The results showed that berry development is concomitant with transcriptome profile changes, which was more pronounced at the véraison (V) stage. Despite that the downregulation pattern of gene expression dominated the upregulation through berry development, the C5 genotype maintained higher expression levels. Comparative transcript profiling allowed the identification of 94 differentially expressed genes with potential relevance in regulating fruit secondary metabolism, including 18 transcription factors and 76 structural genes. The genes underlying the critical enzymes in the modification reactions of polyphenolics biosynthetic pathway, including hydroxylation, methylation, and glycosylation were more pronounced during the immature stages of prevéraison (PrV), V, and postvéraison (PoV) in the C5 genotype, resulting in more accumulation of biologically active phenolic/flavonoid derivatives. The results suggested that muscadine grapes, as in bunch grapes (Vitis sp.); possess a similar mechanism that organizes polyphenolics accumulation; however, the set of total flavonoids (TFs) and structural genes coordinating the pathway varies between the two species.Muscadine grapes accumulate higher amounts of bioactive phenolics compared with other grape species. To identify the molecular events associated with polyphenolic accumulation that influence antioxidant capacity, two contrasting muscadine genotypes (C5 and C6) with varied phenolic/flavonoid content and antioxidant activity were investigated  Vitis that is divided into two subgenera with a varying number of somatic chromosomes (Euvitis (bunch grapes) displays a large group that is classified based on the geographical origin into three groups, including the European (V. vinifera), the East Asian (V. amurensis), and the North American species  , unlike other Muscadinia species, are commercially valuable growing mainly for fresh fruit and wine production but with lesser popularity than the European grapes  . Howeverpopenoei . The musn grapes . Howevern grapes . Addition grapes . Such nun grapes .Polyphenolic compounds are a large group of plants’ secondary metabolites synthesized in multiple sequential pathways, including the pentose phosphate, shikimate, and phenylpropanoid pathways . They haVitis vinifera) has been sequenced and annotated  small) genotypes C5-9-1 (C5) and C6-10-1 (C6), grown at the experimental vineyard of the Florida A&M University . These two breeding lines were developed under the grape breeding program of the Center for Viticulture and Small Fruit Research. They were selected according to their diversity in TPC, TFC, and antioxidant activity  were considered to be expressed (K-means clustering was performed with the Hartigan-Wong Algorithm and based on inputs of log2 relative Transcripts Per Million (TPM) values.The Illumina sequencing of the multiplexed RNA-seq libraries yielded 24 FASTQ files of sequences. Reads quality was checked by FASTQic v0.39 . The triic v0.39 . All theic v0.39 . Differeic v0.39 . For conxpressed –5. The wxpressed . FinallyR2 = 0.8). A module eigengene (ME) value, which summarizes the expression profile of a given module as the first principal component, was calculated and used to evaluate the association of modules with berries biochemical property  and antioxidant activity  of C5 and C6 genotypes at different developmental stages  and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were assigned using the g:Profiler website by applying the Benjamini–Hochberg multiple testing correction method with R < 0.05 . Howeverinterest .MrActin and MrEF1, which showed high stability across the different muscadine genotypes and tissues used. The geometric mean of the selected housekeeping genes was validated as an accurate normalization factor.The DNase-treated RNA (5 μg) was reverse transcribed in a reaction of 50 μl using the High Capacity cDNA Reverse Transcription Kit . The gene-specific primers were designed using Primer Express  . The reaOur previous work built an extensive classification of metabolome profile throughout the six berry developmental stages of contrasting C5 and C6 muscadine genotypes, showing an apparent reduction in the TPC and TFC along with the progression in berry development. However, the differences between the two genotypes were manifested during the developmental stages of V, PoV, and ripening (R). The C5 genotype displayed higher TPC and TFC levels, hence more pronounced DPPH and FRAP antioxidant activities were detected . To idenPFDR < 0.05, log2fold change > 1.5 or < –1.5) of each comparison generated by DESeq2 or EdgeR pipelines were considered (stages) and C6 (C6stages) genotypes resulted in 10,116 and 10,543 non-redundant DEGs, respectively , but roughly 50% of the transcripts during the FS stage . The four pairwise transcriptome comparisons  identified 746, 5647, 2479, and 1996 DEGs, respectively; and 7772 non-redundant transcripts in all comparisons (V-C6V) represented approximately 50% of the total redundant/non-redundant DEGs  in C5stage and C6stage comparisons were shared within the C5stages/C6stages comparisons , the acquired DEGs from each analysis were assigned to each module (r2 ≥ –0.74). However, the ME13 and ME14 modules involving 717 and 3035 DEGs were positively correlated with TPC and DPPH (r2 ≥ 0.79). The ME13 further showed a positive correlation with TFC and FRAP (r2 ≥ 0.75). The ME3, ME6, and ME11 modules exhibited a distinct correlation profile of both negative and positive correlations. For instance, the ME11 was the most extensive module with 5317 DEGs that were positively correlated with TFC (r2 = 0.93) but negatively correlated with the ABTS, NORS, and CUPRAC (r2 ≥ –0.76). A detailed description of the module-trait correlations is presented in The WGCNA system biology approach was utilized to construct co-expression networks based on pairwise correlations of non-lowly expressed genes of the 24 samples. Sixteen modules were identified and labeled in distinct colors shown in a hierarchical clustering dendrogram and network heatmap , 8. Nexth module . Hence, h module . The ME1V. vinifera Ensembl GeneID  with their TPM values, confirming the RNA-seq expression data s  was remarkably evident at the V stage , we decin data s . For conn data s –8. GenerNIC), the arogenate dehydratase/prephenate dehydratase (ADT), and the UDP-phenylpropanoid glucosyltransferases (TOGT)  at the same stages. Interestingly, a strong peak signal of TOGT was detected at the PrV stage of C5, exhibiting ∼175-fold higher transcript abundance and sharply decreased afterward. In general, all genes related to the shikimic and amino acids pathway were more abundant in C5 than in the C6 genotype, particularly at the PrV and V stages, excluding the chorismate mutase2 gene (CM2).For genes involved in the shikimic and amino acids pathway (group-1), 3 out of 12 genes showed distinct accumulation patterns, including the nicotinamidase (s (TOGT) . RelativO-feruloyltransferase2 (HHT2) expression displayed a shifted pattern in C6 genotype toward induction during the late stages of PoV and R. However, the expression pattern of the caffeoyl-CoA O-methyltransferase (CCoAOMT) was indistinguishable between the two genotypes before it peaked by ∼6.5-fold at the R stage of C6.The comparable accumulation pattern was also noted for gene-encoded enzymes involved in the hydroxycinnamic acids pathway (group-2). Only two out of the 14 genes coordinating the pathway exhibited differential expression profiles between the two genotypes . The omeCAD1) and the vestitone reductase (VR) genes displayed high accumulation levels that occurred mainly at the PrV stage, reaching ∼7.5- and ∼3.5-fold higher in C5 genotype, respectively (STS1-3) that manifested higher levels at the V, PoV, and R stages of C6, suggesting the intense activation of the pterostilbene pathway during the late developmental stages of the C6 genotype.Regarding the lignin and pterostilbene pathway (group-3), the cinnamyl alcohol dehydrogenase 1 (ectively . HoweverCHI), 2-hydroxy isoflavanone dehydratase (HID1/2), flavonoid 3′-monooxygenase 1 (F3H1), flavonoid 3′5′-hydroxylase (F3′5′H), flavonol sulfotransferase (FLS), leucoanthocyanidin reductase 1 (LAR1), anthocyanidin 5,3-O-glucosyltransferase-like (RhGT), anthocyanidin reductase (ANR), anthocyanidin 3-O-glucosyltransferase (UFGTs), glutathione S-transferase (GSTU17), and ABC-transporter (ABCs) (CHS), 2-hydroxy isoflavanone dehydratase 3 (HID3), flavonoid 3′-monooxygenase 2 (F3H2), leucoanthocyanidin reductase 2 (LAR2), and flavonoid 3,5-methyltransferase (FOMT). Additionally, the detoxification efflux carrier genes (DTX1/2) responsible for the removal of toxins from the cytosol were also higher in C5 (FS) displayed significantly higher levels in C6, excluding the seeds. These data revealed that the expression of genes associated with flavonoids/anthocyanins biosynthetic pathway mainly during the PrV, V, and PoV stages is probably responsible for the differences between C5 and C6 genotypes, regarding the TPC/TFC accumulation and antioxidant capacity (In the flavonoids/anthocyanins pathway (group-4), several genes exhibited similar accumulation profiles between the two genotypes, considering that both genotypes are colored, albeit higher transcription levels were detected in C5 during PrV, V, and PoV stages such as chalcone isomerase (r (ABCs) . Howeverer in C5 . Howevercapacity .MYB86, MYB17, and SPLs) or positive  regulators of the phenylpropanoid/flavonoid pathway generally displayed a similar accumulation pattern between the two genotypes. However, higher mRNA levels were detected in C5 during PrV, V, and PoV stages albeit a few exceptions. They showed a rapid decline in their transcription levels along with berry development. Nevertheless, three TFs displayed strong mRNA signals detected at one or more developmental stages of the C5 or C6 genotype (MYBF1 and MYB1L), the homeobox-leucine zipper protein anthocyaninless (ANL1), and bHLH1. The MYBF1 sharply peaked at the PoV, reaching ∼75-fold higher in C5 compared with C6 that retained at basal low levels after the PrV  are involved in the turnover of vacuolar phenolic metabolites (O-anthraniloyltransferase (AMAT), the flavin-containing monooxygenase (FMO), and the UDP-rhamnose: rhamnosyltransferase (GRT4s) are enzymes involved in the production of O-methyl anthranilate, formation of the epicatechin 3′-O-glucoside, and metabolism of phenolic compounds, respectively  showed expression induction at the V stage of C5, while the HMA1 was more abundant in the seeds of both genotypes.Finally, the genes in group-6 exhibited a similar expression pattern. The peroxidase proteins  tissue, which is consistent with previous results using different grape cultivars   . The Ente (EntB) . TOGT ene (EntB) . The TOGopanoids . Furtheropanoids . The conopanoids . Certainnt in C6 . The higCHS, HID3, F3H2, F3′5′H, LAR2, and FOMT. The CHS catalyzes the first step in the flavonoid biosynthesis, while HID enzyme activity is a critical limiting factor in the isoflavonoids biosynthesis (FMOT) involved in the flavonoids methylation. The alteration in the enzymes underlying the modification reactions of the flavonoid pathway, such as methylation and glycosylation reactions can modify and enhance the bioactivity of the flavonoids and their derivatives , which catalyze the production of dihydroflavonols, and late biosynthetic genes (LBGs) that lead to the biosynthesis of proanthocyanidins and anthocyanins . FlavonoMYBF1, MYB90, and MYB1L were strongly accumulated in C5. The MYBF1 acts downstream TCP to stimulate the EBGs pathway  of FS in muscadine transcriptome. In V. vinifera, the VvMYBPA1, VvMYBPA2, and VvMYB5a are required for triggering