The prevalence of obesity has reached alarming levels, which is considered a major risk factor for several metabolic diseases, including type 2 diabetes (T2D), non-alcoholic fatty liver, atherosclerosis, and ischemic cardiovascular disease. Obesity-induced chronic, low-grade inflammation may lead to insulin resistance, and it is well-recognized that macrophages play a major role in such inflammation. In the current review, the molecular mechanisms underlying macrophages, low-grade tissue inflammation, insulin resistance, and T2D are described. Also, the role of macrophages in obesity-induced insulin resistance is presented, and therapeutic drugs and recent advances targeting macrophages for the treatment of T2D are introduced. The incidence of obesity is widespread in both developing and developed countries . It is eAmong them, obesity is closely related to the development of insulin resistance. Insulin target organs, such as adipose tissue, skeletal muscle, and liver, are affected by obesity at the morphological, functional, and molecular levels . As one Many molecular mechanisms have been proposed between obesity, insulin resistance, and T2D, including endoplasmic reticulum stress, oxidative stress, lipid homeostasis dysregulation, mitochondrial dysfunction, and hypoxia , but theThis review focuses on the molecular mechanisms between macrophages, chronic inflammation, insulin resistance, and T2D. We discuss the polarization, distribution, and accumulation of macrophages in insulin target organs such as adipose tissue, skeletal muscle, and liver, and summarize the relationship between macrophages and insulin resistance, as well as therapeutic drugs and recent advances targeting macrophages for the treatment of T2D.Macrophages are a type of phagocyte and antigen-presenting cell, which can secrete trophic factors, immune mediators, and effectors, phagocytose pathogens, or cell debris, as well as process antigen and present antigen on the cell surface ,33. DiveIt has also been pointed out that the use of M1 and M2 to classify macrophages is too polarized; the phenotype of macrophages is determined by many stimulating factors and cannot be generalized by limited subtypes . New subInstead of fixed states, M1/M2 polarization is a dynamic process that can be reversed under physiological and pathological conditions . A varie+, CD11b+, CD206+, and CD301+ cells, while human ATMs are characteristically CD14+/CD16− and express markers CD68, CD163, CD204, and CD206. It has been established that ATMs in humans barely express markers for M1 and M2 classification, such as iNOS and arginase-1 [hi, CD11bint, CD68+ cells. Additionally, T-cell immunoglobulin, mucin domain containing 4 (Tim4), and C-type lectin domain family 4 member F (Clec4F) have been described as surface markers specific to KCs [+, Cx3cr1+, Ly6c+, CCR2. When KCs are depleted or tissues are injured or inflamed, monocytes become significant contributors to the liver macrophage pool [Macrophages can also be classified based on their locations and functions. Tissue-resident macrophages are different from monocytes that come from the bone marrow and circulate in the blood . Differeginase-1 . Furtherc to KCs ,59. The age pool . There iage pool .Macrophages in insulin target the organs, including adipose tissue, liver, and muscle, that play a central role in inflammation and insulin resistance . In the + ATMs, which are closely associated with vasculature [+ monocyte-derived ATM subpopulations based on differential expression of MHCII, CD11c, and CX3CR1 [Adipose tissue is a reservoir of fatty acids during fasting. When glucose levels have returned to normal after meals, free fatty acids (FFAs) are released into the circulation by adipose tissue and used by other tissues as an energy source . They arculature ,72. Furtd CX3CR1 ,73,74. TATMs are mainly in the M2 polarization state in the lean state . PreventOne of the hallmarks of obesity-related chronic inflammation is the accumulation of ATMs . MacrophThere are also differences in the distribution of macrophages in adipose tissue between lean and obese states: macrophages are evenly distributed throughout the adipose tissue in the lean state; while in the obese state, macrophages mainly appear in ring-like structures around dying adipocytes, known as crown-like structures (CLSs), to phagocytose the cells and continuously release proinflammatory cytokines . Dying aThe liver is the largest solid organ in the body . It playThe two macrophage populations in the liver include Kupffer cells and monocyte-derived macrophages. During obesity, Kupffer cells are activated to the M1 state, which attracts inflammatory monocytes into the liver and promotes their differentiation into monocyte-derived macrophages ,96, resu3, a specific inhibitor of Kupffer cells, phagocytosis, and other functions of Kupffer cells were inhibited, and whole-body glucose tolerance was increased in mice [Chronic inflammation is known to induce insulin resistance in the liver ,103. Kup in mice . Moreove in mice , and lip in mice .Muscle mass is an important determinant of glucose tolerance, as less muscle mass has been associated with greater insulin resistance ,112. Inc+ macrophages in the skeletal muscle of obese non-diabetic subjects is increased compared to lean subjects [+ F4/80+ macrophage numbers in muscle compared to lean mice [Insulin resistance in skeletal muscle is caused by impaired insulin signaling and many post-receptor intracellular defects including impaired glucose transport, glucose phosphorylation, and reduced glucose oxidation and glycogen synthesis. Macrophage recruitment into skeletal muscle tissue is also involved in promoting insulin resistance ,85,116. subjects . Studiesean mice ,119. Durean mice . The chrean mice ,120, andean mice   also induces insulin resistance through the JNK pathway . LTB4 isIn addition to the JNK signaling pathway, insulin resistance is also closely related to NF-κB activation, which is induced by TNF-α . NF-κB iIL-1β participates in insulin resistance through NF-κB pathway. IL-1β belongs to the IL-1 superfamily and is produced mainly by monocytes and macrophages and regulated by inflammasome activity . The levMoreover, monocytes and tissue-resident macrophages express high levels of PPAR-γ . PPAR-γ IL-6 plays a crucial role in regulating metabolism and immunity. It is also elevated in obese patients compared to lean controls, which is detrimental to metabolic balance . IL-6 woCytokines derived from macrophages, including TNF-α, IL-6, and IL-1β, could influence lipolysis. TNF-α affects lipid metabolism by inducing lipolysis, inhibiting FFAs uptake and lipoprotein lipase activity . StarnesFurthermore, galectin-3  is a member of the galectin family that induces insulin resistance ,193. GalIn addition to cytokines secreted from or related to macrophages, macrophage autophagy is crucial for the regulation of systemic insulin sensitivity . In macrIn addition to macrophages, immune cells residing in adipose tissue and intestines, including cells of innate and adaptive immune systems, are also involved in chronic inflammation caused by obesity. In the process of obesity, immune cells, including Innate lymphoid type 1 cells (ILC1s), Innate lymphoid type 2 cells (ILC2s), eosinophils, mast cells, dendritic cells (DCs), neutrophils, T cells, and B cells, influence the polarization and recruitment of macrophages and play important roles in chronic inflammation and insulin resistance.ILC2s and eosinophils participate in maintaining metabolic homeostasis states partially by maintaining the M2 polarization state of macrophages. ILC2s are a family of innate immune cells that mirror T cells . They seMast cells are first-line responders to invading pathogens by rapid degranulation ability , produci+ and CD8+ T cells by their phenotypic expression of the surface coreceptors. Increased recruitment and differentiation of CD8+ T cells have been found in both animal and human obese WAT [+ T cells, CD8+ T cells are mainly considered cytotoxic. CD8+ T cells secrete perforins, granzymes, and cytokines that regulate the development and activation of macrophages [+ T cells in WAT was found in obese mice, which induces adipose tissue inflammation and attracts macrophage recruitment [+ T cells could lower macrophage infiltration and adipose tissue inflammation and improve insulin resistance [T cells, B cells, and macrophages are all in CLSs surrounding necrotic adipocytes . Accumulbese WAT . T cell bese WAT . The numbese WAT ,216. Adobese WAT . Moreovebese WAT . Treg cebese WAT . Treg cebese WAT . In controphages . Increasruitment . Depletisistance .The impaired adaptive response of B cells leads to inflammation in obese people . B cellsAntidiabetic drugs act on adipose tissue and muscle to enhance insulin sensitivity and target the liver to inhibit glucose production, or stimulate β-cells to release insulin, thereby slowing the development of insulin resistance. However, they are not able to completely prevent or reverse the development of T2D, urgently necessitating new antidiabetic drugs . CurrentTNF-α is identified as a potential target to treat insulin resistance in obesity; it is a classic proinflammatory cytokine and induces M1 polarization of macrophages. Treatment with shRNA to downregulate TNF-α contributes to improving IRS-1 phosphorylation and insulin response, which improves insulin sensitivity . Burska ClinicalTrials.gov identifier: NCT00303394) [ClinicalTrials.gov identifier: NCT01327846) and shows modest improvement in HbA1c, glycemia, and insulinemia [IL-1β is elevated in obesity and induces insulin resistance by inhibiting the insulin signaling pathway ,233. IL-0303394) . Human m0303394) . Canakinulinemia . AdditioIL-6 is another proinflammatory cytokine that participates in the development of T2D. Human monoclonal antibody Tocilizumab could inhibit IL-6 signaling selectively, and treatment with Tocilizumab causes a significant reduction of HOMA-IR and insulin resistance in non-diabetic patients with RA . These sMacrophages could secrete miRNAs encapsulated in extracellular vesicles to exert anti-inflammatory or proinflammatory effects, and miRNAs may also contribute to the development of insulin resistance by regulating macrophage polarization. Since miRNAs are endogenous regulators whose levels are often deregulated in T2D, the therapeutic goal is to restore miRNAs to normal levels . PresentMoreover, therapeutic drugs with folate conjugates could selectively impair FR active macrophages . FR is aAccumulating evidence showed that macrophages play central roles in chronic inflammation and insulin resistance induced by obesity. Macrophages participate in inflammatory pathways including JNK, IKK/NF-κB, and JAK/STAT through secreting inflammatory factors, such as TNF-α, IL-6, IL-1β, and LTB4, thereby inducing chronic inflammation of adipose tissue, liver, and muscle, further leading to insulin resistance. Other immune cells, including ILC2s, eosinophils, mast cells, DCs, neutrophils, T cells, and B cells, affect the polarization state of macrophages, which in turn participate in the induction of insulin resistance and T2D. In recent years, there have been many discoveries in the field of immune metabolism, and direct or indirect targeting of macrophage-secreted factors or macrophage-related markers can improve insulin resistance and reduce the development of T2D by mechanisms including enhancing insulin sensitivity, reducing oxidative stress, and reducing inflammation et al. Current research hotspots include targeted therapy of TNF-α, IL-1β, IL-6, and miRNAs. Although many problems remain unsolved, macrophages may be considered as promising targets for the treatment of T2D.


