Dendritic cells (DCs) are the professional antigen-presenting cells that recognize and present antigens to naïve T cells to induce antigen-specific adaptive immunity. Among the T-cell subsets, T helper type 2 (Th2) cells produce the humoral immune responses required for protection against helminthic disease by activating B cells. DCs induce a Th2 immune response at a certain immune environment. Basophil, eosinophil, mast cells, and type 2 innate lymphoid cells also induce Th2 immunity. However, in the case of DCs, controversy remains regarding which subsets of DCs induce Th2 immunity, which genes in DCs are directly or indirectly involved in inducing Th2 immunity, and the detailed mechanisms underlying induction, regulation, or maintenance of the DC-mediated Th2 immunity against allergic environments and parasite infection. A recent study has shown that a genetic defect in DCs causes an enhanced Th2 immunity leading to severe atopic dermatitis. We summarize the Th2 immune-inducing DC subsets, the genetic and environmental factors involved in DC-mediated Th2 immunity, and current therapeutic approaches for Th2-mediated immune disorders. This review is to provide an improved understanding of DC-mediated Th2 immunity and Th1/Th2 immune balancing, leading to control over their adverse consequences. Dendritic cells (DCs) are the professional antigen-presenting cells (APCs) that play an important role in immune defense by activating the adaptive immune system. DCs were first discovered by Steinman and Cohn in 1973 . Differe+ naïve T or CD8+ T cells  cells, and neutrophils to the site of infection to impart protective Th1 immunity in both humans and mice .,17.+ T hEach T-cell subset originating from naïve T cells takes charge of a specialized function and is recognized by specific characteristics. Th1 cells are characterized by high-level expression of IFN-γ, IL-2, lymphotoxin α, and tumor necrosis factor-beta (TNF-β) in response to IL-12 signaling. Th1 cells induced against intracellular parasites, such as protozoa, bacteria, viruses, and fungi, which recruit macrophages, neutrophils, NK cells, cytotoxic T-cells, B cells, and microglial-like effector cells to eliminate invaders by activating cell-mediated immune responses. Over-activation of Th1 cells leads to organ-specific autoimmune diseases, such as hypersensitivity, arthritis, and type 1 diabetes. The major transcription factors initiating Th1 cell differentiation are T-bet, STAT4, STAT1, Runx 3, Eomes, Hlx, etc. , as summ+ T cells in both humans and mice. Two major signaling pathways, IL-2/STAT5 and IL-4/STAT6, play a crucial role in Th2 differentiation. IL-2/STAT5 signaling implies that IL-2 receptor signaling initiates STAT5 activation, leading to the expression of IL-4 [IL-4 is the key cytokine in Th2 immunity, but for many years, immunologists faced a “Th2 paradox.” Th2 development from naïve T cells requires activation of signal transducer and activator of transcription 6 (STAT6) downstream of the IL-4 receptor-signaling pathway, but the only known source of IL-4 was the Th2 cell itself. Today, however, other cell types, such as basophils ,25, mast of IL-4 . In the  of IL-4 .Basophils and mast cells are the major source of IL-4 inducers in mediating Th2 immunity. Basophils comprise 0.5% to 1% of total blood cells categorized as granulocytes. They contain inflammatory cytokines, such as histamines and heparins, which are known to cause harmful allergic reactions, but also help eliminate parasitic infections. Nakanishi et al. (2010) ,33 propoILCs, which consist of three subsets derived from the same progenitor cells, were recently discovered to be involved in initiating T-cell responses. Among the three groups, only the ILC2 group was found to be involved in Th2 immune responses in response to IL-4, IL-25, and IL-33 ,38. It aECs in the outer barrier of our body constantly encounter invasive or inhaled pathogens and allergens, resulting in the production of thymic stromal lymphopoietin (TSLP), IL-25 (or IL-17E), and IL-33. These cytokines induce APCs, leading to activation of Th2 immune responses. In the thymus, TSLP is important for lymphocyte development, but when secreted by ECs it induces Th2 immune responses .+ T cells toward the Th2 lineage, it has also been found in inhibiting Th1 and Th17 differentiation. In autoimmune inflammation, IL-25 suppresses Th17-mediated inflammation [-/- mouse shows susceptibility to autoimmune encephalitis and severe disease progression, including failure to eliminate the helminth, Nippostrongylus brasiliensis, due to poor Th2 response [TSLP is expressed in several organs, including the intestines, lungs, tonsils, and thymus, and primes DCs to enhance Th2 differentiation while inhibiting Th1-related IFNγ and IL-12 cytokines. TSLP can also activate basophils and mast cells in both humans and mice . TSLP reammation  and gut ammation . In humaammation . The IL-response ,52. Collresponse . IL-33 aresponse ,55. IL-3response  and basoresponse , leadingresponse . The synresponse .Th2 immunity is an important defense mechanism against extracellular parasites and allergens. DCs can generate effector T-cell subsets from naïve T cells specialized for specific immune responses and are therefore considered “master regulators” of immune responses because they have the ability to initiate and control the adaptive immune response. DCs comprise a heterogeneous group of cells originating from bone-marrow hematopoietic stem cells (HSC) and early myeloid progenitors (EMP) in the blood, tissues, and lymphoid organs. They are generally classified on the basis of their locations, functions, and cell surface markers. HSCs give rise to conventional/classical DC1 (cDC1) and DC2 (cDC2), plasmacytoid DCs (pDCs), and monocyte-derived DCs (Mo-DCs) .+ cDC1 subset (CD8α+ cDCs and CD103+ cDCs) is dependent on IRF8, ID2, and Batf3 transcription factors. Development of CD172+ CD11b+ cDC2 subsets is dependent on Klf4, IRF4, ID2, ZEB2, RelB, and Notch2 transcription factors. The IRF8-dependent cDC1 subset presents antigens to CD8+ T (CTL) cells through MHC I, mainly involved in promoting anti-viral and Th1 cell response, whereas IRF4-dependent cDC2 subsets present to CD4+ T cells through MHC II with the ability to favor polarization toward Th2, Th9, Th17, and Treg cells [+ cDC2 group, three subsets  are well established to be associated with induction of the Th2 immune response in mice, as summarized by Na et al. (2016) [+ DCs are a skin-resident DC subset that plays a crucial role in the Th2-mediated contact hypersensitivity response in the skin [+ DCs are enriched in the lung and the draining lymph nodes (dLNs) of the skin and intestine and are involved in allergic inflammation by enhancement of Th2 immunity [+ DCs, however, are involved in the development of Th2 responses from naïve T cells in the regional LN upon activation and CCR7-dependent migration [+ DCs also play an important role in inducing Th2 immunity in intestinal nematode infection [+ DCs in the gut mucosal tissue, which induce Th2 response; CD11b+ CD103+ DCs in the small intestine and CD11b+ CD103- DCs in the colon [+ and CD8+ T cell responses through their cross-presenting capacity in nature. However, when inoculated in vivo, DC vaccines induce different polarization of CD4 vs. CD8 T cell responses in both the human and mouse, depending on the maturation conditions  and pulsed antigens during the DC development even in the presence of same cytokines, like GM-CSF and/or IL-4 [EMPs give rise to monocyte-derived macrophages and long-lived resident macrophages. Development of the XCR1eg cells ,59  [To initiate a Th2 immune response from naïve CD4(CXCL13) ,73; cyto(CXCL13) ,75, IL-2(CXCL13) , OX40/OX(CXCL13) , and TIM(CXCL13) ,79; patt(CXCL13) ; c-type (CXCL13) ,81; RIG-(CXCL13) ; and pro(CXCL13)  are pref(CXCL13) , includi(CXCL13) , neurope(CXCL13) , puriner(CXCL13) , HMGB1 r(CXCL13) , and hea(CXCL13) ,90, are S. mansoni) and HDM, probably due to the reduced populations of the cDC2 subset and IRF4+ pre-DCs [+ cDC2 subset is required for Th2 immunity against a protease allergen and N. brasiliensis infection. STAT5 was also reported to be involved in cDC2-mediated Th2 immunity. Bell et al. found that the DC-specific deletion of STAT5 had no effect on DC development, but impaired Th2-mediated allergic responses in skin and lungs [-/- mice [+ T cells while DC-specific Jagged-1 depletion inhibited Th2 polarization in humans [Among the DC subset, the cDC2 subset plays a major role in inducing Th2 immunity. Several TcFs are required for the proper development of cDC2. In this section, we summarize several TcFs, which are essential for cDC2 development leading to induction of Th2 response. Kruppel-like factor 4 (Klf4) and interferon regulatory factor 4 (IRF-4) are well-established TcFs for cDC2 development from pre-cDCs. CD11c-conditional Klf4 depleted mice showed impaired Th2 response against parasitic infection  pathway [S. mansoni infection and HDM exposure. The DCs lacking in IFN-α receptor (ifnar1-/-) are unable to initiate Th2 response by impairing the optimal DC phenotype, suggesting its critical role in DC-mediated Th2 immunity [Mind-bomb-1 (Mib-1), an E3 ubiquitin-protein ligase involved in regulating cell apoptosis, is a critical regulator of Notch ligands for the activation of Notch signaling, increasing gradually as precursor cells differentiate into DCs in mice. Mib-1-depleted DCs were not effective at stimulating Th2 proliferation in co-culture with T cells , suggest pathway . Webb etimmunity .ζ-chain of TCR and promotes the phosphorylation and activation of central TCR signaling components [+ T cells were hyper-proliferative and polarized toward a Th2 profile under in vitro stimulation [+CD25+Foxp3+ Treg population in OT-II T-cell/DC co-cultures. The severity and rate of development of AD increased in BALB/c mice inoculated with SHB-deficient DCs compared with mice inoculated with normal DCs  infection due to the impairment of Mbd2-mediated H3K9/K14 acetylation, leading to reduced expression of Th2-inducing genes in DCs. Epigenetic factors also contribute to allergic reactions via DC-mediated Th2 response. Alexey et al. (2010)  found thMicroRNAs have been associated with several allergic inflammatory disorders, including asthma, eosinophilic esophagitis, and allergic rhinitis as well as AD. However, most studies have been performed in association with T-cell development. Recently, Zech et al. (2015) reported that microRNA-155 (miR-155)-deficient DCs showed limited Th2 priming capacity and failed to induce airway inflammation in allergen-exposed mice due to impairment of the miR-155-mediated purinergic type 2 receptor (P2R) signaling activation, resulting in inhibition of DC chemotaxis and IL-1beta secretion upon stimulation . This meApart from genetic factors, several environmental or extrinsic factors facilitate the DC-mediated Th2 immune responses. Most environmental factors or variables drive the immune system to determine the fate of DC-mediated T cell responses, i.e., whether Th1 or Th2 immune responses are elicited in the body. In in vitro cultures, the antigen dose and the DC/T-cell ratio in the same culture condition also affect the DC-mediated Th2 response. For example, bone marrow-derived mouse myeloid DCs cultured in the presence of high doses of antigen induce Th1 cell development, whereas low antigen doses induce Th2 cell development . Human M+ iDCs present antigens to T cells after exposure to HDMs and induce Th2 immune responses that lead to features of asthma in mice [+CD11b+) than cDC1 types (CD11c+CD103+) in the mucosa [Allergens, such as pollens and HDMs, can lead to the induction of Th2 immune responses. Exposure of innate immune cells, i.e., eosinophils, basophils, and inflammatory DCs (iDCs), in the mediastinal draining lymph nodes (dLNs) to HDMs activates the TLR4/MyD88-dependent pathway, which recruits the IL-4 competent Th2 immune response. FcεRI in mice . A study in mice ,119. Ano in mice . The rol in mice . This DC in mice . In a che mucosa , which me mucosa . In addie mucosa . It meanS. mansoni, lays soluble eggs in mice that contain an antigen with a glycosylated T2 ribonuclease, termed as omega-1, which activate DCs to induce Th2 immune responses [Schistosoma’s soluble egg antigen [PAMP molecules in invading microbes are recognized by innate immune cells, such as DCs, via specific pattern recognition receptors (PRRs) and activate the immune system . Studiesesponses . Omega-1esponses . Similaresponses . Apart fesponses . A nematesponses ,131. Sim antigen . Tissue  antigen . Adjuvan antigen .More than one billion people worldwide are suffering from parasitic infections, helminths, and allergic disorders, such as asthma, allergic rhinitis, food allergies, and eczema ,134,135.