Sulforaphane, a potent dietary bioactive agent obtainable from cruciferous vegetables, has been extensively studied for its effects in disease prevention and therapy. Sulforaphane potently induces transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated expression of detoxification, anti-oxidation, and immune system-modulating enzymes, and possibly acts as an anti-carcinogenic agent. Several clinical trials are in progress to study the effect of diverse types of cruciferous vegetables and sulforaphane on prostate cancer, breast cancer, lung cancer, atopic asthmatics, skin aging, dermatitis, obesity, etc. Recently, the protective effects of sulforaphane on brain health were also considerably studied, where the studies have further extended to several neurological diseases, including Alzheimer’s disease (AD), Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, multiple sclerosis, autism spectrum disorder, and schizophrenia. Animal and cell studies that employ sulforaphane against memory impairment and AD-related pre-clinical biomarkers on amyloid-β, tau, inflammation, oxidative stress, and neurodegeneration are summarized, and plausible neuroprotective mechanisms of sulforaphane to help prevent AD are discussed. The increase in pre-clinical evidences consistently suggests that sulforaphane has a multi-faceted neuroprotective effect on AD pathophysiology. The anti-AD-like evidence of sulforaphane seen in cells and animals indicates the need to pursue sulforaphane research for relevant biomarkers in AD pre-symptomatic populations. Sulforaphane (1-isothiocyanato-4-methylsulfonylbutane) is a potent dietary bioactive agent obtainable from cruciferous vegetables, such as broccoli, watercress, kale, cabbage, collard greens, brussels sprouts, bok choy, mustard greens, and cauliflower . When thHelicobacter pylori infection, the nervous system, the cardiovascular system, liver, lungs, skin and even mortality. According to clinicaltrials.gov, clinical trials of 159 conditions to study the effects of sulforaphane on human health and diseases have been conducted or are in progress. In 1994, structural analogues of sulforaphane were synthesized, but none showed superior activity compared to sulforaphane [The study of the pharmacological effects of sulforaphane and cruciferous plants was started in 1992 by Zhang, Y. et al. . They cooraphane , and no clinicaltrials.gov, there is a clinical trial underway in China using sulforaphane in patients with prodromal to mild AD (NCT04213391).The study on the neuroprotective effects of sulforaphane began in 2004 with studies showing the protective effects on neurons  and micrAlzheimer’s disease is a slowly progressive neurodegenerative disease that currently has no effective treatment. The most discernible pathology that can identify AD is the extracellular formation of plaques produced by the accumulation of amyloid-β (Aβ) protein , and theSince there is no suitable treatment for AD, the overall goal of AD management is to reduce the incidence of disease in the target population and to ensure pre-symptomatic disease does not proceed to a later stage . SeveralV97L mice, single-mutant transgenic model harboring PS1V97L, as described in Sulforaphane has been found to prevent Aβ production in the brains of AD-like animal models . In the In the brains of APP/PS1 mice, sulforaphane decreased the increment of Aβ plaques and the mechanism underlying these effects was suggested to be associated with increased expression of p75 neurotrophin receptor (p75 NTR) via reducing the expression of histone deacetylase (HDAC) 1 and 3 . p75 NTRIn the hippocampus of 3×Tg-AD mice, oral gavage of sulforaphane reduced the monomeric and polymeric forms of Aβ . In this2 groups  in Aβ and forms three different 1:1 complexes of Aβ+sulforaphane [Aβ aggregation studies with sulforaphane showed that Aβ is less prone to aggregate when sulforaphane is present . Mass sporaphane . Together, the above observations suggest that sulforaphane not only inhibits Aβ production by decreasing the expression of BACE1 and PS1  but alsoV97L mice. In the study using 3×Tg-AD mice, sulforaphane treatment reduced tau and p-tau at the protein level, but not mRNA, suggesting post-translational modifications in tau expression [Tau is the main component of neurofibrillary tangles in AD and is a causative factor for AD ,55. Sulfpression . Sulforapression ,56. In mpression . Although further studies are required to understand how sulforaphane affects tau biology, Aβ, Nrf2 lack, oxidative stress, and inflammation are known factors to promote tau phosphorylation ,58,59,60V97L mice resulted in decreased levels of interleukin (IL)-1β in brain tissue and tumor necrosis factor alpha (TNF-α) in the plasma [Sulforaphane also affects inflammatory biomarkers associated with AD, as below in AD-like cell and animal models . For exae plasma . Sulforae plasma . In mouse plasma . Aβ actie plasma . Mice the plasma ,63. Thuse plasma . microRNe plasma ,65 and se plasma . 2+ levels and blocking NF-κB to rescue the decrease in Mer tyrosine kinase (MerTK) activity that is necessary for amyloid-stimulated phagocytosis [Meanwhile, it has been published that sulforaphane stimulates phagocytosis of immune cells ,67. Accoocytosis . Thus, socytosis . As actiocytosis ,69, sulfV97L mice and AD-like rat induced by Aβ, sulforaphane increased GSH levels and decreased MDA levels [Oxidative damage to brain tissue caused by accumulation of Aβ and tau proteins impairs the function of neuronal synapses, induces neuronal degeneration, and eventually causes the symptoms of memory loss seen in AD . SulforaA levels ,37. CrudA levels . TogetheSulforaphane inhibits the levels of AD-associated biomarkers of synaptic damage and neurodegeneration in AD-like models, as well as inhibiting Aβ-induced neuronal cell death . For exaSeveral studies have reported that sulforaphane prevented Aβ-induced cell death in neuron-like cells  32,35,4,41,43. TV97L mice [Sulforaphane ameliorated cognitive impairment in several transgenic AD-like animal models, including 5×FAD mice , 3×Tg-AD97L mice . Additio97L mice ,37. AlthThe anti-AD-like activity of sulforaphan has been identified in six different animal models and eight different cell models . In animIt has been reported that AD already has pre-symptomatic pathologies in the brain 10–15 years before cognitive decline . In orde® (a sulforaphane-producing dietary supplement) was studied to investigate (1) the dose-response evaluation of sulforaphane, (2) the bioavailability and mucosal bioactivity in healthy subjects, (3) the treatment effects for youth with ASDs, (4) the effects on autistic people living in New Jersey, USA, and (5) the effects on schizophrenia [In one recent clinical trial, in which schizophrenic patients were administered sulforaphane-rich broccoli sprout extract , three tablets of sulforaphane per day (consisting of 30 mg of sulforaphane-glucosinolate per tablet) for 8 weeks resulted in improved cognitive function in schizophrenic patients . In anotophrenia .Helicobacter pylori [In-depth human studies are needed on the medical benefits of a sulforaphane-rich broccoli sprout diet. The association between the Keap1 and Nrf2 genes and cognitive function in patients with schizophrenial was studied, with an epistatic interaction between Nrf2 and Keap1 gene variants on cognitive impairment  . Since sr pylori  and sulfr pylori . It has r pylori . In 2003, the World Health Organization (WHO) International Agency for Research on Cancer (IARC) published a chapter on cruciferous vegetables as recommendations for cancer prevention. Whether in the form of a sulforaphane supplement or in large amounts of cruciferous vegetables, WHO recommended not to consume large amounts. It is generally not advisable for individuals to consume a diet that focuses on only one food type and should be encouraged to consistently consume a variety of cruciferous vegetables as part of their diet. On the other hand, several clinical trials showed little to no side effects when reasonable amounts of cruciferous vegetables containing sulforaphane were consumed . Evidences from several animal- and cell-based studies indicate the need to pursue sulforaphane research in pre-symptomatic AD patients, which is challenging. The discovery and development of practical and inexpensive biomarkers that can diagnose pre-symptomatic AD patients is essential. Genetic polymorphism, intestinal microbiome, and body mass index (BMI) may affect the bioavailability of sulforaphane from cruciferous vegetable consumption ,81,82,83


CCL19, CD3D, CD8A, CD22, LY9, IL12RB1, C1S, C7, AMICA1, TIAM1, TIGIT, THY1 was associated with longer OS (p < 0.05). In addition, multiple genes that encode a component or a regulator of the extracellular matrix such as MMP2 and COL1A2 were identified post-treatment as being associated with longer RFS and OS. In all baseline tissues, high TIS scores were associated with longer OS (p = 0.0166). Also, downregulated expression of cell proliferation-related genes such as CUL1, CCND1 and AAMP at baseline was associated with pathological and radiological response (unadjusted p < 0.01). In conclusion, we identified numerous genes that play roles in multiple biological pathways involved in immune activation, immune suppression and cell proliferation correlating with pathological/radiological responses following neoadjuvant immunotherapy highlighting the complexity of immune responses modulated by immunotherapy. Our observations suggest that TIS may be a useful biomarker for predicting survival outcomes with combination immunotherapy.Patients with locally/regionally advanced melanoma were treated with neoadjuvant combination immunotherapy with high-dose interferon α-2b (HDI) and ipilimumab in a phase I clinical trial. Tumor specimens were obtained prior to the initiation of neoadjuvant therapy, at the time of surgery and progression if available. In this study, gene expression profiles of tumor specimens (N = 27) were investigated using the NanoString nCounter® platform to evaluate associations with clinical outcomes , and overall survival (OS)) and define biomarkers associated with tumor response. The Tumor Inflammation Signature (TIS), an 18-gene signature that enriches for response to Programmed cell death protein 1 (PD-1) checkpoint blockade, was also evaluated for association with clinical response and survival. It was observed that neoadjuvant ipilimumab-HDI therapy demonstrated an upregulation of immune-related genes, chemokines, and transcription regulator genes involved in immune cell activation, function, or cell proliferation. Importantly, increased expression of baseline pro-inflammatory genes  Stage III melanoma encompasses regional lymphatic and/or lymph node involvement, for which the current standard of care is surgery followed by adjuvant therapy. The first adjuvant therapy approved for melanoma was high dose interferon α-2b (HDI) which demonstrated reduced risk of relapse or death , 2. Moreet al. demonstrated that pre-treatment biopsies of patients with metastatic melanoma that responded to IL-2 therapy displayed a pattern of immune activation [In addition to the adjuvant use of immunotherapeutic agents, neoadjuvant therapy has emerged as a promising therapeutic option with the potential to improve surgical resectability, organ preservation, and overall survival, as well as to further reduce the risk of relapse in patients –17. A nutivation . In a retivation . Furthertivation , 25. Rectivation .h1 polarization), leading to increased T-cell infiltration and immune surveillance to control tumors [Previously, we conducted a clinical trial using combination immunotherapy with HDI and ipilimumab as neoadjuvant therapy in locally/regionally advanced operable melanoma (NCT01608594) . The stul tumors . It has l tumors , 28–31, l tumors , 33. Thu; each of which contains 770 genes relevant to cancer progression, cancer pathways, and the immune response to the tumor. After accounting for overlap of genes between the panel, the samples were profiled with 1956 unique genes.In this study, our aim was to investigate potential biomarkers for response to this combined immunotherapy regimen, comparing gene expression in pre-treatment biopsies to specific response