Ovarian cancer (OC) ranks first in cancer-related deaths out of all female reproductive malignancies with high-pitched tumor relapse and chemoresistance. Several reports correlate cancer occurrences with exposure to xenobiotics via induction of a protein receptor named aryl hydrocarbon receptor (AhR). However, the effect of AhR on OC proliferation, expansion, and chemoresistance remains unrevealed. For this purpose, OC cells A2780 and A2780cis cells were treated with AhR activator, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and the effects were determined by Real-Time Cell Analyzer, clonogenic assay, flow cytometry, immunoblotting and wound healing assay. Our results showed that activation of AhR by TCDD in A2780 cells induced the PI3K/AKT pathway followed by induction of anti-apoptotic proteins BCL-2, BCL-xl, and MCL-1. In addition, a significant increase in stemness marker aldehyde dehydrogenase (ALDH1) was observed. This effect was also associated with an accumulation of β-catenin, a Wnt transcription factor. Moreover, we observed induction of epithelial to mesenchymal transition (EMT) upon AhR activation. In conclusion, the results from the current study confirm that AhR mediates OC progression, stemness characteristics, and metastatic potential via activation of PI3K/Akt, Wnt/β-catenin, and EMT. This study provides a better insight into the modulatory role of AhR that might help in developing novel therapeutic strategies for OC treatment. Ovarian cancer (OC), globally the most lethal gynecological malignancy, ranks fifth in terms of cancer-related deaths . RegardlAhR is a cytoplasmic ligand-activated transcriptional factor associated with several cellular and physiological processes in the cell . Upon biTherefore, in our study, we examined the role and involvement of AhR in OC progression and chemoresistance and the underlying mechanisms of AhR in the pathogenesis of ovarian cancer. We observed that AhR induction by a potent activator TCDD promoted cell proliferation in A2780 cells by activating the PI3K/AKT pathway. We also observed an upregulation of anti-apoptotic and stemness markers further confirming AhR mediated induction of chemoresistance (by evading apoptosis) and stemness-like characteristics. Moreover, AhR induction also promoted EMT in A2780 cells. The results from our study provide a better insight into the modulatory role of AhR that might help in developing novel therapeutic strategies for OC treatment.We first identified the basal expression of AhR and its regulatory proteins, CYP1A1 and CYP1B1, in all seven human OC cells. The TCDD-inducible expression levels of AhR and BCL-2 proteins were analyzed by immunoblotting in three OC OVCAR3, SKOV3, and A2780 cells, which showed the highest basal expression, treated with TCDD . Initially, we examined the impact of AhR induction using TCDD on the cell viability of both wild-type A2780 and resistant A2780cis cells. For this purpose, both cells were treated with wide doses of TCDD ; thereafter, RTCA analysis and clonogenic assay were conducted. RTCA analysis A and cloNext, we assessed the effect of AhR activation by TCDD 10 nM for 24 h on the expression patterns of AhR and BCL-2 genes in both wild-type and resistant cells. Interestingly, we observed a differential expression of AhR and its regulatory proteins in A2780 and A2780cis cells. At the basal levels, To explore the mechanisms of AhR-mediated chemoresistance in OC, we investigated TCDD-mediated effects in modulating apoptosis, cell cycle phases, and level of DNA damage in n both cells. Flow cytometric analysis revealed that treatment of A2780 cells with TCDD 10 nM decreased the percentage of cells undergoing early/late apoptosis to 21.4% A and incTo investigate whether AhR induction would regulate the PI3K/Akt/Cyclin D1 pathway, we measured the protein expression of PI3K, AKT, and cyclin D1 in both A2780 and resistant A2780cis cells. As shown in To further explore whether AhR is mediating CSCs properties, self-renewal, and maintenance, in OC A2780 and A2780cis cells, we examined the expression of three proteins, GLI-1, NOTCH1, and β-catenin, the downstream proteins for Hedgehog, NOTCH, and WNT pathways, respectively ,19. FiguWe then inspected the involvement of EMT and invasion in response to AhR activation by measuring the epithelial/mesenchymal markers and the metastasis potential. As illustrated in Despite the fact that most patients respond to primary therapy in OC, a higher rate of relapse due to chemoresistance is unfortunately observed at a surprising 70% occurrence rate in OC, leading to an inevitable death rate of 90% of patients with relapse . EnvironSeven human OC cell lines have been utilized in the current study to initially investigate and correlate the expression patent of AhR and regulated genes with the aggressiveness of cancer. Among these cells, OVCAR3, SKOV3, and A2780 cells exhibited higher protein expression levels of AhR, CYP1A1, and CYP1B1, which was associated with a proportional increase in the expression of anti-apoptotic proteins, BCL-2, BCL-xl, and MCL-1. To the best of our knowledge, this is the first report that describes the crosstalk between AhR and BCL-2 in OC and proposes a role of the AhR/CYP1 pathway in OC chemoresistance. The anti-apoptotic role of AhR was previously reported in breast cancer , UVB-irrThe availability of resistant OC cells (A2780cis) allows us to further assess the involvement of AhR in the chemoresistance of OC cells which showed a differential expression of the downstream proteins of AhR and BCL-2 pathways in cisplatin-resistant A2780cis. AhR induction by TCDD in wild-type A2780 cells induced both AhR as well as BCL-2 family proteins, suggesting crosstalk between AhR and BCL-2 in chemoresistance. These results were further confirmed by our observations that AhR induction reduced the proportion of apoptotic OC cells, increased the cells in the G0–G1 phase, and decreased the DNA double-strand breaks. Surprisingly, A2780cis cells were resistant to the anti-apoptotic effect of AhR activation as evidenced by no induction of the levels of BCL-2 proteins, cell cycle phases, and DNA strand break. Although the mechanisms of resistance are not fully understood, this effect could be correlated to the property of drug efflux mechanisms carried out by chemoresistant cell lines . Perhaps the most interesting part of the study is exploring the molecular mechanisms modulating the AhR chemoresistance in OC. In this study, we specifically explored three pathways, PI3k/Akt, stemness, and EMT. The PI3K/AKT pathway is one of the most frequently upregulated pathways in several cancers and is found to contribute to cancer cell proliferation, invasion, metastasis, and drug resistance ,27. In lA previous study in breast cancer MCF-7 cells showed that activation of AhR/CYP1A1 increased the chemoresistance by activating the Wnt/β-catenin and ALDH pathways , pathwayTCDD was procured from Toronto Research Chemicals . The antibodies against AhR, CYP1A1, CYP1B1, BCL−2, MCL−1, BCL−xl, and ALDH1A1 were purchased from Santacruz Biotechnology , while antibodies against Bax, Oct−4, Nanog, CD−133, E−Cadherin, Vimentin, β−Catenin, PI3K, phospho−PI3K, AKT, phospho−AKT, cyclin D1, GLI−1, NOTCH−1, and GAPDH were bought from Cell Signaling Technologies . All kits for flow cytometric analysis were obtained from BD Biosciences . 2- 95% air atmosphere. Human OC cell lines, PA-1 (ATCC CRL-1572TM), OVCAR-3 (ATCC HTB-161TM), CAOV-3 (ATCC HTB-75TM), SKOV-3 (ATCC HTB-77TM), SW 626 (ATCC HTB-78TM) were obtained from the American Type Culture Collection . A2780 (ECACC 93112519), and cisplatin resistant A2780cis (ECACC 93112519) human OC cells were ordered from European Collection of Authenticated Cell Cultures . The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20% serum (FBS), and 1% antibiotic solution (penicillin 100 mg/mL/streptomycin 10 mg/mL). The cell lines were cultured at 370C in a humidified 5% COhttps://imagej.nih.gov/ij/ accessed on 1 May 2022).Protein extraction was carried out as described previously . Fifty mThe real-time cell analyzer was used to analyze the cell index of untreated and TCDD treated A2780 and A2780cis cells. Briefly, 50,000 cells were seeded in electrodes containing E-Plate 16 as reported before . After 2v/v) solution. The colonies in each well were then stained with crystal violet (0.5% w/v) for 1 h. The colonies were then washed gently in the water, dried, and imaged by a charged-coupled device camera (Nikon D3200).Approximately 4000 cells/well seeded in 12-well plates, with and without varying concentrations of TCDD, and grown for two weeks for developing colonies. Upon incubation, the wells were first washed gently with PBS followed by fixing using glutaraldehyde  as per the manufacturer’s protocol on BD LSRFortessa analyzer (BD Biosciences) as explained previously .A2780 and A2780cis cells untreated and treated with TCDD were