the EBGs , and F3′5′H , methylation (via FOMT), and glycosylation (via UFGT) are highly activated in C5, resulting in the production of more biologically active flavonoid derivatives.The transcription activators  pathway . Consistelopment . Howeverthe EBGs . The VvM and LAR . Interesd F3′5′H . In bothepressor . Concord the SLP . It coulPRJNA775666.The original contributions presented in the study are publicly available. This data can be found here: National Center for Biotechnology Information (NCBI) BioProject database under accession number AI: conceptualization, methodology, validation, formal analysis, software, writing—original draft preparation, and visualization. AD: methodology, investigation, formal analysis, and writing—review and editing. MP: methodology, formal analysis, and software. PG: methodology, formal analysis, and investigation. VT and KS: conceptualization, and writing—review and editing. IE-S: conceptualization, methodology, validation, formal analysis, resources, data curation, writing—review and editing, visualization, supervision, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A deeper understanding of these molecular interactions might provide the rationale for novel and more effective therapeutic combinations to treat sarcomas.Aberrant bioactivity of the insulin-like growth factor (IGF) system results in the development and progression of several pathologic conditions including cancer. Preclinical studies have shown promising anti-cancer therapeutic potentials for anti-IGF targeted therapies. However, a clear but limited clinical benefit was observed only in a minority of patients with sarcomas. The molecular complexity of the IGF system, which comprises multiple regulators and interactions with other cancer-related pathways, poses a major limitation in the use of anti-IGF agents and supports the need of combinatorial therapeutic strategies to better tackle this axis. In this review, we will initially highlight multiple mechanisms underlying IGF dysregulation in cancer and then focus on the impact of the IGF system and its complexity in sarcoma development and progression as well as response to anti-IGF therapies. We will also discuss the role of Ephrin receptors, Hippo pathway, BET proteins and CXCR4 signaling, as mediators of sarcoma malignancy and relevant  The IGF system regulates a variety of physiological processes including aging, glucose metabolism, growth, and differentiation ,2,3. AltThe IGF system includes multiple membrane receptors (IGF1 receptor (IGF1R), the insulin receptor (IR), the mannose-6-phosphate receptor M6P/IGF2R), and the insulin receptor-related receptor (IRR)), three ligands , and six circulating IGF binding proteins (IGFBPs) R, and th,13,14. TDownstream responses elicited by each receptor strongly depend on specific ligand affinities. The IGF1R shows the highest affinity for IGF1 > IGF2 >> insulin. The IR-A displays the highest affinity for insulin > IGF2 >> IGF1 while the IR-B displays the highest affinity for insulin >> IGF2 > IGF1 .The other two receptors of the family are the IGF2R and IRR. The IGF2R is a type-1 transmembrane glycoprotein which lacks intrinsic kinase activity and acts by scavenging extracellular IGF2, thus titrating IGF2 levels and suppressing IGF signaling . On the IGF2 transcript stability at post transcriptional level by modulating IGF2 production in embryos and tumor cells [The three ligands of the system have high homology, with 67% identity between IGF1 and IGF2 and 45–52% sequence homology between IGF1/2 and insulin. However, they are regulated by distinct secretion mechanisms and elicit different properties . Insulinor cells . Importaor cells . IGFBPs or cells ,28. In aor cells ,30. Notaor cells .Ligand binding to the extracellular subunits of IGF1R, IR, or HR induces tyrosine transphosphorylation on the intracellular tyrosine kinase domain ,17. PhosIn addition, IGF1R can directly phosphorylate the Janus kinases (JAK)-1 and -2, which are involved in cytokine-mediated signaling ,35,36. PGH-R) gene, are protected from cancer development [The IGF system is involved in the onset and progression of hematopoietic, mesenchymal and epithelial tumors ,37,38. Eelopment . On the elopment . In addielopment . In epidelopment ,44. Howeelopment ,46,47,48Collectively, the IGF axis modulates major cancer hallmarks  includinThe evidence that various cellular and viral oncogenes require an active IGF1R for neoplastic transformation  supportsIGF1R gene, and its association with oncogenesis and cell survival, has been discovered in subsets of osteosarcoma, a percentage of breast tumors, melanoma, pancreatic adenocarcinoma and gastrointestinal stromal tumors [FN1-IGF1R, which has strong promoter activity for the kinase domain of IGF1R, has been identified in a subset of inflammatory myofibroblastic tumors [IGF1 and frameshift indel in IGFBP5 genes has been identified in osteosarcoma (please refer to [IGF2R is present in various tumors (please refer to [IGF2 imprinting (LOI), leading to biallelic IGF2 expression and enhanced bloodstream secretion levels in Wilms’ tumors and other tumor types including hepatoblastoma, glioma, testicular, colorectal cancers and others, driving malignant processes . Loss-orefer to ) and favrefer to . Epigenerefer to ,46). A rsed IR-A .IGF1R and IR in tumor cells [TMPRSS2-ERG, EWS-WT1, and PAX3-FKHR, sustain IGF1R expression in prostate cancer, desmoplastic small cell tumor and alveolar rhabdomyosarcoma, respectively [EWS-FLI favors IGF1 but inhibits IGFBP3 transcription in Ewing sarcoma [WT1, BRCA1, VHL, lead to enhanced IGF1R gene expression [TP53 mutations upregulated IGF1R expression, as well as INSR, IGF2 and IGFBP3 [IGF2 transcription, as a potential mechanism for tumorigenesis [IGF2 transcription, particularly in cells expressing mutant p53 protein [Alterations of the IGF axis often depend on aberrant expression or activity of transcription factors. Overexpression of HMGA1 and Sp1 drives enhanced expression of the or cells ,67,68. Cectively ,70,71,72 sarcoma . Mutatiopression ,75,76. Td IGFBP3 ,78,79. Iigenesis ,81. Acco protein ,81. In a protein . Accordi protein . This fi protein .INSR exon 11 skipping thereby inducing IR-A expression [IGF2 and IGF1R occurs via the de novo expression of the oncofetal RNA-binding proteins IGF2BPs  in different tumor types [Alterations in the IGF system depend also on dysregulated post-transcriptional mechanisms. Aberrant alternative splicing is associated with prevalent expression of the IR-A vis à vis the IR-B in cancer cells ,83. For pression . As recepression . In addipression . Enhanceor types ,87,88,89Aberrant IGFBPs modulation leads to increased IGFs bioavailability and binding to the IGF1R or the IR-A and contributes to IGF dysregulation in cancer. Abundance of the metalloproteinase ADAM28 in tumor cells contributes to cell proliferation and metastases through the selective proteolysis of IGFBP3 and the consequent enhanced availability of IGF1 . SimilarPTEN) gene include both loss-of-function (deletion on chromosome 10) and gain-of-function (PTENΔ51) mutations [IGF1 promoters and increases IGF1 production [IGF activity in cancer is also altered by increased expression or constitutive activation of downstream signaling. Alterations in the tumor suppressor phosphatase and tensin homologue  anti-IGF1R monoclonal antibodies (mAbs); (ii.) tyrosine kinase inhibitor small molecules; (iii.) IGFs neutralizing antibodies and ligands TRAP. A major limitation in the use of these agents is still the lack of a clear rationale in patient recruitment, which may have significantly contributed to poor clinical results. Accordingly, most of the studies were performed in unselected patients further demonstrating that identification of predictive biomarkers to guide patients’ selection still represents an urgent need to optimize the use of anti-IGF agents .mAbs against the IGF1R not only block the binding of IGFs to IGF1R but also downregulate IGF1R levels by inducing receptor internalization and degradation, thereby inhibiting receptor downstream signaling ,102,103.Of note, hyperglicemia represents the major side effect correlated with the use of IGF1R mAbs . Indeed,The compensatory activation of other RTKs is a common mechanism by which tumor cells develop intrinsic or acquired resistance, thus limiting the antitumor effects of these agents. A phase II study evaluating the efficacy and toxicity of figitumumab in patients with squamous cell carcinoma of the head and neck reported a 41% incidence of hyperglycemia and no clinically significant activity of the mAb in patients . ImportaGanitumab and teprotumumab are currently being tested in clinical trials. Importantly, due to overall lack of IGF1R mAbs efficacy as monotherapy, ganitumab is being tested in combination with other drugs including the Src family knase (SFK) inhibitor dasatinib NCT03041701), the CDK4/6 inhibitor palbociclib (NCT04129151), and metformin (NCT01042379). An ongoing phase I trial is testing the efficacy of microdevice delivery of different compounds, including ganitumab, in sarcomas (NCT04199026). Teprotumumab is being tested in clinical trials in non-malignant disorders 1701, the. InteresSeveral tyrosine kinase inhibitor (TKI) small molecules were developed to target the kinase activity of the IGF1R. These agents are divided into ATP-competitive and non-ATP-competitive inhibitors, depending on the ability to block the ATP binding cleft. Based on the high sequence homology between the IGF1R and the IR kinase domains, particularly in the ATP binding cleft (100%) , ATP-comNon-ATP-competitive inhibitors, such as the cyclolignan picropodophyllin , interact with the substrate binding site, thus inhibiting IGF1R signaling without affecting IR action . In precNeutralizing IGFs ligands represents an alternative therapeutic strategy to block the IGF system. MEDI-573 is a fully human monoclonal antibody that neutralizes both IGF1 and IGF2 and inhibits both IGF1R and IR-A downstream signaling without affecting insulin-dependent IR activation . PreclinAmong the IGF-targeting strategies, an emerging area of research is currently focusing on IGF-Trap. The IGF-Trap is a heterotetramer, composed of the extracellular domain of the IGF1R fused to the Fc portion of human IgG1 ,134. TheSarcomas represent a rare clinical and molecular heterogeneous group of mesenchymal neoplasms, categorized as bone and soft-tissues tumors . DespiteThe IGF system is linked to the pathogenesis and progression of osteosarcoma, the most common primary tumor of bone in children and adolescents ,136. EarOsteosarcoma is characterized by the simultaneous amplification of oncogenes, loss of tumor suppressors and inactivation of DNA-repair genes ,148. AccIGF1R gene with corresponding increased IGF1R transcription and downstream activation of the PI3K/Akt/mTOR pathway [IGF1R amplification might be considered for future clinical trials.Whole exome genome analysis in osteosarcoma has recently indicated that actionable somatic alterations in IGF signaling genes actually occur in a small subset of cases. In particular, somatic copy number variations of the IGF gene family occurr in about 7% of cases ,153 whilOther events have been reported to govern IGF1R expression in osteosarcoma, further demonstrating the complexity which surrounds the IGF axis in this tumor. For instance, recent data demonstrated that the mediator of EMT process cysteine-rich protein 61 (CYR61/CCN1), which correlates with osteosarcoma aggressiveness in preclinical model and in patient tumor samples, increased IGF1R and IGF1 expression in osteosarcoma cells, thereby modulating cell–cell interactions and motility . Non-codEWSR1 to a member