signatures in post-treatment patient samples. An additional goal of this study was to characterize the performance of the Tumor Inflammation Signature (TIS), an 18 gene expression signature that measures a peripherally suppressed adaptive immune response in the TME , 34. TISPatients with locally advanced, regionally advanced or recurrent melanoma were enrolled in this study (N = 30) and were treated with 3 or 10 mg/kg ipilimumab and HDI . InclusiClinical efficacy was assessed as tumor response evaluation per modified World Health Organization (mWHO) criteria using imaging studies conducted at baseline, 6–8 weeks after initiation of therapy (before surgery), and every 3 months thereafter. Based on best overall response as assessed pre-operatively, radiologic responses were categorized as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Due to planned definitive surgery, responses were not possible to confirm radiologically based on mWHO criteria. Pathologic complete response (pCR) was assessed at the time of definitive surgery and defined as no visible malignant cells on hematoxylin and eosin staining by histological assessment. In addition, OS and RFS data were obtained for all patients.Tumor biopsies were obtained at baseline (before initiating neoadjuvant therapy), at the time of surgery (resected tumor at 6–8 weeks following the initiation of neoadjuvant therapy), and at the time of progression (if available). Archival samples collected from primary tumors at the time of diagnosis were also available.S1 Table). Each panel was combined with a custom 30-gene Panel-Plus codeset to profile additional genes related to TIS, immune cell type profiling, and immunometabolism (S1 Table). Therefore, a total of 1,948 unique genes were profiled in this study. The data will be provided upon request from the corresponding author.Formalin-fixed paraffin-embedded (FFPE) tissue samples were collected from 23 primary tumors, 22 baseline metastases, 25 post-treatment metastases, and 5 metastases from progressive disease. Total RNA was extracted using the Qiagen FFPE RNeasy kit. RNA concentration was measured by UV spectrophotometry and each sample was adjusted to 20 ng/μL. 100 ng total RNA from each sample was profiled with the NanoString nCounter® PanCancer Immune Profiling, PanCancer Pathways, and PanCancer Progression panels  with a linear model using the base R p-values derived from the log-rank test [p-values are from the log-rank tests from the Cox proportional hazards model fitted to the data.Associations between gene expression or signature scores and OS or RFS was assessed by fitting a univariate Cox proportional hazards model for each gene or signature, with ank test . For anaS2 Table summarizes demographics and baseline disease characteristics of the patients who participated in the clinical trial. Among 30 patients enrolled, 27 had adequate tumor tissue for gene expression profiling analysis. Among 27 patients, 11 patients relapsed, of whom 5 died. Based on mWHO criteria, radiological response was observed in 10 patients  while 10 showed stable disease and 7 patients showed radiologic progression of disease. In this cohort, 8 patients (29.6%) achieved pathologic complete response.S1 Fig).Gene expression signatures were analyzed and compared between FFPE tumor samples obtained prior to neoadjuvant therapy (baseline) and resected tumor tissues at the time of surgery, normally 6–8 weeks following the initiation of neoadjuvant therapy. These analyses of 48 samples (24 patient-matched pre- and post-treatment samples) demonstrated some clustering of gene expression signatures specific to baseline vs. post-neoadjuvant treatment as shown in heatmaps , and no gene expression signatures were significantly different between primary tumor and baseline metastasis samples . Due to the minimal differences between primary tumor and baseline metastasis tissues, baseline metastasis samples were used as a baseline sample, if possible, otherwise primary tumor samples were used in this study.To characterize and compare primary tumors to baseline metastasis samples, the expression patterns of individual genes as well as gene signatures were analyzed. In these tissue samples (n = 13), only four genes CUL1 gene was significantly downregulated in baseline samples of patients with pCR as compared to those with residual tumor . CUL1 is a scaffold protein in the SCF E3 ubiquitin ligase complex that mediate the ubiquitination of proteins involved in cell proliferation in melanoma [CCND1 and AAMP (unadjusted p < 0.01), which are also linked to cell proliferation and migration . CCND1 encodes the cyclin D1 protein, which is complexed with cyclin-dependent kinases (CDKs) and regulates the cell cycle to induce cell migration and angiogenesis, linked to melanoma and metastatic progression [Differential expression analysis of individual genes was performed on available baseline samples from 8 patients with pCR and 18 patients with residual tumor. The melanoma . Other ggression , 40. AAMgression , 42.Fig 1C).To determine if there are more robust indicators of biological activity, gene signatures rather than individual genes were analyzed in the cohort using previously developed gene signatures that describe TIS, cell typing, and other particular biological pathways , 34, 35.TGFB3 and SH2B3 were highly expressed in radiologic responders . TGFB3 encodes transforming growth factor-β3 (TGF-β3), which is a multifunctional cytokine expressed in melanoma cells [SH2B3 encodes a lymphocyte adaptor, reported as a tumor suppressor gene in leukemia [EZH2, STAG2, SLC35A3, RAF1, and MKI67 . EZH2 is an epigenetic-modifying histone methyltransferase, known to regulate melanoma growth and metastasis by silencing tumor suppressors and also induce resistance to immunotherapy [p < 0.05, Fig 2D and 2E).Next, we analyzed the correlation of the baseline gene expression with radiographic response by comparing radiologic responders (PR+CR) to patients with stable or progressive disease (SD+PD). ma cells . SH2B3 eleukemia , 45. Sevotherapy –48. MoreFig 3A). Analysis of differential gene expression associated with OS identified numerous genes  compared to pathologic or radiologic response . The correlations of gene expression and signatures to OS were plotted with respect to their individual hazard ratios . Many genes differentiated the survivors from non-surviving patients, encompassing aspects of tumor, immune, and stromal gene expression. Among the genes associated with longer survival included TNFSF10, also known as TRAIL, TNS1 , FLT3LG (controls the development of dendritic cells), and TGFB3 (TGF-β3) . In colorectal cancer, TNS1 is identified as a potential biomarker for tumor cell proliferation and invasion [Fig 2. Immune-related genes whose expression was associated with longer OS were CCL19, CD3D, CD8A, CD22, LY9, IL12RB1, C1S, C7, AMICA1, TIAM1, TIGIT, and THY1. In addition, expression of extracellular matrix-related proteins and molecules  was correlated with longer OS . COL1A2 encodes a type I collagen, one of main extracellular matrix proteins, which is controlled by its degrading matrix metalloproteinases (MMPs) and implicated in cancer development and progression [Among 27 evaluable patients, 22 patients survived with the median follow-up of 32 months while 5 patients died. Principal component analysis was performed to look for distinct gene expression patterns in baseline vs. post-treatment samples by outcome, which indicated that pre-treatment samples were relatively distinct when grouped by OS  are also known to have several biological functions. For example, SMC3, SMARCA4, SET, H3F3C, and H3F3A  are involved in regulation of gene expression by controlling the chromosome, chromatin, or histones. CD2AP (CD2 associated protein) and RAC1  play a role in actin cytoskeleton remodeling and cell growth. IRAK1 (IL-1 receptor-associated kinase 1) is responsible for IL-1-induced upregulation of the transcription factor NF-κB. Expression of PRKAR2A  as well as the signal transducer SMAD2, which modulates multiple signaling pathways including TGF-β pathway, was strongly associated with unfavorable outcome .Genes that were associated with shorter OS  signatures which are related to T cell/lymphoid activation and functions . Other genes and signatures that were correlated with longer OS include Treg, TIGIT (T cell immunoreceptor with Ig and ITIM domains), PD-1, MAGEs (melanoma antigen genes), NK cells, MHC2, B cells, and tumor inflammation signature (TIS) . This is concordant with the previously reported prognostic abilities of TIS in metastatic melanoma [p-value of 0.0166 .As observed in gene expression analysis, many genes and signatures correlated with longer OS, including CD8 T cell, T cells, lymphoid, Tmelanoma . As tumop < 0.05, there were 24 genes positively associated with each outcome: ACVRL1, BAP1, CCND1, CD36, CHRDL1, CUL1, DCN, DPT, FBLN1, ISLR, LEPR, LUM, MEOX2, MIF, MMRN2, PDGFRA, PRKAR2A, PTK2, SH2B3, SLC3A2, TIE1, TNFSF10, VASH1, and WEE1.We further identified genes that were significantly associated with both OS and pathologic response. With a significance threshold of S3A and S3B Fig). Expression of TNS1 and TNFSF13B in samples from longer RFS  supports the gene expression data of TNS1 and TNFSF10, another TNF superfamily member, being correlated with longer OS . Notably, there was a trend of extracellular matrix-related proteins  expression with longer RFS  as observed with longer OS .Median follow-up for 16 patients who have not relapsed was 32 months. In this cohort, differentially expressed genes and signatures were assessed for a correlation with RFS . The other gene SETD2 (SET domain containing 2) encodes a histone lysine methyltransferase that plays a role in chromatin structure modulation. Expression of two immune-related genes CD2AP (CD2-associated protein) and CD46 (complement regulatory protein) showed a statistically significant correlation to shorter RFS .Increased expression of p < 0.05, S3B Fig. All of these were also correlated to OS , suggesting the importance of these biological functions in patient survival. As analyzed in the OS cohort, the change in TIS in the RFS cohort was also followed up over the course of therapy. For all available pre-treatment biopsies, stratified TIS scores demonstrated a trend of being associated with RFS, but it was not statistically significant .Gene expression and signature analysis identified an association of MAGEs, CD8 T-cells, and PD-1 with longer RFS . It is intriguing that C7 expression was also associated with longer OS . C7 encodes a complement protein C7 that forms a membrane attack complex with other complement components and is recognized as a potential tumor suppressor [KLRK1, IL2RG, IL10RA, JAK3, CD4, CD27, CD48, CD84, CD96, PTPRC, LCP1, ITGAL, SELPLG, SLAMF7, PECAM1, CCR1, ISG20, LILRB1, IRF2), transcription regulators , and TNF-α function (TNFRSF1B). After therapy, only ERBB3 (Erb-B2 Receptor Tyrosine Kinase 3) gene was significantly downregulated based on a cutoff at 10% FDR . This gene encodes a tyrosine kinase for epidermal growth factor receptors (EGFRs), which play a role in development of various types of cancers [To investigate gene expression changes in response to ipilimumab/HDI combination therapy, we compared the gene expression data between baseline biopsy and post-treatment samples from patients for whom data from both time points were available . After treatment, expression of C7 and CD79A was most dramatically increased  across multiple genes and signatures related to an activation of T-cell activity, including lymphoid, MHC2, cytotoxicity, B-cells, CD45, CD8 T-cells, cytotoxic cells, exhausted CD8, macrophages, T-cells, CTLA-4, IL-10, PD-L2, TIGIT, as well as TIS . High expression of these genes and signatures indicates an overall increase in immune activity and lymphocyte infiltration.Analysis of therapy-induced changes in gene expression signatures detected a significant increase . In patients with pCR, NFKBIA (NF-κB inhibitor α), TNFAIP3 (TNF-α induced protein 3), and MARCO (macrophage pattern recognition scavenger receptor) were highly expressed after treatment . Both NFKBIA and TNFAIP3 proteins inhibit NF-κB transcription factor activity to regulate genes responsible