+ DCs, the Th2 immune responses are interrupted in infections of the helminths, Heligmosomoides polygyrus and S. mansoni [+ DCs are involved in Th2 immune responses against N. brasiliensis in a mouse model [H. polygyrus and N. brasiliensis, but not T. muris, produce excretory-secretory products and thereby suppress the production of IL-12p40 from DCs in mice [T. muris in a mouse model [-/- DCs can maintain the expression of costimulatory molecules in response to the endotoxin, and elicit Th2 immune responses in mice [Parasitic infections, especially helminthic infections, occur in almost one-quarter of the world’s population . These h mansoni ,150,151.se model ,59. Mean in mice . This case model . Therefose model . When mise model . However in mice . It meancDC2s play a vital role in generating Th2-mediated immune responses in asthma, including HDM-mediated asthma in a mouse model ,158,159.AD, also known as eczema, is one of the most common forms of skin ailments. It is characterized by IgE-mediated hypersensitivity to environmental or food allergens and by dry and inflamed skin in humans . In pati+ T cells by mDCs (myeloid DCs) expressing lower levels of the ICOS ligand, which is a costimulatory molecule important for DC-T-cell crosstalk. Although ICOS is linked to Th1 responses, its expression is significantly reduced in patients with allergic rhinitis [+naïve T cells within the LNs. Therefore, it has been suggested that the OX40L and TSLP may be a therapeutic target for AR patients [Also known as hay fever, AR is a nasal inflammation that occurs when the body is exposed to an airborne allergen. It manifests as nasal discharge, sneezing, and ocular discharge with redness and swelling of the eyes in human . In AR, rhinitis , whereaspatients ,174,175.Parasitic infections or allergen invasion stimulate Th2 immunity by expressing Th2 associated cytokines. However, prolonged secretion of Th2 cytokines occasionally leads to adverse allergic reactions in humans, characterized by high serum levels of IgE in allergic disorders, such as AD, asthma, rhinitis, and hay fevers. Thus, the secretion of Th2 cytokines needs to be tightly controlled. Although a detailed therapeutic mechanism is beyond the scope of this review, we briefly discuss the current therapeutic approaches, which are focused on the Th1/Th2 immune balancing from Th2 skewing immunity. The idea behind developing and designing any drug is mainly based on three approaches: First, by blocking the key factor required for secretion of Th2 cytokines; second, by targeting the important signals required for differentiation and survival of Th2 immune cells; and third, by activating Th1 immunity to restore the Th1/Th2 immune balance. For example, eosinophils, a well-known myeloid lineage, rapidly infiltrate into the regions of inhaled allergens or parasitic infections, leading to the conscription of other immune cells, such as DCs, mast cells, basophils, and NKT cells, to enforce Th2 responses. Targeting these intermediate pathways could be the best approach to designing Th2 drugs. In this section, we reviewed the current therapeutic approaches for the treatment of Th2 immune disorders in the aspect of pharmacology, biologics, and molecular targets  Table 2Table 2.Tacrolimus (FK506) and Cyclosporine-A are the well-known immunosuppressive drugs, which have been used successfully in the treatment of AD, and organ transplantation to prevent graft-versus-host diseases by targeting NFAT and AP1 proteins, leading to inhibition of calcineurin and IL-4 production in both humans and treated animals ,216. ParSeveral anti-interleukins are being used successfully in the treatment of Th2 mediated immune disorders . In addiMicroRNAs are recently counted on the list of treatment categories for Th2-mediated immune disorders, which are summarized briefly in In addition to biological and pharmacological treatment, hygiene hypothesis theory recently gained more attention towards preventing allergic disease ,228,229.As professional antigen presenting cells, DCs play a major role in controlling and maintaining the immune homeostasis in the body by means of antigen recognition and presentation to the naïve T cells to induce specific immune responses against harmful antigens. This review described the current understandings of the Th2 immunity induced by specific DC subsets, especially the cDC2s, and their roles in determining the specific pathways that drive and determine the Th1 versus Th2 immune responses. Th2-specific antigens or allergens readily recognized and presented by DCs eventually lead to the induction of the Th2 immune responses, resulting in the clearance of the antigen or may cause Th2-skewing chronic inflammatory disorders. In this review, we have discussed the DC-mediated Th2 polarization in connection with intrinsic, i.e., genetic factors, such as certain gene-related mutations or certain DC-specific genetic aberrations, and extrinsic, i.e., environmental factors, such as allergens, PAMPs, DAMPs, TLRs etc. The genetic factors required for the cDC2 development are essential for the induction of the DC-mediated Th2 immunity while specific environmental factors, such as the allergens or the extracellular parasites, that drive DCs to elicit Th2 polarization in the body. The detailed mechanisms underlying the induction of DC-mediated Th2 immunity to each antigen remain to be further elucidated, which will provide a better understanding of the DC-mediated Th2 immunity, eventually making progress in the field of drug development and therapeutic interventions for Th2-biased chronic inflammatory disorders.


Background: Little is known about the prognostic significance of somatically mutated genes in metastatic melanoma (MM). We have employed a combined clinical and bioinformatics approach on tumor samples from cutaneous melanoma (SKCM) as part of The Cancer Genome Atlas project (TCGA) to identify mutated genes with potential clinical relevance.Methods: After limiting our DNA sequencing analysis to MM samples (n = 356) and to the CANCER CENSUS gene list, we filtered out mutations with low functional significance (snpEFF). We performed Cox analysis on 53 genes that were mutated in ≥3% of samples, and had ≥50% difference in incidence of mutations in deceased subjects versus alive subjects.Results: Four genes were potentially prognostic . We identified 18 additional genes  that were less likely to have prognostic value (FDR < 0.4). Most somatic mutations in these 22 genes were infrequent (< 10%), associated with high somatic mutation burden, and were evenly distributed across all exons, except for RAC1 and MAP2K1. Mutations in only 9 of these 22 genes were also identified by RNA sequencing in >75% of the samples that exhibited corresponding DNA mutations. The low frequency, UV signature type and RNA expression of the 22 genes in MM samples were confirmed in a separate multi-institution validation cohort (n = 413). An underpowered analysis within a subset of this validation cohort with available patient follow-up (n = 224) showed that somatic mutations in SPEN and RAC1 reached borderline prognostic significance . Somatic mutations in SPEN, and to a lesser extent RAC1, were not associated with definite gene copy number or RNA expression alterations. High (>2+) nuclear plus cytoplasmic expression intensity for SPEN was associated with longer melanoma-specific overall survival (OS) compared to lower (≤ 2+) nuclear intensity (p = 0.048). We conclude that expressed somatic mutations in infrequently mutated genes beyond the well-characterized ones , such as RAC1 and SPEN, may have prognostic significance in MM. NRAS and BRAF  using the reference GRCh37/hg19 genome assembly. Somatic mutation calls were annotated by the UNCseq™ pipeline (version 2016.07): snpEFF (v4.3)  and ExAC (v0.3) to obtain the allele frequency of the variants.The Variant Call Format (VCF) files were retrieved from the TCGA access-controlled data portal , n = 133 ]; third, only genes that were more or less frequently mutated in deceased vs. living subjects at the time of last follow-up were considered. By convention, these genes were defined by the fold of the mean frequency of samples mutated in deceased vs. living patients  against the hg19 assembly] were obtained to perform UNCeqR (v0.05) analysis , and conThis multi-institution cohort consists of published datasets whose MM tumors were subjected to whole exome –20, genoTo investigate protein expression of SPEN in relation to several clinical and histopathologic features in MM, we performed single-color immunohistochemical analysis of the SPEN protein expression in MM tissue samples from patients who were treated at the UNC-CH Melanoma Program. Collection of MM tumor tissues, clinicopathologic data, and clinical follow-up were allowed under the UNC-CH IRB approved protocol 09-1737. These MM tissue samples were different from those included in the UNC-CH UNCseq™ MM cohort, which was described above. Representative 0.7-mm diameter tissue cores from each MM specimen were spotted in duplicates or triplicates in the UNC-CH MM tissue microarray (TMA).A commercially available anti-SPEN antibody   was optiwww.r-project.org). Kaplan-Meier curves were constructed using GraphPad Prism  to estimate the melanoma-specific overall survival (OS) in patients with high (>2+) nuclear plus cytoplasmic nuclear-only signal vs. high (>2+) nuclear-only signal vs. low (≤ 2+) expression of SPEN protein by melanoma cells. Given the multiple replicates per patient, the average of the expression intensity was considered. In addition, a specimen was classified as having both nuclear and cytoplasmic signal if at least one of the replicate cores exhibited nuclear plus cytoplasmic localization for the SPEN protein.Histopathologic analysis was performed using the 0, 1+ (< 25% of melanoma cells with nuclear stain), 2+ (25–80% of melanoma cells with nuclear stain), and 3+ (>80% of melanoma cells with strong stain) semiquantitative scale (PBG). Each tissue core was also evaluated for the presence or absence of any small-diameter mononuclear cells with stippled chromatin and indistinctive cytoplasm suggestive of lymphocytes. Contingency tables and the chi-square test were used to assess the association between expression intensity of SPEN protein by melanoma cells and the presence vs. absence of tumor-infiltrating lymphocytes , and stop-gain NF1 gene mutations. We found only 4/303 total hotspot mutations fell outside the quality filtering criteria , the unfiltered MM (n = 357), and the filtered MM (n = 356). Incidence of ultraviolet signature mutations (C>T substitutions) is significantly higher in unfiltered primary melanomas compared to MM samples . Overall, C>T transitions, C>T transitions at a dipyrimidine site, and CC → TT accounted for 79.4%, 77.8%, and 0.11% of the total 5,322 mutations, respectively. Our filtering strategy found a significantly increased incidence of ultraviolet signature in retained vs. filtered mutations in the MM subgroup . Nevertheless, mutations in other previously reported genes  were also retained  had sufficient follow-up; only 7 (4%) vs. 33 (19%) of the censored subjects had follow-up for < 1-year vs. < 2-years, respectively. Of these, 53  and 57  genes were mutated ≥1.5 times or ≤ 0.67 times in deceased subjects compared to living subjects. Gene