of the ETS family of transcription factors, mainly FLI1 (85% of cases) [IGFBP3, while favoring the transcription of IGF1, which is a critical step in Ewing sarcoma tumorigenesis [Ewing sarcoma is the second most common pediatric bone tumor. It is characterized by a recurrent somatic translocation which fuses f cases) . The resf cases) . The IGFf cases) . Conversigenesis ,158. As igenesis . Pharmacigenesis ,161,162.igenesis . R1507 gigenesis . Figitumigenesis  or, in aigenesis . Robatumigenesis . To dateigenesis ,165,167.igenesis . In Ewinigenesis  pretreatThe lack of response to IGF agents in Ewing sarcoma likely depends on an aquired resistance mechanisms developed by these cells to adapt to IGF1R depletion. Enhanced expression of the IR-A and its mitogenic ligand IGF2 is a major mechanism of resistance to anti-IGF1R therapy in Ewing sarcoma. In resistant cells, an IR-A/IGF2 axis activated upon blockade of canonical IGF1R/IGF1 signaling constitutes an alternative autocrine loop sustaining cell growth via MAPK pathway . AccordiIGF1R promoter leading to IGF1R upregulation. Accordingly, combination of trabectedin with anti-IGF1R mAb AVE1642 promoted antitumor activity in Ewing sarcoma xenografts [Combinatorial approaches with anti-IGF1R inhibitors in association with either other drugs or chemotherapy are currently under investigation in order to avoid pathway crosstalks and acquired resistance. Mechanism-based evidence provided the rationale to combine anti-IGF1R agents with the mTOR inhibitor temsirolimus , as in fnografts .PAX3 and FKHR (FOXO1) genes. An additional but rare chromosomal translocation t results in a fusion between PAX7 and FOXO1. The PAX3/7-FOXO1 fusion protein acts as a more potent transcriptional activator than PAX3/7 alone. Importantly, it drives pathogenesis and is associated with increased aggressiveness and poor prognosis [IGF2 mRNAs are directly regulated by IGF2BP2 in those embryonal cells, thereby suggesting a putative post-transcriptional mechanism of IGF2 dysregulation in this tumor [IGF1R and enhances receptor expression [IGF2 and IGF1R are both highly expressed in rhabdomyosarcoma cell lines, xenografts, and human tumor samples [Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma of the muscle . Two majrognosis . Dysregurognosis . Mechanirognosis ,177. Therognosis . Interesis tumor . Importapression . Microar samples . As demo samples . Overall samples . Accordi samples . It also samples . In addi samples . Further samples . Anti-IG samples . Similar samples . Additio samples . Several samples . Constit samples . It is i samples . Accordi samples . The com samples  and the  samples , prevent samples . The effIn parallel, preclinical studies demonstrated that IGF1R inhibition with either mAbs or TKIs activated YES kinase, a member of the SRC family tyrosine kinases (SFKs), as a resistance pathway in rhabdomyosarcoma cell lines and xenografts . AccordiIGF2 DNA, producing antibodies cross-reacting with mouse IGF2, were partially protected from a subsequent injection of RMSp53Neu-5 rhabdomyosarcoma cells, showing a significant 60% decrease in the number of lung metastases when compared to untreated controls [The interruption of IGFs autocrine rhabdomyosarcoma circuits through passive or active immune neutralization of IGF2 has been recently investigated in a preclinical setting. BALB-p53Neu male mice, injected to generate IGF2-dependent rhabdomyosarcoma, were treated with anti-IGFs antibodies and the results indicated a significant delay in the sarcoma onset and prolonged overall survival . Interescontrols .SS18 (SYT), which encodes a SWI–SNF complex component, to one of the homologues SSX genes , which encodes instead a transcriptional repressor [IGF2 via altered methylation at the IGF2 locus [Synovial sarcoma is a rare soft tissue malignancy, accounting for 8% of all soft tissue tumors and affecting young adults . The genepressor ,189. Theepressor . Howeverepressor ,191. Amoepressor . MicroarF2 locus . From a F2 locus ,193. TheF2 locus . PreclinF2 locus . In the F2 locus , while tF2 locus .Overall, the limited efficacy of a therapeutic approach based on a single RTK inhibition is not an unexpacted result. On one side, the complexity surrounding the IGF system in synovial sarcoma might limit the efficacy of anti-IGF agents as monotherapy. For example, synovial sarcoma cell lines and patients all display elevated levels of the IGF1R-interacting partner SRC, which is activated and promotes cell survival and proliferation . In vitrEWS gene to the 3′ exons of the WT1 gene [EWS-WT1 gene fusion generates a chimeric protein which acts as aberrant transcription factor. Various target genes of EWS-WT1 have been previously described including c-MYC and RTKs coding genes like PDGF, FGFR, EGFR and IGF1R [IGF1R gene, thereby significantly enhancing its transcription. Despite a substantial paucity of in vitro functional studies, the EWS-WT1/IGF1R axis likely constitutes one of the major mechanisms for the etiology and/or progression of desmoplastic small round cell tumor malignancy [IGF2, contributing to its over expression [Desmoplastic small round cell tumor is a rare primitive soft tissue sarcoma of children and young adults, mostly confined to the abdome . The driWT1 gene . The EWSnd IGF1R ,198. In lignancy . Publishlignancy ,200. Amolignancy ,202. In lignancy ,202. Recpression . Clinicapression . In addipression . Similarinteractors with a reported role in sarcoma that might work as “druggable” targets in cancer.The IGF system operates in a network of complex interactions that might limit the efficacy of therapeutic agents targeting this axis in sarcomas. A better understanding of these connections might provide the rationale for designing of novel therapeutic combinations to test in the clinic. This section will provide novel perspectives of additional interactions with the IGF system and define their impact on tumor cells. Particularly, we will focus on those The Ephrin (Eph) receptors represent the largest family of RTKs and, in association with their plasma membrane-bound ligands (Ephrin), regulate cell–cell communication, cell proliferation, migration and cell adhesion . Ehp recEphB4, thereby promoting cell proliferation and migration responses, which were sentitive to the PI3K inhibitor LY294002 [Functional interactions between the IGF and the Eph axis have been demonstrated in tumor cells. In preclinical breast cancer models, the IGF1/IGF1R/Akt axis sustained mRNA expression of LY294002 . In maliLY294002 . AccordiLY294002 ,47. An iLY294002 . Upon grLY294002 . OverallTo date, a functional interaction between the IGF system and the Eph axis has not been described in sarcoma models. However, Eph and IGF1 signaling pathways cross-talk to regulate bone and skeletal muscle cells function ,212 and In myoblast cell lines, the EphrinA1/EphA axis suppresses Ras-ERK1/2 signaling, thus favoring IGF1-induced myogenesis . EphA4-kEphA2 is up-regulated in osteosarcoma, Ewing sarcoma and rhabdomyosarcoma ,219,220.Genome-wide microarray and immunohistochemistry studies demonstrated de novo expression of EphA2 in osteosarcoma patients as compared to healthy bone tissues ,218. FroEphB4 has a role in tumorigenesis and the metastatic process of synovial sarcoma  and the Major connections between the IGF system and Ephrin receptors in cancer are summarized in CTGF or Cyr61 , resistance to apoptosis (BCL2), EMT (Snail) and inflammatory response (IFNγ) [Bromo- and extra-terminal domain (BET) proteins, such as BRD2, BRD3, BRD4, BRDT, represent a family of major epigenetic regulators connecting chromatine modifications to transcriptional activation of genes. These proteins are characterized by two tandem bromodomains that recognize acetylated-lysine residues in nucleosomal histones, thus activating transcription by recruiting the multiprotein mediator complex and positive transcription elongation factor b (P-TEFb) . BET proe (IFNγ) ,237. Whie (IFNγ) . Accordie (IFNγ) .IGF1R and INSR genes are direct targets of BRD4 in several cancer models [IGF1R and INSR genes in HeLa, HEK293T as well as MM.1S cells, a multiple myeloma cell line [IGF1R and INSR TSS, downregulated their mRNA expression and inhibited the PI3K pathway and Akt activation [There is evidence in the literature connecting BET protein action and the IGF system in cancer ,241,242.r models . In partell line . Treatmetivation . Accorditivation . The effIGF1 [A rationale for co-targeting the IGF1R/Akt axis and BET proteins was provided in Ewing sarcoma where the expression of a constitutively active Akt significantly decreased sensitivity of Ewing sarcoma cells to the BET inhibitor NHWD870, suggesting a crucial role of the IGF1R/Akt cascade in resistance of Ewing sarcoma cells to BET inhibitors . AccordiIGF1 . As receIGF1 . AccordiIGF1 . BET inhIGF1 . AccordiIGF1 .IGF2 and IGF1R mRNA translation in tumor cells [The BET inhibitor JQ1 inhibited mRNA and protein expression of the RNA-binding protein IGF2BP3 in Ewing sarcoma and B cell acute lymphoblastic leukemia ,245. Wheor cells  but any Major connections between the IGF system and the BET proteins in cancer are summarized in The G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is the membrane receptor for the chemokine CXCL12 and was originally identified in peripheral blood leukocytes and other cell types like hematopoietic stem cells, stromal fibroblasts and tumor cells ,247. LigCrosstalk between the CXCR4 and IGF axis has been documented in embryonal development and cancer ,248. In CXCR4 plays an important role in tumor progression and metastases of several sarcoma subtypes . EvidencIn synovial sarcoma, CXCR4 mRNA and protein expression levels are upregulated in tumor as compared to non-tumor tissues . In addiCXCL12 and CXCR4 mRNA levels [In osteosarcoma cases, high levels of CXCR4 mRNA and protein expression were correlated with tumor progression, metastatic disease, poor metastasis-free and overall survival . Anti-CXA levels . CollectA levels . In vivoA levels .CXCR4 as well as other major SP-target genes including the IGF1R, c-met and PDGFRα, which might represent putative targets for combinatorial treatments [In rhabdomyosarcoma, the CXCL12/CXCR4 axis is crucial for the metastatic process and high CXCR4 expression is associated with poor outcome . CXCR4 oeatments .Major connections between the IGF system and CXCR4 signaling in cancer are summarized in Dysregulation of the IGF system drives cancer cell proliferation, migration, EMT, and drug resistance. Accordingly, multiple anti-IGF therapeutic strategies have been developed to block this pathway, including IGF1R monoclonal antibodies, tyrosine-kinase inhibitors, IGFs neutralizing antibodies or the new IGF-TRAP and IGF1R—targeted antibody—drug conjugates. After decades of research in the field, clinical relevance of therapeutic agents targeting the IGF system in cancer appears limited to subsets of sarcomas. This is in line with the exceptional dependence of these diseases on IGF-driven processes. Still, even in those tumors, molecular profiling to identify patients who would better respond to anti-IGF-based treatment is still an urgent clinical need. In addition, any therapeutic strategy solely targeting the IGF axis is likely insufficient to block tumor growth. The majority of the information obtained to date shows the complexity of the IGF axis regulation and the multiplicity of interactions with other cancer-relevant pathways, which either potentiate or compensate molecular signaling mediated by the IGF system. A better understanding of this network of signaling pathways might contribute to the identification of novel and more effective therapeutic combinations for sarcoma treatment.