for innate and adaptive immune response. MARCO gene expression is normally associated with immunosuppressive pathways, enabling the establishment and persistence of solid tumors as well as metastasis in cancer [CCL14, CXCL2, CXCL4, NFATC1, JAK1, and CD84 . In addition to these immune-related genes, other genes detected in post-therapy samples from patients with pCR play important role in cell cycle (RGCC), autophagy (ATG7), transcriptional regulation , and extracellular matrix . Another upregulated gene corresponding to therapy was FOS proto-oncogene, which regulates cell proliferation, differentiation, and transformation, in some cases, associated with apoptotic cell death.The gene expression change between baseline and post-treatment biopsy from pCR vs. those with residual tumor was examined to investigate if any changes correlated with pathologic response. Principal component analysis revealed that post-treatment samples grouped by pathologic response were most distinct from others outcome groups  . SLAMF7 is a surface marker of NK cells, T cells, and B cells and is involved in the regulation of NK cell functions in addition to other innate and adaptive immune response functions [CD79A, CD164, C7, CCR1, and JAK3. CD164  is involved in the regulation of cell proliferation and apoptosis, and the overexpression is detected in cancer patients [ITGA6 and STMN1 in pathologic responders; ADAM15, BAD, SMURF1, and GNAQ in non-responders .Among pathologic non-responders, one of the differentially expressed genes after treatment was unctions –59. Othepatients . Genes tp < 0.05, Fig 5D). In non-responders, multiple gene signatures including lymphoid, B cells, cytotoxic cells, macrophages, T-cells, CD45 and PD-L2 were increased after treatment . When these signatures were compared between these two cohorts, TIGIT was significantly highly expressed in pathologic responders while lymphoid, B cells, macrophages, and PD-L2 signatures were high in non-responders .Next, treatment-induced gene expression changes that associated with pCR were analyzed for gene signatures. Four gene signatures, TIGIT, CD45, cytotoxic cells, and T cells, were highly expressed in patients who responded to therapy , and TANK (TRAF family member associated NF-κB activator) demonstrated increased expression that were associated with radiologic response . Both NFKBIA, and TANK are involved in NF-κB activation, and common polymorphisms in NFKBIA is associated with susceptibility to cancer [MARCO, NFKBIA, RGCC, CCL14, CXCL2, PIK3R1, and ETS2 was correlated to both radiologic response and pathologic response to therapy .Lastly, changes in post-treatment gene expression were evaluated between samples available from radiologic responders (n = 8) vs. non-responders (n = 16). In radiologic responders, more than 10 genes such as o cancer –63. Chito cancer . DiffereIL2RG (IL-2 receptor subunit gamma) was differentially expressed after treatment, which was previously identified to contribute in pancreatic cancer growth [SLAMF7, CD38, C7, and CD79A was associated with lack of radiologic response , and increased expression of these genes was also associated with pathologic non-responders . Other highly expressed genes in non-responders after treatment were CD22, CD27, CD53, ST6GAL1, JAK3, ICAM3, LCP1, RAC2, LILRB1, and ADA; all these genes play a role in immunity or immune-regulation. Correlated to radiologic response, a few genes that were significantly downregulated after therapy: PCNA and MAD2L2 in radiologic responders, ADAM15 and EPS8L1 in non-responders . Post-therapy downregulation of ADAM15 was seen in pathologic non-responders as well .In tissue samples from patients with SD or PD, r growth . In postp < 0.05, S4C Fig). In radiologic non-responders, B cells, CD45, lymphoid, T cells, cytotoxic cells, TIGIT, cytotoxicity, CTLA-4, and macrophages were highly expressed compared to responding patients . These data indicate overall increase in immune activity and lymphocyte infiltration post-treatment.In evaluation of gene and signature expression in radiologic responders, only PD-L2 was statistically associated with radiologic response . Analysis of correlation to radiologic response also revealed downregulated expression of genes involved in cell proliferation, cancer progression and recurrence .In this study, we characterized gene expression profiles of patients with locally advanced/regionally melanoma who underwent neoadjuvant ipilimumab and HDI combination treatment. The gene expression data were interpreted using PanCancer analysis to identify the association between gene expression at baseline and after treatment with response, either pathologic or radiologic. Special consideration was given to the performance of the TIS to characterize response and survival of patients. Schematic of experimental strategy and summary of results in this manuscript are shown in CCL19, CD3D, CD8A, CD22, LY9, IL12RB1, C1S, C7, AMICA1, TIAM1, TIGIT, THY1 was associated with longer OS (p < 0.05) . CCL19 gene codes a chemotactic factor that strongly attracts naïve T cells and DCs [As expected, high expression of a number of pro-inflammatory genes at baseline was associated with a benefit in patient outcome. Increased expression of  and DCs . Also, C and DCs . More re and DCs . In cont < 0.05) . CCL19 gSH2B3 and TGFB3) as well as TGF-β signature demonstrated a significant correlation to response . SH2B3 is known as a tumor suppressor in leukemia [TGFB3 gene expression and TGF-β signature with radiologic response was unexpected considering that activation of the TGF-β pathway normally leads to melanoma progression and metastasis [TGFB3 was correlated to longer OS . On the other hand, increased expression of SMAD2 and/or SMAD4 that belong to the TGF-β cascade was strongly associated with shorter RFS and OS . It is known that TGF-β plays a complex role during carcinogenesis by either acting as a tumor suppressor or as an oncogene in a context-dependent manner, as well as having known roles in the immune system that compound any activity already present within the tumor [In terms of radiologic response, upregulation of genes , and MMP2, COL1A2, PCOLCE, and DCN with longer RFS . The extracellular matrix, including collagen, fibronectin, decorin, and related regulatory enzymes (e.g. MMPs), is the most abundant component in tumor microenvironment that can regulate tumor cell progression [Figs S4B), implying a beneficial role of these metalloproteinases in response to ipilimumab and HDI therapy.Another biological category of genes that correlated to patient survival was extracellular matrix-related proteins and molecules. Increased expression of gression –74. WhilEZH2 downregulation with radiologic response in this cohort . Another study by Zingg et al. has demonstrated higher expression of EZH2 is linked to poor patient survival in melanoma [reg cells [Further, genes related to epigenetic regulation were also differentially expressed, including an association of melanoma . Our fineg cells –77.MARCO, RGCC, CCL14, CXCL2, PIK3R1, and ETS2 was associated with both pathologic and radiologic response . These data suggest that regulation of NF-κB signaling or cell cycle as well as chemokine-mediated or transcription regulator-mediated immunoregulatory and inflammatory processes are associated with clinical response in post-therapy samples. Interestingly, there was increased expression of JAK1 (Janus Kinase 1) after treatment in patients with pCR . JAK1 plays a key role in IFN-α/β and IFN-γ signal transduction by phosphorylation of STAT signal transducers and transcription activators, and loss-of-function mutations in JAK1 or JAK2 have been identified as a mediator of primary resistance to pembrolizumab [JAK1 by ipilimumab/HDI combination therapy may improve clinical response by potentiating the immunological environment to respond to subsequent anti-PD-1 treatment.When looking further to determine gene expression changes in response to neoadjuvant ipilimumab and HDI treatment, we observed multiple immune related genes that achieved maximal differential expression before and after treatment. Among those genes, upregulated expression of olizumab , 79. TheFig 3C). Most of these signatures are related to activation and function of lymphocytes  as well as antigen presentation. MAGEs, CD8 T-cells, and PD-1 were associated with longer RFS  and OS . The signatures that were associated with response to ipilimumab and HDI combination therapy were TIGIT, CD45, cytotoxic cells, and T cells , suggesting that efficient T cell infiltration correlates with response.In our cohort, analysis of gene signatures indicated the significant correlation of multiple signatures to OS in baseline biopsy samples (p = 0.0166) . In general, patients experienced a significant increase in TIS score after neoadjuvant ipilimumab and HDI therapy , raising the possibility that this regimen may be effective in priming and enhancing the immunogenicity of less inflamed tumors. However, high TIS score post-therapy was not significantly associated with either pathological or radiologic response , possibly attributable to the lower statistical power with the small sample size of subgroup analysis. This finding raises the possibility that ipilimumab and HDI combination therapy may be priming a naïve immune response or augmenting a pre-existing suboptimal immune response, but at the same time, inducing expression of compensatory immune checkpoints that restrict complete elimination of the tumor. In a study of single-dose pembrolizumab in resectable melanoma, response signature  has been associated with clinical benefit while disease recurrence displayed a resistance phenotype, such as immune suppression, mutational escape, or tumor evolution [Next, we applied TIS algorithm to characterize the inflammatory profile of tumor samples and correlate it to OS. It was observed that TIS scores were associated with OS Click here for additional data file.S2 FigA) Volcano plot of unadjusted p-value vs. log2-fold change of the differential expression between baseline metastasis and primary tumor samples (unadjusted p < 0.01). There were 13 samples for this analysis. B) Forest plot of gene signature scores between low expression and high expression in primary tumor samples. The position of the squared dots denotes the difference of score, and the size denotes the statistical significance. The horizontal lines are the Wald-type confidence intervals. There was no significant p-value in this analysis.(TIF)Click here for additional data file.S3 FigA) Volcano plot of unadjusted p-value vs. log-hazard ratio of the differential expression associated with RFS of treated patients (unadjusted p < 0.01). Among 27 evaluable patients, 26 samples were available for this analysis. B) Forest plot of difference of gene signature scores based on hazard ratio between longer RFS and shorter RFS. The position of the squared dots denotes the difference of score, and the size denotes the statistical significance. The horizontal lines are the Wald-type confidence intervals. The * sign denotes the significance of p-value (< 0.05*). C) The Kaplan-Meier curves of TIS score groups for available samples (n = 26). Patients are stratified into “high” and “low” groups based on the TIS score 50% quantile. The Kaplan-Meier curves show that the high TIS score group demonstrated a trend of higher survival rate than the low TIS score group but statistically significant . The survival time was fit to TIS score group (high vs. low) with Cox proportional hazard model.(TIF)Click here for additional data file.S4 FigA) Volcano plot of unadjusted p-value vs. log2-fold change of the differential expression after treatment, which associate with radiologic response (unadjusted p < 0.01). This analysis only includes data from 8 patients (n = 8) who exhibited radiologic response and for whom both baseline and post-treatment samples were available B) Was examine as in S4A Fig, but only includes data from 16 patients (n = 16) who did not exhibit radiologic response and for whom both baseline and post-treatment samples were available. C and D) Forest plot of difference of gene signature scores post-treatment in radiologic responders (C) and non-responders (D). The position of the squared dots denotes the difference of score, and the size denotes the statistical significance. The horizontal lines are the Wald-type confidence intervals. The * sign denotes the significance of p-value .