Background: FUN14 domain containing 1 (FUNDC1) plays a pivotal role in mitochondrial autophagy (mitophagy), which is closely associated with human immunity. However, the role of FUNDC1 in cancers remains unclear. This study aimed to visualize the prognostic landscape of FUNDC1 in pan-cancer and investigate the relationship between FUNDC1 expression and immune infiltration.Methods: In this study, we explored the expression pattern and prognostic value of FUNDC1 in pan-cancer across multiple databases, including ONCOMINE, PrognoScan, GEPIA, and Kaplan-Meier Plotter. Then, using the GEPIA and TIMER databases, we investigated the correlations between FUNDC1 expression and immune infiltration in cancers, especially liver hepatocellular carcinoma (LIHC), and lung squamous cell carcinoma (LUSC).Results: In general, compared with that in normal tissue, tumor tissue had a higher expression level of FUNDC1. Although FUNDC1 showed a protective effect on pan-cancer, a high expression level of FUNDC1 was detrimental to the survival of LIHC patients. Although different from what was found for LUSC, for LIHC, there were significant positive correlations between FUNDC1 expression and immune infiltrates, including B cells, CD8+ T cells, CD4+ T cells, neutrophils, macrophages, and dendritic cells. Furthermore, markers of infiltrating immune cells, such as tumor-associated-macrophages (TAMs), exhibited different FUNDC1-related immune infiltration patterns.Conclusion: The mitophagy regulator FUNDC1 can serve as a prognostic biomarker in pan-cancer and is correlated with immune infiltrates. As the power plants in the human cells mitochondria generate adenosine triphosphate by oxidative phosphorylation to fuel cellular activities while also producing reactive oxygen species, which damage them. To maintain a proper balance, mitochondria undergo fission-fusion cycles and eliminate damaged and redundant sections by mitochondrial autophagy (mitophagy), a process that requires various molecular interactions during mitochondrial quality control –3.FUN14 domain containing 1 (FUNDC1), which anchors to the outer mitochondrial membrane, is pivotal in mitophagy . FUNDC1 The tumor microenvironment (TME) contains various cells. Among them, infiltrating immune cells account for a large proportion . On the In this study, we visualized the prognostic landscape of FUNDC1 in pan-cancer using databases, including ONCOMINE, PrognoScan, GEPIA, and Kaplan-Meier Plotter. We then explored the potential relationships between FUNDC1 expression and immune infiltration levels using the TIMER and GEPIA databases. The findings from this study indicate that FUNDC1 influences the prognosis of patients with cancers, probably via its interaction with infiltrating immune cells.www.oncomine.org). The thresholds were set as a P-value of 0.001 and fold change of 1.5.The mRNA expression of FUNDC1 in different cancer types was analyzed in the ONCOMINE database (http://dna00.bio.kyutech.ac.jp/PrognoScan/index.html), Kaplan-Meier Plotter , and GEPIA (http://gepia.cancer-pku.cn/)  with the “forestplot” package was utilized to summarize and visualize the survival analysis from PrognoScan. GEPIA is an interactive online platform with tumor sample information from TCGA and normal sample information from the TCGA and GTEx projects. We explored the effect of FUNDC1 expression on OS and DFS in each available cancer type . Kaplan-Meier Plotter is a powerful online tool that can be used to assess the effect of 54,000 genes on survival in 21 cancer types. We analyzed the relationship of FUNDC1 expression with overall survival (OS) and relapse-free survival (RFS) in liver hepatocellular carcinoma (LIHC), lung squamous cell carcinoma (LUSC), bladder carcinoma (BC), head and neck squamous cell carcinoma (HNSC), ovarian carcinoma (OV), breast invasive carcinoma (BRCA), lung adenocarcinoma (LUAD), and rectum adenocarcinoma (READ). Hazard ratios (HRs) with 95% confidence intervals (CI) and log-rank P-values were calculated.The correlation between FUNDC1 expression and survival in pan-cancer was analyzed in PrognoScan (pku.cn/) –23. Spechttp://cistrome.org/TIMER/) and GEPIA databases . These gene markers include markers of B cells, CD8+ T cells, follicular helper T cells (Tfh), T-helper 1 (Th1) cells, T-helper 2 (Th2) cells, T-helper 9 (Th9) cells, T-helper 17 (Th17) cells, T-helper 22 (Th22) cells, Tregs, exhausted T cells, M1 macrophages, M2 macrophages, tumor-associated macrophages, monocytes, natural killer (NK) cells, neutrophils, and dendritic cells. The gene expression level was adjusted with log2 RSEM. FUNDC1 was plotted on the x-axis, while marker genes were plotted on the y-axis. Scatterplots were used to analyze correlations between FUNDC1 and each immune gene marker. Similarly, in GEPIA, gene expression correlation analysis was performed for given sets of TCGA expression data. The Spearman method was used to determine the correlation coefficient. FUNDC1 was plotted on the x-axis, while other genes of interest were plotted on the y-axis.In addition to the general analysis of immune cell type, we also analyzed the correlation between FUNDC1 expression and several immune cell markers to identify the potential subtypes of infiltrating immune cells. Immune gene markers were selected from the website of R&D Systems , breast , colorectal , head and neck , and skin cancers . Meanwhile, FUNDC1 had a protective role in the other 3 cancer types, including bladder , lung , and ovarian cancers .Next, we investigated the prognostic value of FUNDC1 for pan-cancer in different databases. In PrognoScan, we explored the relationships between FUNDC1 expression and the prognosis of each cancer. The results are summarized in P = 0.0022; RFS, HR = 1.56, 95%CI from 1.12 to 2.17, logrank P = 0.0082)   (P = 0.00045). However, FUNDC1 worsened relapse-free survival in bladder cancer   but not relapse-free survival  (P = 0.0029) but did not have a significant effect on overall survival    because the data in PrognoScan are mainly extracted from the gene expression omnibus (GEO) database. Interestingly, we newly identified FUNCD1 as a detrimental prognostic factor in LIHC  . This fi 0.0082) . The fin= 0.019)  and not  = 0.13) . For bla= 0.043) . For bot= 0.095) . For BRC = 0.12) . In addi= 0.033) .P = 0; DFS: total number = 9,496, HR = 0.8, logrank P = 6.6E−09) , as well as the overall effect of FUNDC1 on cancers. In general, FUNDC1 was a favorable prognostic marker in cancers  . Specifi6.6E−09) .n = 246, HR = 1.79, 95% CI from 1.14 to 2.83, P = 0.011), Asian , no-alcohol consumption , no hepatitis virus infection , AJCC T3 stage , and micro-vascular invasion . On the contrary, FUNDC1 expression benefited LIHC patients at AJCC T2 stage . For progression-free survival (PFS), FUNDC1 expression was significantly hazardous to LIHC patients without hepatitis virus infection  but became protective to LIHC patients at stage 2  and grade 3   .Immune cells in the TME can affect patient survival, and the above findings support a prognostic role of FUNDC1 in pan-cancer. Hence, it would be meaningful to explore the association between immune infiltration and FUNDC1 expression. We determined whether FUNDC1 expression was correlated with the immune infiltration level in different cancers by calculating the coefficient of FUNDC1 expression and immune infiltration level in 39 cancer types in TIMER. The results indicated that FUNDC1 expression had significant correlations with tumor purity in 11 cancer types. Furthermore, FUNDC1 expression was also significantly correlated with the infiltration levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells in 10, 20, 16, 16, 19, and 19 cancer types, respectively .R = 0.319, P = 1.42E–09), CD8+ T cells , CD4+ T cells , macrophages , neutrophils , and dendritic cells  , macrophages , neutrophils , and dendritic cells  , while the FUNDC1 expression level in LUSC was positively correlated with tumor purity   . However.49E−07) . In addi.55E-03) . These fTo further explore the potential relationships between FUNDC1 and infiltrating immune cells, we examined the correlations between FUNDC1 and several immune cell markers in TIMER and GEPIA. These markers were used to characterize immune cells, including B cells, CD8+ T cells, M1/M2 macrophages, tumor-associated macrophages, monocytes, NK, neutrophils, and DCs in LIHC and LUSC. We also analyzed the different functional T cells such as Tfh, Th1, Th2, Th9, Th17, Th22, Treg, and exhausted T cells . In TIMEAs shown in Moreover, FUNDC1 expression in LIHC and LUSC also relates differently with CD8+ T cell, Tfh, Th2, Th17, and Treg infiltration. The relationships of FUNDC1 with Th9 cells, Th22 cells, neutrophils, and NK cells were partially different between LIHC and LUSC. Additionally, FUNDC1 in LIHC also had significant correlations with exhausted T cell markers such as PD-1 and CTLA4, while FUNDC1 in LUSC did not have such a link . Hence, Mitochondria are essential for human immunity, and aberrant mitochondrial activity affects immune responses. For example, some studies have demonstrated that viruses  can manipulate mitophagy, which enables viruses to promote persistent infection and attenuate the innate immune responses . FUNDC1 Although FUNDC1 has not been well-studied in immuno-oncology, several studies have been conducted. It is now acknowledged that mitophagy is closely related to human immunity. Moreover, to the best of our knowledge, the relationship between FUNDC1 expression and tumor cell proliferation has been proved in cervical and breast cancers , 29. ThuIn this study, we demonstrated the prognostic value of FUNDC1 in pan-cancer. Compared with a low expression level, a high expression level of FUNDC1 correlated with a better prognosis in lung, ovarian, kidney, and thyroid cancers and a poorer prognosis in brain, skin, and liver cancers. Interestingly, increased FUNDC1 expression can specifically impact the prognosis of LIHC patients with the following characteristics: male, Asian, no alcohol consumption, no hepatitis virus infection, stage 2, grade 3, AJCC T2 and T3, and micro-vascular invasion. Our analysis also revealed that LIHC and LUSC have different patterns with respect to correlations between immune infiltration and FUNDC1 expression. Hence, this study provides insights into the use of the mitophagy regulator FUNDC1 as a prognostic marker in pan-cancer from an immuno-oncological perspective, which could benefit future mechanistic studies and aid in the development of immunotherapies.This study explored