The current coronavirus pandemic has affected, in a short time, various and different areas of medicine. Among these, the obstetric field has certainly been touched in full, and the knowledge of the mechanisms potentially responsible for placental damage from SARS-CoV-2 occupy a certain importance. Here we present here a rare case of dichorionic twins born at 30 weeks and 4 days of amenorrhea, one of whom died in the first few hours of life after placental damages potentially related to SARS-CoV-2. We also propose a brief review of the current literature giving ample emphasis to similar cases described. The SARS-CoV-2 pandemic (Severe Acute Respiratory Syndrome Coronavirus-2) has had a global impact that has affected all different and distinct areas of medicine ,2. AmongThe patient was a 32-year-old woman with an early miscarriage previously suffering from mild hypothyroidism, 165 cm tall and 57 kg in weight. Approximately 2 weeks before labor, she contracted SARS-CoV-2, confirmed with a GeneXpert Dx Xpress SARS-CoV-2 RT-PCR assay (Cepheid). The analytical sensitivity and specificity are reported by the manufacturer as 100% (87/87 samples) and 100% (30/30 samples), with a limit of detection of 250 copies/mL or 0.0100 plaque-forming units per milliliter) [Placentas underwent routine clinical examination consisting of storage at 4 °C prior to fixation, fixation in 10% buffered formalin, photographs of the maternal and fetal surface, measurement, trimmed weight, sectioning, and examination of the cut surface. Sections submitted included 2 of membrane rolls, at least 2 of umbilical cord, 3 maternal surface biopsies, 2 full thickness sections, and representative sampling of any lesions present. Sections underwent routine processing, embedding, sectioning at 5 µm and staining with H&E. Histologic examination was performed by subspecialty perinatal pathologists who were aware of the COVID-19 status. Cases were reviewed by 2 pathologists to confirm the diagnoses. They were observed using an Olympus BX-51 optical microscope equipped with the Olympus DP80 image acquisition system. Anti-SARS-CoV-2 spike S1 glycoprotein monoclonal antibody, Thermo Fisher, Rabbit, was added, at pH 6, diluted 1:800, and the antigen was demonstrated by heat-induced citrate buffer epitope retrieval for enzymatic immunohistochemical (IHC) analysis. In addition, electronic microscopy analysis was made from villi samples of both the chorial discs. At the moment of delivery, samples were immediately fixed in 2.5% gluteraldehyde for 4 hours at 4 °C, and after overnight immersion in phosphate buffer, post-fixed with osmium tetroxide in PBS for 2 h at a temperature of 4 °C. The prepared samples were processed for inclusion in Araldite epoxy resin (M) CY212 . Semithin sections 0.5 µm thick were stained with toluidine blue for microscopic analysis. Ultrathin sections were mounted on nickel grilles with uranium acetate and lead citrate contrast. The semithin sections were observed with a Nikon photomicroscope equipped with a Nikon Coolpix DS-U1 digital camera . The ultrathin sections were observed with a Morgagni 268 electron transmission microscope .The first placental disc weighed 240 grams, measured 14 × 12 × 1.5 cm, had smooth and shiny membranes, and had a 31 cm umbilical cord, normospiralized, with paracentric insertion. The second disc weighed 340 g, measured 15 × 10 × 1.5 cm, had smooth and shiny membranes, and had a 15 cm, normospiralized funiculus with central insertion.The first chorionic disc corresponded to the gestational age and presented very large areas of intervillous fibrinous deposition  with theElectron microscopy showed signs of circular formations with a 100–130 nm diameter, with peripheral electron dense spicules, which are likely viral particles in the cytoplasm of the perivillary histiocytes .Additionally, in the second case, developmental characteristics corresponding to gestational age, large areas of intervillous fibrinous deposition A, and thFollowing the birth, the baby was subjected to a nasopharyngeal swab, which was positive, but which, when repeated after a few minutes, gave a negative result for SARS-CoV-2. Four days after death, a complete autopsy on the newborn was performed. On external examination, no malformations were detected. All-natural orifices were probed and appeared patent. Weight (1322 g) and anthropometric parameters were consistent with gestational age (30 w+ 4 d) in a twin pregnancy. External genitalia were normal and indicative of a male phenotype. By a skin and subcutaneous tissue Y-shaped incision, the thoracic and abdominal cavities were explored, noting that umbilical arteries normally extended on either side of the urinary bladder, along the inner abdominal wall, and the umbilical vein normally coursed towards the liver in the falciform ligament and patent. The domes of the diaphragm were inspected, and their positions were at the fourth rib interspaces bilaterally. After removing the chest plate, it was observed with a diaphanoscope, and four ossification nuclei were detected. Mild pleural, pericardial, and peritoneal effusions were noted. Lungs appeared hypo-expanded and crouched in their natural cavities. The heart only exhibited some subepicardial petechial spots on the anterior ventricular surfaces, and its opening in situ showed no congenital anomalies. After evisceration by the Rokitansky technique, all organs were carefully separated, dissected, and subjected to tissue sampling. At skull examination, normal tension of fontanels was appreciated. After their dissection, the brain was inspected in situ and then removed, revealing reduced tissue consistency due to decomposition and no focal lesions. Tissue samples were also taken from the brain. Histologically, all the organs removed, including the lung D, showedThe new coronavirus-2 COVID-19) pandemic has assumed a global importance that has affected various and distinct sectors of medicine 9 pandemi,4,5. In Our case represents a rarity for two reasons: there was not the very rare maternal–fetal transmission of SARS-CoV-2 despite the placental parenchyma being strongly positive for immunostaining for the spike protein, but at the same time the damage to both placental discs did not allow the second fetus to be able to be adequately oxygenated in the intrapartum, leading to a disastrous outcome. We could sum it all up with “died of COVID without COVID”.


TCOF1) plays a crucial role in multiple processes, including ribosome biogenesis, DNA damage response (DDR), mitotic regulation, and telomere integrity. However, its role in cancers remains unclear. We aimed to visualize the expression, prognostic, and mutational landscapes of TCOF1 across cancers and to explore its association with immune infiltration. In this work, we integrated information from TCGA and GEO to explore the differential expression and prognostic value of TCOF1. Then, the mutational profiles of TCOF1 in cancers were investigated. We further determined the correlation between TCOF1 and immune cell infiltration levels. Additionally, we determined correlations among certain immune checkpoints, microsatellite instability, tumor mutational burden (TMB), and TCOF1. Potential pathways of TCOF1 in tumorigenesis were analyzed as well. In general, tumor tissue had a higher expression level of TCOF1 than normal tissue. The prognostic value of TCOF1 was multifaceted, depending on type of cancer. TCOF1 was correlated with tumor purity, CD8+ T cells, CD4+ T cells, B cells, neutrophils, macrophages, and dendritic cells (DCs) in 6, 14, 16, 12, 20, 13, and 17 cancer types, respectively. TCOF1 might act on ATPase activity, microtubule binding, tubulin binding, and catalytic activity (on DNA), and participate in tumorigenesis through “cell cycle” and “cellular-senescence” pathways. TCOF1 could affect pan-cancer prognosis and was correlated with immune cell infiltration. “Cell cycle” and “cellular-senescence” pathways were involved in the functional mechanisms of TCOF1, a finding that awaits further experimental validation.Treacle ribosome biogenesis factor 1 ( TCOF1) gene is located on the long arm of chromosome 5 at the 5q32-33.3 locus and encodes treacle phosphoprotein [TCOF1 was initially found as a gene related to Treacher Collins syndrome (TCS), a rare genetic disorder characterized by severe craniofacial defects, external ear deformation, and hearing impairment [TCOF1 in TCS has been extensively studied [TCOF1 mutations in TCS [TCOF1 has been reported to play crucial roles in multiple processes, including ribosome biogenesis [TCOF1 participates in several key cellular processes, in this study we aimed to investigate the part it plays in human cancers.The treacle ribosome biogenesis factor 1  and cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) have shown great success in the treatment of several cancer types [TCOF1 in this study, at the pan-cancer level.Cancer remains a major public-health problem worldwide and has been a leading cause of death in the past several decades , 17. Emeer types –20. HoweTCOF1 in pan-cancer based on published data. In addition, mutations to TCOF1 and the gene’s relationships with certain genomic signatures were also explored. We further evaluated TCOF1’s relationships with immune cell infiltration, immune checkpoints, and immunotherapy responses. Finally, potential pathways involving TCOF1 in tumor pathogenesis were also investigated.In this work, we visualized the expression and prognostic landscapes of TCOF1 mRNA expression levels in various cancer types by analyzing TCGA data via Oncomine. The results showed that TCOF1 expression in tumors was significantly higher than in normal tissues in many cancers, including bladder, breast, cervical, colorectal, esophageal, gastric, head and neck, liver, lung, and ovarian, as well as in melanoma and lymphoma. However, in certain studies, TCOF1 was less expressed in brain, central nervous system (CNS), head and neck, kidney, and lung cancers and in lymphoma , breast invasive carcinoma (BRCA), cervical squamous-cell carcinoma (CESC), endocervical adenocarcinoma (ECA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), head and neck squamous-cell carcinoma (HNSC), clear-cell renal-cell carcinoma (CCRCC), papillary renal-cell carcinoma (PRCC), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous-cell carcinoma (LUSC), pheochromocytoma and paraganglioma (PCPG), rectal adenocarcinoma (READ), stomach adenocarcinoma (STAD), thyroid carcinoma (THCA), and uterine corpus endometrial carcinoma (UCEC). Notably, TCOF1 expression in skin cutaneous melanoma (SKCM) metastatic tissue was remarkably higher than in respective primary tumor tissue. Lower expression of TCOF1 in tumor was found only in glioblastoma multiforme (GBM). For certain tumors lacking normal-tissue data in TCGA, we used corresponding normal tissues from the GTEx dataset as controls and compared differences in TCOF1 expression using GEPIA2. As shown in TCOF1 was upregulated in diffuse large B-cell lymphoma (DLBCL), sarcoma (SARC), testicular germ cell tumor (TGCT), and thymoma (THYM). However, we did not see significant differences in other tumors, including adrenocortical carcinoma (ACC), acute myeloid leukemia (LAML), brain lower-grade glioma (LGG), ovarian serous cystadenocarcinoma (OV), and uterine carcinosarcoma (UCS).To further evaluate differential expression of TCOF1 in pan-cancer via the UALCAN portal. Compared with normal tissues, expression of TCOF1 total protein was higher in breast cancer (BC), colon cancer (CC), ovarian cancer (OC), CCRCC, and LUAD, but not in UCEC , testicular (16.67%), pancreatic (11.11%), urothelial (10%), stomach (9.09%), liver (8.33%), endometrial (8.33), ovarian (8.33%), renal (8.33%), and skin cancers , liposarcoma, renal-cell carcinoma (RCC), glioma, meningioma, and colorectal cancer  in kidney chromophobe , PRCC (P = 0.0027), LIHC (P = 0.0037), and mesothelioma  in TCGA datasets. Disease-free survival (DFS) analysis results, shown in TCOF1 was correlated with poor prognosis in BLCA (P = 0.031), KICH (P = 0.033), PRCC (P = 0.019), and LIHC (P = 0.05) in TCGA datasets. However, lower expression levels of TCOF1 were linked to poor OS for STAD  appeared in UCEC patients, with “mutation” as the primary type. The “amplification” type of copy number alteration (CAN) was the primary type in CCRCC cases, showing an alteration frequency of >5%. Notably, all UVM cases with genetic alterations (~2% frequency) had copy number deletion of TCOF1  is displayed in Circos plots  in pan-cancer. As shown in TCOF1 and DNMTs in almost all types of cancer except UCS and CHOL. This indicated that upregulated expression of TCOF1 in different cancers might contribute to DNA methylation.We next examined the relationship between TCOF1 expression level. The results indicated that high TCOF1 expression was positively related to tumor purity in six types of cancer (TCOF1 expression level was significantly associated with infiltration levels of Cluster of Differentiation 8–positive (CD8+) T cells, CD4+ T cells, B cells, neutrophils, macrophages, and