CTNNB1 gene at exon 3 and showed a loss of heterozygosity on chromosomes 18 and 21. By integrating macroscopic and microscopic features, along with their molecular profiles, we advocate that such tumors represent a distinct entity from PDAC and should be considered a new variant of solid pseudopapillary neoplasms. The recognition of this new neoplastic category may have immediate implications not only for tumor taxonomy but also for clinical practice.Hepatoid tumors (HTs) represent a rare group of neoplasms that are histologically similar to hepatocellular carcinoma but arise outside the liver. The current World Health Organization classification recognizes the hepatoid morphology of pancreatic tumors only as a possible variant of pancreatic ductal adenocarcinoma (PDAC). Here, we describe two cases of “pure” HT of the pancreas showing common features and characterized by indolent biological behavior. These tumors were roundish nodules with pushing borders, hyaline globules, and pure hepatoid histology; they were diffusely positive for β-catenin and LEF1 on immunohistochemistry. At next-generation sequencing, both neoplasms harbored only one pathogenic somatic mutation that affected the The online version contains supplementary material available at 10.1007/s00428-022-03317-4. Tumors with hepatoid differentiation represent a rare group of cancers that are histologically similar to hepatocellular carcinoma but arise outside the liver –4. This Although morphology is the only acknowledged criterion for diagnosis, positivity for some immunohistochemical markers such as hepatocyte paraffin-1 (Hep Par-1), CD10, alpha-fetoprotein, and arginase-1 may be helpful in supporting the identification of hepatoid differentiation –8.A recent study investigating with next-generation sequencing the genomic profile of HT from different organs did not reveal any common molecular hallmarks to explain the peculiar hepatoid morphology and concluded that the tissue of origin is the most important factor influencing their molecular landscape . TherefoRegarding pancreatic HT, the current World Health Organization (WHO) classification officially recognizes hepatoid morphology as a possible variant of pancreatic ductal adenocarcinoma (PDAC), named hepatoid carcinoma , 9–13. RHere, we describe two cases of pure HT of the pancreas showing the same morphological, immunohistochemical, and molecular profiles and characterized by indolent biological behavior. These tumors cannot be considered either PDAC or a neuroendocrine variant, thus representing a new potential entity among pancreatic tumors.Two cases of “pure” HT of the pancreas, showing 100% hepatoid morphology, have been identified in the pathology archives of the University and Hospital Trust of Verona (ARC-Net Biobank). All clinicopathological parameters were recorded, including the clinical history updated at the time of the last follow-up.The cases were investigated using IHC at the time of diagnosis. However, in this study, IHC was repeated with the addition of other markers. IHC was performed as previously described , accordiGlobally considered, the following antibodies were used: alpha-fetoprotein , androgen receptor , arginase-1 , CD10 , BAP1 , CD56 , CD117 , CD200 , cytokeratin 7 , cytokeratin 8/18/19 , cytokeratin 20 , cytokeratin AE1/AE3 , β-catenin , BCL10 , chromogranin-A , E-cadherin , Hep Par-1 , KDM6A , LEF-1 , MUC1 , MUC2 , MUC5AC , MUC6 , progesterone receptor , SMAD4 , synaptophysin , trypsin , and vimentin .www.agilent.com), hereafter referred to as CORE, as previously described [DNA extracted from formalin-fixed paraffin-embedded tissues was subjected to NGS using the SureSelectXT HS CD Glasgow Cancer Core assay . Forward and reverse reads from each demultiplexed sample were aligned to the human reference genome (version hg38/GRCh38) using BWA and saved in the BAM file format.CORE panel analysis started with demultiplexing was performed with FASTQ Generation v1.0.0 on the BaseSpace Sequence Hub , SnpSift [https://www.ncbi.nlm.nih.gov/refseq/; last access 11/30/2020). All candidate mutations were manually reviewed using Integrative Genomics Viewer (IGV) version 2.9 [Single-nucleotide variants were identified using Shearwater . Small . Structural rearrangements were detected using BRASS software [Tumor mutational burden and microsatellite instability were derived from sequencing analysis and computed according to the method described by Papke et al. . Copy nusoftware  and visuVariants were classified according to the five-tier classification system recommended by the joint consensus of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) . VariantBoth patients were male; the first patient was 53 years old and the second was 56 years old at the time of diagnosis. Both tumors were roundish with pushing borders and yellow-brownish at grossing Fig. , involviOn histological assessment Fig. , both neBoth patients underwent surgical resection for suspected neuroendocrine tumors. The first patient, who underwent pancreaticoduodenectomy in 2009, did not report any relapse during follow-up: after 12 years and 4 months, the patient was alive and still completely free of disease. The second patient underwent distal pancreatectomy in 2021, and was free of disease after 10 months.Both tumors displayed the same IHC profiles. The tumor cells were positive for arginase-1, CD10 , CD56, cytokeratin 8/18/19, Hep Par-1, β-catenin, LEF1, and androgen receptor (weak positivity). At the same time, they were negative for the neuroendocrine markers chromogranin-A and synaptophysin; for different types of cytokeratin such as cytokeratin 7, cytokeratin 20, and cytokeratin AE1/AE3; for the acinar markers BCL10 and trypsin; for some solid pseudopapillary markers such as CD200, progesterone receptor, and vimentin; for the following mucins: MUC1, MUC2, MUC5AC, and MUC6; and for alpha-fetoprotein, SMAD4, and CD117. Both neoplasms showed a loss of E-cadherin and conserved expression of KDM6A; BAP1 showed a heterogeneous staining pattern in the first tumor and conserved expression in the second neoplasm.CTNNB1 gene. The mutation in the first case was c.102_113del, resulting in an in-frame deletion , and the mutation in the second case was c.109 T > G, resulting in a missense substitution . Both cases were microsatellite-stable; the first case presented a tumor mutational burden of 4.86 mut/Mb, and the second case presented that of 9.19 mut/Mb. Additional VUS were detected for each case  on chromosomes 18 and 21 in both cases, gain of heterozygosity on chromosome 20 in patient 1, and LOH on chromosome 1 (region of 1p36.33-p12) in patient 2 Supplementary file2 (DOCX 16 KB)Below is the link to the electronic supplementary material.


Protein complexes termed inflammasomes ensure tissue protection from pathogenic and sterile stressors by induction of inflammation. This is mediated by different caspase-1-induced downstream pathways, including activation of the pro-inflammatory cytokines proIL-1β and -18, induction of a lytic type of cell death, and regulation of the release of other pro-inflammatory molecules. Aberrant inflammasome activation underlies the pathology of numerous (auto)inflammatory diseases. Furthermore, inflammasomes support or suppress tumor development in a complex cell-type- and stage-dependent manner. In human keratinocytes and skin, NLRP1 is the central inflammasome sensor activated by cellular perturbation induced, for example, by UVB radiation. UVB represents the main inducer of skin cancer, which is the most common type of malignancy in humans. Recent evidence demonstrates that activation of NLRP1 in human skin supports the development of cutaneous squamous cell carcinomas (cSCCs) by inducing skin inflammation. In contrast, the NLRP1 inflammasome pathway is restrained in established cSCCs, suggesting that, at this stage, the protein complex has a tumor suppressor role. A better understanding of the complex functions of NLRP1 in the development of cSCCs and in general of inflammasomes in cancer might pave the way for novel strategies for cancer prevention and therapy. These strategies might include stage-specific modulation of inflammasome activation or its downstream pathways by mono- or combination therapy. Inflammation represents a tissue response induced by many different stress factors ,2. TheseThe IL (interleukin)-1 family consists of 11 members  with either pro- or anti-inflammatory activity. IL-1β is a highly conserved, pleiotropic, and very potent pro-inflammatory cytokine that can induce inflammation and, if the dose is high enough, even a septic shock . IL-1β eCaspase-1 is expressed as an enzymatically inactive precursor molecule (pro-caspase-1) and its activation occurs upon assembly of inflammasomes . InflammIn most cases, inflammasome activation requires a preceding priming step for transcriptional induction of expression of proIL-1β, NLRP3, or AIM2 expression and posttranslational modifications of NLRP3 ,23,24. TInflammasome activation is often equated with proIL-1β activation. Indeed, owing to its fundamental role in inflammation, inflammatory diseases, and cancer , proIL-1β is the most important substrate of caspase-1. However, proIL-18 also plays crucial roles in immunity, particularly in the intestine, where it seems to be more important for repair than IL-1 ,26,27. IInflammasome activation in immune cells leads to pyroptosis, a lytic form of cell death that supports inflammation . In 2015Most proteins with extracellular function possess a signal peptide for secretion by the canonical ER (endoplasmic reticulum)/Golgi pathway. However, IL-1α, -β, -18, and other members of the IL-1 family lack such a signal peptide . They arIn general, chronic inflammation is believed to support all stages of malignant transformation and represents a hallmark of cancer development ,50. In cAIM2 contribute to the development of cancer of the small intestine and CRC  [NLRP3 are associated with Crohn’s disease, a risk factor for CRC, and a gain-of-function mutation results in poorer survival of patients suffering from CRC and increases the risk of developing melanoma [NLRP3, proIL-1β, caspase-1, and IL-1RN are associated with gastric cancer after Helicobacter pylori infection [Polymorphisms of inflammasome genes are associated with different types of cancer ,54,55. F cancer) ,57. Furtmelanoma ,59. In anfection ,61. In mnfection . ProIL-1β and IL-1Ra variants are associated with non-small-cell lung cancer as well [ as well ,64,65,66 as well ,68. The  as well . Surpris as well . reg (regulatory T) cells [+ T cell polarization [IL-1β production in tumors, either by tumor cells themselves or by stromal cells, is associated with a worse prognosis ,70,71,72T) cells . ProductT) cells  and can rization , whereasrization . It seemrization .IL-1β, together with IL-18, can also induce epithelial cells to lose their polarity and adherence and to acquire a migratory and mesenchymal cell-like phenotype, a process termed EMT  . This isIL-1, by inducing VEGF  expression, also has a role in angiogenesis ,61,77,80Currently, several trials are ongoing, studying the potential of canakinumab and anakinra (recombinant IL-1Ra) in