the expression levels of FUNDC1 and visualized the prognostic landscape in pan-cancer using independent datasets in ONCOMINE and PROGNOSCAN and TCGA data in GEPIA and TIMER. In ONCOMINE, we found that FUNDC1, compared with expression levels in normal tissues, was highly expressed in breast, cervical, colorectal, lung, ovarian, pancreatic, and prostate cancers, as well as in leukemia and lymphoma, while only one dataset showed that FUNDC1 had a lower expression level in breast cancer . HoweverAnother major finding from this study is that FUNDC1 expression correlates with diverse immune infiltration levels in cancers, especially in LIHC and LUSC. Our findings demonstrate that FUNDC1 expression has significant relationships with the infiltration level of CD4+ T cells, macrophages, neutrophils, and DCs in LIHC and LUSC . LIHC haRecent studies provide possible mechanisms explaining why FUNDC1 expression correlates with immune infiltration and different prognoses in pan-cancer. Initial theories assumed that immune cells helped resisting tumors. Indeed, it is still acknowledged that in the early phase of carcinogenesis, the immune system attacks tumor cells by activating T cells and macrophages to prevent the development of cancer. However, once a tumor progress past this early stage, the immune TME switches to supporting cancer cells and promoting tumor progression while suppressing immune cell-mediated cytotoxicity . FUNDC1 Apart from interactions among immune cells, tumor cells can influence immune cells by producing lactic acid . This siin vivo/in vitro experiments were not performed. Future mechanistic studies on FUNDC1 at the cellular and molecular levels could help clarify the role of FUNDC1. Fifth, despite the finding that FUNDC1 expression correlates with both immune cell infiltration and patient survival in cancers, we could not prove that FUNDC1 affects patient survival through immune infiltration. Future prospective studies focusing on FUNDC1 expression and immune infiltration in a cancer population could help provide a definitive answer.However, even though we integrated information across multiple databases, this study still had limitations. First, a large proportion of the microarray and sequencing data were collected by analyzing tumor tissue information. Thus, the cell-level analysis of immune cell markers could have introduced systematic bias. To overcome this issue, future studies with a higher resolution, such as with the use of single cell RNA sequencing, should be performed , 39. SecIn summary, FUNDC1 can affect pan-cancer prognosis and correlate with immune infiltration. Specially, for LIHC and LUSC, FUNDC1 is related to different TAM patterns in the TME. FUNDC1 can serve as a prognostic biomarker in pan-cancer. These findings may provide an immuno-based anti-tumor strategy involving manipulating the energy system of either tumor cells or tumor microenvironment infiltrates.www.oncomine.org, http://dna00.bio.kyutech.ac.jp/PrognoScan/index.html, https://kmplot.com/analysis/, http://gepia.cancer-pku.cn, http://cistrome.org/TIMER/.Publicly available datasets were analyzed in this study. This data can be found here: QY, NS, and XC designed this study. NS, JZ, YW, XY, and WM extracted the information from the databases. QY and NS analyzed the data. YL and XC supervised the entire study. QY wrote the manuscript. All authors revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


KIT. Another aspiration gave rise to a second cell line which has recently been re-established (LADR). We queried whether LADR had unique properties for the preclinical study of human mast cell biology. Methods: LADR and LAD2 cells were cultured under identical conditions. Experiments examined proliferation, beta-hexosaminidase (β-hex) release, surface receptor and granular protease expression, infectivity with HIV, and gene expression. Results: LADR cells were larger and more granulated as seen with Wright–Giemsa staining and flow cytometry, with cell numbers doubling in 4 weeks, in contrast to LAD2 cells, which doubled every 2 weeks. Both LADR and LAD2 cells released granular contents following aggregation of FcεRI. LADR cells showed log-fold increases in FcεRI/CD117 and expressed CD13, CD33, CD34, CD63, CD117, CD123, CD133, CD184, CD193, and CD195, while LAD2 cells expressed CD33, CD34, CD63, CD117, CD133, CD193 but not CD13, CD123, CD184, or CD195. LADR tryptase expression was one-log-fold increased. LADR cell and LAD2 cell chymase expression were similar. Both cell lines could be infected with T-tropic, M-tropic, and dual tropic HIV. Following monomeric human IgE stimulation, LADR cells showed greater surface receptor and mRNA expression for CD184 and CD195. Expression arrays revealed differences in gene upregulation, especially for the suppressor of cytokine signaling (SOCS) family of genes with their role in JAK2/STAT3 signaling and cellular myelocytomatosis oncogene (c-MYC) in cell growth and regulation. Conclusions: LADR cells are thus unique in that they exhibit a slower proliferation rate, are more advanced in development, have increased FcεRI/CD117 and tryptase expression, have a different profile of gene expression, and show earlier infectivity with HIV-BAL, LAV, and TYBE when compared to LAD2 cells. This new cell line is thus a valuable addition to the few FcεRI+ human mast cell lines previously described and available for scientific inquiry.Background: Laboratory of allergic diseases 2 (LAD2) human mast cells were developed over 15 years ago and have been distributed worldwide for studying mast cell proliferation, receptor expression, mediator release/inhibition, and signaling. LAD2 cells were derived from CD34+ cells following marrow aspiration of a patient with aggressive mastocytosis with no identified mutations in  KIT mutations, while LAD2 cells do not express mutations in KIT. By contrast, LUVA and ROSAKIT WT cell lines were derived from CD34+ cells of non-mastocytosis donors and do not express KIT mutations. ROSAKIT D816V cells do exhibit activating KIT mutations. All cell lines are available for study of preclinical human mast cell biology in lieu of primary mast cell cultures derived from bone marrow, or peripheral or cord blood precursors [Mast cells are known to play a major role in innate and acquired immunity. Since 1988, a number of cell lines have been described, including HMC-1 (Human Mast Cell leukemia-1), LAD2 (Laboratory of Allergic Diseases 2), LUVA (Laboratory of University of VirginiA), and ROSA cell lines ,4. HMC-1ecursors . The LADKIT were not identified. A second aspiration preserved under liquid N2 gave rise to a second mast cell line LAD1, now re-established and characterized and which we term LADR. As will be shown, LADR cells share some similarities to LAD2 cells while differing in some important aspects of degranulation, surface receptor expression, protease content, gene expression, and susceptibility to infection.LAD2 cells were derived from CD34+ cells following marrow aspiration of a patient with aggressive mastocytosis where mutations in We first expanded and then characterized LADR cells after removing them from liquid nitrogen and testing for cell viability. In culture, LADR cells were larger, more granulated, and slower to proliferate A,B, suggStudies with LAD2 cells previously revealed that binding of monomeric human IgE to FcεRI could affect HIV infectivity following upregulation of T- and M-tropic complement co-receptors . Thus, tcytomatosis oncogene (c-MYC) with its central role in regulation of cell growth, survival, and differentiation -forming enzyme and has been implicated in a wide variety of human diseases that also implicate its many other protease actions. LADR and LAD2 cells do not harbor mutations in KIT, expressed by as much as 80% of patients with systemic mastocytosis (SM), representing a disadvantage of this cell line for studying emerging therapeutics directed at KIT D816V. It should be recognized, however, that KIT-independent pathways and pro-oncogenic hits and lesions may be responsible for disease progression in advanced SM [Our purpose in studying additional LAD cell lines was to compare the characteristics of LADR with those published for LAD2 and determine advantages for using one cell line over the other. To this end, LADR cells offer clear advantages depending on both the research at hand and the human mast cell biology questions being addressed. Compared with LAD2 cells, and other human mast cell lines available, we believe that LADR cells appear to resemble primary human cell cultures, with higher β-hex release upon FcεRI crosslinking, and a higher cellular content of tryptase perhaps due to their slower proliferation rates. HMC 1.1 and 1.2 cells lack functional FcεRI and ROSAanced SM . Thus, fLAD cells were maintained in StemPro serum free media with nutrient supplement, glutamine, penicillin/streptomycin, and 100 ng/mL rhSCF as described . Hemi-deβ-hex release was assayed following crosslinking of FcεRI as described . BrieflyLADR and 2 cells were harvested, washed, and incubated with Aqua for to assess viability. Following additional washing, cells were incubated with either CD13, CD25, CD33, CD34, CD63, CD117, CD123, CD133, CD184, CD193, CD195, or FcεRI and examined. Alternatively, cells were fixed with 4% paraformaldehyde at room temperature for 10 min. After permeabilization with cold methanol for 1 h on ice, cells were stained with anti-tryptase for 30 min. For intracellular chymase, cells were fixed and permeabilized with Foxp3 staining buffer set  and stained with anti-chymase for 30 min. All analyses were performed on at least 10,000 cells. All cells were analyzed by LSRFortessa flow cytometry.6 LAD2 cells using the RNeasy Plus RNA isolation kit  as described [LADR and 2 cells were harvested, and total RNA was extracted from 1 × 10LADR and 2 cells were first incubated overnight with/without monomeric human IgE, washed, and examined for complement receptor expression over 96 h as previously described .LADR cells when compared to LAD2 cells have a slower proliferation rate, are larger, and more granulated. LADR cells have increased FcεRI/CD117 and tryptase expression and express chymase. LADR cells have different gene expression which is currently being investigated. Following monomeric human IgE treatment, LADR express CD184 and earlier infectivity with HIV-BAL, HIV-LAV, and HIV-TYBE. Thus, while LADR and LAD2 cells share many characteristics, in some situations, one cell line may have an advantage over the other.