(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Shigella, sapovirus, Shiga toxin-producing Escherichia coli, and Cryptosporidium were the leading antibiotic-treated diarrhea etiologies. Rotavirus caused 29.2% (24.5–35.2%) of antibiotic-treated cases, including the largest share in both the first and second years of life. Shigella caused 14.9% (11.4–18.9%) of antibiotic-treated cases, and was the leading etiology at ages 24–59 months. Our findings should inform the prioritization of vaccines with the greatest potential to reduce antibiotic exposure among children.Diarrhea is a leading cause of antibiotic consumption among children in low- and middle-income countries. While vaccines may prevent diarrhea infections for which children often receive antibiotics, the contribution of individual enteropathogens to antibiotic use is minimally understood. We used data from the Global Enteric Multicenter Study (GEMS) to estimate pathogen-specific incidence of antibiotic-treated diarrhea among children under five years old residing in six countries of sub-Saharan Africa and South Asia before rotavirus vaccine implementation. GEMS was an age-stratified, individually-matched case-control study. Stool specimens were obtained from children presenting to sentinel health clinics with newly-onset, acute diarrhea (including moderate-to-severe and less-severe diarrhea) as well as matched community controls without diarrhea. We used data from conventional and quantitative molecular diagnostic assays applied to stool specimens to estimate the proportion of antibiotic-treated diarrhea cases attributable to each pathogen. Antibiotics were administered or prescribed to 9,606 of 12,109 moderate-to-severe cases and 1,844 of 3,174 less-severe cases. Across all sites, incidence rates of clinically-attended, antibiotic-treated diarrhea were 12.2 , 10.2 (7.4–13.9) and 1.9 (1.3–3.0) episodes per 100 child-years at risk at ages 6 weeks to 11 months, 12–23 months, and 24–59 months, respectively. Based on the recommendation for antibiotic treatment to be reserved for cases with dysentery, we estimated a ratio of 12.6 (8.6–20.8) inappropriately-treated diarrhea cases for each appropriately-treated case. Rotavirus, adenovirus serotypes 40/41,  Shigella etiology), little is known about real-world diarrhea treatment practices in LMICs. We used data from a study of children experiencing diarrhea and matched, asymptomatic controls residing in LMICs of South Asia and sub-Saharan Africa to understand factors leading to antibiotic treatment of diarrhea cases, and the proportion of cases attributable to various enteric pathogens. We identify rotavirus and Shigella as the predominant causes of antibiotic-treated diarrhea at ages 0–23 months and 24–59 months; additional leading causes include adenovirus serotypes 40/41, sapovirus, Shiga toxin-producing Escherichia coli, and Cryptosporidium. We estimate that approximately 12.6 diarrhea cases receive antibiotics inappropriately for each appropriately-treated case in the study settings. Vaccines against prominent enteropathogens may substantially reduce antibiotic consumption among children in LMICs.Because diarrhea is the second-leading cause of antibiotic consumption among children in low- and middle-income countries (LMICs), effective vaccines against diarrheal pathogens may have the collateral benefit of reducing antibiotic exposure and resistance selection in these settings. Whereas antibiotic treatment is only recommended for diarrhea cases with blood in the stool (which suggests  Prevention of avoidable antibiotic consumption is a central goal of efforts to counter the growing threat of antimicrobial resistance . ChildhoStreptococcus pneumoniae [Randomized and observational studies in high-income settings have reported that vaccines against influenza  and Streeumoniae –11 reduceumoniae , there heumoniae ,13. EvidThe Global Enteric Multicenter Study (GEMS) –17 was uThe clinical protocol of the original study was approved by ethics committees of the University of Maryland, Baltimore, Maryland, United States, and at each of the participating field sites; parents or primary caretakers provided written informed consent for participating children at enrollment. This secondary analysis of the de-identified GEMS dataset was considered to be non-human subjects research and was therefore exempt from full review by the Committee for Protection of Human Subjects of the University of California, Berkeley.GEMS was a multi-site, age-stratified, individually-matched case-control study characterizing the incidence, etiology, and clinical consequences of diarrhea among children at four sites in sub-Saharan Africa  and three sites in South Asia . Participants were children ages 6 weeks to 59 months who belonged to routinely-censused populations, with demographic surveillance systems in place to record all births, deaths, and migrations. Enrollment of cases occurred at sentinel hospitals and health centers where children sought care for diarrhea. Sentinel hospitals and health centers  were selected which provided care to the largest number of children with diarrhea in each study region, and which offered appropriate infrastructure for computerized data management, secure freezer storage, at least intermittent internet access, the ability to ship specimens abroad, and laboratory capacity to perform coprocultures, antigen-detection tests, and nucleic acid-based assays. The design, procedures, and primary outcomes of GEMS have been reported previously –16,18. BThe study included a 3-year primary phase (GEMS-1) enrolling MSD cases as well as a 1-year follow-on phase (GEMS-1A) enrolling both MSD and LSD cases; due to an anticipated low enrollment, LSD cases were not enrolled in the Kenya site. Study personnel aimed to enroll the first 9 children with MSD (in the 3-year primary study) and the first 9 children with MSD or LSD (in the 1-year follow-on study) at each site, over each fortnight, within each of three age strata: infants ages 6 weeks to 11 months, toddlers ages 12–23 months, and young children ages 24–59 months. For each case, investigators aimed to enroll 1–3 matched controls from the community. Controls of the same age (±2 months for cases ages ≤23 months and ±4 months for cases ages 24–59 months), sex, and village or neighborhood were selected at random from demographic surveillance site databases and enrolled within 14 days of the case at home visits by field workers. Controls experiencing diarrhea in the preceding 7 days were ineligible. Standardized demographic, epidemiologic, and clinical information was collected by study personnel at enrollment, in consultation with parents or primary caretakers of children.All treatments that clinicians administered or prescribed to diarrhea cases at the primary enrollment visit were recorded. On the basis of these data, we defined clinically-attended, antibiotic-treated diarrhea cases as MSD or LSD cases who were administered antibiotics during the enrollment visit, or who received an antibiotic prescription at the enrollment visit. From this definition, we excluded cases diagnosed with other conditions that would justify antibiotic treatment, regardless of diarrhea symptoms; we considered these diagnoses to include pneumonia/lower respiratory-tract infection, meningitis or other invasive bacterial infection, and typhoid.Salmonella spp., Shigella spp., Campylobacter spp., Aeromonas spp., Vibrio cholerae, diarrheogenic Escherichia coli, rotavirus, adenovirus (serotypes 40 and 41), norovirus (serotypes GI and GII), sapovirus, astrovirus, Giardia lamblia, Entamoeaba histolytica, and Cryptosporidium spp.Microbiological procedures in GEMS have been described previously . BrieflyE. coli isolates as enteropathogenic, enteroaggregative, and Shiga toxin-encoding enterotoxigenic . Rotavirus, adenovirus, G. lamblia, E. histolytica, and Cryptosporidium spp. were detected via commercial immunoassays, and norovirus, sapovirus, and astrovirus were detected via multiplex reverse transcriptase (PCR).For this study, we used data obtained from both conventional diagnostic procedures  and quantitative molecular diagnostic assays  . Under cStool specimens from up to 300 randomly-selected MSD cases and the first available matched controls for each age stratum and study site from the primary study were retested using a custom TaqMan Array Card  with probe-based quantitative PCR (qPCR) assays for 32 enteropathogens, as described previously ,20. The S1 Text). Analyses were stratified for MSD and LSD cases. We also estimated adjusted attributable fractions using pathogen detection data from conventional assays, as applied in the primary analyses of the GEMS datasets [We used the diagnostic data produced by these assays to estimate the proportion of clinically-attended antibiotic-treated diarrhea cases caused by each pathogen, controlling for the presence and/or abundance of all other pathogens; for each pathogen, we refer to this quantity as the adjusted attributable fraction. We estimated adjusted attributable fractions using models that included the Cq value for each pathogen as covariates, similar to previous analyses ,21. We ddatasets ,15,22. DS1 Text).An initial cross-sectional healthcare utilization and attitudes survey was conducted within random samples of children belonging to the demographic surveillance population of each site, followed by brief serial surveys to document in-migration, out-migration, births, and deaths, as described previously . ParentsShigella etiology, we also estimated adjusted attributable fractions of clinically-attended, antibiotic-treated diarrhea associated with dysenteric and non-dysenteric Shigella infections. In addition, we estimated the ratio of incidence of inappropriate  to appropriate (dysenteric) antibiotic-treated cases in each setting. To understand differences in symptoms and management of diarrheal infections, we further estimated associations of detection of each pathogen with administration or prescription of an antibiotic (among MSD and LSD cases) and with dysentery and hospitalization (among MSD cases only). We detail the statistical approaches for these analyses in the supporting information (S1 Text).Because antibiotic treatment of dysentery is recommended based on the possibility of Data were available from 12,109 cases with MSD seeking care at sentinel hospitals and health centers recruited in the primary and follow-on studies between December, 2007 and November, 2012 . The oneTable 1). Concordant with World Health Organization guidelines [S2 Table). Contrary to current guidelines [In total, 9,606 children with MSD and 1,844 children with LSD were administered or prescribed antibiotics . Trimethoprim/sulfamethoxazole was the most common treatment in African sites for both MSD and LSD, while quinolones were more commonly used for MSD in the South Asian sites. Nearly all antibiotic-treated LSD cases in Bangladesh received azithromycin, whereas this drug was less commonly used in other settings.Predominant antibiotic choices varied by setting . In agreement with this finding, Shigella detection was associated with higher odds of dysentery among MSD cases. Within the South Asian GEMS study sites, Shigella was the only pathogen associated with higher odds of antibiotic administration or prescription, whereas detections of rotavirus, ST-ETEC, or V. cholerae O1 among MSD cases each predicted lower odds of antibiotic administration or prescription. Detections of these pathogens among MSD cases were also associated with lower odds of dysentery and higher odds of hospitalization. However, within the African sites, rotavirus-positive MSD cases were more likely than rotavirus-negative cases to be administered or prescribed antibiotics.Across all sites, adjusted odds of antibiotic administration or prescribing were higher in V. cholerae O1, Aeromonas, Shigella, and rotavirus were each associated with elevated odds of antibiotic administration or prescribing . These findings persisted in analyses stratified by region. In contrast to our observations in