mono- and combination therapy for the treatment of patients suffering from different types of cancer  and reviIL-1α also represents a target for cancer therapy . BermekiBy the suppression of the tumoricidal function of NK cells through the induction of expression of PD1 (programmed cell death 1), IL-18 can also contribute to tumorigenesis .Pro-tumorigenic roles of IL-1 ,89 and iA comparison of chemical-induced tumor incidence in mice lacking expression of IL-1RI ligands revealed that IL-1Ra expression strongly protects from tumors, IL-1α had a weak protective effect, whereas IL-1β strongly supports tumor development . SimilarOther members of the IL-1 family can have roles in tumorigenesis, too. It is believed that IL-33 is biologically active in its pro-form , althougFinally, inflammasome proteins have, at least in part, inflammasome-independent roles relevant for cancer development, adding another level of complexity. This is especially relevant for ASC. ASC was originally identified as TMS1 (target of methylation-induced silencing-1), whose expression is suppressed in different types of cancer ,106. ASCWhen NLRP1 was cloned for the first time, it was believed that the protein played a role in apoptosis ,81. LateThe NLRP1 protein consists of 1473 amino acids and five different domains : an aminNLRP1 is the central inflammasome sensor in human skin and is expressed by human keratinocytes . In addiBacillus anthracis, Nlrp1b but not human NLRP1 is proteolytically activated in the aminoterminal region by the lethal factor, a protease of anthrax lethal toxin [A similar mechanism of activation was identified previously for murine Nlrp1b. In contrast to humans, three different Nlrp1 paralogues are expressed in mice, with Nlrp1b possessing the strongest homology to human NLRP1 . Upon inal toxin ,124. Theal toxin ,123. Thial toxin .However, the roles of human NLRP1 are only poorly conserved between humans and mice, demonstrating that the NLRP1 is a relatively young evolutionary pathway . MechaniNLRP1 gene are associated with different (auto)inflammatory diseases, which mainly affect the skin, including vitiligo, Addison’s disease, and NAIAD (NLRP1-associated autoinflammation with arthritis and dyskeratosis) [NLRP1 cause two rare inflammatory skin diseases, MSPC  and FKLC  [Single nucleotide polymorphisms (SNPs) of the ratosis) ,130,131.hronica) . These dhronica)   originate from keratinocytes and represent the main types  of NMSC (non-melanoma skin cancer), the most prevalent cancer worldwide with further increasing incidence rates . Nearly NLRP1 suffer from chronic inflammation of the skin and have a high risk of developing cutaneous SCCs [It is believed that short-term activation of NLRP1 in keratinocytes by UVB with the subsequent release of IL-1 underlies sunburn in humans ,112,114.ous SCCs . Howeverous SCCs , NLRP1, ous SCCs . Silencious SCCs ,142,143.ous SCCs . Furtherous SCCs  and SCC ous SCCs  and suppous SCCs .The NLRP3 inflammasome contributes to the development of other types of human carcinomas . In oropEvidence for the roles of inflammasomes in skin cancer development also comes from mouse models. Experiments based on chemically induced skin carcinogenesis revealed an important role for IL-1α derived from keratinocytes/tumor cells . IL-1α iNLRP3 and particularly of NLRP1 are associated with the development of melanoma [UV is also the most important risk factor for melanoma, which derive from melanocytes and represent a very dangerous type of skin cancer . SNPs ofmelanoma . As in omelanoma . Howevermelanoma . Furthermelanoma . Later, melanoma . In micemelanoma ,157,158.It is well accepted that inflammasomes play key roles in cancer development. However, IL-1 and other inflammasome effector pathways seem to have distinct and sometimes even opposite functions in carcinogenesis, dependent on the relevant types of inflammasomes and effector pathways, cell types that express and activate these inflammasomes, and the time points at which the activation occurs. In general, IL-1 and inflammasomes are considered tumor promoters, particularly at the early stages of cancer development . ConsequNLRP1 are predisposed to develop cSCCs proves that the NLRP1 inflammasome represents a tumor promoter pathway in the development of NMSC [Although inflammasome activation is believed to be mainly associated with immune cells, the NLRP1 inflammasome is highly expressed by epidermal keratinocytes rather than by immune cells . The fac of NMSC . Most li of NMSC . Similar of NMSC ,111,112. of NMSC . TherefoNevertheless, the expression of not only ASC  but of aIn conclusion, the NLRP1 inflammasome seems to have opposing roles in the early versus late development of cSCCs. IL-1 blockade or NLRP1 inhibition in the skin might be protective and antagonize the early development of cSCCs. This option might be particularly useful for patients suffering from MSPC and FKLC when they start developing skin lesions as a consequence of NLRP1 activation. Indeed, as NLRP1 is expressed at particularly high levels by keratinocytes in human skin, its pharmacological targeting could be achieved by topical treatment, thus without major side effects.TM, which is also an activator of NLRP1, is already used for patients suffering from BCCs [In contrast, NLRP1 or IL-1RI activation could represent useful novel strategies for patients suffering from advanced cSCCs. Indeed, the TLR agonist Aldararom BCCs .


Trichoderma harzianum strain (T673), collected in China, was characterized and annotated as Trichoderma harzianum partitivirus 2 (ThPV2). The genome of ThPV2 consists of a 1693 bp dsRNA1 encoding a putative RNA-dependent RNA polymerase (RdRp) and a 1458 bp dsRNA2 encoding a hypothetical protein. In comparative studies employing the ThPV2-infected strain (T673) and a strain cured by ribavirin treatment (virus-free strain T673-F), we investigated biological effects of ThPV2 infection. While the growth rate of the virus-infected fungus differed little from that of the cured variant, higher mycelial density, conidiospore, and chlamydospore production were observed in the virus-infected strain T673. Furthermore, both the ThPV2-infected and the cured strain showed growth- and development-promoting activities in cucumber plants. In vitro confrontation tests showed that strains T673 and T673-F inhibited several important fungal pathogens and an oomycete pathogen in a comparable manner. Interestingly, in experiments with cucumber seeds inoculated with Fusarium oxysporum f. sp. cucumerinum, the ThPV2-infected strain T673 showed moderately but statistically significantly improved biocontrol activity when compared with strain T673-F. Our data broaden the spectrum of known mycoviruses and provide relevant information for the development of mycoviruses for agronomic applications.A new partititvirus isolated from a  Mycoviruses infect members of all major fungal taxa and oomycetes ,2. So faTrichoderma (syn: Hypocrea) belongs to the class of Sordariomycetes in the phylum Ascomycota . More than 375 Trichoderma species have been reported in various environments including soil, air, and plant surfaces [Trichoderma strains exhibit potent antifungal activity, allowing us to employ these strains in biocontrol programs and possibly to reduce the impact of synthetic chemistries in plant disease control. In addition, Trichoderma strains have been reported to exhibit growth-promoting effects by manipulating plant hormone homeostasis, leading to the enhancement of photosynthesis and carbohydrate metabolism [The genus surfaces . Intrigutabolism ,10,11.Trichoderma strains may be strongly affected by mycovirus infections, which is a concern. However, research on mycoviruses infecting Trichoderma spp. is still in its infancy [Trichoderma spp. isolated in the Chiang Mai province, China [Trichoderma strains isolated from contaminated artificial logs and sawdust bags used for cultivating the shiitake mushroom Lentinula edodes. The size and numbers of bands allowed the categorization of dsRNAs into 15 groups, and the sequencing of randomly selected cDNA clones suggested that two viruses belonged to the family of Hypoviridae [T. atroviride NFCF028 was annotated as Trichoderma atroviride mycovirus 1 (TaMV1) [T. atroviride NFCF394 and T. harzianum NFCF319 identified Trichoderma atroviride partitivirus 1 (TaPV1) and Trichoderma harzianum partitivirus 1 (ThPV1) as novel mycoviruses of the family of Partitiviridae [Both biocontrol and plant growth stimulating activities of  infancy . In the e, China . Interesoviridae . Moreoveiviridae ,17. Moreiviridae , Trichodiviridae , Trichodiviridae , and theiviridae , were idTrichoderma harzianum strain identified in a pool of 120 isolates collected at Xinjiang, Inner Mongolia and Heilongjiang, China. The complete genome of the virus was sequenced, and phylogenetic analyses based on an RNA-dependent RNA polymerase and a hypothetical protein identified this virus as a novel member of the Partitiviridae. Hence, the T. harzianum partitivirus was designated as ThPV2. Subsequent culture-based experiments revealed that this mycovirus affects Trichoderma colony morphology and increases the ability of the fungus to produce spores. Interestingly, despite ThPV2 infection, this Trichoderma strain showed considerable plant development-stimulating and biocontrol activities against several plant pathogenic fungi and an oomycete.In this study, we have characterized a novel dsRNA virus isolated from a Trichoderma isolates from the soil of Xinjiang, Inner Mongolia and Heilongjiang were collected from 2014 to 2016, using the five-points sampling method (w/v) agarose gels.In total, 120 g method . All funhttps://digitalworldbiology.com/blog/bio-databases-2020-viruses-and-covid-19/nar-databases-about-viruses, accessed on 20 March 2020) were used for protein annotation. The sum of contig reads per kilobase per million mapped reads (RPKM) values were calculated for each species and based on the obtained annotations and the RPKM scores. Sequenced contigs with high homologies to mycoviruses were selected for further processing.Prior to dsRNA sequencing, the quality of the isolated RNA was improved by purification with a RNAClean XP Kit  and a Ribo-Zero rRNA Removal Kit . A total amount of >1 µg dsRNA was sequenced at the Biotechnology Corporation Shanghai, China, using high-throughput Illumina hiseq 2000 technology and Illumina Novaseq 6000 . Clean contig sequences were obtained by CAP3 EST and the NAR Databases About Viruses  was used to predict putative open reading frames (ORFs) [https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 10 August 2020) [The ORF finder tool (s (ORFs) . The mycst 2020) , to idenhttps://www.kent.ac.uk/software/mega-7, accessed on 10 August 2020) [https://www.kent.ac.uk/software/mega-7, accessed on 10 August 2020) [A phylogenetic tree was constructed using the maximum likelihood method in Mega 7.0 software (st 2020) . The physt 2020) .T. hrzianum strain T673 was used to establish an isolate cured from mycovirus infection. Strain T673 containing the mycovirus was cultured on potato dextrose agar  amended with 100 µM ribavirin  at 28 °C until sporulation. Spores were