Salivation rate was assessed and the animals were euthanized on day 10 for the analysis of macroscopic and microscopic injury by scores. Tissue samples were harvested for the measurement of neutrophil infiltration and detection of inflammatory markers by ELISA and immunohistochemistry. 5-FU induced pronounced hyposalivation, which was prevented by amifostine (P<0.05). In addition, 5-FU injection caused pronounced tissue injury accompanied by increased neutrophil accumulation, tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β) tissue levels, and positive immunostaining for TNF-α, IL-1β, and inducible nitric oxide synthase (iNOS). Interestingly, amifostine prevented the inflammatory reaction and consequently improved macroscopic and microscopic damage (P<0.05 vs 5-FU group). Amifostine reduced inflammation and protected against 5-FU-associated oral mucositis and hyposalivation.Oral mucositis (OM) is a common and dose-limiting side effect of cancer treatment, including 5-fluorouracil (5-FU) and radiotherapy. The efficacy of the therapeutic measures to prevent OM is limited and disease prevention is not fully observable. Amifostine is a cytoprotective agent with a described anti-inflammatory potential. It is clinically used to reduce radiotherapy and chemotherapy-associated xerostomia. This study investigated the protective effect of amifostine on an experimental model of OM. Hamsters were divided into six groups: saline control group (5 mL/kg), mechanical trauma (scratches) of the right cheek pouch; 5-FU (60 and 40 mg/kg,  Oral mucositis (OM) is a side effect related to radiation and cancer chemotherapy. Patients experience pain, xerostomia, erythema, and ulceration of oral mucosa, which, together with leucopenia, increase the risk of bacteremia and sepsis ,2. OM in5-Fluorouracil (5-FU) is an antimetabolite drug used for tumors of the breast, head and neck, and digestive tract administered in regimens that include irinotecan or oxaliplatin . PatientThe pathogenesis of OM includes the direct damage to the mucosa induced by oxidative stress and the activation of transcription factors, such as nuclear factor-kappa B (NF-κB), which increases the expression of proinflammatory mediators, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) –10. The Amifostine, also known as WR-2721, is a prodrug that is dephosphorylated by alkaline phosphatase and acts as a broad-spectrum cytoprotective agent by scavenging free radicals, protecting cell membranes, and preventing DNA damage. In the clinical setting, amifostine is employed to reduce radiotherapy and chemotherapy-associated xerostomia ,14. ThisIn a systematic review by Gu and co-workers, amifostine reduces severe mucositis and xerostomia . Currentad libitum. Experimental procedures complied with the laboratory animal care and the principles outlined by the National Institutes of Health and were approved by the ethics committee for Animal Experiments (protocol number 01/2011) of the Universidade Federal do Ceará.Male Golden Syrian hamsters (100–150 g) from the animal facility of the Federal University of Ceará were kept in appropriate cages in temperature controlled rooms with 12-h light-dark cycles and received food and water ip) injections on the first and second experimental days, using 60 and 40 mg/kg of 5-FU, respectively. This protocol was based on a model previously described by Sonis and co-workers (ip). The needle was dragged twice in a linear movement across the everted cheek pouch until erythematous changes were noted.Oral mucositis was induced by two intraperitoneal ; Mechanical trauma: animals subjected to scratches  on day 4 and treated with 0.9% saline ; 5-FU: intraperitoneal injection of 5-FU , subjected to scratches  on day 4, and treated with 0.9% saline ; amifostine (AMF): intraperitoneal injection of 5-FU , subjected to scratches  on day 4, and treated with 12.5, 25, or 25 mg/kg AMF, sc, from day 1 to 9. On days 1 and 2, AMF was injected 30 min before 5-FU. The animals were euthanized on day 10 and samples of the cheek pouch were excised for macroscopic analysis, histopathology, immunohistochemistry, and biochemical assays. Hamsters were randomly assigned into six groups (n=6/group): Control group: injected with 0.9 % saline  on days 4 and 10 for evaluation of the salivation rate  who was unaware of the treatments, as follows: Score 0: healthy mucosa and no evidence of erosion or vasodilation; Score 1: presence of erythema and no evidence of mucosal erosion; Score 2: severe erythema, vasodilatation, and superficial erosion; Score 3: presence of ulcers in one or more surfaces of the mucosa, affecting no more than 25% of the area, severe erythema, and vasodilatation; Score 4: ulcers in about 50% of the area of the jugal mucosa; Score 5, completely ulcerated jugal mucosa that made it impossible to expose the tissue .ip) and ketamine  followed by exsanguination through the abdominal aorta. Cheek pouch samples were collected for histopathological analysis. The specimens were fixed in 10% (v/v) neutral-buffered formalin, dehydrated, and embedded in paraffin. Sections (5-μm thick) were obtained for hematoxylin-eosin staining (H&E) and examined under light microscopy (40× and 100×). Inflammatory cell infiltration, vasodilatation, presence of hemorrhagic areas, edema, ulcerations, and abscesses were blindly analyzed by a pathologist (GACB) who was unaware of the treatments, as follows: Score 0: normal epithelium and connective tissue without vasodilatation, absence of or mild cellular infiltration, absence of hemorrhagic areas, ulcerations, or abscesses; Score 1: mild vasodilatation, reepithelization areas, mild inflammatory infiltration with prevalence of mononuclear cells, absence of hemorrhagic areas, edema, ulcerations or abscesses; Score 2: moderate vasodilatation, areas of hydropic epithelial degeneration, inflammatory infiltration with neutrophil prevalence, presence of hemorrhagic areas, edema, and eventual ulcerations, absence of abscesses; Score 3: severe vasodilatation, inflammatory infiltration with neutrophil prevalence, presence of hemorrhagic areas, edema, and extensive ulceration, and abscesses . The absorbance change was recorded, plotted on standard MPO curve, and the values obtained are reported as MPO activity U/mg of tissue. A unit of MPO was defined as the amount of enzyme required to convert 1 µmol/min of H2O2 into water at 22°C (Myeloperoxidase (MPO) was used as an inflammatory infiltration marker. Briefly, samples were homogenized in HTAB buffer  and centrifuged at 2500  at 22°C .2O2 and tetramethylbenzidine; R&D System) were added to the plate, which was incubated in a dark at room temperature for 20 min. The reaction was stopped with 2N H2SO4 and the absorbance was measured at 450 nm. The results are reported as pg/mg of tissue.Mucosal samples were homogenized and processed as described by Melo and colleagues . The mic1) weak staining; 2) moderate, or 3) strong immunostaining.Immunohistochemistry was performed using the streptavidin-biotin-peroxidase as described previously . Samples® software, version 6.0 (USA). The data were submitted to analysis of variance (ANOVA) followed by Newman-Keuls' test (parametric data) or to Kruskal-Wallis followed by Dunn's test (non-parametric data). The results are reported as means±SEM (parametric data) or as median (minimum-maximum) (non-parametric data). Differences between groups were considered statistically significant at P<0.05.Statistical analysis was performed using GraphPad Prismper se compared with the saline control group (P>0.05)As depicted in vs the saline group . Additionally, daily injection of AMF (50 mg/kg) attenuated (P<0.05) MPO activity  the MPO activity, indicating a pronounced neutrophil accumulation, and also augmented tissue levels of TNF-α and IL-1β  ity 64%,  and reduity 64%,  and IL-1ity 64%,  comparedity 64%, . Remarkaity 64%, , indicatIn this study, 5-FU induced oral mucositis, leading to a pronounced inflammatory response and reduction of salivary rate. Interestingly, such parameters of tissue injury were prevented by amifostine treatment.First, an experimental model of mucositis in hamsters was used to evaluate the cytoprotective effect of amifostine . This anCurrent guidelines recommend the use of amifostine for the clinical management of radiation proctitis and esophagitis induced by concomitant chemotherapy and radiation therapy . ConversSeveral studies reported that amifostine prevents inflammation in different animal models, such as acrolein- or ifosfamide-induced hemorrhagic cystitis  and indoCytokines are known inflammatory markers that participate in the pathogenesis of 5-FU-induced oral mucositis ,32. LimaIn summary, we showed that in addition to managing hyposalivation, amifostine prevented oral mucositis by reducing the immunoexpression of inflammatory mediators with a well-known role in the pathogenesis of that side effect. Considering the reduced effectiveness of clinical measures to treat mucositis, amifostine could be an important alternative approach for the management of this condition.


TaARF15 in the A subgenome of wheat exhibited high evolutionary rate and underwent positive selection. Transgenic analyses indicated that TaARF15-A.1 promoted the growth of roots and leaves of Arabidopsis thaliana and was upregulated in the overexpression plants after auxin treatment. Our results will provide reference information for subsequent research and utilization of the TaARF gene family.Auxin response factors (ARFs) are important transcription factors involved in both the auxin signaling pathway and the regulatory development of various plant organs. In this study, 23 TaARF members encoded by a total of 68 homeoalleles were isolated from 18 wheat chromosomes (excluding chromosome 4). The TaARFs, including their conserved domains, exon/intron structures, related microRNAs, and alternative splicing (AS) variants, were then characterized. Phylogenetic analysis revealed that members of the TaARF family share close homology with ARFs in other grass species. qRT-PCR analyses revealed that 20 TaARF members were expressed in different organs and tissues and that the expression of some members significantly differed in the roots, stems, and leaves of wheat seedlings in response to exogenous auxin treatment. Moreover, protein network analyses and co-expression results showed that TaTIR1–TaARF15/18/19–TaIAA13 may interact at both the protein and genetic levels. The results of subsequent evolutionary analyses showed that three transcripts of  Currently, only one wheat expressed sequence tag (GenBank No. AY902381) has been reported, which responds to aluminum stress  of the draft genome of “Chinese Spring” bread wheat (1) as well as a high-quality genome  variants, phylogenetic relationships, and expression patterns of those ARFs were analyzed. In addition, the evolutionary rate and transgene function of TaARF15-A were verified. These results will provide reference information for subsequent research and utilization of the TaARF gene family.The rapid development of sequencing and assembly technologies has led to the completion (ad wheat . In addiy genome  have bee2. The sequences of the predicted wheat ARF proteins were obtained by retrieving the whole-protein sequence data based on the ARF family Hidden Markov Model profiles (Pfam accession number PF00931) and checking the ARF domains using the hmmsearch and hmmscan programs of the nhmmer software (E ≤1e-5 was used. Via their registration number (Supplementary Table S1), the protein sequences of ARFs from Arabidopsis, rice and other species were downloaded directly from the NCBI database .The TGACv1 collection of whole-protein sequences of “Chinese Spring” wheat was downloaded from the Ensembl databasesoftware  program,4; 5; Multiple sequence alignments of the ARFs were performed using Clustal X . PhylogeTaARF gene family members were extracted (via their protein accession number) from the Ensembl database. In reference to previous studies  so that each variant was identified as one of the five categories: intron retention (IR), alternative 5′ splicing (A5SS), alternative 3′ splicing (A3SS), exon skipping (ES), or mutually exclusive exon (MXE). The position information of these members was determined by using the RefSeq v1.0 iteration of the wheat whole genome, which was downloaded from the IWGSC database6, after which the TaARFs were assigned to their corresponding chromosomes. The sequences of wheat microRNAs that may regulate TaARFs were obtained by retrieving tae-miR target sequence data (downloaded from the Ensembl database) using TaARF CDS as queries with a similarity >90%. Then, a professional small RNA target analysis server, psRNATarget , AS variants, and genomic sequences of the  studies , the AS NATarget , was usearget 7; . Putativ, TGTCTC  and TGTC, TGTCTC , were idARF codon sequences were used for constructing phylogenetic trees and in subsequent calculations. The ratio