lists from each of the two OS analyses were similar. Although BRAF, NRAS, NF1, TP53, PTEN, PPP6C, CTNNB1, PDGFRA, IDH1, and KIT were significantly mutated, they did not exhibit >50% change in deceased subjects compared to living subjects. Additionally, no change was seen when analysis was restricted to hotspot mutations previously reported for BRAF, NRAS, NF1, TP53, PTEN, ATM, CTNNB1, and PPP6C genes (Table 6C genes .FGFR1), RAS-related C3 botulinum toxin substrate 1 (RAC1), caspase recruitment domain-containing protein 11 (CARD11), and class II major histocompatibility complex transactivator (CIITA). An additional 10 genes had less significance (FDR 0.2–0.4). When OS time from specimen collection was used for Cox analysis, 26 genes looked promising (FDR < 0.4). With the exception of NTRK1, CUX1, TSC2, and AFF3, all 10 genes that were identified in the OS-original diagnosis analysis were also found to have considerable prognostic significance (FDR < 0.4) in the OS-specimen collection analysis. Eight additional genes  also had prognostic value . Figures CDKN2A, ARID2, and FBXW7 genes . Compared with the MAF of BRAFV600 mutations, the MAFs for most genes from the 22-gene list were significantly lower  specimens. These genes include the 14 identified to be promising for prognostic significance from the OS analysis from original diagnosis of melanoma . Overall, the frequency of mutations in any of these 22 genes was < 10% in the MM samples. Mutated RAC1, FGFR1, AFF3, and NTRK1 genes were more frequently seen in MM samples from patients who died within 5 years from the original diagnosis. In contrast, mutated CIITA was more frequently seen in MM samples from patients who have lived >5 years after original diagnosis . Somatic mutations in the haploinsufficient homeodomain transcription factor gene, cut-like homeobox 1 (CUX1) (p = 0.027).Figure s Figure . All 22 1 (CUX1) , were diNTRK1, EGFR, and GNAS , implying low-to-absent gene expression , although corresponding genes were expressed based on the RNA-Seq by Expectation-Maximization (RSEM) data. Figure UNCeqR analysis revealed that 271 of these 423 mutation events 64.1%) were sufficiently covered with the exception of 4.1% weren = 107) vs. metastatic (n = 417) status. As was the case with the TCGA SKCM cohort, the frequency for the 22 somatically mutated genes in MM was low, with few exceptions . In contrast with the TCGA SKCM cohort, however, more mutated genes were seen in primaries compared to metastases . Of the 9 mutated genes whose mutations were confirmed by RNA-seq, all but one  were equivocally found to be expressed in the other two datasets that reported both RNA and DNA sequencing analysis , whereas mutations in SPEN showed trends of mutations with better prognosis   and all, but one, somatic mutations were not associated with changes in gene expression. However, 22/34 patients with any genetic aberrations in RAC1 (somatic mutations and/or copy number amplifications) were deceased at the time of analysis. The unfavorable outcome of patients with any genetic aberrations in RAC1 is in line with a report on the adverse prognostic significance of high RAC1 protein expression by immunohistochemistry in primary cutaneous melanoma samples  or clear changes in gene expression. To investigate the protein expression pattern in SPEN, we performed immunohistochemical analysis in MM samples procured from 87 patients who were treated at UNC-CH Melanoma Program between 2000 and 2011. Clinicopathologic characteristics of this patient cohort are shown in Table 2 test p = 0.08). At a median follow-up of 15 months (range 1–68 months) and for the patients who had melanoma-specific overall survival data (n = 82) patients with high (>2+) nuclear plus cytoplasmic expression of SPEN by melanoma cells had longer melanoma-specific OS compared with patients with low expression (≤ 2+)   Figure . No signRAC1). Other well-characterized and more frequently mutated genes  were not prognostic in MM, perhaps because such mutations are already found in nevi and primary melanomas ]. Not all somatic mutations are expressed at the RNA level. Validation of expressed somatic mutations detected at the DNA level by RNA sequencing maybe the future in clinical oncology; current commercially available molecular tests that perform targeted panel sequencing identify mutations at the DNA level only .We have analyzed the largest to-date sample cohort for somatic mutations in MM. With available follow-up information from the TCGA, we validated our findings in an independent dataset. In contrast to previous work that defined the landscape of somatic mutations in melanoma irrespective of stage and subtype , 26, 28,RAC1 and perhaps MAP2K1). The lack of mutations with high functional impact does not mitigate the importance of such missense mutations in protein function, as in the case of KEAP1 in lung cancer  may be associated with favorable prognosis because potentially damaging mutations are not ultimately expressed. In contrast, somatic mutations in tumor-promoting genes  may be intuitively associated with unfavorable prognosis.To date, there is limited knowledge about the significance of low-frequency somatic mutations in melanoma. Studies have established that ultraviolet radiation induces mutations, which in turn may potentially yield neoantigens. The neoantigens provoke an immune response, which may account for the immunogenicity of cutaneous melanoma and other cancers . The majg cancer . InsteadBRAFV600 mutations and, systemic treatment type , which may have influenced OS  whereas others were found not to be expressed, irrespective of the RNA-seq validation algorithm used .Our validation cohort was comprised of patient samples with significant heterogeneity with respect to selection for enced OS , 38, andenced OS . Despiten = 245 and 70% of patients had an event  is only 0.64. Nevertheless, we were able to show that RAC1 and SPEN may be promising prognostic factors for OS in MM. RAC1 been previously reported in SKCM and other datasets  and is associated with worse OS (HR = 1.93), the power to detect significant prognostic difference if datasets , 20 and datasets . A recenmutation . Little  cancers . In our  cancers , the hos cancers , and the cancers  may playThis study was carried out in accordance with the guidelines and recommendations from the Genomic Data Commons (GDC) Data Portal of the Cancer Genome Atlas Project. GDC Data Portal is an interactive data system for researchers to search, download, upload, and analyze harmonized cancer genomic data sets, including TCGA.GP, MH, AS, MP, DB, AE, JF, WH, JW, CQ, and SM involved in data collection. XZ, Al, PL, AH, JP, DM, MS, HJ, SJ, KH, DO, NT, PG, FC, CL, HE, NS, WH, JW, CQ, NW, PJ, JT, NH, GM, RL, DJ, RS, DH, and SM involved in data analysis. XZ, GP, KH, CQ, RS, DH, and SM involved in manuscript preparation.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.


Renal cell carcinoma associated with Xp11.2 translocations/TFE3 gene fusions is a rare subtype of renal cell carcinoma. This predominantly occurs in juveniles, but rarely seen in adults with lymph node or organic metastasis and a worsened prognosis.Herein, we presented 3 adult cases of Xp11-RCC. Two patients were in early stage and good condition, and the third patient had lymph node metastasis but showed no recurrence after a 3-month follow-up.Case 1: A 50-year-old female without any lumbago and gross hematuria was incidentally detected by left renal mass by ultrasonography. Case 2: A 31-year-old female with 2-year hemodialysis was detected with right renal carcinoma during preoperative examination of renal transplant. Case 3: A 45-year-old male with right lumbago for 1 month was detected with a mass in the lower pole of right kidney by ultrasonography.The characteristics of these 3 images are not consistent with each other, and showed some differences with the previous ones.All these 3 patients underwent laparoscopic radical nephrectomy, and case 1 patient underwent renal hilar lymphnode dissection at the same time. Immunohistochemistry was performed on all the 3 tumors, revealing that the tumor cells were positive for TFE3 and Melan-A. Case 1 showed lymph node metastasis, and received mTOR inhibitors. The 3 patients had no recurrent and new metastasis in other organs after follow-up for 3 months, 2 months, and 11 months, respectively.Whether the adult-onset Xp-RCC has an aggressive clinical course still remains controversial. Characteristics of the images of the 3 adult cases showed some uniformity but still have some differences. Immunohistochemistry results revealed tumor cell positive for TFE3, but have no consistency in carbonic anhydrase IX, CD117, Ki67, CK8/18 AE1/AE3 and so on. Therefore, the uniform and definitive diagnostic standards of the tumors are uncertain. Hence, more cases and findings are required to elaborate the standards of all the tumor subtypes. Vascular endothelial growth factor-targeted therapy showed some efficacious results in patients with metastasis, but more useful treatments are warranted. Older patients with lymph nodes metastasis usually have worsened prognosis. Herein, we reported 3 cases with Xp11.2-RCC.Renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusion is a rare and new subtype of RCC, and is classified by WHO in 2004.2The study protocol was approved by the Ethics Committees of The First People's Hospital of Changzhou, and all participants provided written informed consent.2.1A 50-year-old healthy female previously revealed the presence of a left renal mass by ultrasound 3 weeks ago. The patient had no significant back pain and gross hematuria. The abdominal contrast-enhanced CT demonstrated the presence of a mixed density mass with a size of 7.9 × 7.6 cm and clear boundary in the upper pole of left kidney Fig. A. A slig2.2A 31-year-old female, who was not married, has been undergoing hemodialysis for more than 2 years without any obvious symptoms underwent CT for preparation of kidney transplantation. CT showed a solid cystic and low-density foci in the right kidney, and a nodular and slightly high-density shadow within the foci , PRCC (1q21), PSF (1q34), NonO (Xq12) and CLTC (17q23).,4 The main clinical manifestations of this type of renal cell carcinoma include hematuria and lumbago, and also frequently occur in the clear cell renal cell carcinoma patients. According to the previous reports, the morphology of this tumor was different from that of the clear cell renal cell carcinoma. It is a solid cystic mass that is located in the cortex and often accompanied by internal hemorrhage and peritumor calcification, and has a clear boundary with surrounding tissues. Patients 1 and 3 had a well-defined solid mass, with hemorrhage and no rim calcification. Patient 2 had a solid cystic mass, which may be associated with her hemodialysis for more than 2 years.XP11.2 translocation /TFE3 gene fusion associated renal cell carcinoma showed a very low incidence. It mainly occurs in children and young people, accounting for about 20% to 40% reported that the imaging findings of XP11.2-RCC may be similar to those of papillary RCC in the existence of morphological overlap between the tumor and Papillary RCC. Previous scholars summed up the CT and MRI reports of 20 cases , which showed tumour attenuation in the CT plain in vast majority of cases are stronger than that in renal parenchyma. But during all enhanced phases, tumor enhancement remained weaker than the renal cortex, but greater than the medulla. On delayed phase, the tumor was weaker than the medulla. On MRI, the tumor remained at the same intensity in T1WI, low signal intensity in T2WI was uneven, and slightly high signal intensity in DWI. Liu et al summarized the CT and MRI reports of 5 patients. MRI of 4 cases showed moderate intensity, high signal intensity on T1-weighted MRI, 3 cases showed low signal intensity on T2-weighted imaging, and the other patient showed a relatively high signal intensity because of tumor hemorrhage. CT of 2 cases showed slightly higher tumor density than that of