transferred to PD broth containing ribavirin (100 µM) and incubated with shaking (200 rpm) at 28 °C. After 18–20 h, the mycelium was collected by filtration and rinsed with sterile distilled water three times, followed by three washes with 0.6 M NaCl. The washed mycelium was protoplasted with lysing enzyme (20 mg/mL) from Trichoderma , as described [3 cells/mL in 1 M Tris-HCl, pH 8.2, containing 1.2 M sorbitol and 1 M CaCl2 (TSC) and allowed to regenerate at 28 °C for 2 days [A ribavirin treatment of protoplasts of  shaking 0 rpm at  shaking 0 rpm at r 2 days . StrainsT. harzianum strains in vitro, six pathogens, i.e., Alternaria solanacearum, Fusarium oxysporum f. sp. cucumerinum, Fusarium pseudograminearum, Botrytis cinerea, Rhizoctonia solani, and Phytophthora capsici, were employed in dual confrontation assays on PDA [To evaluate the antagonistic activities of the mycovirus-infected and cured s on PDA .t-test was used [The strains T673 and T673-F were inoculated onto PDA, corn meal dextrose agar (CMDA), and Czapek–Dox agar . The fungi were cultured in the dark at 28 °C, with 25 replicates per treatment. Mycelial growth rates were recorded daily, and differences in colony morphology were monitored. Furthermore, each of the strains were also inoculated into PD liquid medium and incubated at 28 °C with shaking at 180 rpm for 2 days. To compare differences in biomass accumulation between the two strains, the dry weights of mycelia were determined; 10 replications were performed with each strain. For statistical analyses, a was used .Trichoderma conidiospore production, 5 μL of conidiospore suspensions of strains T673 and T673-F, adjusted to contain 1 × 107 conidia per mL, were inoculated onto PDA and cultured in the dark at 28 °C for 7 days. Generated spores were suspended in 5 mL of sterile water and counted using a hemocytometer. Five μL of a T673 and a T673-F conidiospore suspension, containing 1 × 107 conidiospores per mL each, were allowed to form chlamydospores in the dark at 28 °C for four and five days, respectively. The experiment was repeated three times, with eight plates each. Data were analyzed by t-test [To identify the effects of mycovirus infection on y t-test .Cucumis sativus L. 9930) plants were used in these experiments. Strains T673 and T673-F were inoculated onto PDA plates and cultured in the dark at 28 °C for 5 days until sporulation. Cucumber seeds of similar size were soaked in sterile distilled water for 30 min, and disinfected by soaking in 1% (v/v) sodium hypochlorite for 5 min. After rinsing with sterile distilled water (3–5 times), sterilized seeds were first dipped into 2% (w/v) aqueous carboxymethyl cellulose (CMC) and subsequently dipped into spore suspensions containing 1 × 107 conidia of strains T673 or T673-F per mL. The spore-dipped seeds were placed in a Petri dish with moistened sterile filter paper and cultured at 28 °C in an incubator  for two days. After the seeds had germinated and developed cotyledons and roots, the cucumber seedlings were transferred to a test tube containing 10 mL of 1/8 MS medium and cultured in a greenhouse. Each variant of the experiment was conducted with 8 plants and repeated three times. The growth of cucumber seedlings was monitored at 13, 15 and 35 days post-treatment (dpt). Seedlings not treated with either Trichoderma strain were used as a control. The data analysis was performed by t-test [Cucumber , plants were co-inoculated with strain T673 and Foc or strain T673-F and Foc. The inoculum was adjusted to contain 108 conidia of T. harzianum and/or 107 conidia of Foc per mL. Non-inoculated plants and plants inoculated with Foc alone served as negative and positive controls. Plants were grown in a growth chamber  at 16:8 h light:dark and 23:18 °C rhythm. Each variant of the experiment consisted of 8 plants and was performed in three replicates. The growth and disease index (%age of dead cucumber plants) of cucumber seedlings were observed at 9 and 13 days post inoculation (dpi). Statistical differences were analyzed by t-test [In order to study the effects of mycovirus infection on y t-test .Trichoderma harzinum strain 673 (T673)  A, two ds3 (T673) B,C.High-throughput sequencing of the dsRNAs isolated from strain T673 yielded a total of 39,405 clean reads and six contigs. Primers were designed that matched the internal regions of the two contigs. RT-PCR was performed, and the amplification products designated as contigs 7 and 11 mycovirus cDNAs, respectively . RACE waTo obtain clues as to whether dsRNA1 and dsRNA2 belong to the same virus, we sequenced the positive sense strands of both. Interestingly, the dsRNAs carry the same 5’ terminal sequence (GCCUUUUUGUCUCA), strongly suggesting the same origin of both RNAs. The sequence elements may serve as promoter recognition sites for the viral RNA-dependent RNA polymerase (RdRp) .As outlined above, our data suggest that the T673-derived mycoviral genome consists of two dsRNA fragments, i.e., dsRNA1 and dsRNA2 C. DsRNA1https://blast.ncbi.nlm.nih.gov/Blast.cgi: accessed on 20 August 2020). For the RdRp, the results revealed 14 closely related sequences, i.e., those of Colletotrichum gloeosporioides partitivirus 1  [T. harzianum revealed 12 homologous amino acid sequences of mycoviruses including, again, Colletotrichum gloeosporioides partitivirus 1  [The amino acid sequences of the putative RdRp and the hypothetical protein derived from the mycoviral genome were used as queries for BLASTp searches to identify related mycoviruses (88095.1) , Plasmop88095.1) , Erysiph88095.1)  and Plas88095.1) . BLASTp 88096.1) , as well88096.1) , Phoma m88096.1) , Plasmop88096.1)  and Usti88096.1)  were ideT. harzianum belongs to the Partitiviridae family , and of the hypothetical protein showing 63.9% similarity with Colletotrichum gloeosporioides partitivirus 1 [Partitiviridae. As the virus was isolated from T. harzianum, we designated it as Trichoderma harzianum partitivirus 2 (ThPV2). The nucleotide and predicted amino acid sequences of dsRNA1 and dsRNA2 were submitted to NCBI and are available under the accession numbers OL457022 and OL457023  increased in strain T673, as compared to strain T673-F . Roots of cucumber seedlings were dip-inoculated with spore suspensions of Foc, as well as with spore suspensions of Foc combined with spore suspensions of Trichoderma strains T673 or T673-F. Non-inoculated plants served as controls. At 13 dpi, non-inoculated control plants had formed healthy cotyledons and primary leaves , with Trichoderma species being the most commonly used biocontrol agents. Highlighting the importance of the genus Trichoderma, 30 strains belonging to distinct species are currently used in biocontrol products worldwide [Trichoderma species with the strongest impact comprise T. harzianum, T. atroviride, T. asperellum, T. polysporum, and T. viride [Fusarium oxysporum, Fusarium solani, Fusarium graminicola, Rhizoctonia solani, Botrytis cinerea, Colletotrichum acutatum, Sclerotinia sclerotiorum, and several others [Trichoderma species in combating pathogens include coiling and enzymatic lysis hyphae, the secretion of mycotoxic secondary metabolites, competition for resources, and, importantly, the stimulation of development and induction of resistance in the host [By the year 2020, the volume of pesticides applied worldwide has been estimated at approx. 3.5 million tons annually, with China as the major contributing country, followed by the USA and Argentina . Due to orldwide . Trichod. viride ,51,52,53Trichoderma biocontrol species, including T. harzianum [Cryphonectria parasitica [Castanea dentata) tree populations in the US and in Europe, but, surprisingly, the occurrence of dsRNA viruses belonging to the Hypoviridae family caused a dramatic reduction in virulence in the virus-infected chestnut blight fungus and prevented the European chestnut from eradication [Trichoderma strains may affect the antagonistic activity of these biocontrol fungi as well as their plant growth-modulating activities [Members of all major fungal taxa harbor viruses , and tharzianum ,14,20,50arzianum ,46,47,48rasitica  virtualldication ,54. The dication . Accordidication ,57,58,59T. harzianum, we set out to identify virus-infected strains, to cure a strain from virus infection, and to compare the virus-infected and corresponding cured isogenic strain with respect to plant growth- and development-stimulating activities and corresponding antagonistic effects. From three dsRNA-harboring T. harzianum strains out of 120 that were identified in a previous study [Trichoderma atroviride (Trichoderma atroviride mycovirus 1 [T. harzianum (Trichoderma harzianum bipartite mycovirus 1 (ThMBV1) [Trichoderma asperellum , four m(ThMBV1) , Trichod(ThMBV1) , and Tri(ThMBV1) ), and a  virus 1 ). Of theT. harzianum, a partitivirus-free strain was generated. Interestingly, the virus-containing strain differed slightly from the cured strain in pigmentation and mycelial density and showed more efficient conidiation and chlamydospore formation. Chlamydospores are thick-walled resting spores formed to survive unfavorable conditions. Thus, the fact that formation of these structures is increased in the virus-infected T. harzianum strain is important, as it suggests that the antagonistic inoculum is likely maintained in agricultural environments. Intriguingly, neither the growth- and development-promoting activity nor the antagonistic potential against relevant plant pathogens, i.e., the ascomycetes Alternaria solani, Fusarium oxysporum f. sp. cucumerimun, Fusariumpseudograminearum, and Botrytis cinerea, the basidiomycete Rhizoctonia solani, and the oomycete Phytophthora capsici were attenuated by ThPV2. Likewise, the previously compared virus-containing and virus-free strains 525 and 525F, respectively, of T. harzianum revealed that infection with the as yet unclassified dsRNA virus did not diminish the antagonistic potential against F. oxysporum f.sp. cucumebrium, F. oxysporum f. sp. vasinfectum, and B. cinerea [Pleurotus ostreatus and Rhizoctonia solani was higher in the partitivirus ThPV1-infected strain of T. harzianum NFCF319 than in the corresponding cured strain. As the virus-infected strain exhibited significantly increased β-1,3-glucanase activity, as compared with the cured strain, it can be hypothesized that ThPV1 may regulate β-1,3-glucanase activity and, in turn, virulence [T. atroviride infected by the Alphapartitivirus TaPV1 [In order to examine the effect of ThPV2 on plant development and biocontrol activity of  cinerea . Interesirulence . Howeverus TaPV1 .T. harzianum-infecting partitivirus and compared its effect on growth, sporulation, plant performance, and biocontrol efficacy of its host in detail. Although comparisons between the virus-infected and a cured strain indicated that ThPV2 did not cause adverse effects under defined conditions, it would be interesting to perform comparative greenhouse and field experiments with the ThPV2-infected and the cured strains. Such experiments could highlight environmental effects on the infection with the newly discovered partitivirus ThPV2. The data presented here expand the spectrum of known mycoviruses and provide further information for the development of mycoviruses for agronomic applications.Distinct virus isolates, even if they belong to the same phylogenetic taxa, may differentially affect the phenotype of their host(s). We have completely sequenced the genome of a