trypsinized, washed, and stained with PI/RNAase solution (25 μg/mL), and the cell cycle stages were examined by flow cytometry by BD LSRFortessa analyzer as explained before .A2780 and A2780cis cells untreated and treated with TCDD were fixed, permeabilized, and then stained with 3 μL of pH2AX (pS139)-Alexa Fluor 647 antibody for 30 min. The cells were then washed with FBS and the content and expression of phosphorylated H2AX were determined by the BD LSRFortessa cell analyzer .Approximately 90% of confluent A2780 and A2780cis cells were scratched from one end of the wall to the other using a sterile 200 μL pipette tip. After washing out the detached cells carefully using PBS, the cells were incubated in fresh media for 24 h without and with 10 nM TCDD. Migration of cells was observed at 0 and 24 h using an Evos microscope, and images were captured at 4× magnification. The experiment was performed in triplicate.p ≤ 0.05 was considered statistically significant. Data are shown as mean ± SD from three different experiments.Statistical analysis was carried out using Graph Pad Prism 9.0 software. The differences between control and treatment groups were evaluated using one-way ANOVA, and Dunnett’s test. Taken into account, the current study provides the first evidence that the AhR/CYP1A pathway regulates OC progression via the activation of PI3K/AKT, stemness characteristics via the activation of Wnt/β-catenin and NOTCH pathways, and induces metastasis via inducing epithelial to mesenchymal transition . A furth


KRAS, leading to marked overexpression of WT KRAS protein. We previously demonstrated that SHP2 phosphatase, which acts to promote KRAS and downstream MAPK pathway activation, is a target in these tumors when combined with MEK inhibition. We hypothesized that SHP2 inhibitors may serve as a foundation for developing novel combination inhibitor strategies for therapy of KRAS-amplified GEA, including with targets outside the MAPK pathway. Here, we explore potential targets to effectively augment the efficacy of SHP2 inhibition, starting with genome-wide CRISPR screens in KRAS-amplified GEA cell lines with and without SHP2 inhibition. We identify candidate targets within the MAPK pathway and among upstream RTKs that may enhance SHP2 efficacy in KRAS-amplified GEA. Additional in vitro and in vivo experiments demonstrated the potent cytotoxicity of pan-ERBB kinase inhibitions in vitro and in vivo. Furthermore, beyond targets within the MAPK pathway, we demonstrate that inhibition of CDK4/6 combines potently with SHP2 inhibition in KRAS-amplified GEA, with greater efficacy of this combination in KRAS-amplified, compared with KRAS-mutant, tumors. These results suggest therapeutic combinations for clinical study in KRAS-amplified GEAs.Gastroesophageal adenocarcinomas (GEAs) harbor recurrent amplification of  Gastroesophageal adenocarcinomas (GEAs) were responsible for more than 1 million new cases of cancer in 2020 and an estimated 769,000 deaths, making them, collectively, the fifth most frequently diagnosed cancer and the third leading cause of death due to cancer , making KRAS, which is renowned for activation by missense mutation in many cancers, is recurrently subject to focal high-level amplification without mutation. This KRAS amplification is frequently found in CIN-type gastric cancer  and esophageal adenocarcinoma (10.4%).Recent studies from The Cancer Genome Atlas and other groups reported molecular classification of gastric cancer using different modalities, such as somatic copy-number analysis, whole-exome sequencing, and mRNA-Seq –6. TheseKRAS-amplified (KRAS-amp) GEA and found that these tumors, relative to KRAS-mutant (KRAS-mut) GEA models, are relatively insensitive to MEK inhibition. Deeper study revealed that MEK inhibition led to adaptive resistance resulting from marked activation of KRAS and the resulting induction of the PI3K-AKT pathway. Because we found the induction of KRAS activation in the setting of marked overexpression of WT KRAS mediated this adaptive resistance, we evaluated the potential to target physiologic mediators of KRAS activation to enhance efficacy of MEK therapy. SHP2, encoded by the PTPN11 gene, is a tyrosine phosphatase that acts downstream of RTK and directly promotes physiologic activation of KRAS. SHP2 promotes nucleotide exchange in KRAS proteins, leading to transition from the inactive GDP-bound form to the active GTP-bound form in concert with guanine nucleotide exchange factors such as SOS1 . Prior to the screen, we determined the concentration of SHP099 to be used in the screening, aiming to find a dose with evidence of target engagement and with sufficient proliferation in KRAS-amp models to enable the discovery of secondary targets to enhance activity (https://doi.org/10.1172/jci.insight.152714DS1). Because SHP2 promotes RAS activation  were those directed at SHOC2, a protein that dephosphorylates a critical inhibitory site on RAF kinases   , where l2 fold change (LFC) in guide detection in both KE-39 and HUG-1N data  and 1 patient-derived cell line (CAT12)   . We alsoRASG12D) .BRAF, CRAF (RAF1), and SHOC2 among the top depleted genes (denoting sensitization to SHP2i), and NF1 and PTEN were among the top enriched hits (denoting resistance). We then evaluated the composite group of KRAS-amp models from our secondary screen. Again, NF1 was the top enriched gene when silenced. On average, the top 10 depleted genes were SHOC2, RIC1, GAREM1, CRKL, RAPGEF1, RUNX1, BRAF, TCF7L2, CHD2, and VPS16 in the KRAS-amp cells. These included genes related to the MAPK pathway but also others, including RIC1, GAREM1, CRKL, RUNX1, TCF7L2, CHD2, and VPS16  encoding several RTKs, such as EGFR, ERBB2, SRC, and PTK2 , were relatively depleted in the SHP099-treated cells when compared with vehicle control. Although none of these genes except SHOC2 are universal among the KRAS-amp different cell lines, these results clearly indicate that targets related to cellular signaling both upstream and downstream of KRAS are potential modes of enhancing SHP2 therapy  . By contrast, silencing of distinct RTKs showed minimal effects on GSU when added to SHP2 inhibition. Although these screening results broadly indicated that direct MAPK blockade may be the optimal target to enhance SHP2 efficacy, they did suggest additional targets for evaluation and suggested that upstream RTK inhibition may better complement SHP2i in KRAS-amp, compared with KRAS-mut, GEA.We also compared our results from the RASG12D) . As in tKRAS-amp GEA, starting with those candidate targets upstream of RAS. We specifically chose the tyrosine kinase inhibitors lapatinib, bosutinib, and defactinib to inhibit SHP2 and either ERBB families, SRC families, or FAK, respectively, given that these kinases scored in our screen. We used 10 nM MEK1/2 inhibitor trametinib as a positive control, given the established capacity of this agent to potentiate SHP2 inhibition. Because both ERBB2 and EGFR scored separately in our screen, but we noted variability of the effects of ERBBs inhibition, we chose to evaluate lapatinib, which targets both receptors as either a homodimer or heterodimer. We exposed KE-39 to 1 μM lapatinib (a pan-ERBB inhibitor), bosutinib , and defactinib (an FAK inhibitor) with and without 5 μM SHP099.Next, we examined combination strategies with SHP099 in With SHP099, p-ERK was more substantially suppressed when combined with lapatinib or bosutinib, indicating the possibility of a combination strategy using these inhibitors with SHP2i . In timeKRAS-amp cell lines but that, again, the addition of defactinib to SHP099 had weaker effects. Similarly, defactinib did not show either suppression of MAPK pathway  and found that the combination of SHP099 with lapatinib markedly decreased growth relative to lapatinib or SHP099 alone in multiple KRAS-amp cell lines   . Furtherpatinib) .KRAS-amp GEA, with inhibitors of the ERBB family emerging as lead candidates for combination.No apparent ill effects were observed in these experiments. No significant weight loss was observed . These sNext, we examined combination strategies with SHP2 and targets downstream of KRAS. The CRISPR screening data identified SHOC2 as well as CRAF and BRAF as candidate targets. However, we recognized the limitations of the CRISPR data in this area, given the presence of many paralogs , wherein genetic silencing of individual genes may not phenocopy the effects of inhibitors, which can block pairs of similar targets. Therefore, we decided to more broadly evaluate targets downstream of KRAS.KRAS-amp GEA. Specifically, we evaluated not only trametinib (a MEK1/2 inhibitor) but also LXH254 (a BRAF/CRAF inhibitor) and SCH772984 (an ERK inhibitor). Each agent could, as monotherapy, suppress downstream of MAPK pathway with the use of p-RSK as a complement to p-ERK with the evaluation of SCH772984 effects, because suppression of p-ERK did not last for 24 hours     