the cortex. CT results of these 3 cases are not consistent with each other, and are not exactly the same as those of the above results. In case 1, the tumor attenuation was stronger than the renal parenchyma and medulla during CT plain scan (Fig. The renal cell carcinomas have their own characteristic upon imaging. Kato et al All the 3 cases reported by us showed similar results as mentioned above microscopically (Fig.  showed Vimentin positive, but SMA, CD45 and HMB45 were negative. In one case reported by Henry, AE1/AE3, CK7, EMA, RCA, and CAIX were all positive. Of the two cases reported by Ahluwalia, CD10, Vimentin and EMA were positive, while CK7 was negative. As shown in Table ,14 Choueiri et al divided 15 patients with adult metastatic Xp 11.2 translocation renal cell carcinoma into 3 groups. Of which, 10, 3, and 2 patients received sunitinib, sorafenib, and monoclonal anti-VEGF antibody, respectively. Three patients had partial response, 7 had stable disease and 5 had progressive disease. The median OS of all the patients were 14.3 months. Case 1 patient received sorafenib after the surgery, and showed no recurrence after 3 month follow-up. So, VEGF-targeted therapy showed some efficacy in adult patients of Xp11.2-RCC with lymph node or organic metastasis.Xp11.2 translocation /TFE3 gene fusion associated renal cell carcinoma is usually diagnosed by pathological examination. This is characterized by papillary cell arrangement, abundant cytoplasm, eosinophilic, hyaline nodules, and psammoma bodies.lly Fig. A. Case 2lly Fig. B. All th,16 Kuroda et al  thought that the tumors of children and young adults at advanced stage, including lymph node metastasis, were indolent. According to a case report in 2015, a 17-year-old boy with Xp 11.2 translocation associated with RCC as a second tumor had a long-term survival rate. But as shown by a systematic review and meta-analysis of observational studies, no significant differences were observed in the prognosis between children and adults, and between female and male. Qiu et al and Klatte et al demonstrated that Xp11.2-RCC patients has poorer prognosis whatever treatment was applied. Therefore, there is a dispute whether age and sex have an impact on the prognosis of the tumor. According to several other previous reports, positive expression of TFE3 was associated with clinical paremeters and its prognosis.–22 As reported by Mir et al, 7 of 8 TFE3-positive patients had lymph node metastasis, whereas 5.8% of TFÊ3-negative patients had lymph node metastasis and cava thrombus. At the laboratory of Klatte et al, high TFE3 expression was associated with lymph and organic metastasis, which showed poor prognosis. The adult female patient in case 1 had renal hilar lymph node metastasis, and received sorafenib 400 mg bid for 3months. Results showed no significant distant metastasis. Case 2 and 3 patients showed no metastasis in other organs, and did not undergo any other treatments. Therefore, the prognosis of this tumor was affected by tumor stage, which may be associated with TFE3 expression. The prognosis was affected by many factors, such as age, sex or other immunohistochemical parameters, like CD10, Viminten and so on. These were associated with the prognosis, but cannot determine the prognosis lonely.Xp11.2-RCC mainly occurs in young individuals, and recent studies demonstrated that the prognosis of children was better than that of the adults.Herein, we presented 3 cases of adult RCCs associated with Xp11.2 translocations, and investigated the common characteristics of the tumors with the help of imaging. However, if the patients received hemodialysis or the tumor had necrosis and hemorrhage, the imaging results might differ. The main treatment for localized Xp11.2 RCC was radical resection of renal carcinoma, and for advanced renal cell carcinoma with lymph node metastasis, radical resection of renal carcinoma and renal hilar lymph node dissection are essential. The VEGF-targeted therapy showed some efficacious results in the adult patients of Xp11.2 RCC with lymph node or organic metastasis. After the surgery, immunization including TFE3 expression was necessary, indicating the prognosis of the patient. Detection of the relationship between immunization and clinical parameters needs more cases and research.Data curation: Pengfeng Gong.Formal analysis: Pengfeng Gong.Investigation: Pengfeng Gong, Qianfeng Zhuang, Kun Wang, Renfang Xu, Yiming Chen, Xiaogang Wang, Shuai Yin.Methodology: Pengfeng Gong, Qianfeng Zhuang, Kun Wang, Renfang Xu, Yiming Chen, Xiaogang Wang, Shuai Yin.Validation: Pengfeng Gong, Qianfeng Zhuang, Kun Wang.Visualization: Pengfeng Gong, Qianfeng Zhuang, Kun Wang.Writing – original draft: Pengfeng Gong, Qianfeng Zhuang, Kun Wang, Renfang Xu, Yiming Chen, Xiaogang Wang, Shuai Yin.Writing – review & editing: Pengfeng Gong, Qianfeng Zhuang, Kun Wang, Renfang Xu, Yiming Chen, Xiaogang Wang, Shuai Yin.


UBE2C), is overexpressed in all 27 cancers we investigated. UBE2C expression is significantly higher in late-stage tumors, which might indicate its involvement in tumor progression and invasion. This study also revealed that patients with higher UBE2C levels showed a shorter overall survival (OS) time and worse OS prognosis. Moreover, our data show that UBE2C higher-expression leads to worse disease-free survival prognosis (DFS), indicating that UBE2C overexpression correlates with poor clinical outcomes. We also identified genes with positive correlations with UBE2C in several cancers. We found a number of poorly studied genes  whose expression correlates with UBE2C. These genes might be considered as new targets for cancers therapies since they showed overexpression in several cancers and correlate with worse OS prognosis.Ubiquitination is one of the main post-translational modification of proteins. It plays key roles in a broad range of cellular functions, including protein degradation, protein interactions, and subcellular location. In the ubiquitination system, different proteins are involved and their dysregulation can lead to various human diseases, including cancers. By using data available from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases, we here show that the ubiquitin conjugating enzyme, E2C ( Most proteins post-translational modifications are essential for proper cellular localization, substrate activity, and associations with other proteins. One evolutionarily conserved modification is ubiquitination, which can involve one or multiple defined lysine (K) residues or the N-terminal methionine residue M1)  1,2,3].,3.2,3]..,3.2,3].Ubiquitin, a highly conserved regulatory protein (76 amino acids), plays an essential role in modulating protein functions. Ubiquitination is a post-translational modification mediated by a multi-step process which involves three different enzymes, including E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin-ligating enzymes. This function leads to the proteasomal elimination of its substrate or alteration of the substrate activity, localization, and associations with other partners in its protein networks ,5.UBE2C overexpression has been found in different human cancers, including hepatocellular carcinoma [In the ubiquitin-proteasome pathway, anaphase promoting complex/cyclosome (APC/C) and the ubiquitin conjugating enzyme, E2C (UBE2C), are involved in the initiation of ubiquitin chain formation on APC/C substrates. UBE2C principally create Lys-11 (K11)-linked polyubiquitination on these substrates and then APC/C and another E2 enzyme, UBE2S, elongates and branches the ubiquitin, making more efficient proteolytic degradation signals  for the proteasome receptor, S5A, regulating mitosis progression. Various cellular processes are regulated by the ubiquitin system; therefore, it is expected its dysregulation results in human diseases, including cancers ,7. UBE2Carcinoma , thyroidarcinoma , colon [arcinoma , breast arcinoma , brain [arcinoma , and cerarcinoma . UBE2C overexpression leads to chromosomes mis-segregation and alters the cell cycle process, facilitating cell proliferation [UBE2C overexpression correlates with tumor progression and poor prognosis in many tumors [UBE2C were evaluated in 27 different cancers using data from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases. We provide evidences that UBE2C acts as a proto-oncogene and can be considered as a therapeutic target for most cancers. Our results indicate that UBE2C is overexpressed in 27 studied cancers and its overexpression correlates worsen the overall survival (OS), suggesting its involvement in tumor progression and invasion. Our study also identified a number of genes that are in the UBE2C regulatory network.It has been shown that feration ,16. Morey tumors ,18,19. IUBE2C expression was notably higher in all 27 tumor types compared to matched TCGA normal tissues and GTEx data  subtypes show higher expression compared to normal, with slightly more significant for ccB , followed by type2 papillary RCC (ERG) fusion, speckle type BTB/POZ protein (SPOP) mutation, and ETS translocation variant 1 (ETV1) fusion (UBE2C in metastatic PRAD based on androgen receptor (AR) amplification and ERG fusion, there is no significance difference compared to condition without these changes  fusion . In rela changes .UBE2C was higher in all different subtypes, including triple negative breast cancer (TNBC), HER2-amplification, and luminal subtype  and infiltrating ductal carcinoma , cyclin D1 (CCND1), and Erb-B2 receptor tyrosine kinase 2 (ERBB2) in metastatic breast cancer compared to conditions without amplification indicated no significant correlation with UBE2C expression  compared to non-smokers . This indicates a possible involvement of UBE2C in the initiation of cancer. Furthermore, the expression in BRCA, KIRC, KIRP, LIHC, LUAD, LUSC, and UCEC was higher in late-stage cancers compared to early stages, representing a possible role of UBE2C in cancer progression and invasion .The OS time between UBE2C higher-expression levels led to worse DFS prognosis in comparison to its lower expression in the following tumors: ACC, KIRC, KIRP, LGG, LIHC, PAAD, PRAD, THCA, and UCEC. . These data demonstrate that UBE2C overexpression results in poor clinical outcomes in the above tumors.Regarding DFS time in the TCGA tumor types, data showed that UBE2C has a moderate to very strong positive correlation with other genes in 27 cancers . As seen in UBE2C expression correlations are strong to very strong  for the following genes in all 27 cancers: MYB proto-oncogene like 2 (MYBL2), trophinin associated protein (TROAP), cell division cycle 20 (CDC20), centromere protein A (CENPA), kinesin family member C1 (KIFC1), cyclin dependent kinase 1 (CDK1), kinesin family member 4A (KIF4A), and kinesin family member 20A (KIF20A). In addition, the following genes showed a strong to very strong positive expression in correlations with UBE2C in 26 cancers, but moderate positive correlations , polo like kinase 1 (PLK1), aurora kinase B (AURKB), non-SMC condensin I complex subunit G (NCAPG), cyclin B1 (CCNB1), spindle and kinetochore associated complex subunit 3 (SKA3), and kinesin family member 18B (KIF18B). Moreover, some genes showed strong to very strong positive expression correlations with UBE2C in several cancers while moderate correlations were found in a few cancers among 27 studied cancers as indicated in the UBE2C expression and different genes were observed in the TGCT cancer mentioned in TSPYL2), ATR serine/threonine kinase (ATR), and CYLD lysine 63 deubiquitinase (CYLD).Our study revealed that the expression of UBE2C, we found forkhead box M1  distance of +0.1 kb and +158.3 kb), E2F transcription factor 1 , RAD51 recombinase , and BRCA1 DNA repair associated . It is worthwhile to note that only FOXM1, E2F1, and RAD51 showed positive correlations with UBE2C in all 27 cancers. These three genes showed similar strong to very strong positive correlations with UBE2C in the different cancers given in Regarding genes which have protein products that have