Traditional Chinese medicines (TCM), as the unique natural resource, are rich in polysaccharides, polyphenols, proteins, amino acid, fats, vitamins, and other components. Hence, TCM have high medical and nutritional values. Polysaccharides are one of the most important active components in TCM. Growing reports have indicated that TCM polysaccharides (TCMPs) have various biological activities, such as antioxidant, anti-aging, immunomodulatory, hypoglycemic, hypolipidemic, anti-tumor, anti-inflammatory, and other activities. Hence, the research progresses and future prospects of TCMPs must be systematically reviewed to promote their better understanding. The aim of this review is to provide comprehensive and systematic recombinant information on the extraction, purification, structure, chemical modification, biological activities, and potential mechanism of TCMPs to support their therapeutic effects and health functions. The findings provide new valuable insights and theoretical basis for future research and development of TCMPs. In recent decades, increasing researches have confirmed that the leaves, stems, flowers, fruits, and tubers of TCM contain a variety of bioactive compounds such as polysaccharides, polyphenols, proteins, amino acid, fats, vitamins, and other components , 2. TCM 6H10O5)n . Accordi6H10O5)n . FurtherAt present, the researches on TCMPs mainly focus on theirs extraction, purification, structural characterization and modification, and biological activities . NowadayWith the progress of science and technology, the extraction and purification technologies of polysaccharides have been fully developed. The nutritional and medicinal values of TCMPs have attracted extensive attention. The various traditional and novel extraction methods have already been established and proposed . Table 1via RSM, and then purified crude polysaccharides by DEAE-52 cellulose and Sephadex G-100. The results show that the optimal extraction conditions were extraction time of 3 h, extraction temperature of 87 °C, and liquid-to-solid ratio of 39 mL/g. The yield of polysaccharides from black mulberry was 11.86% ± 0.21%. In addition, two purified fractions of BMP-1-1 with molecular weight of 615 kDa and BMP-2-1 with molecular weight of 405 kDa were obtained  with antioxidant capacity by using HWE and optimized extraction process via GA. The results show that the optimal process parameters were pH of 2.0, extraction temperature of 120°C, and extraction time of 3 h. The yield of LJP was 13.31% ± 0.08%. In addition, LJP showed good antioxidant activity including ORAC, ABTS+ radicals scavenging activity and reducing power, suggesting that LJP can be used as a natural antioxidant , microwave assisted extraction (MAE), enzyme assisted extraction (EAE), and ultra-high pressure assisted extraction (UHPAE) methods have been widely used in the TCMPs extraction to improve theirs yield. Furthermore, the extraction conditions also affect the TCMPs yield to some extent. Thus, to improve the TCMPs yield, some optimization methods, including orthogonal experiments, response surface methodology (RSM), genetic algorithm (GA), and genetic algorithm coupled neural network (GA-NN) are used to optimize the extraction process of TCMPs. Wang et al. prepared polysaccharides from black mulberry by using HWE and optimized extraction process obtained . Zhu et ioxidant . Furtherhinensis –20. To sUAE uses the “cavitation effect” and “mechanical shear effect” produced by ultrasound in the extraction process to destroy the plant cell wall, which can dramatically shorten extraction time, reduce energy and solvent consumption, and quickly achieve the purpose of extracting active ingredients . Thus, Uvia RSM based on single factor experimental results. The results show that the optimal combination of process parameters was as follows: Microwave power of 704 W, extraction temperature of 52.2°C, and extraction time of 41.8 min, and the yield of polysaccharides was 19.9% ± 0.2% under the optimal extraction conditions  with the molecular weight of 38.83 kDa, and evaluated its antioxidant activity. It was found that the optimal process was obtained as follows: Microwave extraction of 74 °C, solid-to-liquid ratio of 1:29 g/mL, and extraction time of 15 min. The yield of polysaccharides was 12.31% under above extraction conditions. Additionally, the antioxidant activity of EULP was significantly better than the crude polysaccharides  via MAE, optimized the extraction process by RSM, purified the crude SMPs by using DEAE-52 and Sephadex G-100 chromatography to obtain a novel the homogeneous fraction (SMP1). The results show that the optimal process parameters are as follows: Microwave power of 1,200 W, liquid-to-solid ratio of 38 mL/g, extraction time of 12 min, and ethanol concentration of 86%. The yield of polysaccharides was 14.11% under above extraction conditions. SMP1 with an average molecular weight of 6.087 kDa was consisted of Glu (1%), Gal (1.67%), and Fru (1.12%). Pharmacological researches display that SMP1 protected from OGD/R-induced ferroptosis by activating Nrf2/HO-1 pathway in PC12 cells, suggesting that SMPs can inhibit iron sagging and reduce oxidative stress damage (Fructus Meliae Toosendan (FMTP) by using MAE, optimized the extraction process via RSM based on single factor experimental results, purified the crude polysaccharides to obtain high-purity FMTP polysaccharide fraction with the molecular weight of 1.288 kDa. It was found that the optimum extraction conditions for FMTP: Solvent-to-liquid ratio of 30 mL/mg, extraction time of 20 min, extraction times of 2, and microwave power of 700 W, and the extraction rate of FMTP was 15.75% under the optimum extraction conditions. Furthermore, FMTP could effectively scavenge free radicals , indicating that FMTP can be used as a potential antioxidant in functional foods and drugs. To sum up, MAE has the advantages of high extraction efficiency, energy conservation, and environmental protection. Nevertheless, microwave causes local high temperature in the extraction solution, resulting in polysaccharides degradation  by using ATPE and evaluated its antioxidant activity. It was found that the optimal extraction conditions are as follows: Extraction time of 30 min, extraction temperature of 21.2°C, and 68.9% K2HPO4-20%[Chol]Cl, and the extraction ratio of 68.53% ± 0.29% under the above conditions. PLPS with the weight of 15.2 kDa was consisted of Ara (1.0%), Xyl (1.5%), Man (3.4%), Glc (25.2%), and Gal (1.1%). Moreover, PLPS could effectively scavenge free radicals and show good antioxidant activity (Lycium barbarum L. fruits polysaccharides (LBPs) by using ATPE and optimized the extraction process. The results show that optimal extraction conditions are as follows: Solid-to-liquid ratio of 1:30 g/mL, extraction time of 4 min, and tissue-smashing power of 8,000 rpm, and the yield of LBPs was 24.79 mg/g under the above extraction conditions. In addition, compared with HWE and UAE, the extraction rate of ATPE was the highest  with the weight of 2,954 kDa was obtained, and it was mainly consisted of Glu, and CGFP was a rod-shaped network structure with smooth surface. Moreover, CGFP could improve the levels of SOD, CAT, GSH-Px, and reduce the levels of MDA, LDH, and ROS, suggesting that CGFP can be used as a natural antioxidant with broad development and application prospects  refers to mixing two polymers or one polymer with an aqueous solution of a salt to form a two-phase aqueous system for the extraction active components . Its priactivity . Hu et a highest . Mao et rospects . Li et aonal HWE . HoweverCornus officinalis fruit, Ziziphus Jujuba cv., Lilium davidiivar. unicolor salisb, and Lilium lancifolium thunb  and ultrasound assisted enzyme extraction (UAEE), have become promising substitutes for the traditional extraction methods of TCMPs. UATPE method has the advantages of environmentally friendly, high extraction efficiency, low solvent and energy consumption . Thus, Uum thunb –43  with the weight of 1.7 kDa. The results show that TOP had a backbone composed of (1 → 4)-β-D-Glcp with branching at O-2 of -β-D-Glcp. Moreover, TOP could effectively scavenge DPPH radicals and inhibit the HepG2 cells proliferation, suggesting that TOP can be used as a new natural antioxidant and anti-tumor drug for disease prevention and treatment (Eucommia ulmoides Oliver leaf. polysaccharides (EUOLPs) by anion exchange chromatography. It was found that a high molecular weight and polydispersity polysaccharides was obtained, and purified polysaccharides belonged to β-type acidic heteropolysaccharide structure with glucan group and high degree of branching  by using DEAE-52 and Sephadex G-100 chromatography. The results display that a homogenous fraction (COPs-4-SG) with the weight of 33.64 kDa was obtained and consisted of galacturonic acid, Ara, Man, Glu, and Gal with the molar ratio of 34.82:14.19:6.75:13.48:12.26 (Lonicerae japonica leaves (LJLP) via DEAE-52 and Sephadex G-100 and evaluated its antioxidant activity. It was found that LJLP was mainly comprised of Gal (32.3%), Glu (20.9%), and Rib (15.2%). In addition, LJLP could improve SOD, GSH-Px, CAT activities, whereas LJLP could decrease the levels of MDA in both serum and liver , monosaccharide composition (MC), glycosidic bond type, glycosidic bond position, monosaccharide sequence, and spatial conformation. Furthermore, the analytical methods for the chemical characterization of TCMPs mainly include gas chromatography (GC), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), methylation analysis, Fourier transform infrared spectroscopy (FT–IR), gas chromatography-mass spectrometry (GC–MS), and Smith degradation analysis  by using citric acid extraction. The results show that LJPA with the weight of 17.12 kDa was consisted of Rha (4.51%), Fuc (20.27%), Xyl (12.43%), Man (12.81%), Glu (10.29%), and Gal (39.69%). Moreover, LJPA showed high antioxidant capacities including ORAC, ABTS+ radical scavenging activity and reducing power (Eucommia ulmoides oliver leaf and purified crude polysaccharides by anion exchange chromatography. The results show that a homogeneous fraction (EULP) was obtained and composed of Rha (7%), Ara (4%), Gal (6%), Glu (14%), Xyl (1%), Man (2%), Glucuronic acid (3%), and Galacturonic acid (1%)  and then purified SMPs by DEAE Sepharose Fast Flow and Sephadex G-100 chromatography. It was found that a novel polysaccharide termed SMP1 was obtained and comprised of Glu, Gal, and Fru in a molar ratio of 1:1.67:1.12, and the molecular weight of SMP1 was 6.087 kDa  by UATPE and purified the crude COPs by DEAE-52 and Sephadex G-100 chromatography in turn. It was observed that COPs-4-SG with molecular weight of 33.64 kDa was obtained and composed of Gal acid (34.82%), Ara (14.19%), Man (6.75%), Glu (13.48%), and Gal (12.26%) , fructose (Fru), fucose (Fuc), mannose (Man), arabinose (Ara), rhamnose (Rha), galactose , and xylose (Xyl) . Lu et ang power . Xu et acid (1%) . Meng et, Glu 10.