of non-synonymous substitutions per non-synonymous site to synonymous substitutions per synonymous site  of branches was computed by using the maximum likelihood method of the branch model in the PAML 4.4 software package .With respect to auxin treatment, the wheat cultivar “Chinese Spring” was planted under a long-day photoperiod . Seedlings with three fully opened leaves were sprayed with 10 μM α-naphthylacetic acid (α-NAA) solution or distilled water (mock treatment), as described previously . The rooAux/IAA1  served ad-change  and the e (-ΔΔCt  methods.TaARFs in different organs at different growth stages of Chinese Spring (8 (9).The expression data of e Spring  were obtpring  were used to amplify the coding sequence of the wheat cDNA, after which the sequence under the control of the CaMV 35S promoter was inserted into a pBI121 binary vector using Gateway BP Clonase enzyme mix . The construct was then transformed into Arabidopsis (Col-0) by Agrobacterium tumefaciens strain GV3101 ; each group containing two or three homeoalleles from the wheat A, B, and D subgenomes was regarded as a member of the ARF family in wheat (TaARF family). These 23 TaARF members were distributed across all chromosomes except 4A, 4B, and 4D and were named TaARF1–TaARF23 based on their chromosome position . The largest number of members (TaARF18–23) were distributed across homologous chromosome 7, while only one member (TaARF14) was found on homologous chromosome 5. With the exception of TaARF23, every TaARF member had three homeoalleles.By retrieving wheat protein sequence data and examining the domains, we detected 68 full-length ARF proteins, which were subsequently used to construct an unrooted tree for revealing phylogenetic relationships. The results showed that these wheat ARF proteins could be divided into 23 groups (Supplementary Table S3). With the exception of TaARF20-A, every TaARF protein contained a DBD for binding the promoter region of target genes. Thirty-eight TaARF proteins contained a CTD, suggesting that those proteins may be inhibited by Aux/IAA proteins. In addition, all of the 68 TaARF proteins contained a MR: 17 of the proteins contained an SPGL-rich MR, 14 contained an SG-rich MR, 14 contained an SP-rich MR, and 23 contained a QSL-rich MR . The first three types may suppress the expression of target genes, while the last one may promote it (The length of the 68 TaARF proteins varied from 354 amino acids (AAs) for TaARF23-D to 1174 AA for TaARF20-B . The homeoalleles for most TaARF members, including TaARF1, 3, 5, 6, 8, 10, 13, 14, 17, 19, 21, 22, and 23, showed analogous exon/intron structures . Furthermore, high sequence similarities (93.76–100%) were observed between several target sequences of wheat microRNAs and the sequences of TaARF1, 5, 7, 17, 21, and 22. Finally, TaARF1-A, -B, and -D and TaARF17-B were predicted to be the targets for tae-miR160 .The full lengths of the TaARF members were found to have 103 AS variants; 16 homeoalleles had only one AS variant, while TaARF19-B had the most (12) AS variants . Furthermore, 33 of the 103 AS variants resulted from two or more AS events, resulting in a total of 149 AS events. Among these AS events, 76 were IR events, which were the most abundant (51.7%), followed by A5SS (29), A3SS (23), ES (20), and MXE (1) events . On the whole, the AS variants in the wheat B subgenome presented the most AS events among all AS types .Furthermore, 41 homeoalleles of 17 in silico expression data revealed that TaARF4, 12, 13, and 22 were highly expressed during seedling, vegetative, and reproductive stages in wheat . The expression levels of TaARF5 and TaARF9 were highest in the root, while TaARF2 and TaARF3 were expressed highly in the spike.In addition, the Supplementary Figure S3). TaARFs were distributed among all groups, and each TaARF member has an orthologous OsARF.We constructed a phylogenetic tree of the protein sequences of 68 TaARFs, 23 AtARFs, 25 OsARFs, and 14 ARFs with known functions from other species. These ARFs can be classified into nine groups: Groups I–IX . Thus, TaARFs may also have functions similar to those of their orthologous OsARFs in the same branch. For example, TaARF9–OsARF1  were upregulated and that two members, TaARF6 and 9, were downregulated in the roots in response to exogenous auxin treatment . Among those TaARFs, the expression levels of TaARF11, 12, 13, 15, 19, and 20 increased gradually within 1.5 h after treatment (HAT) but decreased later, and the expression of TaARF6 was downregulated across all time points. Moreover, in wheat stems, the expression levels of 10 genes  were significantly upregulated by auxin . Among those genes, the expression levels of TaARF1, 4, 10, 12, 19, and 20 were downregulated within the first 0.5 HAT but were continuously upregulated after 1.5–3 HAT, while the expression levels of TaARF9 and 13 were lower at 3 HAT than at 1.5 HAT; the expression of TaARF15 was upregulated across all time points. In addition, in wheat leaves, the expression levels of seven genes, TaARF4, 6, 9, 13, 15, 21, and 23, were significantly upregulated in response to auxin , and TaARF4-21 and TaARF15-23 exhibited similar expression patterns in response to auxin. Overall, in the roots, stems, and leaves of wheat seedling, TaARF9, 13, and 15 responded significantly to auxin treatment.qRT-PCR analysis of TaAux/IAA1 was similar to that reported in a previous study .In addition, the expression level of external control us study , indicatFigure 1A) could bind to TaIAA12, 13, and 19 in both models and that TaTIR1 could also bind to TaIAA proteins and inhibit their function ; these results are consistent with the TIR1/AFB–IAA–ARF interaction model in the auxin signaling pathway .Based on the Arabidopsis and rice interaction network models, 46 proteins expressed in wheat, including 20 TaARF members, 25 TaIAA members , and one pathway . The cluAux/IAAs are regulated by ARFs because Aux/IAAs are the target genes of ARFs. Previous transcriptomic research in rice has shown that the expression levels of TIR1 and most ARF genes increased in response to exogenous auxin stimulation, which subsequently regulated the expression of downstream Aux/IAAs . The results showed that the expression trends of TaTIR1, TaIAA13, TaARF15, TaARF19, and TaARF20 were consistent in wheat roots and that the expression patterns of TaTIR1, TaIAA13, and TaARF15 were similar not only in the stems but also in the leaves . In addition, the TaIAA13 promoter contains AuxRE elements that are the binding sites for the DBD of ARF proteins . Therefore, we inferred that TaTIR1–TaARF15/19/20–TaIAA13 could also interact at the gene expression level.At the protein level, by interacting with CTDs, Aux/IAAs inhibit ARFs. However, at the gene expression level, Aux/IAAs . In thisTaTIR1–TaARF15–TaIAA13 showed the same expression trends in the roots, stems and leaves. In addition to those of TaARF15-A, TaARF15-B, and TaARF15-D, the transcripts of TaARF15 can exist as two or one additional AS variant in the A and B subgenomes, respectively. These six transcripts of TaARF15 and their orthologs in both wheat ancestral species and rice were used to construct a phylogenetic tree. The result showed that ARF genes from wheat and its ancestors clustered into the same group . Among those genes, TaARF15-D is located in the same branch as its ortholog AetARF in A. tauschii, which is the donor of the wheat D subgenome. Furthermore, TaARF15-A is located in the same branch as its orthologs TdARF-A and TuARF from the ancestral species of the A subgenome. However, TaARF15-B is distantly related to its ancestral ortholog TdARF-B.On the whole, in response to auxin treatment, TaARF15-A.1–3 (ωa = 0.66) and TaARF15-B.1 (ωb1 = 0.99) were significantly higher than those of the background branch . Additional analyses revealed that only branch-an underwent positive selection; the specific site was the 717L–736S segment of the TaARF15-A.1 protein-coding sequence, which is located on the exon 12 of the gene. TaARF15-A.2 also contains a positive selection site, but its translation is terminated prematurely because of an IR event within intron 12. In addition, TaARF15-A.3 lost a positive selection site in exon 12 because of an A5SS event. These examples may represent techniques used by wheat to regulate genes in different organs or stages.Based on the two-ratio model, the evolutionary rates of TaARF15 showed that TaARF15-A.1 was more highly expressed than TaARF15-A.2, while the expression of TaARF15-A.3 was not detected. Because of the response to exogenous auxin and the more complete gene structure among TaARF15-A transcripts, TaARF15-A.1 was transformed into Arabidopsis for functional verification. The results showed that the root length of the overexpression lines were significantly higher than those of the Col control lines . Additionally, the leaf area of the overexpression lines exceeded those of the control lines . Furthermore, qRT-PCR showed that TaARF15-A.1 was upregulated in the overexpression plants in response to exogenous auxin treatment. Thus, it can be inferred that TaARF15-A.1 participates in the development of roots and leaves during the vegetative growth stage of the plants .qRT-PCR analysis for three AS variants of T. urartu (21), A. tauschii (23), and Triticum turgidum (25); gramineous grasses such as barley (25), Brachypodium distachyon (28), and rice . Phylogenetic analyses  revealed the poor sequence homology between TaARFs and AtARFs, which indicated that the ARF family differentiated after the divergence of mono- and dicotyledons . Among those paralogs, TaARF12-13 are both located on the long arm of wheat chromosome 3 at a close genomic distance, suggesting that those genes may have undergone a tandem duplication event. TaARF12-13 had homologous gene pairs in allied Triceae species but only one common ortholog in A. thaliana, rice, and B. distachyon, indicating that the tandem duplication occurred after the divergence of the Triceae ancestor , A3SS (15.4%), ES (13.4%), A5SS (19.5%), and MXE (0.7%) events, occurred. IRs still represented the most common AS event in the TaARF splicing variants, and 18 of 77 IR events resulted in premature termination codons (PTCs). These PTC transcripts are often recognized and degraded by the nonsense-mediated mRNA decay (NMD) mechanism to avoid cell toxicity resulting from the accumulation of truncated protein products -based regulation of TaARF expression ; AtARF2 , the only ARF sequence reported in wheat , A5SS (29), A3SS (23), ES (20), and MXE (1) events. Thirty-seven (54%) TaARF protein sequences have a DBD, MR, and CTD. Twenty TaARF members are expressed among different organs and tissues. In response to auxin treatment, the expression of nine, 10, and seven TaARF members significantly differed in the roots, stems, and leaves of wheat seedlings, respectively. Overall, TaARF9, 13, and 15 responded significantly to auxin treatment in all the three organs. In addition, TaTIR1-TaARF15, 18, 19, and 20-TaIAA12, 13, and 19 were predicted to be interactive proteins, and TaTIR1–TaARF15/19/20–TaIAA13 exhibited similar expression patterns at the genetic level. TaARF15-A.1 is likely involved in the regulation of roots and leaves of A. thaliana.The TaARF family has a total of 23 members, and each member except two LQ, ZC, and JZ conceived and designed the experiments. LQ, WZ, XZ, XL, HG, and YR performed the experiments. LQ analyzed the data. XyL and LQ contributed reagents, materials, and analysis tools. LQ and LZ wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The proximal femoral head separation (FHS) or epiphysiolysis is a prevalent disorder affecting the chicken femur epiphysis, being considered a risk factor to infection which can cause bacterial chondronecrosis with osteomyelitis in broilers. To identify the genetic mechanisms involved in epiphysiolysis, differentially expressed (DE) genes in the femur of normal and FHS-affected broilers were identified using RNA-Seq technology. Femoral growth plate (GP) samples from 35-day-old commercial male broilers were collected from 4 healthy and 4 FHS-affected broilers. Sequencing was performed using an Illumina paired-end protocol. Differentially expressed genes were obtained using the edgeR package based on the False Discovery Rate (FDR < 0.05).Approximately 16 million reads/sample were generated with 2 × 100 bp paired-end reads. After data quality control, approximately 12 million reads/sample were mapped to the reference chicken genome . A total of 12,645 genes were expressed in the femur GP. Out of those, 314 were DE between groups, being 154 upregulated and 160 downregulated in FHS-affected broilers. In the functional analyses, several biological processes (BP) were overrepresented. Among them, those related to cell adhesion, extracellular matrix (ECM), bone development, blood circulation and lipid metabolism, which are more related to chicken growth, are possibly involved