or modes, 10.44) , dependi, 10.44) .KRAS-amp GEA.No apparent ill effects were observed in these experiments. No significant weight loss was observed , A and BKRAS, without activating missense mutations, has been described in a variety of cancers but most commonly within the CIN-type GEAs. The oncogene KRAS has been heavily studied as a frequently mutated gene in various cancers, including colorectal, pancreatic, and lung cancers. By contrast, there has been minimal study of the effects of marked overexpression of the WT KRAS protein that occurs with genomic amplification. Our group previously reported  and GDP (inactive) status. Beyond the substantial overexpression of KRAS, this plasticity is distinct from that of typical KRAS-mut cells such as KRASG12D, which leads to defective GAP-mediated GTP hydrolysis. In our prior study .Our results show some parallels to efforts to establish combinations for otherapy , 31 thanoncogene . HoweverKRAS-mut cancers . Furthermore, a phase I/II trial of adagrasib (MRTX849) in combination with TNO155 in patients with advanced solid tumors with KRASG12C mutation is ongoing . Besides TNO155, another SHP2i, RMC-4630, is in clinical trials with cobimetinib (an MEKi) in relapsed or refractory solid tumors, and with osimertinib in EGFR+, locally advanced, or metastatic non–small cell lung cancer . Given the ability of SHP2 inhibition to inhibit signaling into the MAPK pathway, which can both attenuate basal signaling into the MAPK pathway and abrogate cellular adaptations in response to targeted inhibitors, SHP2 has emerged as a highly promising target for combinatorial therapies in tumors with RAS and/or RTK driving alterations.There is already a host of trials evaluating SHP2is in distinct combinations for cancer, including a newer, more potent derivative of SHP099, TNO155 , and RMCThere are several limitations to this study. First, we could not clarify whether the effects of lapatinib are mediated by inhibition of EGFR kinase activity, HER2 kinase activity, or both. However, genomics of GEAs shows that both ERBB2 and EGFR are subject to amplification in GEA. Moreover, ErbB receptors, including EGFR and HER2, form both homo- and heterodimers, providing a high degree of downstream signaling diversity  and the KRAS-amp GEA tumors, highlighting the potential to develop effective SHP2-based combinations in these tumors without the need to include inhibitors of the MAPK pathway. This could directly lead to additional trials of effective yet clinically tolerable combinations for this deadly group of cancers.This study highlights that tumors with amplification of WT KRAS represent an especially promising area for development of SHP2 inhibition. Furthermore, we demonstrated the efficacy of SHP2–CDK4/6 co-inhibition and SHP2–pan-ERBB co-inhibition as highly promising candidate strategies against l-glutamine, and maintained at 37°C in a 5% CO2 incubator. All cells were routinely tested for Mycoplasma and found to be free of contamination.KE-39, HUG1-N, YCC1, and GSU cell lines were provided by the Dana-Farber Cancer Institute Belfer Institute, which had obtained them directly from commercial sources and authenticated them using standard short tandem repeat analysis. CAT12, a patient-derived xenograft, was generated at the University of Chicago, after obtaining written patient consent from a 72-year-old male patient with a poorly differentiated cT3N3M1 esophagogastric adenocarcinoma. KE-39, CAT12, HUG1-N, and GSU cells were grown in RPMI 1640 medium supplemented with 10% FBS. YCC1 cells were grown in DMEM and supplemented with 10% FBS. All cell lines were supplemented with 1 mM penicillin/streptomycin and 2 mM SHP099, LXH254, and ribociclib (LEE011) were provided by the Novartis Institutes of Biomedical Research. Lapatinib  was purchased from LC Laboratories. Bosutinib, defactinib, SCH772984, and trametinib were purchased from Selleck Chemicals. All inhibitors were dissolved in DMSO before use.g for 2 hours in 12-well plates with 3 million cells per well. Fresh medium was added at a 1:1 ratio after centrifugation, and cells were transferred to flasks the next day. Infected cells were selected with 2 μg/mL puromycin from days 4 to 8, expanded for another 4 days to reach the desired cell number, and split into parallel cultures with DMSO or 5 μM SHP099 treatment. At least 40 million cells were used for each treatment condition to keep the minimum presentation number for each sgRNA at greater than 500 cells. Cells were exposed to drug treatment for 2 weeks (HUG1-N) or 3 weeks (KE-39), and genomic DNA was collected using pH 7.9 Phenol/Chloroform/IAA  according to the manufacturer’s protocols and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). PCR and sequencing were performed as described previously . P values per gene were calculated as the average –log10 of individual sgRNAs. The average LFC per gene also was calculated to assess the magnitude of effect.Pooled lentiviruses for the genome-wide sgRNA library  and an additional 46 genes, given druggability and biologic interest, were integrated into the library, which comprised 509 genes in total  or RealTime-Glo MT Cell Viability Assay  according to manufacturer’s instructions.5 to 3 × 105 cells in 6-well plates. Cells were exposed to DMSO or inhibitors with medium, and drugs were replaced every 3 days. After 7 to 10 days, cells were fixed in 1% paraformaldehyde for 15 minutes at room temperature, then were washed with PBS and stained with 0.1% crystal violet solution for 15 minutes at room temperature. After staining, plates were air dried and scanned to image.We plated 1 × 105 cells were plated in a 6-well plate. After overnight incubation, cells were exposed to DMSO or indicated inhibitors for 24 hours and then fixed with 70% ethanol at 4°C for 30 minutes. Fixed cells were washed with PBS containing 1% FBS and then were stained with PI/RNase Staining Solution  for 30 minutes at room temperature. DNA content was measured by LSR II flow cytometer and analyzed by using ModFIT LT software.A cell cycle assay was performed as previously described . BrieflyThe RAS G-LISA assay was performed using the RAS G-LISA activation kit  according to the manufacturer’s instructions. Protein (20 μg) from whole-cell lysates was added in triplicate in a 96-well plate, and activated RAS was bound to a RAS–GTP binding protein linked to each well. Bound, active RAS was detected with an RAS-specific Ab and quantified by measuring the relative absorbance at 490 nm with a Tecan Infinite 200Pro plate reader.6 CAT12 and 5 × 106 KE-39 cells were prepared in a 1:1 ratio of Matrigel to medium  and injected subcutaneously into the flanks of NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ female mice (6–8 weeks old) purchased from Jackson Laboratory. Treatment was initiated when tumors reached 100–150 mm3. Electronic calipers were used for tumor measurements twice a week and tumor volumes were calculated using the following formula: volume = length × width2 × 0.5. Mice were sacrificed when tumors reached the endpoint. Dissected tumors were snap-frozen and stored at –80°C or were fixed in 10% buffered formalin for histopathologic process.KE-39 and CAT12 cells were prepared in a 1:1 ratio of Matrigel to medium and 3 × 10Fixed xenograft tumors were embedded in paraffin (FFPE). Unstained sections were stained with the following Abs purchased from Cell Signaling Technology: p-ERK1/2 T202/Y204  and p-Rb S807/811 . The staining kit for Ki67  was used per manufacturer’s instructions. Representative images were taken using a DM1000 LED light microscope camera (Leica Camera).t test between 2 groups or 1-way ANOVA among 3 or more groups, unless otherwise indicated. For xenograft experiments, statistical comparisons were performed using 2-way ANOVA followed by Tukey’s multiple comparison, unless otherwise indicated. All animals were randomized to treatment group before drug administration. Animals were excluded from analysis if they were sacrificed because of health reasons unrelated to the tumor volume endpoint. P values are denoted in figures by asterisks .Statistical analyses were performed using Prism, version 9.1 (GraphPad Software). Data are presented as the mean ± SD unless otherwise noted. For in vitro experiments, data were analyzed by the 2-tailed Student’s All animal experiments were conducted in accordance with IACUC-approved animal protocols (no. 11-009) at Dana-Farber Cancer Institute in compliance with NIH guidelines.TL, OK and AJB designed experiments and wrote the manuscript. TL, OK, JZ, YW, KB, BP, and YZ performed experiments. TL, OK, PG, AK, JGD, and ZVH analyzed data. DVTC provided imperative advice in designing experiments and provided critical insight. DVTC provided the CAT12 patient-derived cells. AJB conducted scientific direction. The authorship order between co–first authors was determined by flipping a coin. All authors approved the manuscript.