%, and Ga(12.26%) .W of TCMPs is the critical parameter to determine its chemical properties, and theirs biological activities are closely related to the average MW of TCMPs. Currently, the MW of TCMPs is usually determined by HPLC, HPLC–ELSD, high performance gel permeation chromatography (HPGPC), light scattering, gel permeation chromatography (GPC), size exclusion chromatography, and viscosity methods (Grifola frondosa by HWE and then purified the crude polysaccharide via DEAE-52 and Sephadex G-100 to produce the purified polysaccharide (CGFP). The results show that CGFP was mainly consisted of Glu, and the MW of CGFP was 2,954 kDa , Ara (2.33%), Xyl (2.85%), Man (0.69%), Glc (3.05%), and Gal (1.54%). Moreover, AGP showed significant inhibition α-glucosidase activity (Glycyrrhiza glabra polysaccharides (GPN) by using DEAE-cellulose 52 column and Sephadex G-100 column. The results show that the molecular weight of GPN was 38.7 kDa, and GPN was composed of Glc, Man, Ara, and Gal , and then identified its structure via methylation and NMR analysis. It was found that the molecular weight of TOP was 1.7 kDa, and TOP had a backbone composed of (1 → 4)-β-D-Glcp with branching at O-2 of -β-D-Glcp. Moreover, TOP showed good antioxidant and antitumor activities  with molecular weight of 44.7 kDa was obtained and mainly composed of (→ 6)-Glc--Ara--Man-(1 → and → 6)-Gal-(1 →). Additionally, LPIIa showed immunomodulatory activity by stimulating macrophages , Glu (17.6%), Gal (33.7%), and galacturonic acid (15.9%), and the backbone consisted of (→ 5)-l-Araf--d-Glcp--d-Galp--d-GalpA-(1 → and → 6)-d-Galp (1 →). Moreover, PLFP showed good ability to inhibit glycosylation reaction in vitro (Laminaria japonica to obtain the two homogenous fractions (LJP12 and WPS-2-1) and identified their structures. It was found that LJP12 with molecular weight of 2.31 kDa was composed of Ara (1.0%), Xyl (0.17%), Man (1.54%), Glc (2.64%), and Gal (0.18%). LJP12 was suggested to be 1,4-linked and 1,3,6-linked while glucose was linked by 1,6-glycosidic bond. Additionally, WPS-2-1 with molecular weight of 80 kDa was composed of Man, Rha, and Fuc in a molar ratio of 1.0:2.3:1.2, and WPS-2-1 had a backbone of array by (1 → 4)-glycosidic linkages  and evaluated its anti-pancreatic cancer activity. It was found that the molecular weight of ROH05 was 16.7 kDa, and it was composed of 1, 4-linked β-D-Gal and 1, 4, 6-linked β-D-Gal. Furthermore, ROH05A could inhibit BxPC-3 and PANC-1 cells growth in a dose-dependent manner . The sulfated derivative (S-PD), phorphorylated derivative (P-PD), and acetylated derivative (Ac-PD) showed good antiherpetic activity. The results suggest that the types of functional groups are very important for the anti-herpetic activity of polysaccharides. Song et al. prepared the acetylated Pleurotus geesteranus polysaccharides (AcPPS) and evaluated its anti-inflammatory, antioxidant, and lung protection. AcPPS could decrease the levels of lung index, TNF-α, IL-1β, IL-6, p-IκBα, ROS, MDA, TC, TG, LDL-C, HDL-C, and VLDL-C, whereas AcPPS could increase the levels of SOD, GSH-Px, CAT, and T-AOC. These results imply that the acetylated polysaccharides may be suitable as a natural food to prevent or alleviate liver injury  and acetylated derivatives (AC-CPS). The results imply that the carboxymethylation substitution of polysaccharide might be C-6, C-2, and the acetylation modification might be C-3, C-6 inferred from NMR analysis. In addition, CM-CPS and AC-CPS exhibited good α-glucosidase inhibitory activity. Hence, the change structures of polysaccharides have a certain impact on the biological activities. The degree of substitution and the position of substituent are the factors that affect the biological activities of polysaccharides (Schisandra polysaccharides (CSP), purified CSP by using DEAE-52 column to obtain the main fraction (CSPP), and then investigated its structural feature and biological availability. It was found that CSPP with the molecular weight of 1.698 × 104 g/mol was comprised of Man, Glu, and Gal at ratio of 1: 44.8: 3.7. The water solubility of CSPP was significantly higher than that of unmodified polysaccharides. Additionally, CSPP showed higher immunomodulating activity than unmodified polysaccharide, indicating that CSPP can be used as an effective regulatory immunosuppressant (Craterellus cornucopioides polysaccharides and then then modified polysaccharides by carboxymethylation modification to produce two fractions (CCPs-1 and CCPs-2). The results show that CCPs-1 and CCPs-2 were consisted of Rha, Fuc, Ara, Xyl, Man, Glu, and Gal with different molar ratios. CCPs-1 and CCPs-2 were primarily connected by Man with (1 → 3)-linked. In addition, CCPs-1 and CCPs-2 showed good antioxidant activity (Tremella fuciformis (ATP) and modified ATP structure. It was found that the degree of substitution (DS) of the four carboxymethylated derivatives varied with the molar number of chloroacetic acid. With the increase of DS content, their water solubility and biological activities increased, suggesting that carboxymethylation modification can effectively improve the potential biological properties of polysaccharides . The results show that pCIPS was observed the polysaccharides surface features. Additionally, the anti-DHAV activity of pCIPS enhanced after phosphorylation modification  and evaluated its antiviral activity. The results observe that the pRCPS1-4, pRCPS7, and pRCPS9 showed significant anti-viral activity. In addition, phosphorylation modification could be used as a method to improve the antiviral activity of polysaccharides  and modified GUP structure via selenide modification to produce the selenized GUP (SeGUP) and evaluated its antioxidant activity. The thermal stability and particle size of SeGUP were significantly different from those of GUP. Moreover, SeGUP showed greater antioxidant activities in vitro and in vivo when compared to GUP. These results show that selenide modification can significantly enhance the antioxidant activity of SeGUP, and SeGUP has the potential to be developed as a natural antioxidant  in RAW264.7 macrophages , stimulate the production of IgG and IgM in serum, activate the immune system and enhance immune regulation function , and then purified TPP by using Sephadex G-100 column to produce the homogeneous component (TPP-1), and evaluated its hypoglycemic activity. It was found that TPP-1 was consisted of Ara, Man, Glc, and Gal with a molar ratio of 1.00:3.27:4.26:6.01. Moreover, TPP-1 could reduce the blood glucose levels in hyperglycemia mice. TPP-1 could increase significantly the contents of insulin and total superoxide dismutase of the hyperglycemia mice, whereas TPP-1 could decrease the levels of biochemical indexes, including MDA, creatinine, triglyceride, LDL-C, and blood urea nitrogen  with the molecular weight of 1,036 kDa, and determine its antioxidant and hypoglycemic activities. The results show that PSP-1 was comprised of Fuc (18.45%), Ara (2.15%), Gal (19.06%), Glu (1.89%), Xyl (16.07%), Man (1.00%), Galacturonic acid (5.74%), and Glucuronic acid (20.09%). PSP-1 showed good DPPH and OH radicals scavenging activities. Additionally, PSP-1 could remarkably inhibit α-amylase and α-glucosidase activities, improve glucose consumption, glycogen synthesis and the activities of pyruvate kinase and hexokinase in insulin-resistance HepG2 cells, suggesting that PSP-1 can be used as a potential antioxidant and hypoglycemic agent for disease prevention and treatment  using EAE, and then purified the crude LEPs to obtain two homogeneous polysaccharides fractions (LEPs-1 and LEPs-2). These results observed that LEPs-1 and LEPs-2 could markedly inhibit the proliferation of HCT-116 and HeLa cells, indicating that LEPs can be used as a potential natural anti-tumor candidate  induced colitis in mice, and myeloperoxidase (MPO) activity and MDA content in colon tissue decreased and protected colitis in mice by regulating potential targeted proteome and key protein metabolism. Carboxymethyl Poria cocos polysaccharides could interfere with the down regulation signaling pathways of NF-κB, myosin light chain kinase (MLCK), and phosphorylated myosin light chain (p-MLC) in TNF-α injured Caco-2 cells. In addition, polysaccharides could enhance the expression levels of intestinal tight junction protein and effectively prevent colitis   through MAPK pathway, which could effectively protect HaCaT keratinocytes from skin damage caused by UVB, and significantly inhibit UVB induced cytotoxicity  . Poria ciostasis . Li et atoxicity . OverallMany diseases, such as malignant tumor, low immunity, gastroenteritis, alcoholic liver, fatty liver, depression, hyperglycemia, etc. induced by poor diet, work, environment and other factors, have aroused widespread concern about traditional Chinese medicines. In recent years, the various active functions of traditional Chinese medicines have been deeply explored, which has great potential in the development of food, drugs and health products. TCMPs combined with Chinese herbal medicines, anticancer drugs, and vaccines will be the research hotspot. The synergistic effect and structure-activity mechanism of TCMPs and other active ingredients deserve further exploration. Different edible parts, sources, storage, and processing methods of traditional Chinese medicines lead to certain differences in the content, structure, and biological activities of TCMPs. Strengthening the quality control of traditional Chinese medicine is the key to keep the chemical structure and biological activities of TCMPs reliable and consistent in food and pharmaceutical applications.TCMPs have poor water solubility, low yield, difficult separation and purification, and low biological activities. The water-soluble TCMPs produced by modification can enhance the original biological activities or produce new biological activities. Hence, the structural modification of TCMPs is the main research direction of scholars at home and abroad. Currently, carboxymethyl TCMPs are used to develop adjuvant drugs for cancer treatment and put into the market. Great breakthroughs have been made in the market development of TCMPs. However, the study on the relationship between the structure and bioactivity mechanisms of TCMPs is relatively limited due to the limitations of spatial structure testing technology, which limits its development and application. Hence, the quality of TCMPs derivatives can be controlled by further standardizing the quality of TCMPs, deeply studying the various biological activities of TCMPs, and clarifying the fine structure and structure-activity relationship of TCMPs. Especially, the chemical modification degree and reliable pharmacokinetic study of TCMPs derivatives can promote the development of new polysaccharides or health food.HX and PL: formal analysis (supporting), investigation , and writing-original draft . JB, YG, and YS: formal analysis (supporting) and investigation . JT: funding acquisition , project administration , and writing-review and editing . 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.