with the onset of FHS. On the other hand, BP associated to apoptosis or cell death and immune response, which were also found in our study, could be related to the consequence of the FHS.Genes with potential role in the epiphysiolysis were identified through the femur head transcriptome analysis, providing a better understanding of the mechanisms that regulate bone development in fast-growing chickens. In this study, we highlighted the importance of cell adhesion and extracellular matrix related genes in triggering FHS. Furthermore, we have shown new insights on the involvement of lipidemia and immune response/inflammation with FHS in broilers. Understanding the changes in the GP transcriptome might support breeding strategies to address poultry robustness and to obtain more resilient broilers. The poultry meat industry is extraordinarily efficient in supplying high-quality and affordable food for human and animal consumption. Chicken is one of the main sources of animal protein for humans, and nowadays is the second most widely consumed meat in the world . In the Zuidhof et al.  showed tA common disorder affecting the proximal femur epiphysis is the proximal femoral head separation or epiphyseolysis (FHS), which is the separation of the growth plate (GP) from the articular cartilage (AC) resulting in disarticulation of the coxofemoral joint , 14. DurThe FHS is a predisposing factor for infection and osteomyelitis , which aHigh-throughput RNA sequencing was used to generate a whole characterization of the proximal femur GP transcriptome of health and FHS-affected broilers. The multi-dimensional scaling (MDS) plot provided a spatial representation of data . The average reads mapped across samples was 98.99% and a total of 12,645 genes were expressed in the GP transcriptome. Out of those, 314 were DE between normal and FHS-affected broilers (FDR < 0.05), being 154 upregulated and 160 downregulated  that are mostly involved in blood circulation and angiogenesis , extracellular matrix , tissue homeostasis  and immune response  ; PLCB2, ITPKA, ITPK1 and ITPR3; 2) OSBPL1A, ITIH5, C1QTNFB and GIMAP5; 3) H1F0, TRIM23, TUBB1, KIF26B, KIF1A and SMAP2; 4) PNPLA2, ACSL6, SCD, ALDH1A1, DDC and ALDH6A1 and 5) NMNAT1, NT5C3, ADA, CECR1, AMPD3 and BTBD10  were differentially expressed (P < 0.05) in the proximal femur GP between normal and FHS-affected groups. Furthermore, all of these genes kept the same expression profile observed in the RNA-Seq analysis  gene can be highlighted since it catalyzes the oxidative deamination of peptidyl lysine residues, promoting the formation of lysyl-derived crosslinks in collagens and elastin [LOXL2 and the other genes could reduce the cell adhesion, which could facilitate the separation of cartilage and bone tissues. Furthermore, this gene is also connected with immune biological responses, blood coagulation and lipid metabolism functions   and aminoglycan metabolic processes involved in the ECM metabolism. The CHST1 (Carbohydrate Sulfotransferase 1) is a membrane bound sulfotransferase potentially involved in the production of L-selectin ligands, important for the recognition of various immunological receptors involved with the recruitment of macrophages to the tissue inflammation sites [CHST1 differential expression pattern between health and FHS-affected group was also confirmed by qPCR (Table Adiponectin (ADIPOQ), Paired related Homeobox 1 (PRRX1), ABI family member 3 NESH binding protein P (ABI3B) and COL8A1 genes were also downregulated in FHS-affected group by qPCR, confirming their involvement with ECM, cell adhesion and bone formation.Many biological processes related to ECM and also cell adhesion were enriched, such as ECM organization, glycosaminoglycan metabolic process, aminoglycan metabolic process, extracellular structure organization, connective tissue development, biological adhesion, regulation of cell adhesion, cell adhesion, cell-matrix adhesion, and others  Figs. . Here, aon sites . In addion sites . CHST1 dFN1 (Fibronectin 1), FBLN7 (fibulin 7), DPT (dermatopontin), GPC4 (glypican 4), BCAN (brevican), FMOD (fibromodulin), FBN2 (fibrillin 2), VIT (vitrin), CHPF (chondroitin polymerizing factor), SDK2 (sidekick cell adhesion molecule 2), DBN1 (drebrin 1), SDC3 (syndecan 3), ITGA11  and OGN (osteoglycin) , COL6A2 , COL6A3 , COL8A1 , COL14A1 , COL18A1  and COL28A1 . Here, the FN1 gene can be highlighted, because it is a multidomain glycoprotein with multiple adhesive properties binding cells and their extracellular matrices, and was recognized to be the target for a large number of bacteria [FN1, Col8A1, Col18A1 and MFGE8 are also involved with angiogenesis  Fig. . Also, sbacteria . The FN1Moreover, most of the DE genes in our study presented binding molecular functions (MF), such as binding to ions, protein, heterocyclic compound and organic cyclic compound Fig. . The molADA, AGT, CBS, CEBPB, EPB42, GFI1B, IL7R, IRF1, JAK2, NPR3, PDGFRA, RSAD2 and RHAG, and downregulated genes in the affected group, such as ADIPOQ, COL8A1, FN1, GLI2, HSPB1, LOXL2, MEOX2, MFAP5, MYH15, PTCH1, PRRX1, SFRP1, SREBF1, THBS1 and TNMD  was upregulated in broilers with FHS. The angiopoietins are proteins with important role in vascular development and angiogenesis. However, this gene was not grouped in any BP using DAVID. In this study, Angiopoietin-like 5 (ANGPTL5) was one of the top 10 downregulated genes (Table ANGPTL5 in FHS-affected broilers by qPCR. Angiopoietin-like proteins (ANGPTLs) are a family of proteins structurally similar to the angiopoietins, with functional roles in lipid metabolism, glucose metabolism, inflammation and hematopoiesis [opoiesis .ABCA8, ALDH6A1, APOA1, CEBPB, OSBPL1A, P2RX7, PNPLA2, AGT and PDK4 genes were upregulated in affected broilers while SREBF1, SFRP1, PTCH1, THBS1 and ADIPOQ were downregulated , a member of the purinoceptors for ATP family [AMPD3 and ADA, and purine-containing compound salvage, including genes CECR1, AMPD3 and ADA. Here, adenosine monophosphate deaminase 3 (AMPD3) and adenosine deaminase (ADA) were upregulated in FHS-affected broilers and the CECR1, also named adenosine deaminase (ADA2), was downregulated. Also included in the purinergic metabolism is the CHAT2 gene, the most upregulated gene in the FHS-affected broiler (Table An important candidate gene to FHS condition in broilers is the P family . This geG0/G1 switch gene 2 (G0S2), a candidate gene for metabolic disorders, was upregulated in broilers with FHS. This gene has been considered a potent endogenous inhibitor of adipose triglyceride lipase (ATGL) revealing a unique mechanism governing lipolysis and fatty acid availability [G0S2 acts as a master regulator of tissue-specific balance of triglycerides storage, mobilization, partitioning of metabolic fuels between adipose and liver, and of the whole-body adaptive energy response.In this transcriptome study, the lability . Zhang alability  evidenceAPOA1, PNPLA2, ABCA8, P2RX7, OSBPL1A, ALDH6A1, CEBPB, AGT and PDK4. Some of them also interact with genes involved in the ossification and inflammation , another interesting gene involved in the defense response to bacterium BP, was one the most upregulated in the femur of affected broilers, suggesting a potential role in the resilience of chickens. The AVD is a biotin-binding glycoprotein expressed under inflammation in several chicken tissues [Gallinacin-9 (GAL9), another chicken defense peptide, in plasma of FHN-affected 35 day-old commercial broilers compared to normal broilers.The  tissues , acting  tissues . Packial tissues  found elC-C motif chemokine ligand 19 (CCL19) was one of the most expressed genes in the affected chickens  are not separated by strict boundaries sharing molecular effectors and signaling ways . In our P2RX7 gene can be highlighted because its activation stimulates downstream events, triggering the caspase cascade and leading to apoptotic death of the target cell [TP53 apoptosis effector (PERP2), which was not recognized in BP with DAVID database. According to KEGG p53 signaling pathway, the p53 activation is induced by a number of stress signals, including DNA damage, oxidative stress and activated oncogenes. The p53 protein is employed as a transcriptional activator of p53-regulated genes resulting in cell cycle arrest, cellular senescence or apoptosis. In our study, PERP2 was one of the most downregulated genes in the GP transcriptome, being 3.04 times less expressed in the affected than in the normal group. This expression profile was confirmed by the qPCR analysis, with PERP2 being 16.1 times less expressed in FHS-affected than in normal broilers  under protocol number 012/2012. Commercial male broilers (Cobb500) with 35 days of age from a poultry farm located in Concórdia, Santa Catarina State, Brazil, were used. The chickens were housed according to optimal industry growing standard practices for this line and raised with free access to both feed and water. To reduce environmental effects, broilers used in this study were sampled from the same flock, in a Darkhouse system managed by a high standard producer. Based on visual observation, a total of 29 individuals, including 14 healthy and 15 lameness-affected chickens, were sampled and transferred to the Embrapa Swine and Poultry National Research Center, located in Concordia-SC, for tissue sample collection. The body score was evaluated in all animals prior to necropsy. Broilers were euthanized by cervical dislocation followed by bleeding.The proximal femoral head was classified according to the presence or absence of different levels of BCO, based upon clinical examination of the growth plate (GP) separation from the articular cartilage (AC) by visual observation of compatible necrosis lesions, according to Wideman et al. . FemoralTotal RNA was isolated from 100 mg of the femoral head tissue using Trizol Reagent  following the manufacturer’s instructions. The RNA cleanup was performed using RNeasy mini kit  also following the manufacturer’s instructions. Total RNA was quantified in Nanodrop spectrophotometer  and the integrity was assessed using an Agilent 2100 BioAnalyzer . Samples with RNA integrity number (RIN) higher than 8 were considered for further analysis. A total of eight samples of GP, four normal and four FHS-affected were prepared for RNA-sequencing. Barcoded Illumina sequencing libraries for gene expression profile were obtained for each of the 8 individuals using the TruSeq™ RNA Sample Prep Kit v2 , according to the manufacturer’s recommendations, with 2 μg of total RNA. Libraries were submitted to the Functional Genomics Center, ESALQ, University of São Paulo, Piracicaba, São Paulo State, Brazil for sequencing in Illumina HiSeq2500 equipment , all in the same lane, following the 2x100bp paired-end protocol.Gallus gallus, assembly 5.0) available in Ensembl 87 (www.ensembl.org) using the BWA-MEM software [MDS) plots using R. Based on DE genes, a heatmap was generated to check the consistence between samples using R. The FASTQ files obtained in this study were deposited in the SRA database, with BioProject number PRJNA352962.Raw FASTQ reads were submitted to quality control using the SeqyClean tool  for remosoftware . The cousoftware . The difsoftware . The DE software . To verihttp://david.abcc.ncifcrf.gov/tools.jsp) was used to improve the biological processes (BP) obtained in the Blast2GO software, where the expressed genes in this study were set as background. Once obtained the BP, the GO numbers from DAVID were clustered by the REViGO tool [The functional annotation analyses were performed based on the DE genes with FDR < 0.05. The FASTA files of these genes obtained in the Ensembl 87 were submitted to the Blast2GO software  for the iGO tool  for easiiGO tool , 80.lipoprotein receptor-related protein 1B (LRP1B), Collagen type XXVIII alpha 1 chain (Col28A1), Family with sequence similarity 180, member A (FAM180A), TP53 apoptosis effector (PERP2), Carbohydrate sulfotransferase 1 (CHST1) and Perilipin-1 (PLIN1). Those genes were selected based on their LogFC, FDR and function. Sequences were obtained from Gallus gallus on Genebank (http://www.ncbi.nlm.nih.gov/gene/) and Ensembl (www.ensembl.org). Primers used for each gene (Table http://www.premierbiosoft.com/netprimer/). The Ribosomal protein 4 (RPL4) and Ribosomal protein 30 (RPL30) genes were used as reference [Quantitative PCR analysis was used to confirm the expression pattern of six DE genes in the GP transcriptome using 20 samples of 35 day-old broilers . The genes selected were: Low density eference . The qPCeference ) that apAdditional file 1. Multi-Dimensional Scaling Plot (MDS-plot) measuring the similarity of the samples into 2-dimensions .Additional file 2. An excel file with the expressed and DE genes between normal and FHS-affected broilers with 35 days of age.Additional file 3. GO biological process enriched based on the differentially expressed genes between normal and FHS-affected broilers.