dendritic cells (DCs) in 14, 16, 12, 20, 13, and 17 cancer types, respectively , leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), CD244, lymphocyte activation gene 3 (LAG3), inducible T-cell costimulator (ICOS), CD40 ligand (CD40LG), CTLA4, CD48, CD28, CD200 receptor 1 (CD200R1), CD80, programmed cell death protein 1 (PDCD1), transmembrane and immunoglobulin domain containing 2 (TMIGD2), programmed cell death 1 ligand 2 (PDCD1LG2), CD27, TIGIT, CD86, and tumor necrosis factor receptor superfamily 9 (TNFRSF9). As shown in TCOF1 had significant correlations with most of the immune checkpoints in KICH, CCRCC, and LIHC. Notably, in 20 of 33 types of cancer, CD276 expression was remarkably associated with TCOF1. Based on these results, we hypothesized a synergistic effect of TCOF1 and immune checkpoints in cancers. However, based on the data from TISIDB, we did not observe any significant difference in TCOF1 expression level between immunotherapy responders and non-responders , postmeiotic segregation increased 2 (PMS2), mutS homologue 2 (MSH2), and mutS 6 (MSH6). The epithelial-cell adhesion molecule (EPCAM) gene has also been identified as a key MMR gene [TCOF1 expression and MSI, a strong mutator phenotype with a deficient MMR system, in different types of cancers and found that TCOF1 was significantly and positively correlated with MLH1, MSH2, MSH6, and PMS2 in almost all types but negatively correlated with EPCAM in CESC, KICH, and THYM  but negatively correlated with TMB in THCA and THYM cohorts  DNA sequences distributed throughout the human genome and prone to replication errors, which can normally be repaired by the mismatch repair (MMR) system . The MMRMMR gene . We analand THYM . In addiotherapy .TCOF1 in tumor pathogenesis, we investigated genes and proteins related to TCOF1 expression and conducted pathway enrichment analyses thereof. Using STRING, we identified TCOF1-binding proteins supported by experimentally available evidence. The interaction network of these proteins and TCOF1 is shown in TCOF1 expression in tumor data from TCGA projects. As shown in TCOF1 expression was most positively significantly correlated to expression of deleted in azoospermia-associated protein 1 (DAZAP1), heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB), Ly1 antibody reactive (LYAR), DNA topoisomerase II binding protein 1 (TOPBP1), interacting checkpoint and replication regulator , and polo-like kinase 1 (PLK1). The corresponding detailed heatmap data of the various cancer types is displayed in TCOF1-binding proteins and GEPIA2-based TCOF1-correlated genes showed three common members: dyskerin pseudouridine synthase 1 (DKC1), nucleolar protein 56 (NOP56), and NOP58  activity, microtubule binding, tubulin binding, catalytic activity (acting on DNA), and other functions  and participate in tumorigenesis through “cell cycle” and “cellular-senescence” pathways , transcription initiation factor 1 alpha (TIF1-α), selectivity factor 1 , and DNA polymerase I (Pol I) [UBF, Pol I, and Nopp140 to the rDNA promoter [UBF, an important regulator in the transcription of rDNA [UBF and Pol I and the resulting inhibition of rRNA transcription [NOP56 (TCOF1 leads to inhibition of rRNA transcription and attenuated cell proliferation [DDX21) by relocating it from the nucleolus to the nucleoplasm [DDX21 has been reported to promote gastric-cancer (GC) proliferation and tumor growth [Treacle acts as a key regulator in the biogenesis of ribosomes, one of the most important cell processes and essential for nearly 95% of total transcription . Ribosom (Pol I) , 31. Trepromoter . In addi of rDNA . Insufficription , 32. Durn [NOP56  to regulleoplasm . DDX21 hr growth .Rad3-related (ATR) kinases, and transcriptional silencing is then induced to save energy and to prevent collision between catalyzing complexes of transcription and repair [NBS1) and TOPBP1, the key adaptor proteins of ATM and ATR kinases, respectively [NBS1 mediated by treacle is crucial for stopping rRNA transcription in the DDR process, and NBS1 overexpression is reportedly associated with chemoresistance and with tumor development and metastasis [NBS1 and thereby contribute to cancer development. Furthermore, overexpression of TCOF1 has been associated with radioresistance in acinar progenitor cells of rat salivary glands, but attenuated TCOF1 expression can sensitize human osteosarcoma cells to irradiation [TOPBP1 in the DDR process and the latter confers radioresistance in osteosarcoma [TOPBP1 might mediate the effect of treacle on sensitivity to radiotherapy. However, the potential contribution of TCOF1 to cancer progression and resistance to therapy needs to be elucidated in future studies.Due to recombination between rDNA sequences from different chromosomes, the genes encoding rRNA are unstable and prone to damage . DDR is d repair . Treacleectively , 37, 41.tastasis , 42–44. adiation . Since tosarcoma , TOPBP1 TCOF1 in pan-cancer, our study had some limitations. First, we analyzed tumor tissue information mainly based on microarray and sequencing data, meaning that cellular-level analysis results might be systemically biased. Future studies using high-resolution methods, such as single-cell RNA sequencing [TCOF1 expression and patient prognosis only, but in vivo/vitro experimental evidence on the cellular or molecular level is still needed to help clarify the role of TCOF1 in tumors. Third, although we found significant correlations among immune cell infiltration levels, survival outcomes, potential pathways, and TCOF1, we failed to identify TCOF1 as friend or foe in cancers due to conflicting results from different databases. Since tumorigenesis is a complex process involving multiple pathways, our study provided only preliminary findings on the oncogenic role of TCOF1; its exact role in certain type of cancers should be evaluated and validated more precisely and comprehensively.Although we integrated information from different public databases to present a broad view of quencing , should TCOF1’s role in pan-cancer. TCOF1 was upregulated in most types of cancers, and we believe it might serve as a prognostic biomarker depending on cancer type. Infiltration levels of several types of immune cells, including CAFs and MDSCs, were highly correlated with TCOF1 expression, suggesting underlying mechanisms involving TCOF1 and immunity in tumorigenesis that should be further explored. Future studies should focus on potential regulation of TCOF1 by multiple oncogenic-signaling pathways.In summary, we presented a broad view of http://cancergenome.nih.gov) is a prestigious cancer genomics project funded by the National Cancer Institute , which has characterized >20,000 primary cancer and matched normal-tissue samples from various cancer types [https://www.ncbi.nlm.nih.gov/geo) is a publicly available genomics-data repository containing array- and sequence-based data [http://commonfund.nih.gov/GTEx) is a commonly funded data resource and tissue bank containing tissue-specific gene expression data [https://proteomics.cancer.gov/programs/cptac), also funded by the NCI, is a comprehensive database that aims to identify proteins in cancer genomes and related biological processes and that provides genomic and proteomic data from >1100 cancer patients [The Cancer Genome Atlas  were 0.01 and 1.5, respectively. Next, we used the “Gene_DE” module of Tumor IMmune Estimation Resource 2 , an online tool for systematical analysis of immune infiltrates across diverse cancer types [TCOF1 in pan-cancer from TCGA data. For certain cancer types without normal-tissue data, we matched and compared them with corresponding normal-tissue data from GTEx via the “Expression DIY” panel of Gene Expression Profiling Interactive Analysis 2 , a portal for analyzing ribonucleic acid (RNA) sequencing expression data from the TCGA and GTEx projects [P-values and |Log2FC| were 0.01 and 1, respectively. TCOF1 protein expression analysis in six cancer types based on CPTAC data was conducted on UALCAN , a web portal for analyzing cancer omics data [Oncomine is a cancer microarray database and web-based data-mining platform aimed at facilitating discovery from genome-wide expression analyses . In thiser types , to visuprojects . Cutoffsics data .https://www.proteinatlas.org/) is an online portal that contributes to the mapping of human proteins in tissues. We obtained protein expression levels and immunohistochemical (IHC) staining results of TCOF1 protein in 20 types of cancer samples from HPA.The Human Protein Atlas  to summarize the results and drew a forest plot of them. In addition, given that PrognoScan is a collection of cancer microarray datasets, we then performed survival analysis based on TCGA data using the “Survival Analysis” panel of GEPIA2. The significance level was set to 0.05.PrognoScan  cancers . We condhttp://www.cbioportal.org), an open-access resource for interactive exploration of multidimensional cancer genomics datasets [TCOF1 in different tissues. We chose data from 10,967 samples in 32 studies from TCGA and determined the frequency, types, and sites of TCOF1 mutations in multiple kinds of cancer. Regulome Explorer (http://explorer.cancerregulome.org) is an online tool to search, filter, and visualize analytical results generated from TCGA data. We used this tool to explore the correlation between TCOF1 expression and certain genomic signatures. The filter of associations was set as follows: −Log10(p) ≥2; Correlation ≥0.4; Max results = 200. We calculated the relationship between TCOF1 expression and four DNA-methyltransferases (DNMTs) and presented the results as a circular plot using SangerBox (http://www.sangerbox.com/tool), a powerful computerized online tool for bioinformatics analysis.We employed cBioPortal , MCPcounter (https://github.com/ebecht/MCPcounter), CIBERSORT (https://cibersort.stanford.edu/), Epigenomics of Plants International Consortium , and quanTIseq (http://icbi.i-med.ac.at/software/quantiseq/doc/index.html). We analyzed the associations among microsatellite instability (MSI), tumor mutation burden (TMB), and TCOF1 expression using SangerBox. Finally, we evaluated mutational and expression differences of TCOF1 between immunotherapy responders and non-responders using the Tumor and Immune System Interaction Database , a web portal for such interactions [We used TIMER2 to explore the relationship between ractions .https://string-db.org) is a database of functional protein association networks [TCOF1-binding protein network based on the experimental evidence, with a minimum interaction score of 0.15. In GEPIA2, the “Similar Genes Detection” pane was used to search for the top 100 genes similar to TCOF1 in TCGA tumors. We computed the correlations between TCOF1 and the top 5 similar genes in all types of cancers and presented them in scatter plots using the “Correlation Analysis” pane in GEPIA2. The results were also presented as a heatmap plot using the “Gene_Corr” module of TIMER2. Intersection analysis results of STRING and GEPIA2 results were displayed as a Venn diagram using the “VennDiagram” package in R studio. Next, we combined two sets of data to conduct Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) enrichment analysis. For both types of analyses, we used the “GO/KEGG clusterProfiler” module in Hiplot (https://hiplot.com.cn), a comprehensive web platform for visualizing scientific data. P- and Q-value thresholds were set at 0.01 and 0.05, respectively.The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; networks . We usedThe studies involving human participants were reviewed and approved by all the research data based on the bioinformatic analysis of the open resources from the TIMER, Oncomine, TCGA, and GEPIA databases. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.http://timer.cistrome.org, http://gepia2.cancer-pku.cn, http://ualcan.path.uab.edu, http://dna00.bio.kyutech.ac.jp/PrognoScan/index.html, http://www.cbioportal.org, https://hiplot.com.cn, https://string-db.org, http://cis.hku.hk/TISIDB, http://www.sangerbox.com/tool. However, the Oncomine database has been closed and it’s no longer available. The original contributions presented in the study are included in the Supplementary Material. Further inquiries can be directed to the corresponding author.Publicly available datasets were analyzed in this study. The data can be found here: Supplementary Figures 1 and 3Supplementary Figure 2Supplementary Figure 4Supplementary Table 1Supplementary Table 2Supplementary Table 3