Pleioblastus pygmaeus. Phylogenetic analysis indicates that 183 SPLs from eight plant species can be classified into nine subfamilies, and the 28 PpSPLs are distributed among eight subfamilies. Homologous analysis shows that as many as 32 pairs of homologous genes were found between P. pygmaeus and rice, and 83 pairs were found between P. pygmaeus and Moso bamboo, whose Ka/Ks values are all <1. MiRNA target prediction reveals that 13 out of the 28 PpSPLs have recognition sites complementary to miRNA156. To screen the SPLs involved in the reproductive growth of bamboo plants, the mRNA abundance of the 28 PpSPLs was profiled in the different tissues of flowering P. pygmaeus and non-flowering plants by RNA-Seq. Moreover, the relative expression level of eight PpSPLs is significantly higher in flowering P. pygmaeus than that in non-flowering plants, which was also validated by RT-qPCR. Combined with phylogenetic analysis and homologous analysis, the eight significant, differentially expressed PpSPLs were identified to be associated with the reproductive process and flower organ development. Among them, there are four potential miRNA156-targeting PpSPLs involved in the flowering process. Of significant interest in the study is the identification of 28 SPLs and the exploration of four key flowering-related SPLs from P. pygmaeus, which provides a theoretic basis for revealing the underlying functions of SPLs in the reproductive growth of bamboo plants.SQUAMOSA Promoter-Binding Protein-Like (SPL) family is well-known for playing an important role in plant growth and development, specifically in the reproductive process. Bamboo plants have special reproductive characteristics with a prolonged vegetative phase and uncertain flowering time. However, the underlying functions of SPL genes in reproductive growth are undisclosed in bamboo plants. In the study, a total of 28 SPLs were screened from an ornamental dwarf bamboo species,  Poaceae plants, such as rice. Therefore, extensive research is essential to reveal the flowering mechanism of bamboo plants, which contributes to their cultivation and conservation.As one of the most potential and most efficient renewable evergreen species for bioenergy and carbon fixation, bamboo has the advantages of rapid growth, a large biomass, and wide use ,2. BamboAntirrhinum majus and have been successively identified from many plant species, such as Arabidopsis thaliana (17 members), Physcomitrella patens (13), Zea mays (31), Oryza sativa (19), Ananas comosus (15), Brachypodium distachyon (17), Sorghum bicolor (19), Phyllostachys edulis (32), etc. [Transcription factors (TFs) are characterized by the fact that they contain one or more DNA-binding domains (DBDs) through which they bind to specific promoter sequences to enhance or block the expression of their downstream target genes ,7. A gena patens , Zea may comosus , Brachypthaliana  members,2), etc. ,19,20,21SPL2 was proved to control reproduction process and flower organ development by activating ASYMMETRIC LEAVES 2 (AS2) in Arabidopsis [OsSPL16 promotes cell division and grain filling, which has positive consequences for grain width and the yield of rice [SPL8 and miR156-targeted SPL genes redundantly regulate gynoecium differential patterning by interfering with auxin homeostasis and signal transduction in Arabidopsis [SPL1 or SPL12 can improve the heat tolerance of Arabidopsis and tobacco during the reproduction process [Arabidopsis SPL3, SPL4, and SPL5 can enhance the FLOWERING LOCUS T (FT) -FD module by targeting a few flowering regulators such as AP1, LFY, and FUL in the flowering pathway [miRNA156-SPLs act as a regulatory hub in the age pathway, which controls the flowering time and phase transition in Arabidopsis [miRNA156-SPL3 prevents early flowering by translational inhibition in Arabidopsis [miRNA156 and its target SPLs also participate in vegetative growth. For instance, miR156-SPL10 was proved to modulate lateral root development, branching, and leaf morphology in Arabidopsis [SPL genes mainly play essential roles in plant growth and development, specifically in reproductive growth ,14. An ibidopsis . OsSPL16 of rice . In addibidopsis . Overexp process . The mos process ,28. As a pathway ,30. In pbidopsis . For exabidopsis . In addibidopsis .P. pygmaeus is a kind of ornamental dwarf bamboo species with a high ecological and economic value in China [P. pygmaeus flowered in the bamboo garden of Nanjing Forestry University, which provided a great opportunity for validating the underlying functions of SPL genes in bamboo plants [P. pygmaeus, and 13 of them were predicted to be potential miRNA156-targeting PpSPLs. In addition, we profiled the expression pattern of the 28 PpSPLs in the different tissues of flowering P. pygmaeus and non-flowering plants by RNA-Seq. A total of eight PpSPLs were found to be highly expressed in flowering P. pygmaeus, which was also validated by RT-qPCR. Moreover, we conducted phylogenetic analysis and homologous analysis of the 183 SPL genes from P. pygmaeus and seven other plant species. Combined with theoretic analysis, the eight significant, differentially expressed PpSPLs were identified to participate in reproductive growth and flower organ development, and four of them are potential miRNA156 targets involved in the flowering process, which is our research emphasis in the future.in China ,35. Durio plants ,35. In tP. pygmaeus and named as PpSPL1-28. The conserved domain of the PpSPLs contains 75 amino acid residues, including two zinc finger motifs and a highly conserved NLS  and PpSPL1 is the weakest (−0.268). Subcellular localization prediction shows that all 28 PpSPLs are more likely to exist in the nucleus . The 12 SPLs, including PpSPL26, PpSPL18, PpSPL8, PpSPL28, PpSPL25, PpSPL9, PpSPL27, PpSPL10, PpSPL7, PpSPL22, PpSPL21, and PpSPL20 belong to the second type, which are highly expressed in the germinated shoots (FM). The third type consists of seven PpSPLs, including PpSPL2, PpSPL1, PpSPL23, PpSPL13, PpSPL5, PpSPL17, and PpSPL15, which are highly expressed in the flower buds (FL). Among them, the relative expression level of eight PpSPLs in flowering P. pygmaeus was significantly higher than that in non-flowering plants, including two highly expressed genes in FE (PpSPL14 and PpSPL16), three in FM , and three in FL .We profiled the expression pattern of 28 PpSPLs in P. pygmaeus, their relative expression level in different tissues was further confirmed by RT-qPCR. As shown in PpSPLs, quantified by RT-qPCR, are generally consistent with RNA-Seq.To validate the expression pattern of the eight significant, differently expressed P. pygmaeus and profiled their expression pattern in the different tissues of flowering P. pygmaeus and non-flowering plants. We also conducted phylogenetic analysis and homologous analysis of the 28 PpSPLs, which provides a functional and comprehensive reference of the single SPL gene in bamboo plants.As a major plant-specific TF family, the SPL family plays an important role in plant growth and development. In particular, many SPL genes are well-known for regulating the plant reproductive process, such as vegetative phase change, flora transition, and flowering time ,28,31. DP. pygmaeus, whose genome information was lacking up to now, a phylogenetic tree of 28 PpSPLs and 155 SPLs from seven other plant species was constructed in the study. Here, we focus on the phylogenetic analysis of the SPLs between P. pygmaeus and the model plant Arabidopsis. There is a total of 19 SPLs from Arabidopsis, which are distributed in the eight subfamilies, except for VII. The 28 PpSPLs are classified into the eight clades, except for VIII. However, Arabidopsis has the most SPLs in the clade-VIII, including AtSPL3–6, which are post-transcriptionally regulated by miRNA156 [AtSPL6 has a positive function in regulating defense genes in innate immunity [AtSPL3–5 were proved to promote reproductive transition by regulating a few flowering regulators, such as AP1, LFY, and FUL [PpSPLs indicates functional evolution following speciation and gene duplication in this clade. The largest subfamily, clade-IV, occurs three times of gene duplication events that P. pygmaeus all participates in, contributing to the most PpSPL members, including PpSPL3, 4, 11, 12, 13, 15, and 19. Arabidopsis has only two identified SPLs in this clade, AtSPL13A and AtSPL13B, which have been implicated in the regulation of post-germinative transition from