The suppressor of cytokine signaling (SOCS) family of intracellular proteins has a vital role in the regulation of the immune system and resolution of inflammatory cascades. SOCS1, also called STAT-induced STAT inhibitor (SSI) or JAK-binding protein (JAB), is a member of the SOCS family with actions ranging from immune modulation to cell cycle regulation. Knockout of SOCS1 leads to perinatal lethality in mice and increased vulnerability to cancer, while several SNPs associated with the SOCS1 gene have been implicated in human inflammation-mediated diseases. In this review, we describe the mechanism of action of SOCS1 and its potential therapeutic role in the prevention and treatment of autoimmunity and cancer. We also provide a brief outline of the other JAK inhibitors, both FDA-approved and under investigation. Cytokines are small glycoproteins secreted by a variety of immune and non-immune cells. These molecules govern a range of processes including, but not limited to, hematopoiesis, inflammation, cell proliferation, survival, apoptosis, and chemotaxis. As such, intricate modulation of cytokine signaling is required for maintaining immune system homeostasis and regulation of inflammatory responses. Cytokine receptors typically belong to one of the following families: IL-1 receptor superfamily, TNF-receptor family, IL-17 receptor superfamily, G-protein coupled receptor (GPCR) superfamily, transforming growth factor superfamily, receptor tyrosine kinase superfamily (RTK), and type I and II cytokine receptor superfamily. Among these, only type I and II cytokine receptors are physically associated with JAKs . JAKs ar+ cells that SOCS1ΔNLS has impaired ability to inhibit NF-κB-induced inflammation as compared to the complete SOCS1 protein   . The SOCSuppressor of cytokine signaling 1 not only modulates JAK/STAT pathways, but it can also regulate TLR signaling. TLRs are pattern recognition receptors that can identify conserved microbial molecules and upregulate immune response against them . SOCS1 r-/- mice die of perinatal autoinflammatory disease or lymphoid deficiencies, develop polycystic kidneys, and inflammatory lesions. While these mice can be partially saved by IFNγ deletion, these mice still develop fatal inflammatory diseases later , which functions through IFNGR1/IFNGR2 and JAK1/JAK2 . Additioes later . SOCS1 dd asthma . SOCS1-/diseases . Moreovepulation ,b.SOCS1 deficiency is the use of SOCS1 mimetics. A SOCS1 mimetic peptide containing only the n-terminal kinase inhibitory region (KIR 53- DTHFRTFRSHSDYRRI-68) domain has gained attention due to its effectiveness in JAK1/2 and TYK2 inhibition activity   is a dermatological disease marked by plaques and erythematosus on the skin. Histological analyses reveal excess keratinocyte proliferation (acanthosis) and lymphocyte infiltration into the epidermis . Five tybitor A) . Even thbitor A) . In a stcteremia . Certaincteremia . LL37 cacteremia . Since Scteremia . Interesin SOCS1 , 2017.+ Th1 cells, NK cells, and neutrophils to aggravate inflammation during early stages of the disease  in response to TLR7 ligation . Kim et TLR4 expression is upregulated in PBMCs in human patients with psoriasis, and variants of TLR4 are implicated in both plaque-type psoriasis and psoriatic arthritis . TLR4 caAnomalous gene expression by cancer cells can lead to cell transformations. Such transformed cells can sustain unimpeded growth, evade contact inhibition, ignore apoptotic signals, undergo metastasis and angiogenesis, and evade the immune response . While tSuppressor of cytokine signaling 1 has been shown to regulate, directly or indirectly, a number of molecules and pathways that have been implicated in cancer – CDK2, CDK4, Cyclin D1, MAPK/p38, PDL1, STAT1, STAT3, STAT6, p53, p21, FAK, E-cadherins, Met tyrosine kinase, type I and II IFN, and numerous proinflammatory cytokines , a ligand for PD1 expressed by T cells. The interaction between PD1 and PDL1 can cause anergy or even programmed cell death in T cells . Anti-PDe cancer . Hence, STAT3 gene expression and its phosphorylation-mediated activation resulting in the transcription of STAT3 target genes  is DNA-binding, an intracellular signaling protein that has pleiotropic effects on embryogenesis, oncogenesis, tumor suppression, cell differentiation, growth, and both innate and adaptive immunity , 2009. Iet genes . The actet genes . Intrigund ICAM1 . In addind ICAM1 . Under pnd ICAM1 . Mouse fnd ICAM1 . Moreovend ICAM1 . STAT3 pnd BIRC5 . Irradiand BIRC5 . STAT3 h and p21 . Furtherco gene] . Constitco gene] . It is wco gene] . Variousco gene] . Moreoveco gene] . SOCS1 ico gene] . This paaneously . Activataneously . Part ofaneously . Moreoveaneously . On the g review . This scFocal Adhesion Kinases (or protein tyrosine kinase 2) reside within focal adhesions where the cell cytoskeleton contacts the extracellular matrix. FAK activation can result from receptor signaling via receptor tyrosine kinases (RTK), cytokine receptors, growth factor receptors, G-protein coupled receptors, and integrins through FERM domain interactions  which is a major cause of morbidity in SLE patients . These pCCL2 can be upregulated by macrophages in response to IFN γ  and since then a number of kinase inhibitors have been approved by FDA for cancers . The sucThe first generation of jakinibs were pan-inhibitors, i.e., they blocked multiple JAKs. The shortcoming of the first generation of jakinibs is (1) they would also block JAKs required for anti-inflammatory cytokines, like IL-10, to signal (2) they could expose the patient to infections (3) they can block hematopoietic cytokines that typically signal through JAK1/JAK2 from functioning and cause cytopenia, which may not be desirable. Tofacitinib, a first generation JAK1/3 inhibitor, was the first jakinib to be approved for autoimmunity in humans . It is aSuppressor of cytokine signaling 1 KIR, a SOCS1 mimetic containing only the KIR domain, acts as a pseudosubstrate for JAK1, JAK2, and TYK2, with no known interaction with JAK3 . HoweverSuppressor of cytokine signaling 1 is an essential molecule for maintaining immune homeostasis and subverting inflammation. Disorders arising from excess inflammation or SOCS1 deficiency can be potentially treated with SOCS1 mimetics . While SJS and JL wrote the manuscript. JS designed the figures. JL reviewed the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


POD genes (MePODs) and classified them into six subgroups using phylogenetic analysis. Conserved motif analysis demonstrated that all MePOD proteins have typical peroxidase domains, and gene structure analysis showed that MePOD genes have between one and nine exons. Duplication pattern analysis suggests that tandem duplication has played a role in MePOD gene expansion. Comprehensive transcriptomic analysis revealed that MePOD genes in cassava are involved in the drought response and postharvest physiological deterioration. Several MePODs underwent transcriptional changes after various stresses and related signaling treatments were applied. In sum, we characterized the POD family in cassava and uncovered the transcriptional control of POD genes in response to various stresses and postharvest physiological deterioration conditions. These results can be used to identify potential target genes for improving the stress tolerance of cassava crops.The class III peroxidase (POD) enzymes participate in plant development, hormone signaling, and stress responses. However, little is known about the POD family in cassava. Here, we identified 91 cassava  Howeverhneideri . Cassavahneideri ,33. Unfohneideri . ROS prohneideri . The mecAccording to the 211 POD protein sequences from Arabidopsis and rice genome databases, 91 POD members were predicted from the cassava genome using BLAST and HMMER methods. After conserved domain detection was confirmed, these cassava POD proteins (MePODs) were named MePOD01 to MePOD91. The full length of these putative cassava POD proteins ranges from 153 (MePOD63) to 422 (MePOD46) amino acid residues, and their relative molecular weight varies from 16.64 (MePOD63) to 46.12 kDa (MePOD46), with isoelectric points ranging from 4.43 (MePOD26) to 9.63 (MePOD39) .POD genes in cassava were determined by performing multiple sequence alignments. Then, the radiation phylogenetic tree of the 91 POD genes was constructed using the neighbor-joining (NJ) method with a bootstrap value of 1000 using MEGA 5.1 . The phylogenetic analyses indicated that MePOD genes can be divided into six subgroups on the basis of the observed genetic distance and bootstrap support  were noted as POD protein motifs, which are an essential feature of the peroxidase family. On the basis of the motif analyses, 83 MePODs were assigned to one of five subgroups (A–E). Each of these 83 MePODs contains at least nine POD motifs, except for MePOD57 (in subgroup D) and MePOD69 (in subgroup B), which have seven and five motifs, respectively. The presence of these motifs suggests that the identified proteins are characteristic of the POD family . SubgrouPOD genes were analyzed. Subgroup F is exon-rich, with five to nine exons, whereas other subfamilies have fewer (between one and four) exons, except for MePOD02 (in subgroup B), which has five exons , suggesting that tandem duplication played a significant role in POD family expansion in the cassava genome  and synonymous (Ks) ratios to understand the modes of evolutionary selection for the duplicated volution .MePOD genes in different tissues were investigated by performing RNA-Seq analysis on the storage roots, stems, and leaves of a cultivated variety (Arg7) and wild subspecies (W14). The resulting expression data covered 59 and 56 MePOD genes in the transcriptome dataset of Arg7 and W14, respectively  in stems, leaves, and storage roots, respectively. The number of MePODs with high expression (log2-based > 4) in the stems, leaves, and storage roots of W14 was 9 (16%), 7 (13%), and 10 (18%), respectively. Notably, MePOD5 in subgroup E and MePOD89 in subgroup F were strongly expressed (log2-based fold change > 4) in the three diverse tissues of Arg7 and W14. These POD genes may play a molecular role in the development and function of different cassava tissues.The expression levels of ectively . Of thesPOD genes , whereas 7 (10%) and 29 (41%) were down-regulated (log2 based fold change < −1) in the leaves and roots, respectively. After SC124 was subjected to drought stress, 9 (13%) and 4 (6%) MePOD genes were upregulated (log2-based fold change > 1) but 6 (8%) and 29 (41%) were downregulated (log2-based fold change <−1) in the leaves and roots, respectively. After the W14 subspecies was exposed to drought, 13 (18%) and 21 (30%) MePOD genes were induced (log2-based fold change > 1), whereas 11 (15%) and 9 (13%) were depressed (log2-based fold change < −1) in the leaves and roots, respectively. MePOD13 (subgroup A) and MePOD16 (subgroup B) were upregulated (log2-based fold change > 1) by drought stress in the leaves of all three genotypes. The above data reveal that more MePOD genes were upregulated in response to drought treatment in W14 than in Arg7 and SC124.To study the possible role of MePODs in the cassava response to drought stress, water was withheld from a wild subspecies (W14) and two cultivated varieties (Arg7 and SC124) for 12 days. The leaves and roots of these samples were then collected to perform RNA-Seq. Of the transcriptome data, the expression data were obtained for 71 out of the 91 cassava OD genes . After dMePOD