transcription factor binding sites on both the promoter and enhancer regions of UBE2C and tumor suppresser genes including BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B), BRCA1, BRCA2 DNA repair associated (BRCA2), checkpoint kinase 2 (CHK2), ATM serine/threonine kinase (ATM), ATR, tumor protein p53 (TP53), CYLD, and TSPYL2, the positive correlation between BUB1B and UBE2C was seen in 26 cancers, but not in TGCT cancer , meiotin breast  and ovarn breast ), familyy in GBM ), and PO bladder , brain [UBE2C expression in the 27 cancers . Proteins in this network are involved in different pathways, including cell cycle, oocyte meiosis, p53 signaling pathway, double-strand break repair, oocyte development and differentiation, FoxO signaling pathway, ubiquitin mediated proteolysis, cellular senescence, and progesterone-mediated oocyte maturation among others  are also involved in the UBE2C network. As described in TSPYL2 was under-expressed in most cancers  subtype [UBE2C is a common feature of all 27 human cancers tested in this study, suggesting it acts as a proto-oncogene. Moreover, the current study revealed the significant UBE2C overexpression across the histological and molecular subtypes of different tumors mentioned in the results and also possible associations between this expression and different patient conditions, such as drinking and smoking habits in PAAD and LUAD, respectively. UBE2C expression, based on patients’ pathological stages, showed that UBE2C overexpression can be involved in tumor progression and invasion. Our data broaden the observations of previous reports [The current study shows, for the first time, a global analysis of g cancer ,11,12,13 subtype , and our reports ,30,31.UBE2C had a shorter overall survival time and worse prognosis, and UBE2C higher-expression levels also resulted in worse DFS prognosis in many cancers, confirming that UBE2C overexpression results in poor clinical outcomes in many tumors. As an example, Qin et al. found that the UBE2C upregulation was associated with poor prognosis in breast cancer [We showed that patients with higher expression of t cancer , and ourUBE2C expression in all cancers , with roles in mitotic spindle maintenance, chromosome segregation, and microtubule depolarization [AURKB as a regulator of chromosome segregation during mitosis [TROAP (a member of the cell adhesion molecule complex) with a role in centrosome integrity during cell cycle progression [TPX2 (a microtubule-associated protein) having a key function in mitotic spindle formation [PLK1 as a regulator of cell division and maintenance of genome stability and spindle assembly, and DNA damage response) [CDK1 (a cyclin-dependent kinase), involved in cell division with important functions in mitosis and driving cells into the S phase [CENPA (a key component of the inner kinetochore plate), playing a role in chromosome segregation during oocyte meiosis) [CDC20 as an activator of APC/C during mitosis for mitosis progression [MYBL2, involved in cell cycle progression, cell survival, and cell differentiation [BUB1B as a vital component of the mitotic checkpoint complex [CCNB1, having an essential role in the transition of the cell cycle from G2 phase to mitosis [NCAPG (a mitosis-related chromosome condensation protein) involved in the condensin I complex [SKA3, controlling the correct exit from meiosis, migration of meiotic spindle, and stability of anaphase spindle [Furthermore, our study identified coexpression genes associated with the UBE2C protein network. Those genes with strong and very strong positive correlations with  cancers  are invorization ,33; AURK mitosis ; TROAP  ,38; CDK1 S phase ; CENPA (meiosis) ; CDC20 agression ; MYBL2, ntiation ; BUB1B a complex ; CCNB1,  mitosis ,45; NCAP complex ; SKA3, c spindle . Our datUBE2C coxpressed genes the transcription factors, FOXM1, E2F1, and RAD51, which have their binding sites on the UBE2C promoter and enhancer regions, suggesting they are transcriptional regulators of this gene. Our results are in agreement with a previous report of a positive association of FOXM1 with UBE2C in normal tissues and in tumors [Interestingly, we also highlighted as n tumors . The iden tumors , and main tumors ,51,52,53UBE2C and other important regulators of the cell cycle, including CDC20, which was previously identified as being co-expressed with UBE2C [MTFR2, MND1, FAM72D, and POC1A as genes whose expression correlates with worse OS prognosis, suggesting their possible involvement in tumor progression and invasion. We also found a significant correlation between th UBE2C . These dUBE2C can be considered as a general tumor marker and study of its related pathways can help to discover common therapeutic targets for cancers. However, further functional studies are required to clarify the role of UBE2C in cancers.In conclusion, the current study showed that http://gepia.cancer-pku.cn), UALCAN  [https://string-db.org/) [In the current study, our investigations were performed using different bioinformatics tools and databases, including GEPIA  (a webseuab.edu) , and STRdb.org/) ).UBE2C expression levels across all cancers and their subtypes, its differential gene expression analysis at different pathological stages, correlation between its expression and cancer prognosis in cancers, overall survival (OS) and the disease-free survival (DFS) analysis on the basis of UBE2C gene expression, investigation of genes with similar UBE2C expression patterns, and their associations in the UBE2C protein network.Here, we report the following investigations: UBE2C expression across 27 human tumor types compared to normal matches, we used the GEPIA webserver. One advantage of GEPIA is that it also uses normal data from the GTEx project to provides a reliable baseline for comparison. In most cancer research, normal tissues are prepared from areas adjacent to tumors, but they may be pre-cancerous tissue and not truly normal, healthy tissue. UBE2C expression between tumors, their matched normal, and data from the GTEX database in 27 tumor types were compared. These tumors included ACC, BLCA, BRCA, COAD, DLBC, ESCA, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, READ, SKCM, STAD, TGCT, THCA, THYM, UCEC, and UCS. Six tumor types were excluded from the study  due to their small numbers (13 and lower) or lack of normal samples, including cholangio carcinoma (CHOL), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), mesothelioma (MESO), pheochromocytoma and paraganglioma (PCPG), sarcoma (SARC), and uveal melanoma (UVM). Regarding parameter options, we used the ANOVA statistical method for differential gene expression analysis, selected log2(TPM + 1) transformed expression data for plotting, TCGA tumors compared to TCGA normal and GTEx normal for matched normal data in plotting, |log2FC| cutoff of 1, and a q-value cutoff of 0.01. Also, for cancers with different subtypes and conditions, we analyzed them using the UALCAN webserver.To investigate UBE2C expression box plots on the basis of patients’ pathological stage  in TCGA cancer types, we used the UALCAN webserver to get data from TCGA. In this analysis, cancers without normal matches or below two numbers in each stage were excluded from analysis.To provide UBE2C gene expression. Regarding hypothesis test, the GEPIA considers the Log-rank test. For this, we selected a hazards ratio (HR) based on the Cox PH model and also the 95% confidence interval information to show the 95% confidence interval (CI) as the dotted line. The UBE2C expression threshold of 50%  was determined to split the UBE2C high-expression and low-expression cohorts. Therefore, samples with UBE2C expression levels higher and lower than 50% were applied as the high-expression cohort (cutoff-high) and the low-expression cohort (cutoff-low), respectively.Overall survival (OS) and the disease-free survival (DFS) analysis were also performed on the basis of UBE2C and other genes was performed by pair-wise gene expression correlation analysis with the expression data of TCGA and GTEx, using the method of the Pearson correlation coefficient. At first, we searched for moderate, strong, and very strong UBE2C expression correlations (the Pearson correlation coefficient between 0.4 and 1) with other genes on average in all 27 caners. Then, we investigated the UBE2C expression correlations with each gene individually in each cancer to see the exact correlation. We considered the following correlation coefficients: 0.00–0.19 as very weak, 0.20–0.39 as weak, 0.40–0.59 as fairly strong , 0.60–0.79 as strong, and 0.80–1.0 as very strong. Correlation analysis between UBE2C (extracted from TCGA cancer types using GEPIA) and also some important tumor suppresser proteins, such as ATR, ATM, BUB1B, BRCA1/2, CHK2, and CYLD, were searched in STRING.To provide the UBE2C protein network, the STRING database was used and most genes with medium to very strong correlations with http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAup.cgi) and RPISeq (http://pridb.gdcb.iastate.edu/RPISeq/), respectively. Also, to search for a D-box (one of the recognition amino acid sequences to be identified by APC/C in the ubiquitin–proteasome pathway) in TSPYL2 protein, we used the GPS-ARM tool (http://arm.biocuckoo.org/down.php). To predict the RNA–RNA association and RNA–protein interaction between TSPYL and UBE2C, we used the RNAup webserver (


Sle1 and Sle1-TLR7 transgenic (Sle1Tg7) murine models, respectively. Elimination of IRAK4 signaling prevented all pathological traits associated with murine lupus, including splenomegaly with leukocyte expansion, detectable circulating antinuclear antibodies and glomerulonephritis, in both Sle1 and Sle1Tg7 mice. The expansion of germinal center B cells and increased effector memory T cell phenotypes that are typical of lupus-prone strains, were also prevented with IRAK4 kinase elimination. Analysis of renal leukocyte infiltrates confirmed our earlier findings of an expanded conventional dendritic cell (cDC) within the kidneys of nephritic mice, and this was prevented with IRAK4 kinase elimination. Analysis of TLR7 at the protein level revealed that the expression in immune cells is dependent on the TLR7-transgene itself and/or autoimmune disease factors in a cell-specific manner. Increased TLR7 protein expression in renal macrophages and cDCs correlated with disease parameters such as blood urea nitrogen (BUN) levels and the frequency of leukocytes infiltrating the kidney. These findings suggest that controlling the level of TLR7 or downstream signaling within myeloid populations may prevent chronic inflammation and severe nephritis.The global increase in autoimmunity, together with the emerging autoimmune-related side effects of cancer immunotherapy, have furthered a need for understanding of immune tolerance and activation. Systemic lupus erythematosus (SLE) is the archetypical autoimmune disease, affecting multiple organs, and tissues. Studying SLE creates knowledge relevant not just for autoimmunity, but the immune system in general. Murine models and patient studies have provided increasing evidence for the innate immune toll like receptor-7 (TLR7) in disease initiation and progression. Here, we demonstrated that the kinase activity of the TLR7-downstream signaling molecule, interleukin-1 receptor associated kinase 4 (IRAK4), is essential for mild and severe autoimmune traits of the  The immune system provides a crucial barrier against invasive pathogenic challenges. Defects can lead to opportunistic infections, cancer or the development of autoimmunity. Systemic lupus erythematosus (SLE) is the archetypical autoimmune disorder, whereby the immune system attacks itself in a systemic manner. It is complex and is driven by both genetic and environmental factors with no effective prevention or cure , 2.TLR7 mRNA expression is higher in PBMCs from SLE patients, with levels correlating with the expression of IFNα (SLE is characterized by the development of anti-nuclear antibodies (ANAs), particularly dsDNA- and RNA-reactive autoantibodies. These