IL1RN, GALNT12, ADH6, SCN7A, CXCL1, FGF18, SOX9, ACACB, PRRX1, MZB1, SLC22A3, CNNM4, LY6E, IFITM2, and GDPD3. Among them, IL1RN, ADH6, SCN7A, ACACB, MZB1, and GDPD3 exhibited statistically significant survival differences, whereas limited studies were conducted in CRC. Based on the enrichment results of the “Gene Ontology“(GO) and “Kyoto Encyclopedia of Genes and genomes “(KEGG) as well as documented findings of key genes, we further emphasized the potential of IL1RN and PRRX1 as markers of immune infiltrates in CRC and confirmed our hypothesis by compiling data from the UALCAN, Tumor Immune Estimation Resource, and TISIDB databases for these two genes. The above-mentioned genes might offer a valuable insight into the diagnosis, immunotherapeutic targets, and prognosis of CRC.The extensive morbidity of colorectal cancer (CRC) and the inferior prognosis of terminal CRC urgently call for reliable prognostic biomarkers. For this, we identified 704 differentially expressed genes (DEGs) by intersecting three datasets, GSE41328, GSE37364, and GSE15960 from Gene Expression Omnibus database, to maximize the accuracy of the results. Preliminary analysis of the DEGs was then performed using online gene analysis datasets, such as DAVID, UCSC Cancer Genome Browser, CBioPortal, STRING, and UCSC Cancer Genome Browser. Cytoscape was utilized to visualize the protein perception interaction network of DEGs, and the bubble map of GO and KEGG enrichment function was demonstrated using the R package. The Molecular Complex Detection (MCODE), Biological Network Gene Oncology (BiNGO) plug-in in Cytoscape, was applied to further screen the DEGs to obtain 15 seed genes, which were  Colorectal cancer (CRC) remains the third most prevalent cancer globally, with the most recent data estimating its incidence to be the second highest mortality rate . SmokingBiomarkers are signposts for early cancer detection and individualized CRC treatment . Most noIL1RN and PRRX1 in CRC. Furthermore, mapping of DEGS function enabled us to identify more precise key genes accompanying with a deeper perception of the role of IL1RN and PRRX1 in CRC.Hence, here, we obtained the differentially expressed genes (DEGs) of CRC by interacting multiple Gene Expression Omnibus (GEO) gene microarray datasets, furthermore, using diverse bioinformatics tools (DAVID and Tumor Immune Estimation Resource [TIMER]) to explore the possible mechanisms linking DEGs to CRC. These include GO and KEGG pathway enrichment analyses, and protein perception interactions (PPI) network as well as immune infiltration were also included. It also reveals the potential critical role of http://www.ncbi.nlm.nih.gov/geo) database is known as a freely accessible database containing gene expression information, such as gene chips, high-throughput gene expression data, and gene microarrays [GEO  [P < 0.05, logFC (fold change) >1 or logFC <−1 for DEG screening [http://bioinformatics.psb.ugent.be/beg/tools/venn-diagrams) [Comparison of target datasets filtered from GEO database was performed by GEO2R (o/geo2r) . Furthercreening . During iagrams) .http://david.ncifcrf.gov) [P < 0.05 considered statistically significant [GO  and KEGGcrf.gov) , which ccrf.gov) , 27. Thenificant , 28.http://string-db.org), an online gene interaction database, and the PPI interactions network was constructed using a “combined score > 0.4” as a screening condition [http://www.cytoscape.org; version 3.6.1) for visualization [kScore = 2. The major functions of the seed genes were searched via NCBI (https://www.ncbi.nlm.nih.gov/gene) and were presented in a table format.The DEG interactions were analyzed using STRING  [http://genome-cancer.ucsc.edu) [https://www.cbioportal.org/) [GO enrichment analysis and KEGG pathway analysis of the hub gene were performed through the DAVID Gene Analysis Online database  . The anacrf.gov) . Construcsc.edu) . Overallal.org/) .http://ualcan.path.uab.edu/), an online data site that integrates RNA-seq and clinical data from 31 malignancies of TCGA , was used to perform the analysis of IL1RN expression levels with PRRX1, including differences in tissue type , and GC staging  in COAD as well as READ [UALCAN ( as READ .https://cistrome.shinyapps.io/timer/) was used to systematically analyze the level of immune infiltration in various malignancies [IL1RN and PRRX1 expression and tumor-infiltrating lymphocytes (TILs) expression in COAD and READ was investigated through the TIMER gene module. Furthermore, the interaction between CRC and the immune system, immune cells, was studied through the online platform TISIDB (http://cis.hku.hk/TISIDB/index.php) [IL1RN and PRRX1 with 28 TILs, 45 immunostimulants, and 24 immunosuppressive agents in CRC.The TIMER2.0 (gnancies . The reldex.php) . This inhttps://www.proteinatlas.org/) allows online access to human proteins mapped in cells, tissues, and organs through the integration of a variety of histological techniques (including antibody-based imaging and transcriptomics) [IL1RN and PRRX1 expressions in normal colorectal tissues and CRC tissues were retrieved from the HPA database.The HPA database (ptomics) . IL1RN aAfter data validation analysis, 1452 (GSE41328), 3424 (GSE37364), and 6037 (GSE15960) DEGSs were obtained, and a total of 704 DEGs were found to be present in all three datasets using online Venn diagram analysis .IL1RN  and myeloid dendritic cells , HAVCR2 , PDCD1LG2 , IL-10 , HAVCR2 (RHO = 0.508 READ), and PDCD1LG2 . IL1RN is also closely linked to immunostimulatory factors, as shown by CD86 , TNFRSF9 , CXCR4 , CD86 , and CD80 , ENTPD1 , TNFSF4 , CXCR4 , and TNFSF13B  were significantly associated with PRRX1 expression, as well as with immunosuppressive factors, TGFB1 , KDR , HAVCR2 , TGFBR1 , and PDCD1LG2  were similarly the same , we discovered that IL1RN, ADH6, SCN7A, ACACB, MZB1, and GDPD3 were very limitedly studied in the context of CRC. Among them, IL1RN was the hub gene with the most edges found in this study, despite the fact that its overall survival or disease-free survival showed differences between the cancer-bearing and normal populations, but online databases suggested that the results were not statistically significant. Ma et al. had explored the ability of antagonizing IL-1 to inhibit CRC liver metastasis, but did not dive into the prospects of IL1RN in the context of CRC [IL1RN might provide novel orientations in curing glioma [IL1RN polymorphisms have similarly been proven to reduce the population risk of thyroid cancer risk [IL1RN is closely related to tumor immunity and tumor metabolism, of which further studies are needed to investigate its value in the prognosis of immune infiltration. According to our study, a strong correlation was shown between IL1RN and CD274, which is well-known as PD-L1 [IL1RN and CD274 implies that IL1RN is likely to be involved in PD-L1 targeted therapy in the future. In addition, we noted PRRX1 possessed the astonishing potential to be a prognostic biomarker correlated with immune infiltrates in CRC. Currently, a few publications have described the capacity of PRRX1 to induce the EMT process in CRC cells, which in turn facilitates distant CRC metastasis [PRRX1 in alternative tumor contexts also focus mostly on its induction of the EMT process in tumor cells, which leads to implications of proliferation, migration, and invasion of tumor cells [PRRX1 to interact with tumor immune infiltration and thus affect patient prognosis. By compiling data from online database, we found a remarkable correlation between PRRX1 and TILs. PRRX1 showed significant correlations with both immunostimulators and immunoinhibitors. For instance, the correlation coefficient between PRRX1 and CD86, a co-stimulatory molecule on antigen-presenting cells that had been demonstrated to act as a pivotal role in tumor immunity in pancreatic and bladder cancers, reached 0.683 [PRRX1 in CRC tissues gradually increased. All these evidences pointed to the promising potential of PRRX1 as a marker of tumor immune infiltration. Although current study initially reveals the utility of IL1RN and PRRX1 as markers of immune infiltration, what is lacking is that this conclusion relies only on data from online databases and lacks specific experiments to validate this unique potential.t of CRC . Wang etg glioma . IL1RN pcer risk . Existinas PD-L1 . A bruncas PD-L1 . CD274 pas PD-L1 . Targetias PD-L1 . The cortastasis . Moreoveor cells , 52. Howed 0.683 , 54. In Furthermore, although our study identified hub genes as affecting CRC and revealed promising potential for being biomarkers, subgroup information, such as tumor location as well as staging, was not precisely pinpointed. Along with the disclosure of increased sequencing data, precisely targeting biomarker function to location-specific, stage-specific features would be much more likely.SCN7A, ACACB, and MZB1 showed statistically significant prognostic results, further biological experiments are required to investigate their roles in the context of CRC.Similarly, whereas the aforementioned IL1RN and PRRX1 as markers of tumor immune infiltration in CRC tissues.In conclusion, 704 DEGs with specific expression in CRC were identified by bioinformatics means, basing on which 15 selected genes with enhanced differential properties were further identified, all of which may operate as diagnostic markers for CRC. Furthermore, 15 seed genes were further characterized to identify initially the potential of


FOS orthologues, fosaa, fosab, fosb, fosl1a, fosl1b, and fosl2, which show high conservation in key regulatory domains and post-translational modification sites compared to their equivalent human proteins. During embryogenesis, zebrafish fos genes exhibit both overlapping and distinct spatiotemporal patterns of expression in specific cell types and tissues. Most fos genes are also expressed in a variety of adult zebrafish tissues. As in humans, we also found that expression of zebrafish FOS orthologs is induced by oncogenic BRAF-ERK signalling in zebrafish melanomas. These findings suggest that zebrafish represent an alternate model to mice for investigating the regulation and functions of Fos proteins in vertebrate embryonic and adult tissues, and cancer.Members of the FOS protein family regulate gene expression responses to a multitude of extracellular signals and are dysregulated in several pathological states. Whilst mouse genetic models have provided key insights into the tissue-specific functions of these proteins in vivo, little is known about their roles during early vertebrate embryonic development. This study examined the potential of using zebrafish as a model for such studies and, more broadly, for investigating the mechanisms regulating the functions of Fos proteins in vivo. Through phylogenetic and sequence analysis, we identified six zebrafish  The mammalian FOS proteins, c-FOS, FOSB, FOSL1, and FOSL2, belong to the activator protein-1 (AP-1) family of transcription factors that contain evolutionarily conserved basic leucine zipper (bZIP) domains ,4,5. AP-FOS family members are implicated with the pathogenesis of various diseases . Aberranc-Fos deficiency perturbs normal development of bone, cartilage, and the haematopoietic system [Fosb deficiency impairs brain development, leading to nurturing defects in the adult female mouse, while transgenic overexpression of Fosb2 in the thymus disrupts T-cell differentiation [Fosl1 and Fosl2 in mice caused embryonic lethality, while their overexpression resulted in abnormalities in bone and ocular development [Fosl1 deletion also led to placental defects associated with the aberrant differentiation of trophoblasts [Genetic knockout and transgenic mouse models have shown that individual Fos proteins have specific in vivo functions ,27. c-Foc system , whereasc system . Fosb dentiation . Deficieelopment ,27. Foslhoblasts ,32.FOS homologues, fosaa, fosab, fosb, fosl1a, fosl1b, and fosl2, which encoded proteins showing high sequence conservation in key regulatory domains and post-translational modification sites compared to the equivalent human proteins. Spatiotemporal expression analysis revealed both overlapping and distinct patterns of fos gene in specific cell types and tissues during early embryonic development. Our data thus suggest that the zebrafish represents a valuable vertebrate model for investigating Fos protein regulation in vivo and defining their functions during embryogenesis.Despite providing important insights into the in vivo functions of specific FOS proteins in normal and disease states, the use of mouse models to investigate the mechanisms regulating the activities of individual FOS isoforms in vivo is challenging. In addition, mouse models have