genes during PPD  at all points. Conversely, MePOD-7, -8, -9, -12, -23, -38, -50, -70, -71, and -88 were downregulated (log2-based fold change < −1) at all tested times. These results suggest the possibility that MePODs play a role during PPD in cassava.Transcriptome analyses were conducted at different postharvest periods of the storage roots of SC124 to examine the possible function of ring PPD . TranscrMePOD genes upon exposure to methyl jasmonate (MeJA), salicylic acid (SA), abscisic acid (ABA), H2O2, salt, osmotic stress (by mannitol treatment), cold stress, and Xanthomonas axonopodis pv manihotis (Xam), nine genes  that were induced by drought stress in at least two tissues in all three varieties were selected for quantitative real-time PCR (qRT-PCR) analysis  was induced, with particularly high levels expressed 24 h after treatment. ABA treatment resulted in increased transcript levels of MePOD-17 and -85. With SA treatment, the expression levels of MePOD-13, -16, -19, -23, -68, -85, and -86 were amplified. H2O2 treatment led to the induction of MePOD-13, -17, -23, and -85. Salt treatment induced the expression of MePOD17 after two and six hours, and three days of treatment, but the gene was repressed after 14 days of treatment. Under osmotic stress, MePOD-13, -17, -19, -23, -85, and -86 were upregulated, among which MePOD-85 was induced throughout the entire treatment time. In response to cold treatment, MePOD17 was upregulated at 2 and 15 h. Exposure to the pathogen Xam led to the upregulated expression of six POD genes  at a minimum of two time points. Together, these results demonstrate that the POD genes of cassava respond to multiple stresses and related signals  in cassava harbor more than one exon (POD genes in Pyrus bretschneideri and Zea mays (PbPRX (90/94) and ZmPRX (89/107), respectively) [Given the significant role of PODs in various physiological processes, including responses to biotic and abiotic stresses, it was necessary to scientifically investigate the potential functions of a genome ; thus, chneideri ,28,29,30hneideri . Most ofone exon , which iPOD genes in cassava, we first identified the chromosomal locations of the MePOD genes. Chromosomal mapping revealed that these genes are widely distributed among 17 chromosomes and one scaffold   , which ihneideri ,27,29,30 process C. These POD genes responding to drought (log2-based fold change > 1) was greater in both the roots and leaves of W14 than that in Arg7 and SC124, suggesting the comprehensive activation of PODs in response to drought in W14  to further examine their expression levels after various treatments  were induced by six treatments ; MePOD13 was upregulated by five treatments ; and MePOD23 and MePOD86 were upregulated by four treatments . Of these, MePOD13 and MePOD23 were induced after H2O2 treatment in cassava leaves  were upregulated by MeJA and SA treatments but downregulated by ABA treatment  for 0, 2, 5, 15, and 48 h; 100 μM SA for 0, 2, 6, 10, and 24 h; 200 mM mannitol (to induce osmotic stress) for 0 h, 2 h, 6 h, 3 days, and 14 days; 100 μM ABA for 0, 2, 6, 10, and 24 h; 10% H2O2 for 0, 2, 6, 10, and 24 h; or Xam for 0, 2, 6, 12, and 24 h. Ten-month-old cassava storage roots (CSR) were cut into 5-mm-thick slices and placed into Petri dishes containing wet filter paper for 0, 6, 12, and 48 h to study the expression changes in MePOD genes via RNA-Seq during CSR deterioration. All samples were frozen immediately in liquid nitrogen and stored at −80 °C for RNA-Seq and qRT-PCR.Three cassava genotypes, W14, SC124, and Arg7, were planted in the greenhouse of the Chinese Academy of Tropical Agricultural Science . Their characteristics were described in our previous studies ,55. StemMePOD genes were identified in cassava on the basis of homology with 73 POD protein sequences from the Arabidopsis genome database (available online: http://www.arabidopsis.org/index.jsp) and 138 POD protein sequences from the rice genome database (available online: http://rice.plantbiology.msu.edu/index.shtml) [http://hmmer.wustl.edu/) profile of these confirmed POD proteins was constructed to search the cassava genome hub (available online: http://www.phytozome.net/cassava.php) [http://www.ncbi.nlm.nih.gov/cdd/) and PFAM (available online: http://pfam.sanger.ac.uk/) after being checked by BLAST analyses [http://smart.embl-heidelberg.de/) [x.shtml) ,57. The ava.php) . Finallyanalyses ,60. All erg.de/) . Multiplerg.de/) . The phyerg.de/) .http://web.expasy.org/protparam/) was used to predict the properties, including amino acid numbers, molecular weights (MW), and isoelectric points (pI), of all presumed POD proteins [http://meme-suite.org/tools/meme), in which the maximum number of motifs was set to 10, the optimum width of motifs was set to 15–50 amino acid residues, and the other settings were kept at default values [http://www.ebi.ac.uk/Tools/pfa/iprscan/) [http://gsds.cbi.pku.edu.cn/) using each MePOD’s genomic DNA sequence and its corresponding CDS sequence, which were retrieved from the cassava genome database [The ProtParam database  . The gendatabase .MePOD genes were mapped to different chromosomes. On the basis of the calculated value of nucleotide sequence similarity and the phylogenetic relationship of cassava POD genes, paralogous genes were identified. The gene duplication pattern of paralogous MePOD genes was determined by the following two criteria: (1) the identity of the aligned region was >90% and (2) the alignment covered >90% of the longer gene. Circos software  was used to draw the duplication events of MePOD genes [According to the results of BLASTN in the Phytozome 12.0 cassava database, OD genes ,69. The OD genes . Ka/Ks rOD genes .MePOD genes. Total RNA was isolated from frozen stems, leaves, roots, and storage roots using the plant RNeasy extraction kit  and quantified with a NanoDrop 2000c . Total RNA (3 µg) of each sample was used to construct the cDNA library according to the Illumina instructions and then sequenced using an Illumina GA II . The original data processing and analysis methods were described in our previous study [RNA-Seq was used to determine the expression of cassava us study .2O2, or Xam treatments were collected to perform qRT-PCR analysis. Total RNA (1 µg) from each sample was used to synthesize the first-strand cDNA using oligo-dT primer by SuperScript reverse transcriptase . The cDNA product was diluted to 50 ng·μL−1, and 1 μL was used for qRT-PCR. The qRT-PCR reaction mixtures (20 μL) contained 0.6 μL of each gene-specific primer (300 nmol·μL−1), 10 μL of 2× FastFire qPCR PreMix , and 7.8 μL of RNase-free water. The qRT-qPCR thermal cycling included cDNA denaturation at 95 °C for 1 min, with 40 cycles of 95 °C for 5 s and 60 °C for 15 s in the Mx3005P Real-Time PCR System  with the SYBR green method. The β-tubulin gene of cassava was chosen as an internal control. All qRT-qPCR experiments were performed in triplicate, and the gene-specific primers used in expression analysis are listed in P < 0.05) after fold treatment with the 2Ct−△△ method.Leaf samples of the Arg7 variety subjected to MeJA, SA, ABA, NaCl, low temperature, mannitol, HMePOD genes  were found to be transcriptionally upregulated after multiple different treatments, suggesting that these genes are good candidates to target for cassava improvement. These findings increase our understanding of POD-mediated stress and hormone responses and storage root deterioration in cassava, laying a foundation for the genetic improvement of cassava.In this study, we identified 91 PODs from the cassava genome and studied their basic classification, protein motif, gene structure, chromosomal distribution, and duplication pattern. Comprehensive transcriptional level analyses revealed the involvement of MePODs in biotic and abiotic stress responses, hormone responses, and storage root deterioration. Several


Nicotiana benthamiana induced cell death, whereas SlLYK12 overexpression did not. Based on our results and comparison with rice OsCERK1, we hypothesize that OsCERK1 orthologs in tomato underwent gene duplication, leading to the subfunctionalization of immunity and symbiosis.Arbuscular mycorrhiza (AM) is a widespread symbiotic relationship between plants and fungi (Glomeromycota), which improves the supply of water and nutrients to host plants. AM symbiosis is set in motion by fungal chitooligosaccharides and lipochitooligosaccharides, which are perceived by plant-specific LysM-type receptor kinases (LYK). In rice this involves OsCERK1, a LYK also essential for chitin triggered innate immunity. In contrast in legumes, the CERK1 homologous gene experienced duplication events resulting in subfunctionalization. However, it remains unknown whether this subfunctionalization is legume-specific, or has occurred also in other dicot plant species. We identified four CERK1 homologs in tomato  and investigated their roles in chitin signaling and AM symbiosis. We found that knockdown of SlLYK12 in tomato significantly reduced AM colonization, whereas chitin-induced responses were unaffected. In contrast, knockdown of SlLYK1 resulted in reduced responses to chitin, but did not alter responses to AM fungi. Moreover, ectopic overexpression of SlLYK1 and SlLYK13 in  N-acetyl-D-glucosamine (GlcNAc)-containing molecules are important microbial signaling factors, and include chitin from pathogenic fungi, peptidoglycan from pathogenic bacteria, Nod factors from symbiotic rhizobia, and Myc factors from symbiotic arbuscular mycorrhizal (AM) fungi. These molecules mediate the initiation of either plant innate immune functions or symbiotic pathways , which has an inactive kinase domain  between 6 and 8 elicit plant immune responses. Such immune responses include the elevation of cytosolic calcium, production of reactive oxygen species (ROS), induction of defense-related gene expression, callose deposition, and pathogen growth restriction . In Arabe domain . After pe domain . In ricee domain . Recentlentified . Thus, CL. japonicus, and LYK3-NFP (NF PERCEPTION) complex in Medicago truncatula  can act as signaling molecules triggering symbiosis; for example, Nod factors from rhizobia. Nod-LCOs are also recognized by the LysM-containing receptors, called NFR1-NFR5 (NF RECEPTOR1 and 5) complex in uncatula . Similartivation .Parasponia andersonii, the only non-leguminous plant that can establish symbiosis with rhizobia, is required for both AM and rhizobial symbioses  for AM symbiosis, a widespread symbiotic relationship occurring between fungi in the phylum Glomeromycota and 80% of terrestrial plant species under phosphorus- or nitrogen-limiting conditions . The AM ymbioses . Similarymbioses , whereasymbioses . Interesymbioses . Howeverymbioses . This raSlLYK12 significantly reduced AM colonization; however, the chitin responses of these plants were similar to those of controls. In contrast, knockdown of SlLYK1 resulted in reduced responses to chitin, but normal responses to AM fungi. In addition, we found that ectopic overexpression of SlLYK1 and SlLYK13 in Nicotiana benthamiana caused cell death; however, SlLYK12 overexpression did not. Taken together, these results suggest a hypothesis whereby an ancestor of CERK1 with dual function in both immunity and symbiosis gave rise to multiple molecules during evolution through gene duplication in tomato, among which SlLYK1 and SlLYK12 were sub-functionalized for a role in immunity and symbiosis, respectively.In this study, we identified the orthologs of the CERK1 subclade in