MSD, detections of rotavirus were associated with higher odds of antibiotic administration or prescribing in LSD in South Asian sites as well as African sites.Among LSD cases, detections of Tables 2 and 3). Rotavirus was associated with the greatest share of LSD cases resulting in antibiotic treatment across all ages, and with the greatest share of MSD resulting in antibiotic treatment during the first year of life . Although a minor contributor to clinically-attended, antibiotic-treated diarrhea during the first year of life, Shigella accounted for 23.4% (17.9–29.7%) and 23.7% (15.3–34.2%) of cases at ages 12–23 months and 24–59 months, respectively, and was thus the second-leading and leading etiology of antibiotic-treated cases at these ages (Table 2). In contrast to rotavirus, Shigella was associated with >4-fold greater fractions of clinically-attended, antibiotic-treated MSD cases than LSD cases, and was the leading cause of clinically-attended, antibiotic-treated MSD at ages 12–23 months as well as 24–59 months . In total, we estimated that 14.9% (11.4–18.9%) of clinically-attended, antibiotic-treated diarrhea was attributable to Shigella (Table 3). Our findings that rotavirus and Shigella were the leading causes of diarrhea did not differ in analyses based only on conventional diagnostic data (S9 Table), although qPCR data yielded higher adjusted attributable fraction estimates for these and most other pathogens .Rotavirus was the leading clinically-attended, antibiotic-treated diarrhea etiology overall and during both the first and second years of life, accounting for 34.2%  and 28.8% (22.9–37.2%) of cases in these age groups, respectively (Table 2). Of these pathogens, only ST-ETEC was a prominent cause of clinically-attended, antibiotic-treated diarrhea across all ages; we estimated that ST-ETEC accounted for 12.4% (0.0–29.4%) and 6.3% (3.2–12.4%) of cases at ages 12–23 months and 24–59 months, respectively. In contrast, by ages 24–59 months, adenovirus 40/41 and sapovirus each accounted for <3% of all clinically-attended, antibiotic-treated diarrhea.Adenovirus serotypes 40/41, sapovirus, and ST-ETEC were also important etiologies of antibiotic-treated diarrhea during the first year of life, accounting for 23.1% (0.0–43.3%), 21.4% (1.2–63.1%), and 10.3% (5.9–17.5%) of cases in this age group, respectively (Shigella was associated with roughly 2-fold greater fractions of cases in Bangladesh and Pakistan as compared to other sites (Tables 2 and 3). Additionally, V. cholerae O1 accounted for 3.3% (1.7–5.2%) and 5.4% (1.2–15.0%) of clinically-attended, antibiotic-treated diarrhea in India and Pakistan, respectively, but <1% of cases in each African site (Table 3). In contrast, estimates of the etiologic fraction for Cryptosporidium tended to be larger in the African sites than the South Asian sites.We noted several regional differences in etiologies of clinically-attended, antibiotic-treated diarrhea. Overall and within most age groups, Table 4). Whereas 52.4% (19.5–70.4%) and 24.5% (13.2–41.5%) of incident clinically-attended, antibiotic-treated diarrhea cases in Bangladesh and Pakistan experienced dysentery, the fraction was below 10% in all other settings. On this basis, the ratio of inappropriate to appropriate treatment of diarrhea cases with antibiotics ranged from 0.9 (0.4–4.1) in Bangladesh to 29.8 (16.6–75.3) in Mali. Accounting for the absolute incidence rates of clinically-attended, antibiotic-treated diarrhea in each setting, we estimated that the number of inappropriately-treated episodes per 100 children annually ranged from 1.5 (0.9–2.3) in Pakistan to 8.4 (6.2–11.2) in India.We estimated that dysentery occurred in only 17.5% (11.2–22.5%) of clinically-attended, antibiotic-treated diarrhea cases across all settings  and 12.2% (7.3–20.1%) of all clinically-attended, antibiotic-treated diarrhea cases at ages 12–23 months and 24–59 months, respectively (Table 5). This finding was consistent in all settings except Bangladesh and Pakistan, where the majority of antibiotic-treated, Shigella-attributable cases were dysenteric.Non-dysenteric cases accounted for over half of Fig 3). Consistent with this pattern, incidence rates declined with older age within each setting. Overall and within each age group, incidence rates were highest in India and lowest in Pakistan, where the fewest cases received antibiotics (Table 1).Across sites, we estimated incidence rates of 12.2 (9.0–17.8), 10.2 (7.4–13.9), and 1.9 (1.3–3.0) clinically-attended, antibiotic-treated diarrhea cases per 100 child-years at risk at ages 6 weeks to 11 months, 12–23 months, and 24–59 months, respectively . A similar pattern appeared in age-specific incidence of clinically-attended, antibiotic-treated diarrhea attributable to sapovirus and ST-ETEC, albeit less consistently. In contrast, Shigella-attributable incidence was highest among children ages 12–23 months in all settings. Similarly, incidence attributable to Vibrio cholerae O1 peaked at ages 12–23 months in India and Pakistan, where burden of this pathogen was greatest.Incidence rates of clinically-attended, antibiotic-treated diarrhea attributable to rotavirus, adenovirus 40/41, Fig 4). However, the relative contributions of MSD and LSD varied by pathogen. Point estimates suggested rotavirus, adenovirus 40/41, sapovirus, ST-ETEC, Cryptosporidium, and norovirus GII were each associated with a greater share of less-severe than moderate-to-severe episodes of clinically-attended, antibiotic-treated diarrhea. In contrast, we estimated that Shigella caused higher incidence of moderate-to-severe than less-severe cases overall and within each age group.Within each age group, LSD cases accounted for the majority of clinically-attended, antibiotic-treated diarrhea incidence  per appropriately-treated dysentery case in The Gambia, Mali, Mozambique, and India. A lower but substantial proportion of antibiotic-treated episodes were non-dysenteric in Bangladesh and Pakistan. Whereas this finding reflected lower rates of inappropriate antibiotic treatment in Pakistan, in Bangladesh this finding was instead driven by high prevalence of dysentery, as the proportion of non-dysenteric MSD and LSD cases receiving antibiotics resembled observations in other settings.Shigella detection was strongly associated with antibiotic administration or prescription in all settings. Left untreated, shigellosis is associated with severe adverse outcomes including mortality and growth faltering, independent of dysentery [Shigella infections exceeds the proportion with dysentery, current guidelines for antibiotic treatment of only dysenteric cases may be overly restrictive [Shigella could offer synergistic benefits, for instance by reducing the incidence of shigellosis cases that drive antibiotic use as well as reducing clinical suspicion of shigellosis and resulting empirical antibiotic treatment of diarrhea attributable to other causes. Moreover, expansion of multidrug-resistant Shigella lineages, most notably in South Asia, has resulted in a diminishing number of practical treatment options [Shigella.ysentery –33. Becatrictive ,33, infl options . The emeWhile general limitations of GEMS have been addressed previously , severalOur findings suggest that even a moderately effective rotavirus vaccination program could substantially reduce antibiotic consumption. For instance, 40–70% reductions in rotavirus diarrhea incidence reported in African settings after vaccine implementation  would trAntibiotic use endpoints merit inclusion in trials of vaccines against enteric pathogens to demonstrate whether prevention of diarrhea meaningfully reduces overall antibiotic consumption . ClusterS1 TextThe supporting information document includes further elaborations of methods (sections 1–5), 11 supplemental tables, and one supplemental figure, and citations to references in the supplemental text, as listed in the Table of Contents (page 1).(PDF)Click here for additional data file.


In particular, pharmacologically mediated stimulation of human γ-globin gene expression, and increase of fetal hemoglobin (HbF) production, have been suggested as potential therapeutic strategies for these hemoglobinopathies. In this article, we screened a small chemical library, constituted of 150 compounds, using the cellular biosensor K562.GR, carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and β-globin gene promoters, respectively. Then the identified compounds were analyzed as HbF inducers on primary cell cultures, obtained from β-thalassemia patients, confirming their activity as HbF inducers, and suggesting these molecules as lead compounds for further chemical and biological investigations.The screening of chemical libraries based on cellular biosensors is a useful approach to identify new hits for novel therapeutic targets involved in rare genetic pathologies, such as  In this context, these tools can be important in identifying new hits for novel therapeutic targets involved in rare genetic pathologies, such as β-thalassemias are inherited hematological genetic disorders, caused by more than 300 mutations of the human β-globin gene, causing reduced or absent synthesis of adult β-globin, and a consequent excess of α-globins, which induces ineffective erythropoiesis and severe anemia [β-thalassemias can be grouped in different classes, depending on the β-globin gene mutations  and on the transfusion dependency . In the case of the most severe forms of β-thalassemia, regular blood transfusions and chelating therapies are the conventional therapeutic approaches [The e anemia . The anne anemia . The β-tproaches ,3,4,5. Wproaches , severalproaches , gene edproaches , and treproaches .SCD is a rare monogenic, hematological, and multi-organ disorder, associated with acute and chronic illness, and progressive organ damage . The disγ-globin, absent after birth. In this case, even a small increase in the percentage of fetal hemoglobin (HbF), replacing the deficient adult hemoglobin (HbA), could ameliorate the clinical picture, reducing anemia and transfusion burden in β-thalassemia, and inhibiting polymerization of sickle hemoglobin (HbS) in SCD [β-thalassemia and SCD [The clinical severity for these two hemoglobinopathies could be reduced by reactivating the expression of ) in SCD ,13,14. T and SCD ,16,17. A and SCD ,20,21,22β-thalassemia, in spite of its verified benefits in certain cases, demonstrated by the reach of a transfusion-independent phenotype in patients with β-thalassemia intermedia [β-thalassemia are lacking. On the other hand, adverse effects and potential toxicity in the case of long-term treatment are expected, and have been described for HU [Currently, hydroxyurea (HU) is the only FDA-approved therapeutic treatment for the induction of HbF in SCD patients ,23. Withtermedia ,25, specd for HU ,27. Furtd for HU ,28; therγ-globin and β-globin gene promoters, respectively [γ-globin and β-globin genes in erythroid cells in an efficient and reproducible way [β-thalassemia patients. This assay has been widely used to validate the HbF-inducing potential of molecules identified using large or middle-scale screening approaches [In this work, we have screened a small chemical library constituted of 150 compounds, randomly selected, and exhibiting heterogeneous chemotypes, using our cellular biosensor K562.GR carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human ectively . This syible way ,30. Thisproaches ,32,33.β-globin gene promoters, respectively, and it has been validated as a high throughput screening (HTS) system in previous publications [β-globin promoter, no fluorescence is detected. Despite the fact that in this study we focused on HbF inducers, it should be underlined that the system would also allow the identification of transcription activators of the β-globin promoter, in the case RFP is increased instead of EGFP [In order to discover novel fetal hemoglobin (HbF) inducers from the collected chemical library, we performed a first screening based on the K562.GR reporter system. The clone K562.GR contains a reporter construct with EGFP (enhanced green fluorescent protein) and RFP (red fluorescent protein) genes under the control of the human γ-globin or ications ,30. With of EGFP ,30.For each compound of the chemical library, four concentrations  were administered to K562.GR cells for 5 days, and then FACS analysis was performed.