H19, HOTAIR, PVT1, UCA1, XIST, CRNDE, FOXD2-AS1, ANRIL, HOXA11-AS, TP73-AS1, and DANCR. On the other hand, MEG3, GAS5, CCASC2, and TUSC7 are tumor suppressor lncRNAs, which are downregulated. While most studies reported oncogenic effects for MALAT1, TUG1, and NEAT1, there are some controversies regarding these lncRNAs. Expression levels of lncRNAs can be associated with tumor grade, survival, treatment response (chemotherapy drugs or radiotherapy), and overall prognosis. Moreover, circulatory levels of lncRNAs, such as MALAT1, H19, HOTAIR, NEAT1, TUG1, GAS5, LINK-A, and TUSC7, can provide non-invasive diagnostic and prognostic tools. Modulation of expression of lncRNAs using antisense oligonucleotides can lead to novel therapeutics. Notably, a profound understanding of the underlying molecular pathways involved in the function of lncRNAs is required to develop novel therapeutic targets. More investigations with large sample sizes and increased focus on in-vivo models are required to expand our understanding of the potential roles and application of lncRNAs in glioma.Glioma is the most common malignant central nervous system tumor with significant mortality and morbidity. Despite considerable advances, the exact molecular pathways involved in tumor progression are not fully elucidated, and patients commonly face a poor prognosis. Long non-coding RNAs (lncRNAs) have recently drawn extra attention for their potential roles in different types of cancer as well as non-malignant diseases. More than 200 lncRNAs have been reported to be associated with glioma. We aimed to assess the roles of the most investigated lncRNAs in different stages of tumor progression and the mediating molecular pathways in addition to their clinical applications. lncRNAs are involved in different stages of tumor formation, invasion, and progression, including regulating the cell cycle, apoptosis, autophagy, epithelial-to-mesenchymal transition, tumor stemness, angiogenesis, the integrity of the blood-tumor-brain barrier, tumor metabolism, and immunological responses. The well-known oncogenic lncRNAs, which are upregulated in glioma, are  Glioma is the most common malignant central nervous system (CNS) tumor with significant mortality and morbidity . GlioblaNon-coding ribonucleic acids (RNAs), comprising more than 97% of the human genome with various functions in physiological and pathological conditions, play a major role in glioma tumorigenesis . Non-codLack of optimal treatment options in addition to specific and sensitive biomarkers  necessitin-vivo were investigated.In this review, after providing a brief background on lncRNAs and their functions, we reviewed their role in various oncogenic processes. We also assessed their role in determining treatment response, survival, and prognosis. Lastly, the diagnostic and prognostic value of circulatory lncRNAs and potential therapeutic applications of modulation of lncRNAs expression LncRNAs are non-protein-coding RNAs with more than 200 nucleotides that are transcribed mainly by RNA polymerase II. As a result, lncRNAs, like messenger (m)RNAs, are typically polyadenylated and capped . HoweverLncRNAs can be categorized into six groups according to their location on the genome, namely (a) sense, (b) antisense, (c) bidirectional (d) intronic, (e), and (f) enhancer lncRNAs Figure 1via vying with mRNAs for mi-RNA and act as mi-RNA sponges. Moreover, lncRNAs can recruit mRNA degradation-associated proteins or act as decoys for RNA binding proteins involved in mRNA decay machinery. lncRNAs can affect translation through interacting with ribosomes or modifying mRNAs to activate their translation. lncRNAs are also involved in a variety of post-transcriptional modifications, most importantly phosphorylation and ubiquitination  can modulate mi-RNA availability tination , 10.MALAT1), also known as nuclear enriched abundant transcript (NEAT)2, is an intergenic lncRNA located on chromosome 11q13. Originally, MALAT1 was introduced as a prognostic marker in non-small cell lung cancer. It is associated with several cancers such as breast, ovarian, prostate, pancreatic cancers, and leukemia  axis  by sponging miR-101 in glioma (miR-199a in glioblastoma (hibition  and induB2) axis . Nano coe arrest . Accordin glioma  and miR-blastoma , respectblastoma .MALAT1 induced tumor proliferation in U87 and U252 cell lines, potentially by suppressing the extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway  in glioma  70 (MALAT-1 knockdown resulted in lower expression of Bax and higher expression of Bcl-2 via regulating the miR-199a/ZHX1 axis (n glioma . Inhibitp1B axis  and the p1B axis . Moreove(HSP) 70 . AdditioHX1 axis .Autophagy: Although autophagy may induce cytotoxic effects, it has also been suggested to promote the progression and viability of glioma in stressful environments (MALAT1 is found to promote tumor progression by enhancing autophagy. Sponging miR-101 not only enhanced tumor proliferation by upregulating Rap1b (MALAT1 acts as a sponge for miR-384 as well. Inhibition of miR-384 activity induced autophagy by putatively interfering with Golgi membrane protein 1 (GOLM1) and led to increased migration and invasion of glioma cells  regulated MALAT1 expression through the non-canonical Wnt signaling pathway 2 2 .Stemness: MALAT1 overexpression promoted proliferation of glioma stem cells  and proliferation of glioma stem cell lines by activating the ERK/MAPK signaling pathway, which is a key pathway in tumor development : MALAT1 knockdown led to enhanced BTB permeability and reduced expression of tight junction proteins in glioma endothelial cells via upregulating miR-140. The effect of miR-140 on BTB is mediated by inhibiting expression of nuclear factor YA (NFYA), a regulator of BTB integrity, resulting in increased expression of tight junction proteins  axis resulting in a reduced inflammatory response  classification and tumor size -resistant glioblastoma cells. Its knockdown reduced TMZ resistance both in vivo and in vitro -associated protein 1 (MRP1), a drug efflux pump associated with TMZ resistance  (The expression of h grade) , 46 and h grade) , 48.Cell cycle and proliferation: Knockdown of H19 inhibited glioma cell growth, indicating that H19 interacts with the cell cycle and enhances glioma proliferation /Unc-51 like autophagy activating kinase 1 (ULK1) axis by inducing increased ULK1 phosphorylation and inhibiting mTOR phosphorylation  process: EMT, a major role player in tumorigenesis by promoting metastasis, tumor stemness, and chemoresistance, is characterized by increased expression of epithelial markers, such as E-cadherin, and decreased mesenchymal markers, such as N-cadherin, vimentin, and ZEB1/2  through inhibiting the Wnt/β-catenin pathway activity  and promoted stemness  (an angiogenic factor)  , and the factor) . Furtherulations , and itsigations .Circulatory biomarker: While circulatory H19 levels were a reliable prognostic indicator, to the best of our knowledge, their diagnostic value has not been investigated in glioma , and ATP-binding cassette subfamily G member 2 (ABCG2) , also known as gene-trap locus 2 (GTL2) in mice, is a maternally imprinted intergenic lncRNA (like H19) located on chromosome 14q32.3. MEG3 has shown anti-tumoral effects in several cancers, such as lung, breast, liver, gastric, colorectal, ovarian, and cervical, in addition to glioma  gene, is involved in several cellular protective mechanisms, including inducing cell cycle arrest, DNA repair, and apoptosis (MEG3 expression due to DNA (cytosine-5)-methyltransferase 1 (DNMT1)- mediated hypermethylation inhibited the p53 pathway in glioma  expression, negatively regulating the phosphoinositide 3-kinase (PI3K). miR-19a is found to have repressive effects on PTEN expression. MEG3 acted as a ceRNA for miR-19a, recovering its inhibitory effects on PTEN expression. It resulted in decreased cell proliferation, cell cycle arrest at the G1/S phase, and increased apoptosis  by downregulating miR-96-5p  of the invasion .MEG3 also modulated Wnt/β-catenin signaling, leading to enhanced tumor proliferation following MEG3 downregulation in glioma  and increased expression of E-cadherin in U87 and U251 cells , isocitrate dehydrogenase (IDH) wild-type, tumor recurrence, and poor overall survival  reduced the abnormal MEG3 promoter hypermethylation and prevented low MEG3 expression (pression . Moreovepression . Notablypression .HOTAIR), an oncogenic lncRNA located on chromosome 12q13.13, is the first identified trans-acting lncRNA with widely explored roles in breast, lung, cervical, colorectal, and bladder cancers, and glioma , as well as glioma cell lines (U867 and U251), had higher n tissue –94. Inven glioma , also inpromoter .Cell cycle and proliferation: HOTAIR is required for the formation of glioblastoma ] activity, which leads to chromatin condensation by binding to the PRC2 complex  in glioblastoma , leading to increased growth and proliferation of glioma stem cells 1 (r (USF)1 .Metabolism: HOTAIR regulated glutamine metabolism, which is essential for glioma progression, by sponging miR-126-5p  were also associated with more prolonged overall survival in patients with WHO grade III anaplastic oligodendroglioma  expression is associated with chemoresistance putatively through HK-2 mediated lactate production and mitochondria permeability transition pore opening  is an intergenic lncRNA located on chromosome 8q24, a well-known cancer-associated region. The role of PVT1 has been explored in several cancers such as leukemia, colon, hepatocellular, breast, lung, and ovarian cancers  expression  axis resulting in inhibition of the bone morphogenetic protein (BMP) signaling pathway and tumor growth , an oncogenic factor in glioma , which is a key role player in the nonsense-mediated mRNA decay (NMD) (pathways . PVT1 si pathway . Moreoveof PTBP1 . In anot miR-424 . PVT1 knbehavior . PVT1 upay (NMD) .Angiogenesis: Glioma vascular endothelial cells had a higher PVT1 expression. PVT1 overexpression promoted angiogenesis via degrading miR-186, resulting in upregulated Atg7 and Beclin1 expression , located on chromosome 19p13.12, is an intergenic lncRNA involved in several cancers, such as lung, breast, gastric, and colorectal cancers, as well as glioma  axis (B2) axis . NotablyB2) axis .Circulatory biomarker: Diagnostic values of circulatory UCA1 has been reported in other cancers, such as bladder, gastric cancer, and colorectal cancer  to cisplatin and TMZ in U87 and SHG139 cells. This effect was attenuated by inhibiting the Wnt/β-catenin signaling pathway. Notably, TMZ sensitivity increased after UCA1 knockdown  axes, respectively. XIST also induced tumor proliferation and angiogenesis via inversely regulating miR-429  and Plexin C1 via targeting miR-222, which led to inhibition of tumor progression  and phosphotyrosine interaction domain containing 1 (PID1)  . Direct nd Bcl-2 . Furthern glioma .MATN1-AS1 is controversial in glioma. Han et al. found lower expression of MATN1-AS1 in glioblastoma tissue compared to the adjacent tissue, acting as a tumor suppressor , which is a treatment response predictor in glioma  is the only lncRNA approved as a diagnostic biomarker for prostate cancer in clinical practice (LncRNAs hold promise for developing novel biomarkers and therapeutic targets. To the best of our knowledge, prostate cancer antigen 3 (practice . All in practice . Two maipractice . While opractice , 263.in-vitro assessments highlighting the need for further validation by nude animal models and clinical trials. However, the low conserveness of lncRNAs among different species may hinder using animal models since the function of a certain lncRNA can be different between humans and animal models. As a result, engineered models may be required (Despite considerable attention drawn to lncRNAs in cancer and the growing evidence, there are several gaps in the literature. The findings of some studies are not sufficiently reliable due to small sample sizes. Moreover, the majority of investigations are required .in-vivo investigations and studies with larger sample sizes, it is not elucidated whether disruption in expression of lncRNAs is a culprit or consequence of the malignancy. Future studies need to address this question, particularly by investigating upstream regulators of the expression of lncRNAs. Moreover, the development of specific panels of lncRNAs for diagnostic or prognostic applications can lead to increased specificity and sensitivity. Several studies have already taken this approach and sought to find specific signatures of lncRNAs for glioma (MALAT1 in addition to TMZ in vitro and in animal models (via classification models (Our study shed light on several directions for future studies. In addition to the need for increased r glioma –266. Addl models . Additiol models . Lastly,l models . Applyinn models .in-vivo models are required to expand our understanding of the potential roles and application of lncRNAs in glioma.To conclude, regulating diverse cellular signaling pathways and the expression of various proteins involved in different stages of tumor formation, proliferation, and invasion, lncRNAs are potential candidates for developing novel diagnostic, prognostic, and therapeutic approaches. The so far discovered associations between their expression in the tissue or circulatory exosomes with treatment response and prognosis boost hopes for their potential use in clinical practice. However, despite substantial advances, the role of lncRNAs in glioma remains fairly unknown. It is not well known whether the disruption in the expression