provided limited insight into the functions of FOS isoforms during early vertebrate development. The zebrafish has emerged as a robust in vivo model for early developmental studies, as its embryos are transparent and develop externally, facilitating easy observation of key processes . The zebFOS gene family consists of FOS, FOSB, FOSL1, and FOSL2 [FOS genes, which synteny analysis indicated were single fosb and fosl2 orthologues, but duplicated fosaa/fosab and fosl1a/fosl1b paralogues  . Key regIn contrast to c-FOS and FOSB, human FOSL1 and FOSL2 lack potent transactivation domains . The ovefosaa transcripts were detected in 1-cell embryos (0 hpf), and by 12 hpf (6-somite stage), zygotic expression was observed in the brain , presomitic mesoderm, somites, and tailbud  and formation of diverse cell lineages including melanocytes, smooth muscle, craniofacial bone and cartilage, cranial neurons, and glia. The pattern of fosab expression was similar to fosaa in early embryos  and cell lineages .Wholemount in situ hybridisation (WISH) analysis was used to establish the pattern of expression of each zebrafish Fos isoform during early embryonic development. Maternal  tailbud A, and at tailbud A,A’. Frocontrols . These r embryos B but per embryos D–F’. The embryos , and the embryos , while a embryos . These ofosb was expressed from 8 hpf (75% epiboly stage) , heart, hatching gland, and tailbud (Zebrafish y stage) C, with sy stage) G,G’. At  tailbud , while a tailbud .fosl1a transcripts were not detected at early time points, but were observed in the forebrain, pancreas, and tailbud at 24 hpf   E. At 24 ng gland M,M’, whing gland N,N’. Thufosl2 showed the highest early embryonic expression being detected at 12 hpf in the eyes, somites, presomitic mesoderm, mid and hindbrain, and tailbud , eye, otic vesicle, somites, and tailbud   .fosaa, fosab, fosb, fosl1a, fosl1b, and fosl2 was similar in adult zebrafish female tissues, but with modest variations compared to males  and the Zebrafish Information Network . The FASTA sequences of human FOS mRNAs were obtained from the NCBI database and used for Basic Local Alignment Search Tool (BLAST)  analysis to identify the zebrafish orthologues which were further crosschecked with the ZFIN database and confirmed by sanger sequencing.Zebrafish orthologues of human FOS gene loci, including syntenic genes, was determined using NCBI Map Viewer. Information on regulatory phosphorylation sites in human FOS proteins was obtained from the Phosphosite database . Synteny analysis for zebrafish and human FOSL1 and FOSL2 genes was performed using the Genomicus database [Sequence comparisons of human and zebrafish FOS protein orthologues were determined using pBLAST. Multiple sequence alignments (MSA) were performed using Clustal X version 2.1 software and bootstrapped phylogenetic trees of 1000 replicates generated using the neighbor-joining algorithm , then vidatabase .w/v) 1-phenyl-2-thio-urea (PTU), a pigment inhibitor into the E3 media from 9 h post fertilization (hpf).Zebrafish were maintained under standard husbandry practices , followifosaa , fosab , fosb , fosl1a , fosl1b , fosl2 , and actb . Standard and unknown samples were assayed in triplicate using the following thermocycle profile conditions: initial incubation at 95 °C for 5 s, 58 °C for 10 s, and then 72 °C for 20 s. Data was normalized to actb and the relative fold changes in adult male and female zebrafish tissue gene expression were determined by comparison with the expression in the male eyes  by using the ddct method [Adult zebrafish were euthanised with benzocaine and their tissues were isolated by dissection. Total RNA was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s guidelines. qRT-PCR was performed using the iTaq™ Universal SYBRGreen Supermix  according to the manufacturer’s guidelines with the following primers: fosaa , fosab , fosb , fosl1a , fosl1b , and fosl2 . PCR reactions were performed under the following conditions for 35 cycles: 2 min at 95 °C, 30 s at 95 °C, 30 s at 55 °C, 1.5 min s at 72 °C, and 10 min at 72 °C. RT-PCR amplified cDNA products of fosaa, fosab, fosb, fosl1a, fosl1b, and fosl2 were isolated, purified and cloned into pGEM-T Easy vectors. Purified plasmids with the gene insert were linearised, followed by transcription using either T7 or SP6 polymerase and DIG RNA labelling mix (Roche) to generate DIG-labelled anti-sense and sense probes, which was followed by their purification using Probe-Quant G-50 micro columns (Cytiva). At different time points during development, embryos were collected, dechorionated, and anesthetized with 0.4 mg/mL benzocaine before fixing with 4% w/v paraformaldehyde (PFA) at 4 °C. WISH was performed using digoxygenin (DIG)-labelled RNA probes as described in [Total RNA was isolated from zebrafish embryos using TRIsure (Bioline) according to the manufacturer’s instructions and cDNA synthesis was performed using an iScript cDNA synthesis kit (Promega). RT-PCR was performed to amplify cDNA by using the Go Taq Green Master Mix  using the following primers: ribed in  and the V600E (MiniCoopR mitfa:BRAFV600E) was microinjected along with 25 pg of Tol2 transposase mRNA into one-cell stage tp53 knockout embryos. The animals were monitored weekly for the presence of visible tumours. The tumours were dissected and RNA extracted for subsequent qRT-PCR analysis to determine the expression of zebrafish fos genes.Zebrafish melanoma formation was studied as described previously . Briefly


ACAP1 plays a key role in endocytic recycling, which is essential for the normal function of lymphocytes. This study aimed to assess the expression and function of ACAP1 in lymphocytes. In this study, we revealed that ACAP1 is expressed primarily in lymphocytes and is necessary for the normal function of lymphocytes. Its expression is regulated by promoter DNA methylation and the transcription factor SPI1. ACAP1 levels positively correlate with the infiltrating levels of tumor-infiltrating lymphocytes (TILs) across a broad range of solid cancer types. Moreover, ACAP1 deficiency is associated with poor prognosis and immunotherapeutic response in multiple cancer types treated with immunotherapy. Thus, this study demonstrates that ACAP1 is a novel lymphocyte marker. We propose a widely applicable indicator to predict response to immunotherapy that may guide cancer patient stratification for appropriate therapy.Background: ACAP1 plays a key role in endocytic recycling, which is essential for the normal function of lymphocytes. However, the expression and function of ACAP1 in lymphocytes have rarely been studied. Methods: Large-scale genomic data, including multiple bulk RNA-sequencing datasets, single-cell sequencing datasets, and immunotherapy cohorts, were exploited to comprehensively characterize ACAP1 expression, regulation, and function. Gene set enrichment analysis (GSEA) was used to uncover the pathways associated with ACAP1 expression. Eight algorithms, including TIMER, CIBERSORT, CIBERSORT-ABS, QUANTISEQ, xCELL, MCPCOUNTER, EPIC, and TIDE, were applied to estimate the infiltrating level of immune cells. Western blotting, qPCR, and ChIP-PCR were used to validate the findings from bioinformatic analyses. A T-cell co-culture killing assay was used to investigate the function of ACAP1 in lymphocytes. Results: ACAP1 was highly expressed in immune-related tissues and cells and minimally in other tissues. Moreover, single-cell sequencing analysis in tumor samples revealed that ACAP1 is expressed primarily in tumor-infiltrating lymphocytes (TILs), including T, B, and NK cells. ACAP1 expression is negatively regulated by promoter DNA methylation, with its promoter hypo-methylated in immune cells but hyper-methylated in other cells. Furthermore, SPI1 binds to the ACAP1 promoter and positively regulates its expression in immune cells. ACAP1 levels positively correlate with the infiltrating levels of TILs, especially CD8+ T cells, across a broad range of solid cancer types. ACAP1 deficiency is associated with poor prognosis and immunotherapeutic response in multiple cancer types treated with checkpoint blockade therapy (ICT). Functionally, the depletion of ACAP1 by RNA interference significantly impairs the T cell-mediated killing of tumor cells. Conclusions: Our study demonstrates that ACAP1 is essential for the normal function of TILs, and its deficiency indicates an immunologically “cold” status of tumors that are resistant to ICT.  Cancer is a significant public health burden with an ever-increasing incidence rate worldwide. ICT has revolutionized cancer treatment, resulting in an outstanding clinical benefit for certain patients, especially for advanced-stage cancers. It is currently the first-line treatment for advanced melanoma and lung cancer. However, most patients have limited or no response or clinical benefits from ICT . FurtherACAP1 , also named CENTB1 (Centaurin beta1), negatively regulates the function of ARF6. It also interacts with Akt to form a protein complex, acting as an adaptor for the endocytic recycling of intracellular cargos such as transferrin receptor, cellubrevin, integrin β1, and Glut4 (glucose transporter type 4) ,9,10,11.This work systematically explores the genetic alterations, expression patterns, prognostic significance, and transcriptional regulation of ACAP1 across various cancer types. Furthermore, we investigate the relationship of ACAP1 expression with TILs and the ICT response of patients. We identify ACAP1 as a lymphocyte-specific gene and show that ACAP1 is indispensable for the normal function of lymphocytes. Its deficiency ubiquitously correlates with the “cold” immune phenotype and indicates resistance to immunotherapy in solid tumors. Based on these results, we identified a pan-cancer predictor of TILs and immunotherapy response, which could help clinicians optimize treatment regimens for cancer patients.Gene expression for ACAP1 in normal human tissues was queried through the GTEx (Genotype-Tissue Expression) portal, a large-scale database containing 54 types of normal tissues . Gene ex2(x + 1) transformed. The CpGs located on the ACAP1 promoter were identified using xena-browser [Clinical data, RNA sequencing (level 3 HTSeq-FPKM), DNA methylation data, and copy number variation (CNV) data across 33 tumor types were obtained from TCGA (The Cancer Genome Atlas). The clinical data analyzed included T, N, M, pathologic stage, and survival information. The TCGA cancer types and corresponding abbreviations are seen in -browser . The proOne glioblastoma dataset (‘Neftel cohort’ ), two prEight immunotherapy-related datasets were acquired for subsequent analyses of the relationship of ACAP1 expression with immunotherapy response and the prognosis of immunotherapy-treated patients. These datasets were: (I) four melanoma cohorts, including “VanAllen 2015” , “Gide 2The protein levels of ACAP1 in tumors and corresponding paracancerous tissues were compared using CPTAC  datasets through UALCAN.  All avaiTo confirm the association of ACAP1 levels with prognosis in cancer patients from TCGA, four datasets, including ICGC-LIRI-JP , GSE6846The ChIP-sequencing data was analyzed using Cistrome and visualized with the UCSC Genome Browser. The SPI1 datasets used were: GSM1681425 , GSM1703https://jaspar.genereg.net/; accessed on 1 July 2022). All SPI1 motifs in the JASPAR database were used for binding enrichment analysis using a significance threshold of 0.90. The SPI1-binding sites surrounding the ACAP1 promoter region were predicted using JASPAR  and “survival” . The patients were dichotomized into high and low groups based on an optimal cutoff value, which was determined with the “survminer” package.Survival analyses were ascertained using Kaplan–Meier methodology and analyzed by the log-rank test with the R  packages “survminer”  and summarized in Spearman’s correlation analyses of ACAP1 with all other genes were performed across TCGA tumors. The calculated correlation coefficient in each tumor type was used for GSEA analysis using GSEA software (version 4.1.0) with the following parameters: database = c5.go.bp.v7.4.symbols.gmt, number of permutations = 1000, enrichment statistic = weighted, max size = 500, min size = 3. The genes involved in the main associated pathways were obtained from the MsigDB . Recombinant human IL-2  was added to the TALL-104 culture media with a final concentration of 100 units/mL. A-498 was cultured in MEM + 10% FBS. HCT116, HT-29, SW480, and SW620 were cultured in DMEM + 10% FBS. Cells were obtained from Shanghai Cell Bank  or ATCC.