β-thalassemia and SCA treatment [β-globin gene promoter activation was analyzed (Hydroxyurea (HU) was selected as a positive control A, considreatment . As expeanalyzed B. After analyzed E,F; the analyzed C,D. InteIn order to identify the compounds able to induce the γ-globin gene, we selected nine potentially active molecules, with values of median fold over control, for EGFP signal, greater than 1.35 in at least one of the four tested concentrations , showing no induction activity at the other concentrations, some of them, such as 56, 58, and 59, also showed a relevant activity at low concentrations (1 µM). The 1.35 value was identified as a minimum cut-off in previous articles, where the same K562.GR cellular biosensor was validated using well-known fetal hemoglobin inducers, such as 4,4′,6-Trimethylangelicin (TMA) and HU ,30,34.In addition, the experiments shown in Using methanol/DMSO, three independent experiments on K562.GR clones were performed , in ordeThe specificity of the induction activity of the nine compounds was determined by performing the same treatments on wild type K562 cells without the reporter construct. As reported in After this first screening, the effects of the selected compounds were investigated on cell growth, and on hemoglobin production by the K562 cell line. This cell line is a very useful experimental model system, since it synthesizes hemoglobin at very low levels when untreated, but significantly increases the expression of embryo-fetal globins after treatment with inducers of erythroid differentiation .50 values (relative to the effects on cell proliferation) and the percentages of benzidine-positive cells calculated after 5 days of treatment. The analyzed compounds can be divided into three classes based on the relative IC50. The first one comprises 56, 58, and 59, showing the lowest values: 0.85 μM, 0.69 μM, and 0.10 μM, respectively. The second class includes compounds 6, 17, and 22, exhibiting intermediate values, between 1.2 and 5. Finally, for the other three molecules it was not possible to determine the IC50 value, since in our experimental conditions we did not detect significant antiproliferative effects at the maximum tested concentration (10 μM).50 (because it was greater than 10 μM) generally showed differentiation values dependent on the administered dose, even reaching quite high percentages of positive cells . In contrast, the three compounds with lower IC50 showed lower differentiation values, between 1 and 6%.As far as the hemoglobin production analyzed using a benzidine test , the samβ-thalassemia patients, evaluating the HbF production by HPLC. In total seven β-thalassemia patients were recruited, and their ErPCs isolated and treated as described elsewhere [The results obtained in K562 cell lines, prompted us to determine whether the nine molecules were able to induce the expression of γ-globin genes in primary cultures of erythroid precursor cells (ErPCs) obtained from lsewhere .2 was detected.A representative example of HPLC analysis after 5-day treatment of these isolated ErPCs is reported in For each compound, we compared the HbF fold induction over untreated control values to those obtained after HU treatment (used as positive control) .β-thalassemia patients, while the other compounds reported fold induction values greater than that of HU in one or two patient cultures. This result confirmed the erythroid differentiation ability of compounds 6 and 17 demonstrated in K562 cells  without cytotoxicity effects. In addition, compound 63 deserves consideration in future experiments, focusing on its mechanism for activity as a HbF inducer  are among the most frequent monogenic diseases, originally characteristic of the tropics and subtropics, but now commonly distributed worldwide, mainly due to migration . Hydroxytermedia ,25, specopathies ,23,38. Iucer see .These data represent the first step of a process of drug discovery, that involves the identification of novel hits , and the development of a program of medicinal chemistry, in order to produce analogues, to increase the affinity, selectivity, efficacy/potency, metabolic stability, and oral bioavailability of the identified lead compounds. In addition, these data represent the starting point for the future structure–activity relationship (SAR), in order to analyze the chemical structure/biological activity correlation, and identify the specific chemical group responsible for HbF increase in our cellular models. The SAR investigation will permit synthetizing new derivatives of the bioactive compounds 6, 17, and 63, modifying their chemical structure to improve their biological effect and potency with low toxicity.β-thalassemia patients with different mutations of the β-globin gene, different HbF-related polymorphisms, and different starting levels of endogenously produced HbF are necessary to verify the therapeutic potential of the identified compounds. In addition, since SCD is expected to greatly benefit from production of anti-sickling HbF, ErPCs from SCD patients should be considered for experimental validation. Furthermore, combined treatments using compounds identified in the present study administered together with HbF-inducers employed in clinical trials  might help to verify whether synergisms using different molecules can occur in inducing HbF. Finally, treatment with HbF inducers (including those described in this study) might also be combined with other strategies aimed at augmentation of HbF, for instance (a) inhibiting the activity of γ-globin gene transcriptional repressors  [Further experimental efforts on ErPCs isolated from B7A/LRF) ,41,42,43B7A/LRF) ,45,46.The chemical library collects 150 chemical compounds developed at the University of Ferrara.Each compound was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM, with a minimum degree of purity of 90%, evaluated by HPLC or mass analysis.In the first screening of K562.GR clone, the compounds were tested in DMSO, while for the following experimental cultures they were diluted in methanol + 5% DMSO.1HNMR spectra and found (M+H) + ESI mass) of the reference compounds of the manuscript are reported in Analytical data . The number of K562 cells containing hemoglobin was assayed by benzidine staining as elsewhere described .For the determination of fluorescence intensity of treated K562 and K562.GR clones, BD FACSCanto™ II instrument and BD FACSDiva™ Software  were used. After a PBS washing step, 30,000 cells were analyzed by the fl1 channel to detect green fluorescence. The results were expressed as median fold over control, that is the ratio between the median of fluorescence intensity values obtained by cells in the presence, and in the absence, of treatment.α-MEM , 10% FBS, 1 µg/mL cyclosporine A , 10% conditioned medium from the 5637 bladder carcinoma cell line and 10 ng/mL stem cell factor . After seven days incubation at 37 °C and in an atmosphere of 5% CO2, the non-adherent cells were harvested, washed, and cultured in fresh medium composed of α-MEM, 30% FBS, 1% deionized bovine serum albumin , 10−5 M β-mercaptoethanol , 2 mM L-glutamine , 10−6 M dexamethasone , 1 U/mL human recombinant erythropoietin , and 10 ng/mL SCF. After seven days of incubation, the cells were seeded at 1 × 106/well in a 12-well plate, and treated with compounds for five days before the HPLC analysis to determine the proportion of HbF . In particular, a Beckman Coulter instrument System Gold 126 Solvent Module-166 Detector was used, in combination with a Syncropak CCM 103/25 column eluting the samples in a solvent gradient using aqueous sodium acetate-BisTris-KCN buffers, as previously reported [Blood samples were collected from the Thalassemic Day Hospital (DHT) of the University Hospital “Sant’Anna” . Written informed consent was obtained from each patient, and peripheral blood samples were collected just before the blood transfusion. The primary cell cultures were obtained using a two-phase liquid culture procedure, as previously described ,48. Brie


Zea mays L., ZmADFs) ADF genes were identified using Hidden Markov Model. Phylogenetic analysis indicated that the 36 identified ADF genes in Physcomitrella patens, Arabidopsis thaliana, Oryza sativa japonica, and Zea mays were clustered into five groups. Four pairs of segmental genes were found in the maize ADF gene family. The tissue-specific expression of ZmADFs and OsADFs was analyzed using microarray data obtained from the Maize and Rice eFP Browsers. Five ZmADFs (ZmADF1/2/7/12/13) from group V exhibited specifically high expression in tassel, pollen, and anther. The expression patterns of 13 ZmADFs in seedlings under five abiotic stresses were analyzed using qRT-PCR, and we found that the ADFs mainly responded to heat, salt, drought, and ABA. Conclusions: In our study, we identified ADF genes in maize and analyzed the gene structure and phylogenetic relationships. The results of expression analysis demonstrated that the expression level of ADF genes was diverse in various tissues and different stimuli, including abiotic and phytohormone stresses, indicating their different roles in plant growth, development, and response to external stimulus. This report extends our knowledge to understand the function of ADF genes in maize.Actin-depolymerizing factor (ADF) is a small class of actin-binding proteins that regulates the dynamics of actin in cells. Moreover, it is well known that the plant ADF family plays key roles in growth, development and defense-related functions. Results: Thirteen maize ( Arabidopsis [Arabidopsis, rice, and tomato, has been reported, but not with the maize ADF gene family. Generally, according to phylogenetic analysis of their amino acid sequences, the ADF protein in higher plant can be divided into four subclasses (I–IV) [AtADF1, -2, -3, and -4 comprise subclass I and are expressed at a relatively high level throughout the plant except pollen, whereas AtADF7 and AtADF10 which belong to subclass IIa exhibit pollen-specific expression patterns. AtADF8 and AtADF11 (IIb) are expressed specifically in root epidermal cells. Subclass III (AtADF5 and AtADF9) and subclass IV (AtADF6) exhibit universal expression in different tissues, but AtADF9 is more strongly expressed in root subapical region, trichomes, shoot apical meristem (SAM), and callus [AtADF5 is limited to the root tip meristem [In eukaryotes, actin-depolymerizing factor (ADF) is conserved and performs an essential function in actin dynamics which is important for cell motility, development, differentiation, signal transduction cytokinesis, and maintenance of eukaryotic cell surface structure ,2,3. ADFbidopsis , 27 in bbidopsis , 14 in pbidopsis , and 11 meristem .AtADF1 significantly elongates or shortens organ length, such as that of roots, hypocotyls, cotyledons, and stems. Moreover, downregulation of AtADF1 expression significantly delays flowering time, without affecting that in overexpressing plants [GhADF1 is a constitutive expression gene in cotton, while abundantly in fiber cells, and the downregulation of GhADF1 alters the cell wall morphology and elongates the length of fiber cells [AtADF2 and AtADF4 knockdown lines [AtADF4 causes hypocotyl elongation under dark growth condition [AtADF4 shows significantly enhanced resistance against powdery mildew fungus [AtADF2 knockdown leads to cellular defects but successfully prevents nematode infection [AtADF3 is required for positively controlling GPA (green peach aphid) infestation [Currently, there are only a few reports on the analysis of plant ADF genes. The ADF gene family is well known to make a special contribution to the processes of plant growth and stress responses, including pollen tube development ,14,15,16g plants ,26. GhADer cells ,18. Wavywn lines . In addiondition , whereasondition . Recentlw fungus , while Anfection . The phlestation . TherefoAtADF5, but not AtADF9, makes an important impact in the formation, organization, and maintenance of actin bundles [AtADF7 which significantly suppresses the pollen tube growth [AtADF10 is a pollen-specific ADF, however, AtADF7 and AtADF10 show different spatial profiles, indicating their unique functions in the pollen tube for regulating actin dynamics [AtADF9 in the root tip meristem, trichomes, and SAM is increased by 2,4-D, IAA, kinetin, GA3, and ABA, and the knockout mutant of AtADF9 shows early flowering and small plant size under long-day conditions [During pollen germination and pollen tube growth,  bundles . The lose growth  and AtADdynamics . The expnditions .TaADF3 is induced by ABA, drought and cold, and negatively regulated wheat resistance to Puccinia striiformis f.sp.tritici (Pst) [OsADF3 is reduced by salt stress and induced by exogenous ABA and cold, and OsADF3-transgenic Arabidopsis shows increased drought stress tolerance [TaADF4 is also upregulated by JA and ABA, but differs from TaADF3, which positively modulates wheat resistance to Pst [TaADF4, TaADF7 also positively contributes to wheat resistance to Pst [ci (Pst) . Whereasolerance . TaADF4 e to Pst . Similare to Pst . The TaAe to Pst .LiADF1 [ZmADF1/2 are expressed only in pollen which function in pollen actin reorganization [ZmADF3 is expressed in every examined maize tissue except pollen which is a vegetative gene and, furthermore, ZmADF3 binds quantitatively to both monomeric and polymeric [In maize, three ZmADFs  have been cloned and their functions have been discovered. Similar to LiADF1 , ZmADF1/nization ,33, wherolymeric . Howeverhttp.