bind to self-antigens, resulting in the formation of immune complexes (ICs) which deposit in tissues, initiating, and promoting inflammation, tissue destruction, and often culminating in organ failure . The ICs of IFNα .TLR7/TLR8 associations with SLE across multiple ethnicities  locus is essential for the rapid-onset of severe disease in Yaa-associated strains (Sle1 background (Sle1Tg7) is sufficient for the progression to severe disease  mutant mice . ABIN1 iyaa locus  mice and backcrossed these with Sle1 and Sle1Tg7 mice to generate Sle1IRAK4KD/KD and Sle1Tg7IRAK4KD/KD mice. The model in our studies uses a low copy TLR7-BAC transgenic system, therefore we were able to examine genomic vs. disease effects on TLR7 protein expression in individual leukocyte compartments. Our data shows that IRAK4 kinase activity is essential for the development of all known autoimmune traits in both models, including splenomegaly, leukocyte activation, antibody production, and renal leukocyte recruitment with accompanying kidney disease. TLR7 is known to play a critical role in the onset of lupus, yet studies reporting comparative expression at the protein level are limited  mice bearing NZM2410-lupus susceptibility intervals and the generation of the conditional BAC-Tg7 for increased TLR7 expression have been previously described  and the concentrations were normalized to urine creatinine measured with a colorimetric assay as per manufacturer's instructions . Blood Urea Nitrogen (BUN) was assessed using the QuantiChrom Urea Assay Kit (BioAssay Systems). To detect GN, kidneys were fixed in formalin and embedded in paraffin. Three micrometer sections of formalin-fixed, paraffin-embedded kidney tissues were stained with hematoxylin and eosin and with periodic acid–Schiff (PAS). Microscopic morphologic analysis was performed by an independent pathologist according to the International Society of Nephrology / Renal Pathology Society (ISN/RPS) 2003 classification of lupus nephritis ; Class I®488 secondary antibody (Abcam). HEp-2 were imaged at 100X using the Zeiss LSM 800 upright confocal microscope and processed with Zeiss Zen (Blue edition) software. HEp-2 staining patterns were evaluated by two independent investigators according to the International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP). Luminex was used to quantify antibody subsets and subtypes including: IgA, IgM, IgG1, IgG2a/c, IgG2b, and IgG3 . This panel is designed to detect IgG2a (from BALB/c mice), which cross-reacts with IgG2c from mice on the B6 background, which we have designated as IgG2a/c  and anti-RNA (1:100 dilution) using established ELISAs , 47, 51. IgG2a/c .Frozen OCT embedded spleens were sectioned, fixed with 4% paraformaldehyde and stained with anti-CD4 (Ebioscience), anti-GL7 (BD Horizon) and anti-IgD (BD Pharmingen) antibodies. Confocal images of splenic germinal centers were collected as described above.+sIgMhiCD23−), T2 (CD93+sIgMhiCD23+), and T3 (CD93+sIgMloCD23+) cells. Splenic T cells: CD45, CD3, CD4, CD8, CD44, CD62L, CD69, CXCR5, ICOS, and PD-1; splenic pDCs: CD11b, CD11c, mPDCA1, SiglecH, MHCII, CD86, CD9, CCR9, Gr-1, CD3, and B220. Splenic and kidney DCs & macrophages were stained with CD45, CD3, B220, CD8, CD11b, CD11c, Gr-1, F4/80, CD64, CD86, and MHCII. For the analysis of kidney B cells, T cells and pDCs and TLR7 expression in splenic and kidney subsets, the cells were stained with CD45, EpCAM, B220, CD44, CD62L, ICOS, CD138, CD4, CD8, NK1.1, Gr-1, mPDCA1, SiglecH, CD11b, CD11c, CD115, F4/80, MHC-II, and TLR7. All samples were stained with a live/dead fixable dye . For intracellular TLR7 staining, the BD Cytofix/Cytoperm kit was used as per manufacturer instructions. Samples for the majority of panels were analyzed using a BD LSR Fortessa, a BD LSR II or BD Symphony with Flowjo 7.6. Antibodies were purchased from BD Biosciences, eBioscience, Biolegend, or Life Technologies.Single cells suspension from both spleen and kidney were prepared as described previously . SplenocP values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 for ANOVA. Additional 2 group comparisons were completed for single gene changes where indicated, using a two-tailed Student's t-test  or Mann-Whitney u-test (non-parametric); #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001. Correlations were assessed by computing Pearson's coefficient r and a two-tailed t test was used to establish if the correlations were significant (P < 0.05). For the comparison of survival curves, the log-rank (Mantel-Cox) test was performed and differences were considered statistically significant at P < 0.05 (*).Statistical analysis was performed with Graph Pad Prism 7.0. Normal distribution of the data was assessed by the Kolmogorov-Smirnov test. Data comparing the 4 strains was analyzed using a 1-way ANOVA followed by the Bonferroni's multiple comparison tests for normally distributed data, while the Kruskal-Wallis test with post hoc Dunn's multiple comparisons test was used for non-parametric data. Sle1Tg7 mice develop severe autoimmune traits including splenomegaly and kidney disease at around 6 months of age (KD/KD) mice by engineering a mutation at position-213 from Lysine (K) to Alanine (A) using a plasmid with puromycin resistance as a selective marker  in mination . Furthercontrols . The eli/KD mice , howeverSle1Tg7 mice develop higher titers of anti-nucleosome, anti-dsDNA and anti-snRNP autoantibodies than their mild lupus-prone Sle1 counterparts  in Sle1Tg7 mice compared to Sle1 controls which was prevented in IRAK4KD/KD mice , 51Sle1Tody data , plasma of IRAK4 . Moreoveterparts . Therefoterparts . The dec strains , 51, was strains . Additio/KD mice . Taken t+ T follicular helper cells  were fewer in IRAK4 KD/KD mice compared to either Sle1 or Sle1Tg7 , 51+ T f/KD mice .+ DCs were completed using the gating strategies in + and Gr1− CD11b+ cells in Sle1Tg7 mice were completely prevented by IRAK4 KD/KD  and the associated changes in MHCII and CD86 expression, were also prevented by IRAK4KD/KD (+ cDCs and plasmacytoid DCs (pDCs) frequencies showed a minor effect of disease or IRAK4KD/KD , CD11b+ cDCs (F4/80−CD11c+MHCII+) and their putative precursor (CD11c+MHCII−) , 5125, 5activity . Moreoveependent . CD11b+G+MHCII−)  (51). Coe  increases TLR7 expression in both B cells and plasmablasts. Since expression in Sle1Tg7 is higher than the respective Sle1Tg7IRAKKD/KD mice, it confirms that the inflammatory environment of severe autoimmunity and signaling through IRAK4 contributes to the increase in TLR7 expression  monocytes and macrophages were augmented by the Tg7 and severe disease , eliminating influences of other loci. In these studies, we were able to confirm that the elimination of proteinuria and splenomegaly seen in BXSBYaa.IRAK4KD/KD mice was not due to the dilution of susceptibility loci present in the BXSB background by the introduction of the B6 non-autoimmune genome (Sle1Tg7IRAK4WT/KD mice developed severe disease in a similar manner to Sle1Tg7IRAK4KD/IRAK4KD homozygotes (data not shown). The increased disease in the BXSB F2 heterozygous highlights additional loci which contribute to disease and are not present in the B6 background of Sle1Tg7 mice. Similar to the findings of the homozygous BXSBYaa.IRAK4KD/KD male mice shown in the above study, we saw a prevention in the expansion of B cells and plasma cells, with IRAK4 kinase elimination. The splenomegaly by most spontaneous severe lupus models is also characterized by increases in germinal center B cells and T follicular helper cells, together with T cell activation (+ lineage excluded neutrophils and revealed that CD11b+ cDCs and their putative precursors, CD11c+MHC− cells, comprised the majority of this expansion. Our splenic analysis also showed that in the majority of leukocytes, TLR7 was upregulated not only through the genomic contribution of the Tg7-BAC, but also by the intense autoimmune environment observed in the Sle1Tg7 mice.An additional advantage of using B6.e genome . Howevertivation , 26, 51.Sle1 and Sle1Tg7 models, respectively, together with the associated T cell and DC infiltrate. Our examination of TLR7 protein levels using flow cytometry showed that the autoimmune inflammatory environment was able to augment TLR7 expression, with the exception of renal infiltrating pDCs. Moreover, renal CD11b+cDC TLR7 expression was associated with key disease parameters, including BUN levels and the renal leukocyte infiltrate. This may suggest that the development of a soluble TLR7 assay as a urine biomarker could prove to be clinically relevant. However, given the endosomal location of TLR7, it is unclear as to whether this is possible and therefore warrants further investigation.Our work also provides a detailed analysis of the immunological events occurring not only in the spleen, but also in the diseased kidney. Approximately 50% of SLE patients develop GN and obvious challenges restrict comprehensive understanding of the inflammatory events occurring. Given the burgeoning data on the role of TLR7 in SLE pathogenesis, analysis at the protein level within the tissue may prove to be invaluable. Overall IRAK4 kinase ablation prevented both mild and severe kidney disease in the Sle1 mice, including a decreased GC formation, splenic TFH frequencies and serum autoantibody titers (lpr lupus prone mice results in a similar ablation of all autoimmune phenotypes (Sle1 on chromosome is a common susceptibility locus, additionally identified in NZB/W and in MRLlpr mice, and previous work showing TLR7 and MyD88 elimination preventing autoimmune phenotypes, we propose that IRAK kinase perturbations in these polygenic murine models would result in eliminating autoimmunity. Further studies into the signaling, nuclear translocation and cytokine production would shed further light on these mechanisms, particularly given the bias toward TLR7 of our model system.Previous data from a number of groups has shown that TLR7 plays a critical role in the initial stages of autoimmunity and development of ANAs –20. Intey titers , 61, 62.enotypes . In patienotypes –36. Congenotypes . This prenotypes , 64. TakWe and others have previously shown that IFNα and TLR7 ligands, such as RNA-containing viruses and TLR7 agonist R848 can increase TLR7 mRNA expression , 65. Sinlpr models (Over 90 patents for small molecule IRAK4 inhibitors have been filed from 2016 to 2018, primarily for autoimmune diseases and cancer , 69. In r models . Similarr models . In separ models . Howeverr models .Sle1 and Sle1Tg7 models, respectively. The regulation of TLR7 expression is not as simple as one expects, and depends on the pathogenic environment and the individual leukocyte involved. Further studies are required to further elucidate these factors, including cytokine induced nascent expression of TLR7 which may exacerbate BAC-induced TLR7 expression.In summary, the findings in our study show a dependency on IRAK4 kinase activity for increased TLR7 protein levels, together with mild and severe autoimmune phenotypes of the Taken together, our findings show the importance of increased signaling through TLR7 in SLE and targeting this pathway, may lead to a suppression of severe disease development.*STAR. The protocol was approved by the IACUC committee, protocol ID 161176.This study was carried out in accordance with the recommendations of the Biological Resource Center of ATC, HKL, HYL, ST, OZ, WYO, HY, LB, LR, BA, TPT, and A-MF performed the experiments. TC, HKL, DM, LB, LR, TBT, and A-MF analysed the data. A-MF, TC, and HKL wrote the manuscript. A-MF, DMZ, JSM, and JEC designed the research studies. A-MF, DMZ, MFM, JSM, JEC, and LHKL provided reagents, mice and/or statistical support.The authors declare that this study received funding through a joint innovation grant between Merck & Co., Inc. and A*STAR, Singapore. LB and LR are employees of Merck. MFM, JSM, and DMZ were employees of Merck at the time of the study. DM was an employee of Tessa at the time of the study. The remaining 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.