® TRC shRNA library, Sigma-Aldrich, St Louis, MO, USA) and sub-cloned into the pLKO.1 vector. shRNA1: CCGGGCAGGAGATGAGACGTATCTTGGATCCAAGATACGTCTCATCTCCTGCTTTTTG; shRNA2: CCGGTCACGCTAAATACGTGGAGAAGGATCCTTCTCCACGTATTTAGCGTGATTTTTG.For SPI1 overexpression, the coding sequence of the SPI1 genes was amplified by PCR and sub-cloned into pSIN-3×Flag for lentivirus production. For ACAP1 knockdown, the sequences of two shRNAs targeting human ACAP1 were obtained from The RNAi Consortium  to package virus particles. Then, the cells were transduced with recombinant lentivirus suspension (+8 μg/mL polybrene) and selected with 1 μg/mL puromycin to obtain stable cell lines.For the demethylation treatment, SK-HEP-1 and Huh-7 cells were treated with 30 μM 5-aza  or DMSO for five days. Then, total RNA was harvested, reverse-transcribed, and analyzed by qRT-PCR.2 (100 and 200 μM) for 8 h and 24 h. Cells were then harvested for subsequent Western blotting.For the hypoxia treatment, Jurkat cells were treated with vehicle or hypoxia mimetic CoClA ChIP assay was carried out in the Flag-SPI1-overexpressing Jurkat cells using a ChIP kit  as the manufacturer’s instructions. The Flag antibody  was used to enrich the DNA fragments containing the putative SPI1 binding site of the ACAP1 promoter, and the normal IgG  was used as a negative control. Primers used for ChIP-PCR were: 5′-ACTGCCTGGAAGTGTGGGGT-3′ and 5′-ATGCAGGTGGAGGCACTTTCT-3′.Total RNA was harvested, reverse-transcribed, and analyzed by qRT-PCR as described before . The priThe assay was performed as previously described . The priThe assay was carried out as described . Brieflyt-tests were used to compare the difference between two groups, and Wilcoxon signed-rank test or paired t-test was used for paired comparisons. Spearman correlation was used for all correlation analyses. Log-rank test was used for survival analyses. All analyses were conducted in GraphPad (version 9.3.0) or R (version 3.6.0). p-values < 0.05 were considered significant. Wilcoxon rank-sum test or two-sided Student’s We first examined the expression distribution of ACAP1 in normal human tissues using the GTEx portal. ACAP1 exhibited a tissue-specific expression pattern across different tissues. Its expression was enriched in immune-related tissues and cells, including whole blood, spleen, and lymphocytes. It showed a moderate expression level in the small intestine and lung but minimal expression in other tissues A. ExpresTo better understand the expression pattern of ACAP1, we extended the analysis to the single-cell level. Analysis of two melanoma single-cell sequencing cohorts, including GSE72056 and GSE115978, revealed that ACAP1 was highly expressed in lymphocyte cells, including T, B, and NK cells but was barely expressed in malignant cells and other types of immune cells A,B. The Altogether, these collective results suggest that ACAP1 is predominantly expressed in lymphocytes, including T, B, and NK cells, and may be a marker gene for TILs.We then profiled the expression of ACAP1 in tumor samples and corresponding normal samples across 33 TCGA cancer types by integrating the sequencing data from the TCGA and GTEx datasets. It revealed that ACAP1 was significantly decreased in most cancer types (17 of 33) compared to normal samples, including BLCA, BRCA, COAD, DLBC, GBM, KICH, LGG, LIHC, LUAD, LUSC, PRAD, READ, TGCT, THCA, THYM, UCEC, and UCS. Conversely, ACAP1 expression was significantly increased in six cancer types: CHOL, HNSC, KIRC, KIRP, LAML, and PAAD, compared to normal samples A. PairwiTo determine whether ACAP1 expression reflects cancer progression, we assessed ACAP1 expression in different T, N, M, and pathologic stages. It revealed that the low expression of ACAP1 tended to be accompanied by advanced stages in most cancer types . For exaAltogether, the ACAP1 expression in cancers was lower than in normal or paracancerous tissues in the majority of cancer types, although the differential expression between them varies across cancer types. Moreover, low ACAP1 levels were often associated with advanced stages.We next evaluated the prognostic value of ACAP1 mRNA expression for cancer patients by Kaplan–Meier analysis of OS , DSS , and PFI . The OS analyses across TCGA datasets indicated that low ACAP1 levels correlated with shorter survival in patients with BLCA, BRCA, CESC, HNSC, LIHC, LUAD, OV, PAAD, PCPG, SARC, SKCM, STAD, THYM, UCEC, and UCS A–O but mThe DSS analysis in TCGA demonstrated that patients with low ACAP1 levels possessed significantly worse DSS than those with high ACAP1 levels in ACC, BLCA, CESC, DLBC, HNSC, LIHC, LUAD, PAAD, SARC, SKCM, UCEC, and UCS but better DSS in GBM and KIRC . The PFIOverall, these results indicate that, despite the prognostic significance of ACAP1 expression varied by tumor type, low ACAP1 expression was associated with poor clinical outcomes in the majority of tumor types, especially solid tumors.Next, we attempted to explore the underlying mechanisms that regulate ACAP1 expression. DNA methylation is a prominent regulatory mechanism for gene expression. There are four CpG sites, including cg13295242, cg13670306, cg11807006, and cg25671438, in the ACAP1 promoter A. We fouCNV (copy number variation) often positively regulates gene expression. ACAP1 deep deletion and amplification, both of which are CNV, were observed in most cancer types (25 of 32) . SpearmaTranscription factors also play a key role in the regulation of gene expression. As a result, we investigated whether SPI1 (PU.1), a major transcription factor involved in immune cell activity, modulates ACAP1 expression. Spearman correlation analysis indicated a strong positive correlation between ACAP1 and SPI1 expression in the TCGA pan-cancer dataset A and 28 Taken together, our results suggest that ACAP1 expression is positively modulated by CNV and SPI1 but negatively regulated by promoter methylation. To identify the specific pathways associated with ACAP1 expression, we initially performed pathway enrichment analysis using GSEA for each TCGA cancer type based on ACAP1 expression in TCGA cohorts. The analyses suggested that ACAP1 expression was strongly associated with multiple immune-related “Biological Processes”, such as “B cell-mediated immunity”, and “humoral immune response mediated by circulating immunoglobulin”, in almost all tumor types, except two immune-related tumor types (LAML and THYM) . As we have demonstrated that ACAP1 is a lymphocyte marker, combined with these GSEA results, we hypothesized that ACAP1 could be a suitable, sensitive, and broadly applicable indicator of the infiltrating level of TILs. We then systematically analyzed the relationship between ACAP1 expression and the immune infiltrates across diverse cancer types in TCGA, except LAML, via TIMER2, which estimates the abundances of immune infiltrates using eight immune deconvolution algorithms, including xCell, CIBERSORT, CIBERSORT-ABS, EPIC, MCPCOUNTER, QUANTISER, TIMER, and TIDE. All of these immune deconvolution analyses showed that ACAP1 was significantly positively correlated with the level of CD8+ and CD4+ T cells in most cancer types, except DLBC . In addiGiven the high ACAP1 levels in lymphocytes, we postulated that ACAP1 might play an essential role in the immune response of these cells. TALL-104 is a lymphocyte cell line that expresses markers characteristic of both NK cells and cytotoxic T-lymphocytes ,67. The Moreover, we found that ACAP1 expression was strongly positively correlated with cytotoxicity score (CYT) and the expression of GZMA and PRF1, which encode two key cytolytic effectors (granzyme A and perforin 1), in most cancer types . It was Thus, the analyses of immune infiltrates from different methods consistently pinpointed that ACAP1 expression was significantly positively correlated with the level of TILs, especially CD8+ T cells, in the majority of cancer types. Meanwhile, ACAP1 is essential for the cytotoxicity of lymphocytes. http://tide.dfci.harvard.edu/setquery/, accessed on 13 September 2022) to determine the performance of ACAP1 expression in predicting the ICT response. We also compared its predictive power with existing biomarkers, including the TIDE score, the MSI score, tumor mutational burden (TMB), and CD274 (PD-L1), by calculating the AUC (area under the ROC curve). As shown in As ACAP1 expression was strongly correlated with immune cytotoxicity activity and the infiltrating level of CD8+ T cells in tumors at a pan-cancer level, we wondered whether ACAP1 expression could predict the tumor response to ICT and the prognosis of ICT-treated patients. Firstly, we utilized the ‘Biomarker evaluation’ module from TIDE (Tumor Immune Dysfunction and Exclusion) , two lung cancer datasets (“Ruppin 2021” and GSE126044), a kidney cancer dataset (“Miao 2018”), and an mUC dataset (IMvigor210). All sequencing samples were collected before therapy, except for a subset of patients in the “Riaz 2017” cohort. Analysis from “VanAllen 2015” and “Snyder 2014” cohorts showed that ACAP1 expression in the non-response group was lower than in the response group of melanoma patients receiving anti-CTLA-4 therapy. Furthermore, low ACAP1 expression was associated with worse OS and PFS  in these patients A,B. AnalOverall, these results suggest that low ACAP1 levels are associated with a decreased response rate and worse clinical outcomes in ICT-treated patients, highlighting the potential clinical significance of ACAP1 for identifying patients who will benefit from ICT across multiple cancer types, especially for melanoma and lung cancer patients.Previous studies of ACAP1 focused mainly on its role in the endocytic recycling of intracellular cargos. Other biological functions of ACAP1, especially in immune response, have barely been studied. In this study, we found that ACAP1 expression was highly enriched in T, B, and NK cells but at a low level in other cells, indicating that ACAP1 is a novel marker gene for lymphocytes. Its expression is highly correlated with infiltrating levels of TILs, especially CD8+ T cells, and its deficiency defines a “cold” immune microenvironment across all solid tumors. Moreover, ACAP1 is essential for the cytotoxicity of lymphocytes. Consequently, the low expression of ACAP1 indicated resistance to immunotherapy and was associated with poor outcomes in multiple cancer patients following ICT, especially in patients with melanoma or lung cancer. Thus, we excavated a biomarker to predict the response to ICT.Pan-cancer expression analysis of ACAP1 showed that ACAP1 was significantly decreased in most cancer types but significantly increased in several cancer types, such as CHOL, HNSC, and KIRC. We also demonstrated that ACAP1 levels represent the abundance of lymphocytes in tissues. Combining the above results, we speculated that lymphocyte was excluded and decreased in most types of cancer, such as LUAD. However, the abundance of lymphocytes increased in CHOL, HNSC, and KIRC. We speculated that some factors that may attract immune cell infiltration are significantly increased. For example, HNSC is a type of cancer characterized by high TMB ,69, whicAccumulating evidence suggests that a low level of TILs is often associated with poor cancer progression and survival ,71. In tWe compared the predictive ability of ACAP1 level with several other biomarkers for immunotherapy response. We found that its prediction performance is good in lung cancers and melanoma and is comparable to TMB, MSI, CD274, and TIDE scores in gastric cancer, ccRCC, glioblastoma, and metastatic urothelial cancer. In addition, ACAP1 expression does not highly correlate with TMB in the majority of tumors , such asThe presence of TILs, such as T, B, and NK cells, in tumors is the fundamental determinant of the ICT response, as ICT functions through re-activating TILs to recognize and attack cancer cells. Tumors with low ACAP1 levels probably do not respond to ICT. We speculate that ICT combined with TILs infusion might be superior to ICT alone for these patients. However, this should be verified by follow-up preclinical and functional studies. More work is required to optimize the clinical efficiency before it could be considered a practical treatment option.Previous studies showed that ACAP1 acts as an adaptor for the endocytic recycling of intracellular cargos, such as transferrin receptor and integrin β1 CD29) ,9,10,11. ,9,10,11In summary, our data demonstrate that ACAP1 is a marker of TILs, and its expression was regulated by CNV, promoter methylation, and SPI1. Its deficiency is a pan-cancer indicator of immune-cold tumors, conferring resistance to ICT and inferior outcomes following ICT. The quantitation of ACAP1 expression could help determine appropriate therapies for cancer patients, although many preclinical studies need to be done before clinical application.