the cotyledon to vegetative-leaf stages [PpSPLs may have functions in phrase transition. Similar to clade-IV, clade-V also has seven PpSPLs, including PpSPL2, 6, 8, 18, 23, 24, and 26, which occurs two times of gene duplication events. There are three orthologous AtSPLs in this clade, including AtSPL2, AtSPL10, and AtSPL11, which were reported to control plant morphology, plant reproduction, and flower organ development by targeting related genes [AtSPL7 and supports the role of PpSPL7/10/21/22/27 in regulating copper homeostasis [AtSPL14 and AtSPL16 exist for the function annotation of PpSPL25/28 in the clade-I. AtSPL14 was proved to delay the transition from juvenile to adult and improve fumonisin sensitivity, while AtSPL16 is a non-functional duplication of AtSPL14 [PpSPL5 and AtSPL8. AtSPL8 has a positive effect on pollen sac development and seed set, which is also reported in AtSPL9 and AtSPL15 [AtSPL9 and AtSPL15 are classified into subfamily VI, which indicates either functional conservation or independent recruitment of SPL genes. The two AtSPLs were also reported to regulate shoot maturation, leaf initiation, and flowering time by miRNA: target gene interactions [PpSPL14/16 in the same group may have similar diverse functions with AtSPL9 and AtSPL15. In the subfamily IX, PpSPL1, PpSPL9, and PpSPL20 are classified with AtSPL1 and AtSPL12, which are involved in plant thermotolerance at the reproductive stage [PpSPLs involved in phrase transition (PpSPL3/4/11/12/13/15/19/25/28), flowering (PpSPL14/16), and flower organ development (PpSPL2/5/6/8/14/16/18/23/24/26). Combined with transcriptome screening of the eight significant, differentially expressed PpSPLs, including PpSPL5/13/14/16/17/21/25/27, PpSPL5/14/16 are likely to be associated with flower organ development, PpSPL13/25 may be involved in phrase transition, and PpSPL14/16 have the potential to participate in flowering.Phylogenetic analysis is of great significance for family evolution and functional divergence, which provides important references for the characterization of unknown genes ,37. To imiRNA156 ,38. Amonimmunity . AtSPL3– and FUL ,30. The f stages . It is ied genes . Functioeostasis . AtSPL14 AtSPL14 ,43. Ther AtSPL15 . Howeverractions ,46. ThisZea mays and triticeae crops, the SPLs from bamboo plants have similar sequence features with those from rice, such as GC content and codon usage. This indicates that bamboo is a close relative of rice, which is also reflected from phylogenetic analysis and motif composition. To explore the biological functions of SPLs from P. pygmaeus, we focused on the homologous analysis of OsSPLs and PpSPLs. In the study, as many as 32 pairs of orthologous genes were found between rice and P. pygmaeus. Specifically, there are five homologous pairs including OsSPL3/12, OsSPL4/11, OsSPL5/10, OsSPL14/17, and OsSPL16/18 involved in the reproduction growth in Oryza sativa [OsSPL16/18 are positive regulators of cell proliferation and contribute to the grain weight and yield of rice, indicating the two genes function in floral organs and seeds [OsSPL16/18, PpSPL13/15 are highly expressed in FL. According to the studies of Yang et al. [OsSPL5/10 have similar functions to OsSPL16/18, although they are classified into a different clade than OsSPL16/18. PpSPL5 is the orthologous genes of OsSPL5/10, which also indicates its role in tiller number, setting rate, heading date, and germination rate [PpSPL5 is significantly higher in FL than in NL. PpSPL17 is also highly expressed in FL, and its homologous gene, OsSPL7, was reported to regulate the tiller number, panicle architecture, and grain size [PpSPL5/13/15/17 may function in flowering and/or flower organ development. OsSPL14 was well-known for defining plant architecture and promote panicle branching and grain productivity in rice [OsSPL17, plays a role in panicle size, plant architecture, and root development [PpSPL14/16 have a close homologous relationship with OsSPL14/17. The phenomenon that new shoots of P. pygmaeus bloom in a flowering forest indicates that flora transition has been activated at shoot bud stage in bamboo plants [PpSPLs are highly expressed in FE, which implies that PpSPL14/16 participate in reproductive processes. The homologous pairs OsSPL3 and OsSPL12 are highly expressed in the leaves and panicles, respectively, which regulate heading date and panicle size, respectively [OsSPL3 and OsSPL12, PpSPL2/18/23 and PpSPL6/8 adopt different spatial expression patterns with the two OsSPLs. The expression of the five PpSPLs displays no significant difference between flowering P. pygmaeus and non-flowering plants. OsSPL4 and OsSPL11 are a pair of homologous genes in rice. OsSPL4 affects heading date, grain size, and grain yield, while OsSPL11 affects setting rate and tiller number [PpSPL26 is a homologous gene of OsSPL4, which is highly expressed in FM, indicating its role in flowering and reproductive growth. Based on RNA-Seq and RT-qPCR, the relative expression level of PpSPL21/25/27 is highly expressed in FM. Among them, PpSPL21 and PpSPL27 are both homologous genes of OsSPL9, which have been proved to regulate tiller number and the grain yield of rice [PpSPL25 is homologous to OsSPL15, which has no published functional information. However, as shown in the phylogenetic tree, the high expression of PpSPL25 in FM is consistent with the fact that it is homologous to AtSPL14, which plays essential roles in the transition from juvenile to adult. In conclusion, according to the homologous analysis, a total of 14 PpSPLs may participate in the reproductive process (PpSPL2/5/6/8/14/16/18/23/26) and flower organ development (PpSPL5/13/14/15/16/17/21/26/27). In particular, the 14 PpSPLs contain all eight significant, differentially expressed PpSPLs screened from transcriptome profiling, which indicates they are likely to play essential roles in flowering and reproductive growth in P. pygmaeus.The pangenomic era has witnessed a great advance in clade-specific homology inference with increasing genome sequencing of plant species. A few plant species can even have multiple high-quality references . Due to a sativa . OsSPL16nd seeds ,49. As og et al.  and Shaog et al. , OsSPL5/ion rate . Interesain size ,54. The  in rice ,56. Its elopment ,54. PpSPo plants . In partectively . This far number ,57. PpSP of rice ,58. PpSPmiRNAs through mRNA cleavage and/or translational repression [miRNAs can recognize miRNA responsive elements located in the coding regions, 5′ untranslated region (UTR), or 3′ UTR of the target SPLs. Specially, most SPLs have recognition sites complementary to miR156 [miR156-SPLs are conserved throughout the plant kingdom. miR156-SPLs modules play diverse functions in multiple developmental processes such as flowering time, reproductive organ development, leaf morphology, root development, etc., as well as response to various abiotic stresses, such as salt, drought, cold, and pathogen defense [AtSPLs, including AtSPL2-6/9-11/13/15, were reported to be potential targets of miR156, which participate in developmental processes and abiotic stress responses [miR156-SPL3/9 module illustrates a specific flowering regulation process in the age pathway in Arabidopsis [miR156-targeting SPLs from P. pygmaeus, including PpSPL2/3/4/6/8/12/13/14/15/16/17/19/23. Combined with transcriptome screening, four key miR156-targeting PpSPLs, including PpSPL13/14/16/17, were differentially expressed in the flowering and non-flowering P. pygmaeus, which are speculated to participate in flora transition and the flowering process in bamboo plants.Many SPLs have been reported to be down-regulated by pression . In geneo miR156 ,61. An a defense ,54,61,62esponses ,64. In pbidopsis ,31,62. IP. pygmaeus plants were grown in the bamboo garden of Nanjing Forestry University, Jiangsu province . The dormant shoot buds (FE), germinated shoots (FM), and flower buds (FL) from flowering P. pygmaeus and the dormant shoot buds (NE), germinated shoots (NM), and leaf buds (NL) from non-flowering plants were harvested with six biological repeats, respectively. The 6 tissues with respective 3 biological repeats were rapidly frozen by liquid nitrogen and stored at −80 °C for RNA-Seq and RT-qPCR.https://en.novogene.com/, Beijing, China) for RNA-Seq, accessed on 1 June 2020. RNA libraries were constructed by NEBNext® Ultra™ RNA Library Prep Kit for Illumina  with 1.5 μg RNA per sample. The clean sequencing data were obtained from raw reads