Saccharomyces cerevisiae strain, we previously produced two novel phenolic compounds, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid  and N-(E)-caffeoyl-3-hydroxyanthranilic acid , which are endowed with a structural similarity to bioactive oat avenanthramides and stronger antioxidant properties. In this study, we evaluated the ability of these yeast-derived recombinant avenanthramides to inhibit major hallmarks of colon cancer cells, including sustained proliferation, migration and epithelial-mesenchymal transition (EMT). Using the human colon adenocarcinoma cell line HT29, we compared the impact of YAvns and natural Avns, including Avn-A and Avn-C, on colon cancer cells by performing MTT, clonogenic, adhesion, migration, and anchorage-independent growth assays, and analyzing the expression of EMT markers. We found that both YAvns and Avns were able to inhibit colon cancer cell growth by increasing the expression of p21, p27 and p53 proteins. However, YAvns resulted more effective than natural compounds in inhibiting cancer cell migration and reverting major molecular features of the EMT process, including the down-regulation of E-cadherin mRNA and protein levels.Avenanthramides (Avns), polyphenols found exclusively in oats, are emerging as promising therapeutic candidates for the treatment of several human diseases, including colon cancer. By engineering a  N-cinnamoylanthranilate alkaloids or anthranilic acid amides) that are uniquely found in oats, where they act mainly as key phytoalexins in the plant defense mechanisms against certain pathogens, such as fungi [N-(4’-hydroxycinnamoyl)-5-hydroxyanthranilic acid), Avn-B (N-(4’-hydroxy-3’-methoxycinnamoyl)-5-hydroxyanthranilic acid), and Avn-C (N-(3’-4’-dihydroxycinnamoyl)-5-hydroxyanthranilic acid), which are amides of 5-hydroxyanthranilic acid with p-coumaric, ferulic, and caffeic hydroxycinnamic acids, respectively [Avenanthramides (Avns) are low molecular weight phenolic amides  encoding key proteins involved in the biosynthesis of phenolic esters, we have previously produced two novel yeast-derived recombinant Avns, namely N-(4’-hydroxycinnamoyl)-3-hydroxyanthranilic acid (YAvnI) and N-(3’-4’-dihydroxycinnamoyl)-3-hydroxyanthranilic acid (YAvnII) [Considering the wide range of potential therapeutic applications of Avns, specific efforts have been devoted to their economical and sustainable production at scales suitable for industrial applications, including novel approaches based on genetic engineering strategies as eco-friendly alternatives to conventional chemical synthesis or purification from plant sources. Indeed, knowledge of the biosynthetic pathways has now made it possible to synthesize Avns through genetically engineered microorganisms, including euticals ,21,22,23(YAvnII) . These n(YAvnII) .Furthermore, they were shown to possess bioactive properties relevant to biomedical applications, including a potent antioxidant activity related to their capacity of stimulating master regulators of cellular antioxidant responses ,25, as wIn the present study, we compared the impact of yeast-derived recombinant Avns, YAvnI and II, and major natural Avns, Avn-A and Avn-C, on colon cancer cells, showing that YAvns were as effective as natural Avns in inhibiting tumor cell growth and survival, but were more effective in inhibiting EMT and reducing tumor cell migration through transcriptional regulation of E-cadherin, suggesting enhanced functional and nutraceutical properties that might be advantageous with respect to colon cancer management and metastasis prevention.® CRL-1459™) human fibroblast cell line isolated from normal colon tissue  were cultured in 75 cm2 flasks  in RPMI-1640  supplemented with 10% (v/v) fetal calf serum (FCS) , 100 U/mL penicillin G, 40 µg/mL gentamycin sulfate, and 2.5 µg/mL amphotericin B  at 37 °C in a humidified 5% CO2 atmosphere. Cells were harvested twice a week with 0.25% porcine trypsin solution . The HT29 and WiDr human colorectal adenocarcinoma cell lines, and the CCD-18Co , and kept in a humidified incubator for 10 days. Colonies (>50 cells) were fixed and stained with Diff-Quik, counted and photographed.The clonogenic assay is useful for testing the proliferative capability of cultured cells by virtue of the cell’s ability to undergo sufficient proliferation to form a colony, thus being especially suitable for assessing long-term effects of cell treatments . In particular, as the cell metabolic state and clonogenic potential are not necessarily parallel events, the clonogenic assay can add valuable and distinct information to that provided by the MTT assay and may be seen as complementary. Briefly, cells were plated in 6 multi-well plates (700 cells/dish) in medium containing 10% FCS . After 24 h cells were treated with Avn-A, Avn-C, YAvnI and YAvnII (200 μM) or DMSO as a control, in 10% FCS  for 1 h at room temperature, and membranes were then incubated overnight at 4 °C with appropriate dilutions of specific primary antibodies. Membranes were then washed with TBS 0.1% Tween-20, and incubated for 1h at room temperature with horseradish peroxidase-conjugated anti-mouse (1:2500)  or anti-rabbit (1:10.000)  secondary antibody, followed by enhanced chemiluminescence detection system . As an internal control for protein loading, membranes were re-probed with antibodies for housekeeping proteins, including β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Images were finally digitalized with Image Quant LAS4000 . Quantitative determination of immunoreactive bands was performed by densitometry using the ImageJ software , and data were normalized to the levels of internal control. Cells were seeded at the density of 350,000 cells/well in 6 multi-well plates in medium with 10% serum. After adhesion, cells were treated for 48 h with either Avn-A, Avn-C, YAvnI or YAvnII (200 μM). Cell lysates were then obtained and analyzed as previously described . BrieflyPrimary antibodies used in the present study included: anti p27 (1:1000) , anti p21 (1:1000) , anti p53 (1:1000) , anti-focal adhesion kinase (1:1000) , anti P-FAK (1:1000) , anti E-cadherin (1:1000) , anti β-actin (1:1000) , anti GAPDH  . 4 cells/well on glass cover-slips placed into 24 multi-well plates) were maintained in 10% FCS for 24 h. Cells were then treated with Avn-A, Avn-C, YAvnI and YAvnII  and fixed in acetone for 5 min. After blocking of unspecific bindings with 3% bovine serum albumin (BSA), cells were incubated overnight at 4 °C with the primary antibody . Samples were then incubated with a secondary antibody Alexafluor 488, and analyzed by confocal microscopy (Zeiss LSM700) at 60× magnification.Cells  or DMSO as a control. The wells were washed gently with phosphate-buffered saline (PBS) , and adherent cells were fixed and stained with Diff-Quik. Adherent cells were counted in five randomly selected microscopic fields at 200× total magnification.Cells were maintained in 10% FCS and then trypsinized; 5 × 105 cells/mL were incubated in suspension in medium with 0.1% FCS for 48 h in presence of either Avn-A, Avn-C, YAvnI or YAvnII (200 μM), or DMSO as a control. After incubation, the ratio of dead cells stained with trypan blue versus total cells was evaluated by light microscopy. The number of dead cells was reported as a percentage of total cells.Cells at a density of 5 × 104 HT29 cells were added to the upper wells of the chamber and Avn-A, Avn-C, YAvnI and YAvnII (200 μM) or DMSO as a control were added in the lower wells of the chamber in medium with 10% FCS. Migration was measured in triplicate by counting the number of cells that had moved in 18 h across the filter coated with gelatin (1%). Cells were counted in five random fields/well at a magnification of 40×. Data are reported as total number of counted cells/well.Chemotaxis experiments were performed with the Boyden chamber technique. 1.25 × 10http://bioinfo.ut.ee/primer3-0.4.0/). As a housekeeping gene, GAPDH was chosen for its stability and level of expression, which is comparable to the genes of interest and whose expression remained stable after cell treatment.Cells were seeded at the density of 350,000 cells/well in 6 multiwell plates in medium with 10% serum. After adhesion, cells were treated for 48 h with either Avn-A, Avn-C, YAvnI or YAvnII (200 μM). Cells were then lysed, and total RNA was obtained using an RNA Mini kit . cDNAs were synthesized from 1.0 μg total RNA using a High Capacity RNA-to-cDNA Kit  according to the manufacturer’s instructions. Primers  were desPCR reactions were carried out in 96-well optical plates using the StepOnePlus Real-Time PCR System and the Power SYBR Green PCR Master Mix . Three biological replicates for each tested sample were performed in a reaction volume of 20 L. The PCR conditions comprised an initial incubation of 95 °C/10 min, followed by 35 cycles of 95 °C/15 s and 60 °C/1 min. In all experiments, appropriate negative controls containing no template were subjected to the same procedure to detect or exclude any possible contamination. Melting curve analysis was performed at the end of amplification. Amplicons were analyzed by the comparative threshold cycle method, in which ΔΔCt is calculated as ΔCtI-ΔCtM, where ΔCtI is the Ct value for the any target gene normalized to the endogenous housekeeping gene and ΔCtM is the Ct value for the calibrator, which is also normalized to housekeeping gene. t-test, was set at p < 0.05.Data were generated from three independent experiments and expressed as means ± standard deviation (SD). Statistical significance, determined by Student’s To evaluate the effects of natural and yeast-derived recombinant avenanthramides on the viability of colon cancer cells, we treated HT29 and WiDr human colorectal adenocarcinoma cells with different concentrations of either Avns or YAvns (from 50 to 200 μM) and performed the MTT assay at 72 h. Normal colon fibroblasts (CCD18) were used as control. As shown in In light of the outcomes of the MTT assay, we used HT29 cells to comparatively investigate the potential anticancer properties and mechanisms of action of natural and yeast-derived Avns.Firstly, we performed clonogenic assays to compare the efficacy of Avns and YAvns for inhibiting colon cancer cell growth and colony formation. To this end, HT29 cells were incubated with either Avn-A, Avn-C, YAvnI, or YAvnII at the same concentration (200 μM) for 10 days. Experimental outcomes showed that both natural and yeast-derived recombinant avenanthamides were able to significantly restrain the colony formation of HT29 cells A,B, implPrevious studies showed that the inhibitory effects of Avns on cell proliferation may be attributed to their ability to modulate the expression levels of important regulators of cell cycle progression, including the downregulation of cyclin D1 ,29, and Previous work has characterized the process of anchorage-independent growth of cancer cells in vitro as a key aspect of the tumor phenotype, particularly with respect to metastatic potential ,32. To fIt is well known that normal cells die through apoptosis when detached from extracellular matrix. However, cancer cells undergo phenotypic changes, acquiring the ability to survive and grow under anchorage-independent conditions, as well as to leave the original tumor site, migrate through surrounding tissues and establish metastasis to a distant site. To compare the capacity of natural and yeast-derived recombinant Avns to reduce the metastatic potential of colon cancer cells, we first analyzed their effects on adhesion and migration of HT29 cells. As shown in In the attempt to identify the molecular mechanisms underlying