tomato, and investigated their function by knocking down their expression. We found that knockdown of Solanum lycopersicum L. cv Zheza 809 was used for all experiments. For virus-induced gene silencing (VIGS) experiments, seedlings were grown in a plant growth room at 22°C with a 16 h photoperiod. For Rhizophagus irregularis inoculation, plants were grown at 25°C with a 16 h photoperiod.Total RNA was isolated using TRIzol reagent . RNA samples were treated with DNase I to eliminate potential contamination with genomic DNA. qRT-PCR was performed on an Applied Biosystems Plus Real-Time PCR System  using a SYBR premix Ex Taq kit . Primers are listed in Supplementary Table N. benthamiana. All plasmids were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation.The primers used for gene cloning are listed in Supplementary Table GUS , pTRV2-NbPDS , pTRV2-SlLYK1, 12, 13, and pTRV1 were cultivated in YEP medium  for 36 h at 28°C. Cultures were passaged in fresh medium at a dilution of 1:100 and cultivated for a further 8 h. After adjusting the concentration to OD600 = 1.5, each pTRV2 construct was mixed with pTRV1 (1:1) in infiltration buffer . The agrobacterial mix was infiltrated into the abaxial surface of 10-day-old tomato seedlings. Gene silencing efficiency and specificity were determined 4 weeks after agrobacterial infiltration. At least six individual seedlings were analyzed for each construct.Agrobacteria carrying pTRV2-Rhizophagus irregularis was purchased from the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences, and propagated using N. tabacum as the host in 4 L pots containing sand. Plants were watered with a solution without added phosphorous every week. Four months later, the sand containing R. irregularis was harvested and dried to obtain mycorrhizal inoculums. For mycorrhizal inoculation, after VIGS infiltration, each tomato plant was transferred to a 4 L pot containing sand including 4 g of sand-based mycorrhizal inoculums at the base of the roots.Roots were cleared with 10% (w/v) KOH for 1 h at 90°C, acidified with 2% HCl for 5 min, and then stained with trypan blue (0.5 mM). Mycorrhizal colonization was observed under a light microscope . The rate of root colonization was quantified using the grid line intersect method at 3 and 6 weeks after mycorrhizal inoculation, and calculated as the ratio of intersects with hyphopodia, intracellular hyphae, arbuscules and vesicles over all root intersects (100 intersects per plant) × 100 .Leaf disks  were punched and incubated in water for at least 8 h. After addition of water containing 1.25 μM L-012 chemiluminescent probe , 20 μg/mL horseradish peroxidase, and 500 nM chitooctaose , chemiluminescent signals were immediately recorded using a Photek camera  for 30 min.N. benthamiana leaves was detected by trypan blue staining. Excised leaves were vacuum-infiltrated with trypan blue solution (2.6 mM) for 30 min, and incubated for a further 8 h. Leaves were then destained in a solution containing ethanol and glycerol at a ratio of 9:1 at 65°C for 30 min.Cell death in Immunoblot analysis was performed as previously described  using an1.The amino acid sequences of LYK genes are listed in Supplementary Table S. lycopersicum LYKs. The results showed that four genes from S. lycopersicum, SlLYK1, SlLYK11, SlLYK12, and SlLYK13, were clustered into one clade with AtCERK1, similar to the tree constructed by SlLYK1 gene is expressed at similar levels in roots and leaves, and the SlLYK12 and SlLYK13 genes were each primarily expressed in roots and leaves, respectively -treated leaves and AM-inoculated roots; therefore, we did not perform further analysis of the SlLYK11 gene.To identify orthologs of OsCERK1/AtCERK1 in tomato, we generated a phylogenetic tree using the full length amino acid sequences of SlLYK1, SlLYK12, and SlLYK13, we silenced these genes individually using a VIGS approach, which is a powerful tool for the study of AM symbiosis in the tomato  control, but the transcript levels of SlLYK12 and SlLYK13 did not show significant differences . After confirming the silencing effectiveness in roots , roots were inoculated with R. irregularis and grown for another 3 and 6 more weeks. To observe mycorrhizal colonization, roots were harvested and stained with trypan blue. Arbuscules could be observed in plants infiltrated with all constructs . The rate of total root colonization including hyphopodia, intracellular hyphae, arbuscules, and vesicles was calculated using the grid line intersect method . Similarly, the percentage of hyphopodia and arbuscules were significantly reduced in VIGS-SlLYK12- infiltrated plants . Consistent with these results, levels of the SlLYK12 transcript were increased fourfold after R. irregularis inoculation, whereas those of SlLYK1 and SlLYK13 were not .To study the function of e tomato . As the t method . We founSlLYK12 is required for the development of AM symbiosis, we determined the expression levels of a fungal housekeeping gene GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (RiGAPDH), and three AM responsive genes in tomato, including REDUCED ARBUSCULAR MYCORRHIZATION2 , BLUE COPPER-BINDING PROTEIN1 , and PHOSPHATE TRANSPORTER4  .To confirm that al gene) . CompareSlLYK1, SlLYK12, and SlLYK13 are required for chitin signaling, we analyzed their expression after CO8 treatment in 4-week-old leaves and roots. Our results indicated that SlLYK1 gene expression is upregulated sevenfold after CO8 treatment in leaves and threefold in roots. SlLYK13 is also slightly upregulated in leaves after CO8 treatment, whereas SlLYK12 did not show significant upregulation in either leaves or roots . These results suggest that SlLYK12 might not have a role in chitin signaling. Next, we examined the responses of leaves infiltrated with VIGS-SlLYK1, -SlLYK12, -SlLYK13, and VIGS control to CO8 elicitation. First, ROS generation was measured after CO8 treatment, using a luminol-based chemiluminescence detection system. We found that VIGS-SlLYK1-inoculated leaves showed reduced ROS levels after CO8 treatment compared to the VIGS control, whereas plants infiltrated with VIGS-SlLYK12 and -SlLYK13 did not exhibit lower ROS levels . Second, transcript levels of SlWRKY53 (the ortholog of AtWRKY53), a typical chitin response gene . Third, MAP kinase activity was analyzed by immunoblot assay. CO8 treatment causes MAP kinase phosphorylation, as demonstrated using an anti-p42/p44-MAPK antibody in VIGS control plants; however, levels of phosphorylation were significantly reduced in VIGS-SlLYK1-inoculated plants . Similar to the results in leaves, CO8-triggered SlWRKY53 gene expression and MAPK phosphorylation in roots was only reduced in VIGS-SlLYK1 infiltrated plants . Together, these results suggest that SlLYK1 plays a role in chitin signaling.To determine whether nse gene , were deAtCERK1 in N. benthamiana leaves results in symptoms of cell death  cDNA driven by the CaMV 35S promoter, respectively, and the resulting constructs were transiently expressed in N. benthamiana leaves using the same amount of agrobacteria. A positive control, overexpression of AtCERK1-GFP, caused leaf chlorosis and tissue collapse in the entire infiltrated region 3 days after agrobacterial infiltration. Compared with AtCERK1-GFP, overexpression of SlLYK13-GFP resulted in similar levels of cell death . Overexpression of SlLYK1-GFP did not result in obvious tissue collapse; however, dead tissues (dark blue in color) were visible after trypan blue staining . This symptom of cell death was never observed on overexpression of SlLYK12-GFP .The results described above (sections “SlLYK12 Is Required for AM Symbiosis” and “SlLYK1 Is Required for Chitin Responses”) suggest that SlLYK1 is required for chitin signaling, while SlLYK12 is involved in AM symbiosis; hence, we wished to determine the function of SlLYK13. AtCERK1 has a chitin-independent role in cell death , and ectll death ; therefoSlLYK1 (K355E) and SlLYK13 (K328E). Our results showed that SlLYK1 and SlLYK13 lacking kinase activity did not cause symptoms of cell death . All constructs were verified by confocal microscopy observation of the green fluorescence signal and immunoblot analysis to determine protein size . Since tissue collapse leads to degradation of target proteins, SlLYK1- and SlLYK13-GFP proteins could not be detected by either method; however, SlLYK12-, SlLYK1(K355E)-, and SlLYK13(K328E)-GFP all showed green fluorescence signals in the cell periphery as expected for proteins predicted to localize in the plasma membrane . Taken together, our results suggest that SlLYK13 and SlLYK1 have redundant functions in cell death, while overexpression of SlLYK13 could cause more severe symptoms of cell death.We then examined whether SlLYK1- and SlLYK13-induced cell death were dependent on their kinase activities, by generating constructs with inactive kinase domains; Glycine max, G. soja, Cajanus cajan, Lupinus angustifolius, M. truncatula, and L. japonicus), three Solanaceae species , three Cruciferae species , and two Gramineae species (Oryza sativa and Zea mays). Finally, full length amino acid sequences encoded by 48 genes from 14 species were used to construct the phylogenetic tree .To decipher the evolutionary relationships among LYK family proteins correlated with their functions in immunity and symbiosis, we constructed a phylogenetic tree by analyzing the protein sequences of CERK1 homologs from six Leguminosae species . Monocotyledonous maize and rice only have one or two CERK1 paralogs, whereas dicotyledonous species other than the Cruciferae have evolved several homologs, suggesting that the CERK1 family has experienced duplication events in dicotyledonous species during their evolution. The CERK1 homologs in Solanaceae and Leguminosae species are clustered into three clades: a Leguminosae-specific clade (blue), containing the Nod factor receptor NFR1/LYK3 and chitin receptor LjCERK6; a Solanaceae-specific clade (olive), including the immune receptors SlLYK1 and SlLYK13; and a mixed clade (red). SlLYK12, the potential Myc-factor receptor, was assigned to the mixed clade, which contains LYKs from both Solanaceae and Leguminosae species, suggesting that an ancestral gene duplication event occurred before the divergence of the Solanaceae and Leguminosae species. As OsCERK1 has a dual role in immunity and symbiosis, we hypothesize that an ancestor molecule in dicotyledons, which was responsible for both immunity and symbiosis, underwent gene duplication, leading to the sub-functionalization for a role in immunity and symbiosis, respectively.Similar to other reported phylogenetic trees of LysM receptor proteins or kinases , CERK1 hIn this study, we identified the orthologs of CERK1 in tomato, and investigated their function using a VIGS approach. Unlike rice OsCERK1, which has dual roles in chitin and AM symbiosis , we founArabidopsis  is completely independent of chitin signaling , a Solanaceae-specific clade (SlLYK1), and a mixed clade (SlLYK12-LjLYS7-MtLYK8). Consistent with the function of SlLYK12 in AM symbiosis, the SlLYK12-LjLYS7-MtLYK8 clade does not contain genes from rhizobia , suggestanalysis . Given tanalysis . Accordiymbiosis . All theymbiosis , so it irception . TherefoYL designed the research and wrote the manuscript. DL and XS performed most of the experiments. NW contributed to analysis of gene expression. FS designed primers for VIGS constructs and provided technique support for VIGS approach.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.