of lncRNAs has a causal effect on glioma or is a consequence of the malignant process. Moreover, some controversial reports hinder drawing a concrete conclusion. More investigations with larger sample sizes and increased focus on SM: Conceptualization, Investigation, Writing - Original Draft, and Visualization. NR: Conceptualization, Writing - Review & Editing, Supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Solenopsis invicta Buren. Olfactory receptor (OR) complexes consist of odor-specific ORs and OR co-receptors (Orco). Orcos are highly conserved across insect taxa and are widely co-expressed with ORs. Odorant binding proteins (OBPs) can transport semiochemicals to ORs as protein carriers and thus constitute the first molecular recognition step in insect olfaction. In this study, three OBP genes highly expressed in S. invicta antenna, OBP1, OBP5, OBP6, and Orco were partially silenced using RNA interference (RNAi). RNAi SiOBP5- and Orco-injected ants showed significantly lower EAG (electroantennography) responses to fire ant alarm pheromones and the alkaloid, 2,4,6-trimethylpyridine than water- or GFP-injected ants 72 h post injection. Subsequent qRT-PCR analysis demonstrated that the transcript level of the OBP1, OBP5, OBP6, and Orco significantly decreased 72 h after ants were injected with dsRNAs; however, there were no transcript level or EAG changes in ants fed dsRNAs. Our results suggest that S. invicta Orco and SiOBP5 are crucial to fire ants for their responses to alarm pheromones. RNAi knocking down SiOBP5 can significantly disrupt alarm pheromone communication, suggesting that disrupting SiOBP5 and Orcos could be potentially useful in the management of red imported fire ants.Olfaction is crucial in mediating various behaviors of social insects such as red imported fire ants,  Solenopsis invicta Buren, is a well-known global invasive ant species that was introduced into the United States from South America in the 1930s. It is currently found in many southern and western states and Puerto Rico [The red imported fire ant, rto Rico .Insect olfaction is mediated by a subset of specialized cuticular structures called sensilla that are mainly found on the insect antennae and maxillary palp. Olfactory sensilla contain olfactory receptor neurons (ORNs), which are suspended in an aqueous lymph that harbors olfactory receptors (ORs) in their dendritic membranes. OR complexes consist of odor-specific ORs and OR co-receptors (Orco). Orcos are highly conserved across insect taxa, which are co-expressed with ORs. In addition, the sensillum lymph is characterized by water-soluble proteins called odorant binding proteins (OBPs) ,4,5. TheNasonia vitripennis, in which 90 sequences that encode putative OBPs were identified [S. invicta, a 17-gene-member OBP family was identified in both antennal and non-antennal tissue that are specific to the expression of these OBPs [S. invicta, and is identified as the primary alarm pheromone [S. invicta workers [OBPs have been reported in a wide range of insect species, and the number of OBPs varies in different species, ranging from 12 in ants to at least 35 in Drosophila, 44 in silkworms, and more than 100 in some mosquitoes ,9,10,11.ese OBPs . Like otese OBPs . The pheheromone ; however workers . We atteS. invicta colonies was determined using PCR on Gp-9 alleles [Acheta domesticus L., and kept at room temperature with ~70% humidity and a 16:8 dark–light photoperiod.Red imported fire ants were collected from Washington County, Mississippi. The colonies were maintained in Fluon-coated trays and kept in an insect rearing room at 26 °C. The social form of  alleles . All the2-ethyl-3,5(6)-dimethyl pyrazine , E-β-Farnesene (EBF), 2,4,6-trimethylpyridine (TMP), and ylang ylang oil were purchased from Sigma-Aldrich . The purities of these four chemicals were >95%.S. invicta total RNA was used for cDNA synthesis with the SuperScriptTM II Reverse Transcriptase System . One microliter of the cDNA sample was used as a template, and the SiOBP1, SiOBP5, SiOBP6, and SiOrco fragments were amplified using RT-PCR with specific primers  following the manufacturer’s instructions. One microgram of  primers  and conj primers A was use primers B.S. invicta adult workers using a Nanoinjector II System  with a pulled borosilicate needle . The connective membrane between the third and fourth dorsal abdominal segments of each ant was pierced for the delivery of 50 nL of dsRNA (200 ng) in each treatment. The OBP dsRNA (200 ng or 50 nL/ant), Orco dsRNA (200 ng or 50 nL/ant), GFP dsRNA (200 ng or 50 nL/ant) and water (50 nL/ant) were each injected into 50 S. invicta major workers. Five live ants per replicate were tested for EAG, and thus 10 antennae were collected and pooled at each time point post injection or feeding.Different time point experiments were conducted by using dsRNA feeding or injection on the major fire ant worker, with each using the same experimental design and treatments. At 48, 72 and 96 h post injection and feeding, treated worker ants were collected for EAG tests, and, once EAG bioassays were finished, the antennae were immediately immersed in RNALater , and stored at −80 °C in a freezer until RNA extraction. For the dsRNA feeding experiment, treatment replication was achieved over three independent experiments. Fifty microliters each of the OBP dsRNA, Orco dsRNA, and GFP dsRNA were diluted about 20-fold in sucrose water in a 1.5 mL Eppendorf tube, and the final concentration was 0.2 μg/μL. A cotton ball was used to plug the tube and also provided the ants with a feeding platform. For the dsRNA injection experiments, there were four treatments  and three controls , and each treatment had three experimental replicates. The major adult workers were placed in a petri dish , which was placed in a bucket of ice to immobilize the ants for injection. The OBP dsRNA was injected into the hemocoel of v/v) for each odorant stimulus was used to evaluate the EAG changes in the RNAi-treated ants.The procedures for electroantennography (EAG) and odorant delivery were identical to those previously described ,17,18,19S. invicta, was used for initial normalization and was quantified. The real-time quantitative RT-PCR procedures used were identical to those of Zhang et al. [TM II Reverse Transcriptase System . Gene-specific primer pairs were designed using Primer 3  to determine the mRNA expression level of SiOBP1, SiOBP5, SiOBP6, and SiOrco. GAPDH, an internal reference transcript gene of Primer 3 , and 9 µp < 0.05. All statistical analyses were performed using SPSS .The statistical significance of the differences between the RNAi-treated groups for each of qRT-PCR and EAG was determined by using a one-way analysis of variance (ANOVA)—with the least significant difference (LSD) test for qRT-PCR and Tukey’s HSD test for EAG analysis—and significant values were set at S. invicta. Among 17 S. invicta, the OBPs SiOBP1, SiOBP5, and SiOBP6 exhibited antenna-enriched expression [S. invicta. Thus, RNAi-treated ants were fed or injected with the dsRNAs SiOBP1, SiOBP5, SiOBP6, SiOrco, GFP and with water.We aimed to identify which OBP(s) are involved in detecting and transporting the alarm pheromones of We then examined the EAG responses of sham- and RNAi-treated ants to four odorant stimuli: 2-ethyl-3,5(6)-dimethyl pyrazine (EDP), E-β-Farnesene (EBF), 2,4,6-trimethylpyridine (TMP), and ylang ylang oil. To validate the altered EAG responses of the SiOBP1, SiOBP5, SiOBP6, and Orco dsRNA-injected major fire ant workers, EAG responses from five ants of each experimental group  were recorded at 48, 72, and 96 h post injection. The odorant stimuli EDP, TMP, and ylang ylang oil elicited significant EAG responses in non-injected, water-injected, and dsGFP-injected ants, but EBF evoked no significant EAG response A. FurtheOnce the EAG bioassays were finished, we applied a combination of RT-PCR and real-time quantitative PCR (qPCR) to determine the mRNA levels of SiOBP1, SiOBP5, SiOBP6, and Orco in the antennae of RNAi-treated (dsSiOBPs-injected and dsSiOrco-injected) and control  ants using GAPDH as an internal reference transcript gene. The SiOBP dsRNA-injected ants displayed a reduction in SiOBP1, SiOBP5, SiOBP6, and Orco transcript levels  when compared with the water-injected ants (average 96.3%), dsGFP-injected (average 95.6%) ants, and non-injected controls  A. The RTS. invicta to an alarm pheromone EDP; an alkaloid, TMP; and an essential oil, ylang ylang oil. The results indicated that SiOBP5 is the binding protein involved in the capture and transport of EDP, TMP, and the active compound(s) in ylang ylang oil to the receptors in the antennae of S. invicta. Although only a 42–55% silencing of SiOBP1, SiOBP5, SiOBP6, and Orco was achieved 72 h post injection, the partial silencing clearly affected the antennal EAG responses to EDP, TMP, and ylang ylang oil, which suggested that the fire ant olfactory system is remarkably sensitive [S. invicta.In this study, we demonstrated that SiOBP5 and Orco dsRNA injection resulted in a significant reduction in the EAG responses of ensitive . That maS. invicta Orco’s and three highly expressed antenna OBPs’  transcript levels using RNAi injection severely affected the S. invicta olfactory signal transduction and made some ants less mobile than normal, but the RNAi-fed ants were very active even 120 h post feeding. Unfortunately, the ants fed with dsRNA SiOBPs, and Orco did not show any effects on the transcription and EAG levels at all. Feeding with dsRNA was reported to be a successful method for RNAi in some sap-sucking insects, such as the pea aphid (A. pisum) [Bemisia tabaci) [Rhodnius prolixus) [S. invicta, the effect of the regulation of the pheromone-biosynthesis-activating neuropeptide (PBAN) on S. invicta survival has been investigated using RNAi. The PBAN dsRNA interfered with the melanization (cuticle darkening) of pupae after injection and larvae mortality increased when dsRNA was fed in a sucrose solution to nurse ants [The down regulation of the . pisum) ,22, whit tabaci) , and trirolixus) . Even inrse ants . Additiorse ants .S. invicta, and the RNAi silencing the olfactory Orco also reduced the response to pheromones in other insects, including the red palm weevil (Rhynchophours ferrugineus) [Grapholita molesta [Protaetia brevitarsis Lewis [Blattella germanica [gypsy moth [Rhodnius prolixus [Bactrocera dorsalis [Apolygus lucorum [Our results indicate that the RNAi Orco dsRNA-injection resulted in a significant reduction in the transcript level and electrophysiological recording of the response to EDP, TMP, and ylang ylang oil. Insect Orcos are highly conserved, not only in ugineus) , Graphol molesta , Protaetis Lewis  Blattellermanica , aphids ermanica , subterrermanica , gypsy mpsy moth , Rhodniuprolixus , Bactrocdorsalis , and Apo lucorum . Orco siS. invicta antennae. In a total of 17 SiOBPs, SiOBPs 1, 5, 6, 9, 10, and 11 have various orthologous OBPs in different insects, especially SiOBP5 and SiOBP6 [Acromrymex echinatior, and Indian/Jordon jumping ant, Harpegnathos saltator) and the European honeybee (Apis mellifera) illustrated that SiOBP5 is highly conserved with AeOBP6, HsOBP5X1, HsOBP5X2, and AmOBP6, and that SiOBP6 is conserved with AeOBP5, HsOBP7, and AmOBP6/8. Since EDP, TMPs and ylang ylang oil also elicited strong EAG responses in A. mellifera [A. Mellifera. Although EBF could not elicit significant EAG responses in either the SiOBP RNAi-treated or -untreated ants, EBF evoked robust olfactory responses in S. invicta workers and female alates-specific basiconica sensilla in single-sensillum recording (unpublished). Which SiOBPs bind to EBF in S. invicta? The answer is very likely the orthologue of ApisOBP7. In aphid species, ApisOBP7 is the OBP responsible for mediating the transportation of the aphid alarm pheromone EBF [Unlike SiOBP5, silencing SiOBP1 and SiOBP6 did not result in any EAG changes in response to EDP, TMP, and ylang ylang oil. These three SiOBPs were highly expressed in mone EBF ,37. FurtRNAi in combination with EAG approaches are widely used and well-accepted methods for the characterization of OBPs ,39,40,41S. invicta population. The RNAi technique has shown considerable potential for controlling insect pests [S. invicta, having a feasible dsRNA delivery method is a must. Delivery through feeding is desirable because it is easy to incorporate into the current bait technology. A variety of new technologies might offer excellent future prospects for using RNAi for managing ants, such as nanoparticles, engineered microorganisms, transgenic bacteria that express dsRNA, the mass production of siRNA, and advanced formulations [Currently, synthetic insecticides are commonly used in controlling fire ants. However, synthetic insecticides do not always deliver satisfactory control. The RNAi technique aimed at disrupting the olfactory system has the potential to interfere with such critical behaviors as foraging and mate location, ultimately disturbing the reproductive process and decreasing the ct pests . To enabulations .