the superior efficacy of YAvns in reducing HT29 cell migration, we analyzed the expression and activation of focal adhesion kinase (FAK), a protein known to promote malignancy, by regulating the metastatic potential of cancer cells through highly-coordinated signaling networks that drive cell migration and tissue invasion . The HT2Given the important role of E-cadherin downregulation and epithelial-mesenchymal transition (EMT) in phenotypic changes underlying the acquired enhanced motility and invasion of epithelium-derived cancer cells ,16,17,18Overall, our data demonstrate the superior capacity of YAnvs in inhibiting EMT and migratory processes in colon cancer cells, suggesting promising activities against the metastatic potential of colorectal cancer cells that deserve further study using in vivo models.Colorectal cancer (CRC) incidence and mortality rates are rising rapidly in many countries worldwide, often reflecting the adoption of western diets and lifestyles. Multiple interventions, including prophylactic and therapeutic approaches through dietary nutraceuticals, are therefore needed to reduce the number of patients with CRC in future decades ,12,34,35TM , a pharmaceutical drug currently used in Japan and South Korea to treat allergic disorders [Among nutraceuticals with potential medicinal benefits, avenanthramides (Avns), phenolic compounds found exclusively in oats, have growingly become promising as potential therapeutic candidates for the treatment of several important human diseases, such as cancer, diabetes and cardiovascular diseases ,6,7,8,9.isorders ,40. IndeSaccharomyces cerevisiae, generating two novel Avn analogs, YAvn I and YAvn II, which showed structural similarity to Avn-A and Avn-C, respectively, and potent antioxidant properties [In light of this, we previously developed a genetic engineering strategy for producing novel phenolic compounds in operties . Furtheroperties ,26,37. Toperties , structuoperties ,41,42, sOur experimental outcomes showed that YAvns were as effective as natural Avns in inhibiting colon cancer cell proliferation, clonogenicity and anchorage-independent growth, and enhancing cell adhesion, as well as in increasing the expression of major cell cycle regulators, such as p21, p27 and p53 proteins, suggesting that YAvns and natural Avns share a common capacity in preventing and reversing key events involved in cancer development and progression. Consistent with these findings, whereas there is evidence in vascular smooth muscle cells that the inhibition of cell proliferation promoted by Avns implicates the upregulation of the p53–p21 pathway , we prevInterestingly, the enhanced anti-migratory and anti-EMT properties of YAns vs. natural compounds, including Avn-A and Avn-C, might be related to differences in the substitution patterns of the two aromatic moieties, including the hydroxyanthranilic acid moiety (A ring) and the hydroxycinnamic acid moiety (B ring) . Indeed,in vivo models of colorectal cancer.Overall, this study demonstrates that YAvns possess significant anti-proliferative activity against colon cancer cells, and may exert superior anti-migratory and anti-EMT activities as compared to major natural Avns, including the ability to modulate transcriptional factors involved in E-cadherin downregulation and EMT progression, suggesting enhanced functional properties as bioactive nutraceuticals. Taken together with the feasibility and reliability of producing such bioactive nutraceuticals through the yeast-based system, their anti-proliferative activity, shared with natural Avns, and their superior anti-migratory and anti-EMT activities might be highly advantageous for colon cancer management and metastasis prevention, thus paving the way for further studies in


Viruses are commonly found in patients with acute respiratory infections (ARIs). However, the viral etiologies and clinical characteristics of outpatients with ARIs are poorly understood in China. Here, we identified the viral etiologies in outpatients with ARIs in Huzhou, China.Our results indicated that of 426 outpatients, 246 were positive for viruses. Of them, 221 were positive for a single virus, including influenza A, which comprised H3N2 (28.5%) and pandemic H1N1 (2009) (19.0%), enterovirus (10.4%), and influenza B (8.6%). Other single viruses were detected at less than 8.0%. Twenty-five patients were positively coinfected with two viruses. The prevalent viruses in coinfections were rhinovirus and H3N2 virus (28.0%). Viruses were major pathogens in young children (< 5 years) (75.0%). Coinfections were prevalent in older adults (11.9%) and young children (9.5%). Virus-positive outpatients presented higher temperatures and more sore throat, fatigue and shortness of breath than virus-negative outpatients. ARIs and most virus detections peaked during the winter, but enteroviruses emerged between April and September.Viruses are major agents of ARIs among outpatients in Huzhou, China. There was a variation in the distribution of viruses across different age groups and seasons. These findings are beneficial for planning prevention and treatment services for outpatients with ARIs. Acute respiratory infections (ARIs) are common and major public health threats, causing high morbidity and mortality worldwide, particularly in developing countries , 2. ManyCurrently, there are few available vaccines to prevent respiratory virus infections , 6. It iIn this study, we collected clinical and demographic data from outpatients including children and adults with ARIs, and their specimens were tested for 15 viruses. This study aims to provide basic data to direct local disease prevention and control measures for ARIs in Huzhou, China.This study was conducted from January 2015 to April 2016 in two general hospitals in Huzhou city located beside Tai Lake of southeast China. Demographic and clinical data from all enrolled ARIs outpatients of any age were collected, and clinical specimens from the upper respiratory tract of these outpatients were tested for 15 viruses by the multiplex RT-PCR method using a Seeplex® RV15 ACE Detection Kit . Furthermore, a positive sample with FluA virus was discriminated for seasonal H1N1 (sH1N1), seasonal H3N2 (sH3N2) and pandemic H1N1 (2009) viruses by a one-step real-time RT-PCR assay from Shanghai ZJ Bio-Tech Co., Ltd. .Outpatients of any age were enrolled from January 2015 to April 2016 at the Department of Fever Outpatient Clinic of the First People’s Hospital of Huzhou and at the Huzhou Central Hospital, Huzhou, China. Outpatients suffering from fever now or in recent days, such as influenza-like illness (ILI) and ARIs patients, were seen at the fever outpatient clinic. A case definition of ARI was described in a previous report . BrieflyViral nucleic acids were extracted from 200 μl of clinical samples using the RNeasy Mini Kit  and eluted in 50 μl of elution buffer according to the manufacturer’s protocol. cDNA synthesis was conducted with a PrimeScript™ II 1st Strand cDNA Synthesis Kit . The specimens were detected for viruses by the multiplex RT-PCR method using a Seeplex® RV15 ACE Detection Kit  according to the manufacturer’s instructions. The following 15 viruses were tested in three groups: Group A: ADV, PIV-1, PIV-2, PIV-3, and CoV-229E/NL63; Group B: CoV-OC43/HKU1, RhV, RSV-A, RSV-B, and FluA; Group C: BoV, FluB, hMPV, PIV-4 and EV. The full meaning of each of the viruses abbreviated thus was defined as follows: ADV, PIV-1, PIV-2, PIV-3, CoV-229E/NL63, CoV-OC43/HKU1, RhV), RSV-A, RSV-B, FluA, human BoV, FluB, hMPV, PIV-4, and EV. Additionally, samples positive for FluA virus were further discriminated for sH1N1, sH3N2 and pandemic H1N1 (2009) viruses by a one-step real-time RT-PCR assay from Shanghai ZJ Bio-Tech Co., Ltd. .P-values < 0.05 were considered to be statistically significant.Statistical analysis was conducted with SPSS software . Descriptive statistics were used to analyze the seasonal and age distribution and infection rates of different respiratory viruses. Chi-squared tests were used to compare different age groups, gender, and clinical characteristics between age virus-positive and virus-negative outpatients with ARIs. A total of 426 outpatients with ARIs were enrolled from January 2015 to April 2016 in this study. Of them, 246 specimens  were positive for at least one virus, and single infections accounted for 89.8% (221/246) of cases. Coinfections were observed in 10.2% (25/246) of cases . However, the distribution of viruses notably differed among the different age groups . In addition, 90.1% (384/426) of ARIs outpatients suffered from high fever (body temperature ≥ 38 °C), followed by cough (68.5%), sore throat (51.9%), fatigue (45.8%), and other respiratory symptoms. Moreover, a significant difference was observed in clinical symptoms including fever , sore throat , fatigue , and shortness of breath  between virus-positive and virus-negative cases, and the proportion of patients with abdominal pain was greater among virus-negative cases . Additionally, physical examinations showed abnormal lung auscultation (12.9%) and X-rays (6.1%).Demographic and clinical characteristics of outpatients with ARIs are shown in Table The distribution of the viral etiologies of the four age groups is shown in Table The virus detection rates for different seasons are shown in Fig. Viruses are major agents contributing to the high morbidity and mortality of patients with ARIs, particularly in children under 5 years of age , 4, 6. MLaboratory diagnosis of viruses is commonly conducted by conventional methods (such as culture or antigen detections), and real-time and multiplex RT-PCR assays have been considered to be important tools for identifying the etiologies of ARIs , 5, 7, 8The proportion of respiratory viruses notably differed across different age groups; the virus positive rate was the highest in young children under 5 years but was lowest in adults 18~60 years) in this study. These findings indicated that the viruses were the predominant pathogen found in young children with ARIs, and similar morbidity rates have been reported in previous studies 0 years i. The FluMany studies have indicated that viral ARIs are affected by seasonal distributions and occur commonly in spring, autumn, and winter , 27, 29.Our study had some limitations. First, there were only 426 outpatients with ARIs enrolled in the two local hospitals; because this was a small sample size, it may be difficult to estimate the disease severity of outpatient ARIs in detail. More large-scale surveillance for ARIs will performed, and analyses over additional years will provide a more accurate picture of seasonal variation in respiratory virus circulation in this community in the future. Second, we detected only viruses, but did not test for bacterial pathogens in the respiratory tracts of outpatients with ARIs; therefore, our data regarding pathogens causing ARIs were not comprehensive, which also affected our ability to determine the relationships between pathogens and disease severity in outpatients with ARIs. Taken together, our results were valuable to a certain degree for assessing outpatients and clinical treatments.In summary, this study provides important epidemiologic data regarding the clinical characteristics, viral spectrum, age distribution and seasonality of viruses in outpatients with ARIs in Huzhou, China. These findings contribute to evaluating the burden of virus infections in outpatients, including young children and adults. Timely and accurate diagnosis of pathogens in outpatients with ARIs is required to reduce the burden caused by these diseases.