//smart.embl-heidelberg.de/) software was adopted to determine whether the ADF-H domain existed in the candidate ADF genes. Finally, 13 ADF-H domain-containing genes (ZmADF1-ZmADF13) were identified in this study, the number of ADF genes identified in maize (13) was similar to Arabidopsis (11) and rice (11). We also analyzed the gene location, open reading frame (ORF) length, amino acids length, molecular weight, iso-electric point (PI), and grand average of hydropathy (GRAVY)  as the query sequence. SMART ( (GRAVY) . The lenZmADF3, ZmADF5, ZmADF9, and ZmADF11, three genes  on chromosome 5, two genes (ZmADF2 and ZmADF12) on chromosome 2, and chromosomes 4, 6, 7 and 9 have one ADF gene, separately.To show the location of 13 ZmADFs in the maize genome, we manually mapped the 13 ZmADFs on seven maize chromosomes in view of the MapChart results . The resZmADF1/ZmADF12, ZmADF5/ZmADF8, ZmADF6/ZmADF9, and ZmADF7/ZmADF13 gene pairs were found to be segmentally duplicated  for each ZmADF gene . ZmADF4 from cluster Ⅱ at position 26 was T-Thr. ZmADF3 and ZmADF10 from cluster Ⅲ at position 26 were L- Leu. Cluster Ⅳ which included ZmADF9 (K-Lys), ZmADF6 (E-Glu), ZmADF11 (D-Asp), ZmADF3 (K-Lys), and ZmADF8 (Q-Gln) showed various amino acid sequences at position 26. Moreover, cluster Ⅳ also showed motifs divergently, indicating that amino acid 26 and motifs were related to the putative functions of ZmADFs.The gene structure of ZmADF genes was analyzed by aligning the CDS region and the genomic sequence from the maize reference genome database , and five OsADFs also belonged to this group. Group IV also contained 11 members, including eight AtADFs, two ZmADFs (ZmADF3/10), and one OsADFs, followed by group Ⅲ, which included three numbers of ZmADFs (ZmADF6/9/11). Group Ⅱ only contained five ADF members, including ZmADF5 and ZmADF8 in this clade.In addition to the PhpADF (group Ⅰ), the phylogenetic tree classified the ADF proteins into four main groups , consistent with published data of rice, d tomato ,11. In tZmADF4, ZmADF5, ZmADF6, ZmADF10, and ZmADF3 showed relatively higher expression in all tissues, whereas ZmADF9, ZmADF8 and ZmADF11 showed very low levels of expression in most tissues and developmental stages. Interestingly, we found that ZmADF1, ZmADF2, ZmADF7, ZmADF12, and ZmADF13 showed relatively high expression levels in tassel, anther, and pollen, which implied their crucial role in maize tassel development.In an attempt to explore the possible functions of ZmADF genes in maize growth and development, the tissue expression profiles of ZmADF genes were determined in the embryo sac, ovule, embryo, silk, endosperm, SAM, leaf, root, tassel, anther, and pollen. These expression data showed that all ZmADF genes have different expression levels  in 11 tiTo validate ADF genes’ function in different species, we also retrieved the tissue expression profiles of OsADFs from Rice eFP Browser and analyzed their expression. As shown in OsADF1/ZmADF7/ZmADF10 and OsADF6/ZmADF12 showed high expression levels in anther; OsADF2/ZmADF6 and OsADF11/ZmADF9 were highly expressed in root; OsADF5/ZmADF5 showed the same expression pattern, with medium expression levels in leaf; and OsADF10/ZmADF11 showed relatively higher expression in ovule.In this study, we also identified orthologous genes between maize and rice C and aimZmADF1, ZmADF7, ZmADF12, and ZmADF13 from group V and ZmADF5 from group Ⅱ clade) were responsive to all abiotic stress treatments in this study. Moreover, ZmADF1 was significantly upregulated in all abiotic stresses, indicating that ZmADF1 plays an important role in various abiotic stresses. ZmADF2 and ZmADF3 were significantly induced by heat, drought, and ABA treatments. ZmADF2 could also be induced by NaCl. ZmADF4 was upregulated under drought and heat treatments. ZmADF6 was found only to be slightly upregulated under heat treatment.In order to gain insights into the putative functions of ZmADF genes under abiotic stresses, we investigated their expression patterns in five abiotic stresses at different points . The qRTZmADF1/2/3/4/5/7/8/12/13 remained increased after 0.5 h of the heat treatment as compared with the control. However, ZmADF6/9/11 were slightly induced until 4 h, and then decreased. ZmADF10 was insensitive to the heat treatment. For cold-treated samples, first, ZmADF11 was induced by cold treatment at 0.5 h, but then decreased. The expression levels of ZmADF1/2/5/7/12 reached a peak at 1 h, and then decreased. Other ZmADFs were insensitive to the cold treatment. For NaCl-treated samples, the expression levels of ZmADF1/2/5/7/13 were increased at 0.5 h, and then decreased, except for ZmADF13 which continuously increased after 0.5 h and until 12 h of the NaCl treatment as compared with the control. Under drought and ABA treatments, the majority of the ZmADFs had similar expression characteristics. The expression levels of ZmADF1/2/3/5/7/8/12/13 were significantly upregulated. Only ZmADF4 exhibited the opposite expression pattern under drought and ABA treatments, which was downregulated in ABA and upregulated in drought treatment.For heat-treated samples, the expression levels of Physcomitrella patens, Arabidopsis thaliana, Oryza sativa japonica, and Zea mays were clustered into five groups. In addition to PhpADF (group Ⅰ), the ADF proteins from Arabidopsis thaliana, Oryza sativa japonica, and Zea mays were classified into four main groups , consistent with published data of rice, Arabidopsis, and tomato [ZmADF3 and ZmADF10 from group Ⅳ at position 26 were L-Leu and they expressed higher in all tissues, suggesting that amino acid 26 was related to the putative functions of ZmADFs. In addition, OsADFs and ZmADFs, in the same group, possess a similar function. For instance, OsADF1/6/9 and ZmADF1/2/7/12/13 from group V predominately expressed in anther and pollen. OsADF3 was highly induced under drought stress in rice shoot  was expressed in all vegetative tissues examined, whereas it was suppressed in pollen and germinated pollen [ZmADF1 and ZmADF2. Mutants have only been reported on ZmADF3, demonstrating that ZmADF3 binding of F-actin could be spatially distinguished from that of G-actin [ZmADF1 and ZmADF2 in maize two-week-old seedlings is induced by several stresses, especially under drought treated stress, indicating that the pollen-specific expressed ZmADF1 and ZmADF2 not only play an important role in pollen actin reorganization but also function to resist external environmental stresses. It is not known whether their function is more important in reproductive development or in stress response. For example, RNAi and downregulation mutant lines of a meiosis-enriched gene ZmAGO18b, which is specifically expressed in tapetum cells, pollen mother cells (PMCs), and microspores of meiotic tassel [ZmAGO18b in those plant lines resulted in increased height as compared with the control lines [ZmAGO18b could be significantly induced in seedlings by drought treatment [Currently, maize has become one of the most important crops in the world. Unfortunately, during the period of growth and development, maize suffers from numerous stresses. Nowadays, thanks to the publication of the maize genome sequence and the development of bioinformatics, the identification and analysis of gene families has become less difficult. Summarizing the important gene families related to plant growth, development, and stress resistance helps to study the specific function of genes, and therefore provide a theoretical basis for genetic improvement. It can also provide a valuable reference for the study of candidate genes obtained by map-based cloning. The ADF gene family has been reported to play a crucial role in growth, development, and response to abiotic stresses. However, the genome-wide identification of ADF gene family in maize has not been reported. In our study, 13 ZmADF genes were identified from maize B73) genome, and several analyses of bioinformatics and expression profiles were conducted. Consistent with the reports of the ADF family classification in other plants 3 genome,, ADF genABP2, in ) suggest G-actin . In the c tassel , did notol lines . Furtherreatment . Therefohttps.//www.maizegdb.org/, version 2). In total, we identified 13 ADF genes in maize using the InterPro domain accession number IPR002108 (the ADF-H domain) and using BLAST searches of nucleotides in maize reference genome, all redundant protein sequences were discarded in this study. In order to confirm the putative ADF genes in the maize genome, Pfam and SMART  were used to confirm the presence of ADF domains. Protein isoelectric point (PI) and molecular weight (Mw) of each gene product were calculated using ExPasy and parameter was set to average [The whole protein sequences, coding sequences (CDS), mRNA sequences and genome sequences of maize (inbred line B73) were downloaded from MaizeGDB ( average .Arabidopsis and rice from the published article were download [https.//www.arabidopsis.org/) and RGAP (http.//rice.plantbiology.msu.edu/) databases with manual corrections. The non-vascular bryophyte Physcomitrella patens was used to root the tree. Multiple sequence alignment of maize ADF protein sequences and the orthologous genes from Arabidopsis and rice was performed with MUSCLE software (version 3.6) with default parameters [In order to study the phylogenetic relationship, the ADF protein sequences of download , and thirameters . The alirameters . Online rameters . https.//github.com/CJ-Chen/TBtools).The chromosomal locations of the 13 maize ADF genes were determined according to maize reference genome information . The Maphttp.//meme-suite.org/tools/meme, version 5.1.1) was adopted to identify the possible conserved motifs in the complete amino acid sequences of maize ADF proteins setting a maximum number of motifs to 10. Exon and intron organizations for all ZmADF genes were obtained by using the online Gene Structure Display Server   via alighttp.//bar.utoronto.ca/efp_maize/cgi-bin/efpWeb.cgi), which included embryo sac, ovule, embryo, silk, endosperm, SAM, leaf, root, tassel, anther, and pollen. These public data sources are all based on the experimental data from maize inbred line B73. Rice expression data were retrieved from the Rice eFP Blowser (http://bar.utoronto.ca/transcriptomics/efp_rice/cgi-bin/efpWeb.cgi), which included seedling root, anther, inflorescence, mature leaf, young leaf, stigma, seed, shoot, endosperm, SAM, ovary, and embryo. All expression values were normalized by log10 transformed and displayed using a heat map to analyze corresponding genes’ expression pattern in different tissues. The heat map was generated using Tbtools software (https.