations, we launched a large multicentric comparative NGS study of non-pretreated primaries from IBC and non-IBC patients treated in our institutions, pooled with publicly available NGS data . IBC wasTP53 (63%), HER2/ERBB2 (30%), MYC (27%), PIK3CA (21%), BRCA2 (14%), CCND1 (13%), GATA3 (13%), NOTCH1 (12%), FGFR1 (11%), and ARID1A (10%). We identified 96 genes differentially altered between IBC and non-IBC, including 95 more frequently altered in IBC such as TP53, genes involved in DNA repair (BRCA2) and NOTCH pathways, and only one (PIK3CA) more frequently altered in non-IBC. Genes such as EZH2 and SMARCA4, involved in chromatin remodeling, were also more frequently altered in IBCs, providing a rationale for the evaluation of epigenetic modifiers. Interestingly, 37/96 differential genes were more frequently altered in metastases than in primaries of non-IBC patients, suggesting a possible link of these genes with proclivity to metastasize.The 10 most frequently genes we found altered in IBCs were Ninety-seven percent of IBCs displayed at least one AGA. This percentage was higher than in non-IBCs (87%). We analyzed six specific drug classes and functional pathways. In four classes, and even if the percentage of patients with AGAs in IBC did not remain significantly superior  or inferior (class of PI3K/AKT/mTOR inhibitors) to that of patients with AGAS in non-IBC in multivariate analysis, the percentages were high in IBC patients. Such observation suggests that, like non-IBC patients, IBC patients may benefit from these inhibitors. Of note, an expression signature associated with sensitivity to palbociclib showed a higher score in IBCs than in non-IBCs.ATM, ATRX, BARD1, BRCA2, ERCC3, MSH2, MSH6, PMS2, and POLE, confirming recent findings [Two functional pathways gave particularly interesting results. Several genes involved in DNA repair were more frequently altered in IBCs such as findings . The perNOTCH1 was the most frequently altered NOTCH gene in IBC (12%), and NOTCH2, and NOTCH4 were more frequently altered in IBC compared with non-IBC, as reported [Alterations in the NOTCH pathway were almost twice more frequent in IBC (30% vs 17% in non-IBC). reported . NOTCH tbona fide option in this aggressive disease, notably with drugs targeting immune system, DNA repair, NOTCH signaling, and CDK4/6. Functional and clinical validation is warranted and clinical trials are ongoing. But clearly, analysis of larger series of IBC samples with larger-scale NGS , RNA-Seq, epigenomics and other technologies is needed, though collaborative studies we launched. Such analyses will allow better assessment of structural variations, mutational signatures, clonality and epigenetic alterations and could reveal the etiology of this mysterious and devastating disease.In conclusion, the genomic landscape of IBC is different from that of non-IBC, independently from molecular subtypes and stage. The high percentage of patients with AGA suggests that precision medicine is a