Objective: To analyze the clinical features of common autoimmune encephalitis and evaluate the sensitivity of antibodies contributing to focal epilepsy signs and symptoms (ACES) score.Methods: Collecting and analyzing the data of 242 patients with autoimmune encephalitis (AE) diagnosed in the First Affiliated Hospital of Zhengzhou University from August 2015 to December 2020 in this retrospective study. The six items of the ACES score  were screened in patients with complete clinical data.Results: (1) In total, 242 patients were included, with 147 cases of anti-N-methyl-D-aspartate receptor encephalitis, 47 cases of anti-γ-aminobutyric acid type B (GABA-B) receptor encephalitis, and 48 cases of anti-leucine-rich glioma inactivating protein 1 (LGI1) encephalitis. The most common clinical symptoms are cognitive impairment (77%), behavioral changes (79%), and seizures (71%). In total, 129 cases (54%) combined with autonomic dysfunction, such as gastrointestinal dysmotility, sinus tachycardia, and central hypoventilation. Twelve patients had autoimmune diseases, most of which were of thyroid diseases. (2) One hundred and twenty-seven patients with complete clinical data evaluated ACES score, 126 cases of whom  were equal to or >2 points, 1 case  was of <2 points.Interpretation: (1) Cognitive impairment, abnormal behavior, and seizures are the most common manifestations of AE and autonomic symptoms. Thyroid disease is the most autoimmune disease in AE. Clinically, for patients of suspected AE, increasing the knowledge and testing of thyroid function and rheumatism is necessary. (2) ACES score is a simple, effective, and easy-to-operate score, with a certain screening value for most patients suspected of AE. The six items include cognitive symptoms, behavioral changes, autonomic symptoms, speech problems, autoimmune diseases, and temporal MRI hyperintensities. Each factor is assigned 1 point. Antibody testing is recommended when the score is ≥2 points for which the sensitivity is 100%, specificity is 84.9%, and they are highly suspected as the autoimmune causes of epileptic seizures. To verify the sensitivity of the ACES score, 242 definite cases with AE were retrospectively collected, and clinical features of which were analyzed. At last, 127 cases with complete data were selected to validate the score.Since the first case of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis was reported in 2007 , more anBR encephalitis, and 48 cases of anti-LGI l encephalitis. All patients with AE were identified via their medical records. During hospitalization, all the manifested symptoms were observed and recorded. We analyzed their clinical data, such as the demographic characteristics (age and sex), prodromal symptoms , clinical manifestations , co-existing autoimmunity, tumors, previous history, previous and current medication, and modified Rankin Score (mRS). The auxiliary examination included routine and biochemical tests of serum and cerebrospinal fluid (CSF), tests of the virus, thyroid function, tests for rheumatic diseases, 3.0 T brain MRI scanner , CT scan of the thorax, ultrasound of the abdomen, pelvic and reproductive regions, routine electroencephalogram (EEG), and/or Video EEG monitoring. The J Neurol Neurosurg Psychiatry (2021) was the diagnostic criterion for AE , contactin-associated protein-2 (CASPR2), leucine-rich glioma inactivating protein 1 (LGI1), and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR). All patients were positive for the neuronal antibody in the serum and/or CSF. Cell-based assays (CBAs) for clinical laboratory studies were conducted using Euroimmun IIFT kits: Autoimmune Encephalitis Mosaic 1 (FA 1121-1005-1), and/or NMDAR kits (FA112d-1005-51), based on the instructions provided by the manufacturer, which is highly sensitive and specific assay. Tissue-based assays  are used in confirmative tests in addition to CBA if CSF is not available but the serum is available, or serum but not CSF is positive.Several encephalitis-associated antibodies were screened, such as NMDAR, γ-aminobutyric acid type B In total, 242 patients were screened out, of which 127 patients had complete data of 6 items in ACES score were calculated score .n = 63), headache (n = 63), vomiting (n = 25), dizziness (n = 25), fatigue or drowsiness (n = 25), bloating, diarrhea and poor appetite (n = 25), cough (n = 5), and sore throat (n = 5). Ninety-four patients had cognitive problems, 121 patients had mental and behavior changes, 96 patients had seizures, 81 patients had autonomic symptoms (n = 5), hyperthyroidism (n = 5), subclinical hyperthyroidism (n = 1), subacute thyroiditis (n = 1), and psoriasis , lung cancer (n = 2), renal hamartoma (n = 1), sigmoid colon cancer (n = 1), meningioma (n = 1), gallbladder cancer (n = 1), and nasal myofibroblastoma , headache (n = 10), vomiting (n = 7), dizziness (n = 6), bloating, diarrhea and poor appetite (n = 5), fatigue or drowsiness (n = 4), cough (n = 1), and sore throat (n = 1). Forty-three patients had cognitive problems, 40 patients had mental and behavior changes, 39 patients had seizures, 34 patients had autonomic symptoms (n = 1), and ankylosing spondylitis , urothelial carcinoma (n = 1), and esophageal cancer (n = 1).Among the 47 patients of anti-GABAsymptoms , 21 patis n = 1; , 4. Twenn = 2), headache (n = 2), dizziness (n = 8), and fatigue or drowsiness (n = 2). Thirty-three patients had cognitive problems, 29 patients had mental and behavior changes, 37 patients had seizures, 14 patients had autonomic symptoms  scored ≥4 points, 17 cases  scored 2–3 points, and all patients scored ≥2 points. Eighteen cases were excluded, among whom 5 cases were not tested for either thyroid function or rheumatism, nine cases were only normal for thyroid function but did not undergo rheumatism tests, 2 cases had no brain MRI, and two cases were lost to follow-up.Twenty-nine patients  with anti-GABATwenty-one patients  with anti-LGI1 encephalitis were examined for ACES scores. Among them, 2 cases  scored ≥ 4 points, 19 cases  scored 2–3 points, and all patients scored ≥2 points. Twenty-seven cases were excluded, among which 18 cases were only tested for thyroid function, 2 cases were only tested for rheumatism, 2 cases were not tested for either thyroid function or rheumatism, and five cases were lost to follow-up.2O, the white blood cell count was 52 × 106, and the protein was 362.5 mg/L. The titer of the CSF anti-NMDAR antibody was 1:3.2. Two months after he came back, the CSF antibody became negative. The test result showed that white blood cells were 4 × 106, the protein was 453.1 mg/L. There was no seizure and no discomfort at the follow-up at 4 months.The patient was admitted to the hospital with generalized tonic-clonic seizures, fever (37.4–38.4°C), and headache. Eight days before admission, the patient caught a cold and felt uncomfortable. Then the patient had three seizures in a week. The patient had no signs and symptoms of cognitive impairment and abnormal behaviors. The patient was treated with lumbar puncture, routine EEG, brain MRI, and other examinations upon admission. The patient was given antiviral and antiepileptic treatment initially, but the patient still had intermittent headaches. When the tests for CSF autoimmune antibody showed anti-NMDAR antibody was positive, the patient underwent first-line immunotherapy of intravenous methylprednisolone 80 mg daily for 2 weeks. Symptoms of the patient gradually improved and the headache disappeared. Auxiliary examination showed the EEG: abnormal electroencephalogram, diffuse seita-based abnormal slow-wave activity, brain MRI: swelling of the right temporal lobe with abnormal enhancement of pial, inflammation, and chest CT: subpleural inflammation in the right lower lobe, nodular thickening of the right lung pleura. The pressure of CSF was 160 mm HThere has been an increasing knowledge of AE. One study in Minnesota reported an estimated incidence of AE in the United States of America, of 0.8/100,000 person-years . In contBR encephalitis, and anti-LGI1 encephalitis were most the common AE types, which were consistent with the study by Guy et al. these three types of encephalitis account for 80.95, 7.41, and 4.76%, respectively , systemic lupus erythematosus , anaphylactoid purpura (n = 3), vitiligo (n = 3), Sjögren's syndrome (n = 2), chronic urticaria (n = 2), bullous pemphigoid (n = 1), uveitis (n = 1), myasthenia gravis (n = 1), and the coexistence of SLE and anaphylactoid purpura (n = 1)  . In 2019 (n = 1)  reported (n = 1)  reported (n = 1) . In anim (n = 1) , 30. In Viral infection may be a potential cause of AE . Some stn = 1). This patient scored one point had generalized tonic-clonic seizures that did not meet the inclusion conditions of the ACES score, which shows that the results of this article do not conflict with the original text. In this study, 99.2% of patients had a score of ≥2, which is not consistent with all patients  in the original text with a score of ≥2, which may be related to the inclusion conditions. The object of the de Bruijn Marienke AAM's study had focal epilepsy of unknown cause without obvious symptoms of encephalitis, and the number of samples is small (n = 20). This article expanded the scope of the study (n = 127) and included all patients with confirmed AEs, but some patients had no seizures or showed general tonic-clonic seizures. In this study, 99.2% of patients were no <2 points. This indicates that the ACES score is still a simple, reliable, and easy-to-operate screening method for patients with AE.This study reveals that 127 patients were scored, of which 126 patients  had ≥ 2 points, 1 case  had <2 points .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.