//github.com/CJ-Chen/TBtools). To profile orthologous gene pairs’ expression level between maize and rice, we retrieved the orthologous gene pairs from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/index.shtml).To determine the expression patterns of the ADF genes in maize tissues, expression profiles based on microarray data were collected from the Maize eFP Browser (Zea mays L. cvB73) were surface sterilized and repeatedly rinsed with tap water, the seeded in Hoagland nutrient solution after immersion and imbibition for 12 h, cultured in potting sand in the greenhouse conditions at 25 °C with a 16 h light and 8 h dark cycle . Two-week-old seedlings growing identically were processed with heat (40 °C), cold (4 °C), salinity (0.2 M NaCl solution), drought, and abscisic acid (0.1 mM ABA). The seedlings were subjected to abiotic stress treatments basically as described by Zhai et al. (2019) [ZmActin gene was used as the internal control. The primer sequences of ZmADFs and ZmActin are in −∆∆Ct method in Excel was used to calculate the relative gene expression [The selected seeds of maize (. (2019) . We selepression .ZmADF1, ZmADF2, ZmADF7, and ZmADF12) had high expression levels in tassel, anther, and pollen, implying ZmADFs from group Ⅱ could play important roles in the reproductive development stage. However, the expression of ZmADF1, ZmADF7, ZmADF12, and ZmADF13 from group V were also remarkably induced by multiple stresses in this study, suggesting that these genes have much more important tolerance functions in maize, especially in the reproductive stage. The results of our study build the foundation for functional research into the ZmADF genes and also provide a valuable reference for stress-resistant breeding of maize.The plant ADF family plays key roles in growth, development, and defense-related functions. However, there has been a lack of genome-wide information of this family in maize. In this study, a total of 13 ADF genes were first identified in maize. According to the alignment of the protein sequences, the ZmADF proteins were divided into four groups. Subsequently, we used a variety of bioinformatics methods to analyze chromosomal location, gene duplication, and gene structure. Moreover, the expression patterns of ZmADFs in different tissues and abiotic stresses were analyzed by microarray data and qRT-PCR, respectively. The results showed that ZmADF genes from group V (


Pancreatic cancer (PC) is one of the most deadly cancers worldwide. To uncover the unknown novel biomarker used to indicate early diagnosis and prognosis in the molecular therapeutic field of PC is extremely of importance. Accumulative evidences indicated that aberrant expression or activation of immunoinhibitors is a common phenomenon in malignances, and significant associations have been noted between immunoinhibitors and tumorigenesis or progression in a wide range of cancers. However, the expression patterns and exact roles of immunoinhibitors contributing to tumorigenesis and progression of pancreatic cancer (PC) have not yet been elucidated clearly. In this study, we investigated the distinct expression and prognostic value of immunoinhibitors in patients with PC by analyzing a series of databases, including TISIDB, GEPIA, cBioPortal, and Kaplan-Meier plotter database. The mRNA expression levels of IDO1, CSF1R, VTCN1, KDR, LGALS9, TGFBR1, TGFB1, IL10RB, and PVRL2 were found to be significantly upregulated in patients with PC. Aberrant expression of TGFBR1, VTCN1, and LGALS9 was found to be associated with the worse outcomes of patients with PC. Bioinformatics analysis demonstrated that LGALS9 was involved in regulating the type I interferon signaling pathway, interferon-gamma-mediated signaling pathway, RIG-I-like receptor signaling pathway, NF-kappa B signaling pathway, cytosolic DNA-sensing pathway, and TNF signaling pathway. And TGFB1 was related to mesoderm formation, cell matrix adhesion, TGF-beta signaling pathway, and Hippo signaling pathway. These results suggested that LGALS9 and TGFBR1 might serve as potential prognostic biomarkers and targets for PC. The mortality of pancreatic cancer (PC), the fourth widely occurred cancer with poor prognosis, has an overall five-year survival rate lower than 10% . To imprβ signaling exhibited importance in biological signal regulation including cell growth and death, differentiation, angiogenesis, and inflammation [β signaling played a key role in immune response [Previously, the application of the immune system to recognize and eradicate tumors has made significant advance in the clinical use of cancer immunotherapy –9. Notabammation . Severalresponse . UnderstThe occurrence and progression of newly produced strategies, comprising microarray and RNA-sequencing, exerted a positive effect in molecular research and also gave impetus to exploring accurate and safe treatment for PC –27. Herehttp://www.kmplot.com/) is an online database containing microarray gene expression data, and survival information derived from Gene Expression Omnibus, TCGA, and the Cancer Biomedical informatics Grid. Kaplan-Meier (K-M) Plotter database was used to analyze prognostic parameter of expected candidates [P < 0.05.Kaplan-Meier plotter (ndidates . K-M surhttp://string-db.org/) [A functional protein interaction network was constructed as indicated in website (db.org/) . Among thttp://cis.hku.hk/TISIDB) database to detect the relationship between centromere protein expression and clinical stages, lymphocytes, immunomodulators, and chemokines in PC. Gene Expression Profiling Interactive Analysis (GEPIA) [TISIDB is an integrated repository portal for tumor-immune system interactions. The present study used TISIDB ( (GEPIA)  was a po (GEPIA) . Besides (GEPIA) .P < 0.01 was set as the cutoff criterion.Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using DAVID online tool. t-test was analyzed for statistical significance. Statistical analysis was performed by SPSS 21.0 .Student's The present study analyzed the expression pattern of 23 immunoinhibitors in PC using TCGA database, including CD160, CD244, KIR2DL1, KIR2DL3, BTLA, CSF1R, HAVCR2, TIGIT, LAG3, PDCD1, VTCN1, PDCD1LG2, LGALS9, CD96, TGFBR1, TGFB1, CTLA4, ADORA2A, PVRL2, IL10, IDO1, IL10RB, and KDR. As shown in The GEPIA database was used to compare the difference of expression of 9 overexpressed immunoinhibitors in transcription level between cancers and normal tissues . The datFurthermore, the TISIDB database analysis showed TGFB1 was positively correlated to the advanced grades of PC samples . HoweverInterestingly, our data also revealed the correlation between Immunoinhibitors level and stages of PC samples. The results displayed that expression of VTCN1 was raised in grade 2 and grade 3 samples compared to grade 1 PC samples, but expression of VTCN1 was decreased in grade 4 sample after being normalized to that in grade 1/2 PC samples . MeanwhiWe deeply explored the profile of immunoinhibitors implicated in prognostic PC patients. Our data revealed that the increasing level of TGFBR1 , VTCN1 , LGALS9 We evaluated the association of candidate gene expression with immunoinhibitors by Pearson's correlation analysis. The immunoinhibitor-target pair with absolute Pearson's correlation coefficient value > 0.5 was considered significant. The networks were constructed using Cytoscape software. As presented in We finally validated the role of LGALS9 and TGFB1 after the analysis of GO and KEGG in the DAVID system using their genes. After bioinformatics analyzing, LGALS9 was involved in regulating type I interferon signaling pathway, defense response to virus, interferon-gamma-mediated signaling pathway, response to virus, innate immune response, and negative regulation of viral genome replication . KEGG paAlso, we found that TGFB1 was related to mesoderm formation, cell matrix adhesion, substrate adhesion-dependent cell spreading, in utero embryonic development, extracellular matrix organization, outflow tract septum morphogenesis, mitral valve morphogenesis, and Hippo signaling . And KEGPC with a poor prognosis was regarded as one of the most deadly carcinomas . The groIn this study, we analyzed the expression pattern of 23 immunoinhibitors in PC using TCGA database and found that IDO1, CSF1R, VTCN1, KDR, LGALS9, TGFBR1, TGFB1, IL10RB, and PVRL2 were highly expressed in PC tissues. Moreover, the analysis revealed that IDO1, CSF1R, VTCN1, KDR, LGALS9, TGFBR1, TGFB1, IL10RB, and PVRL2 mRNA level was significantly upregulated in patients with PC compared to normal tissues. Kaplan-Meier plotter results demonstrated that the increasing level of TGFBR1, VTCN1, and LGALS9 mRNA was closely pertained to poor OS.β was a primary executor of the stability and tolerance of the internal environment of immune system, including controlling many component functions [β signal could result in inflammatory diseases and tumorigenesis. In addition, TGF-β is also a preliminary immunosuppressor in the tumor microenvironment [β was engaged in tumor immune evasion and adverse reactions to tumor immunotherapy [TGF-unctions . Thus, dironment . Currentotherapy . Neverth+ T cell response as previously described. Studies have indicated VTCN1 expression was positively linked to tumor progression and acted as a candidate for the treatment of cancer [+ regulatory T cells (Tregs) [VTCN1 exists on the surface of antigen-presenting cells (APC) and interacts with ligands that bind to T-cell surface receptors . The actf cancer . The levf cancer . Moreove (Tregs) . Because (Tregs) . Our stuHAVCR2 and expressed on a variety of cells [Transmembrane receptor TIM-3 was encoded by of cells . The expof cells  and alsoIDO1 was responsible for converting tryptophan (Trp) into downstream catabolic product, called caninuria. Emerging studies have shown that IDO1 was expressed in a large number of human cancers. In the transcription level, IDO1 displayed powerful relevance with T cell infiltration .Even though the expression of CSFR1, KDR, IL10RB, and PVRL2 was upregulated in PC samples, which was not associated with the prognosis of PC. CSFR1 was mostly found in aggressive cell models and participated in the invasion and migration of tumor cells, and its expression is related to the poor prognosis of cancer patients. Positive feedback existed between the expressions of CSF1 and EGF in tumors . By blocOf note, this study revealed that LGALS9 and TGFBR1 were upregulated in PC compared to normal tissues. Moreover, we showed LGALS9 and TGFBR1 were significantly associated with the prognosis in PC. Despite the fact that LGALS9 and TGFBR1 were not significantly correlated to the grades, we indeed observed LGALS9 had an upregulated trend and TGFBR1 had a downregulated trend. We thought the limited sample size may contribute to this result. Also, the coexpression plus bioinformatics analysis revealed that immunoinhibitors were involved in regulating multiple inflammatory and immune response-related pathways as previously described. Very interestingly, we found LGALS9 was involved in regulating type I interferon signaling pathway, interferon-gamma-mediated signaling pathway, RIG-I-like receptor signaling pathway, NF-kappa B signaling pathway, cytosolic DNA-sensing pathway, and TNF signaling pathway. We also found that TGFB1 was related to mesoderm formation, cell matrix adhesion, TGF-beta signaling pathway, and Hippo signaling pathway. These pathways had been demonstrated as key regulators of tumorigenesis and immune therapy.Several limitations should also be noted in this study. First, we showed LGALS9 and TGFB1 had a crucial role in PC with a series of bioinformatics analysis. The further experimental validations of their functions in PC could strengthen our conclusion. Second, more clinical samples should be collected to detect the expression of these immunoinhibitors in PC, which could provide more evidences to confirm their prognostic value.Conclusively, our data suggested that immunoinhibitor mRNA level was dramatically upregulated, but negatively correlated with OS for PC. All the data suggested these genes could be used as an emerging prognostic indicator and targets in PC patients. Our findings would give a hint to have a better understanding of the mechanism implicated in PC and stretched out more precise immunotherapeutic treatments for PC prognosis. Nevertheless, more researches and efforts should be contributed to our findings, followed by providing a much more promising clinical strategy for an early diagnosis and prognostic marker in PC therapy.