by RSeQC. A de novo transcriptome assembly was performed by Trinity 2.2 and the transcript sequences were profiled by Corset. The raw sequencing data have been deposited in NCBI SRA with the accession number PRJNA648794. All the information can be referred from our previous study [The 6 tissues with respective 3 biological repeats were sent to Novogene company  and performed a local BLAST to screen SPL genes from the transcriptome of P. pygmaeus, accessed on 6 June 2022. We then used a set of high-quality settings with value < 1 × 10−20 to identify SPL genes based on SBP HMM. The presence of an intact SBP domain in candidate SPL genes were checked against Pfam (http://pfam.xfam.org/), SMART , and NCBI Conserved Domains , accessed on 6 June 2022.We downloaded the Hidden Markov Model (HMM) profile of SBP domain from Pfam database  for physicochemical property analysis and Wolf PSORT  for subcellular localization prediction, accessed on 6 June 2022. MEME  was used to identify the conserved motifs of the SPL family, with repetition number setting as the multiple, maximum pattern number as 20, and optimal width as 6–200 residues, accessed on 7 June 2022.All SPL genes identified from https://phytozome.jgi.doe.gov/pz/portal.html, U.S. Department of Energy, Washington, USA), accessed on 8 June 2022. Multiple alignment was performed using MAFFT V7.407 with default parameters, which was then imported into GeneDoc software for visualization. The phylogenetic tree of the 183 SPL genes from 8 plant species was constructed using the ML method by RAxML(Random Axelerated Maximum Likelihood) with default parameters.The sequences of the other 7 plant species from SPL family members were downloaded from Phytozome v13  protein sequences of orthologous or paralogous pairs were aligned by MAFFT V7.407, accessed on 9 June 2022; (2) multiple aligned amino acid sequences were converted into nucleotide sequences using a homemade perl script based on original CDS sequences; (3) Ka and Ks substitutions were calculated using DnaSP5 software; (4) a sliding window analysis on Ka/Ks ratio was performed with a window parameter of 150 bp and step length of 9 bp.The aligned sequences with the length of ≥300 bp and the identity of ≥40% were regarded as homologous pairs . BLASTN miRNAs were predicted by psRNATarget server  in all 28 PpSPLs, accessed on 9 June 2022. The relationship of miRNAs and their potential PpSPL targets was visualized by Cytoscape v3.3 , accessed on 9 June 2022 [The potential targets of une 2022 .PpSPL genes was explored in the different tissues of flowering and non-flowering P. pygmaeu by RNA-Seq. In order to improve accuracy, we used CLCworkbench software  with full-length sequences of third-generation transcriptomes as a template to remap the clean data of next-generation transcriptomes and profile the mRNA abundance of each SPL gene, accessed on 10 June 2022. The heatmap of the 28 PpSPLs was drawn by R software , accessed on 10 June 2022.The expression pattern of 28 Tubulin (Genbank accession: gi|242385991) was used as a reference gene [PpSPLs were designed using Primer Premier 5.0 . The quantity of RNA was determined by Nanodrop 2000c, and the integrity of RNA was detected by Agilent 2100 and gel electrophoresis. The cDNA was synthesized using PrimerScript RT MasterMix . nce gene . The spemier 5.0 . The relt method . Mean vaP. pygmaeus systematically. Based on RNA-Seq and RT-qPCR, the relative expression level of eight PpSPLs is significantly higher in flowering P. pygmaeus than that in non-flowering plants, including two highly expressed genes in FE (PpSPL14 and PpSPL16), three in FM , and three in FL . Combined with phylogenetic analysis and homologous analysis, the eight significant, differentially expressed SPLs are likely to participate in the reproductive process and flower organ development. In addition, a total of 13 PpSPLs were predicted to have recognition sites complementary to miRNA156. Among them, four potential miRNA156-targeting PpSPLs, including PpSPL13/14/16/17, are highly expressed in flowering P. pygmaeus, which were speculated to participate in flora transition and the flowering process. Of significant interest to the study was the screening of 28 PpSPLs and the exploration of four key flowering related PpSPLs in P. pygmaeus, which fosters substantial insights into the underlying functions of SPLs in reproductive growth in bamboo plants.In the study, we identified and characterized a total of 28 SPLs from


Fusarium oxysporum f. sp. niveum (Fon), the causative agent of watermelon Fusarium wilt, in terms of the phylogenetic relationship, gene/protein structures, and basic biological functions. The SUMOylation components FonSMT3, FonAOS1, FonUBC9, and FonMMS21 are predominantly located in the nucleus. FonSMT3, FonAOS1, FonUBC9, and FonMMS21 are highly expressed in the germinating macroconidia, but their expression is downregulated gradually in infected watermelon roots with the disease progression. The disruption of FonUBA2 and FonSIZ1 seems to be lethal in Fon. The deletion mutant strains for FonSMT3, FonAOS1, FonUBC9, and FonMMS21 are viable, but exhibit significant defects in vegetative growth, asexual reproduction, conidial morphology, spore germination, responses to metal ions and DNA-damaging agents, and apoptosis. The disruption of FonSMT3, FonAOS1, FonUBC9, and FonMMS21 enhances sensitivity to cell wall-perturbing agents, but confers tolerance to digestion by cell wall-degrading enzymes. Furthermore, the disruption of FonSMT3, FonAOS1, and FonUBC9 negatively regulates autophagy in Fon. Overall, these results demonstrate that the SUMOylation pathway plays vital roles in regulating multiple basic biological processes in Fon, and, thus, can serve as a potential target for developing a disease management approach to control Fusarium wilt in watermelon.SUMOylation is an essential protein modification process that regulates numerous crucial cellular and biochemical processes in phytopathogenic fungi, and thus plays important roles in multiple biological functions. The present study characterizes the SUMOylation pathway components, including SMT3 (SUMO), AOS1 (an E1 enzyme), UBC9 (an E2 enzyme), and MMS21 (an E3 ligase), in  SUMOylation is a dynamic and reversible multistep post-translational modification process that attaches small ubiquitin-related modifiers (SUMOs) to substrate proteins . This prArabidopsis [Saccharomyces cerevisiae [Magnaporthe oryzae [Botrytis cinerea, possess two or more SUMOs [S. cerevisiae, the disruption of SMT3 is lethal [SMT3 in Schizosaccharomyces pombe, Candida albicans, Aspergillus nidulans, Aspergillus flavus, M. oryzae, and Beauveria bassiana resulted in growth- and development-related defects [SUMOs are a family of small proteins comprising ~100–115 amino acids with molecular weights of ~11 kDa, and possess flexible amino acid extensions at their N-terminal ,4. Unlikbidopsis , most furevisiae  and MoSMe oryzae ,9. Howevs lethal ; however defects ,13,14.S. cerevisiae, the SUMOylation pathway consists of E1 heterodimer activating enzymes (AOS1/UBA2), an E2 conjugation enzyme (UBC9), and various E3 ligases  [SUMOylation of the substrate proteins involves a sequential concerted action of a cascade of enzymes including SUMO-activating enzymes E1), SUMO-conjugating enzymes (E2), SUMO ligases (E3), and SUMO proteases [, SUMO-cod MMS21) ,16. In td MMS21) . Subsequd MMS21) . E3 assid MMS21) . The SUMd MMS21) ,21,22.S. cerevisiae, SMT3, AOS1, UBA2, UBC9, MMS21, and ULP1 have been found to be crucial for cell viability [C. albicans [A. nidulans, SUMO has been shown to be non-essential for fungal vegetative growth, but indispensable for cellular differentiation [ULPA and ULPB resulted in growth, conidiation, and self-sterility defects in A. nidulans [A. flavus gene, AfsumO, caused significant defects in conidiation [M. orayzae, the deletion of MoSMT3, MoAOS1, MoUBA2, and MoUBC9 led to significant defects in mycelial growth, conidiation, septum formation, cell cycle, conidial germination, appressorium formation, and pathogenicity, and also attenuated the tolerance to nutrient starvation, DNA damage, and other environmental stimuli [Fusarium graminearum [F. graminearum [Fusarium species.The SUMOylation pathway has been shown to modulate a variety of cellular and biochemical processes, including chromosome segregation, DNA replication, cell cycle events, telomere position effect, septin ring, and nuclear pore dynamics ,16. Giveiability . CaSMT3,albicans ,24. In Antiation . The inanidulans ,25. The idiation . In M. o stimuli ,9,26. Siminearum . These iminearum ; howeverFusarium oxysporum f. sp. niveum (Fon), is one of the most destructive fungal diseases. In the present study, we identified the putative SUMOylation pathway components in Fon and revealed their phylogenetic relationship, as well as the gene and protein structures. Further, we investigated their roles in the basic biological functions of Fon by generating the targeted deletion mutants. Our results showed that the SUMOylation pathway is essential for the mycelial growth, conidiation, conidial morphology, spore germination, cell wall integrity, responses to DNA damage or metal ions stresses, apoptosis, and autophagy of Fon, thus highlighting its significance in regulating imperative biological functions in plant pathogenic fungi.Watermelon Fusarium wilt, caused by Fon strain ZJ1 was used as the wild type (WT) strain for the genetic manipulation. The following media were used for the growth and stress sensitivity tests: minimal medium , complete medium , potato dextrose agar (PDA) medium , yeast extract peptone dextrose (YEPD) medium , and mung bean liquid (MBL) medium .Fon, BLASTp was performed using M. oryzae SMT3, UBC9, AOS1, and MMS21 sequences [F. oxysporum genome database FungiDB . To determine the phylogenetic relationship, the retrieved protein sequences of the SUMOylation pathway components in Fon were aligned with those from representative fungal species and humans using the MUSCLE program [http://gsds.cbi.pku.edu.cn/, accessed on 24 October 2022) [http://meme-suite.org/tools/meme, accessed on 24 October 2022) with the default parameters, except for any number of repetitions and a maximum number of motifs of 15 [To identify the SUMOylation pathway components of equences ,9 as que program . A phylo program . For gener 2022) , and thefs of 15 .Fon genomic DNA and fused with the 1349 bp hygromycin gene using the double-joint PCR method. The obtained gene knockout fragments were directly transformed into WT protoplasts [FonSMT3 with Green Fluorescent Protein (GFP) fusion at the N-terminus, under the control of the Rp27 promoter, was generated. The GFP gene was amplified from pYF11 plasmid with primers SMT3-nPYF111p1-F and SMT3-nPYF11p1-R , Sodium chloride , Iron chloride , Zinc chloride , Copper chloride , Driselase (Sigma-Aldrich), Lysozyme , and Cellulase (Shanghai Ryon BioTech). All experiments were performed independently at least three times.The measured . For themeasured . The conescribed . The cheFon strains were washed with sterile ddH2O2 and co-stained with CFW or Hoechst 33342, respectively [Fon strains were treated with/without apoptosis-inducing compound farnesol  at 25 or 50 μM for 4 h at 26 °C. Necrotic cells and nuclei were co-stained with propidium iodide  and Hoechst 33342 , respectively [Fon strains were cultivated in liquid CM for 12 h, and the collected mycelia were rinsed with sterile ddH2O2. After washing, the mycelia were transferred into nitrogen-starved MM medium (MM-N) with/without 4 mM phenylmethanesulfonyl fluoride  for 1 h. The mycelia were stained with the fluorescent dye monodansylcadaverine  for 30 min in the dark, followed by ddH2O2 washing [To analyze septation and subcellular localization, fresh conidia and mycelia of the ectively . For apoectively ,36. To e washing ,38. All v/v) glutaraldehyde. The samples were dehydrated in a graded series of ethanol and then immersed in epoxy resin. The specimens were sectioned into ultrathin sections using an EM UC7 ultramicrotome , and stained with uranyl acetate or lead citrate. The stained ultrathin sections were subjected to TEM .TEM observations for cell wall thickness and autophagy were conducted as previously described . Fresh mFonActin was used as an internal control to normalize the qPCR data for the purpose of comparing the relative transcript abundance of the target genes, and relative expression of the genes was calculated using the 2−∆∆CT method [Total RNA was extracted by immersing pulverized samples in RNA isolator reagent  and treated with RNase-free DNase according to the manufacturer’s instructions. Subsequently, cDNA was synthesized from 1 µg total RNA using HiScript QRT SuperMix . The reaction mixture of qPCR was prepared using AceQ qPCR SYBR Green Master Mix , and qPCR was carried out with three technical replicates using a CFX96 real-time PCR system . T method . The genFonsmt3::GFP-FonSMT3 strain were quickly pulverized in liquid nitrogen and resuspended in 1 mL lysis buffer  containing 40 μL 25× protease inhibitor cocktail. The suspension was then centrifuged at 13000 rpm and the supernatant was collected. Western blotting hybridization was performed as previously described [Fresh mycelia of the Δescribed . The immAll experiments were independently performed three times. Data were normalized when necessary and analyzed using one-way analysis of variance (ANOVA). The significance of the various data was determined by the Fisher’s least significance difference test at a 95% confidence level.Fon, we first identified the SUMOylation pathway components in F. oxysporum, and found all of the orthologs of M. oryzae SUMOylation pathway proteins in F. oxysporum (F. oxypsorum SMT3 and UBC9 exhibited strong sequence similarities (>75%) to those in M. oryzae , M. oryzae (Mo), Colletotrichum graminicola (Cg), B. cinerea (Bc), A. flavus (Af), A. nidulans (An), and Blumeria graminis (Bg) , oomycete Phytophthora infestans (Pi), and Homo sapiens (Hs)  A. Furtheens (Hs) A. The ge members . For insC9 group . HoweverC9 group . These fnd MMS21 A.F. oxysporum SUMOlyation pathway components, which were labeled consecutively as motifs 1–15    C,E. By ce septa) C,E. Spore septa) F. The dee septa) G,H. All nmms21-C A–F. NotaM. oryzae [Fon cell wall integrity, we analyzed the effect of three cell wall-perturbing agents, including CR, CFW, and NaCl [Fonsmt3-C strain grown in potato dextrose broth (PDB) supplemented without or with different cell wall-perturbing agents, including CR, CFW, and NaCl. Western blotting results showed relatively higher levels of GFP-FonSMT3 in ΔFonsmt3-C strain under stressed conditions , MMS (0.13 mg/mL), HU (1 mg/mL), and 4-NQ (10 µg/mL), on the mycelial growth of the WT, deletion mutants, and complementation strains. Phenotypic tests showed that the deletion mutants exhibited increased sensitivity to DNA-damaging agents as compared to WT , Zn2+ (0.5 mM), Ca2+ (0.2 M), and Cu2+ (0.5 mM). The mycelial growth of WT was inhibited by the tested metal cations , and the resulting germ tubes were stained with Hoechst 33342 and PI. At the 25 μM FOH level, the germ tubes of the deletion mutants showed obvious apoptosis- and necrosis-like cell features, such as chromatin condensation and cell death, whereas the germ tubes of WT and complementation strains did not show any obvious change  has been found to be crucial for protein stability and anti-apoptotic action [Fon. On the other hand, overexpression of SUMO1 or UBC9 increased autophagic activation, while the depletion of SUMO1 reduced the ATG12–ATG5 complex in neuroglioma H4 cells [M. oryzae, two important autophagy-related proteins, Snx41 and Atg24, were found to be putative SUMO-substrates [Fon, as previously shown in F. oxysporum [Fonsmt3, ΔFonaos1, and ΔFonubc9 mutants displayed reductions in the formation of autophagosome or autophagic flux under nitrogen-starved conditions , and stress responses to DNA-damaging agents and metal ions in Fon. The expression levels of the SUMO components were significantly upregulated during initial infection, and gradually decreased in the infected watermelon roots at later stages of disease, thus suggesting their potential roles in Fon virulence. This study not only provides experimental evidence supporting the biological functions of the SUMOylation pathway components in F. oxysporum, but also offers an opportunity to investigate the molecular mechanisms of the SUMOylation pathway in regulating growth, development, and virulence of phytopathogenic fungi by further pinpointing the putative SUMOylation substrate proteins.In the present study, we characterized the biological functions of the SUMOylation machinery in


Mycoplasma hyopneumoniae (Mhy) is the cause of porcine enzootic pneumonia (PEN) which, although not lethal, may lead to fatal pneumonia caused by opportunistic bacteria and/or viruses, in concurrence with environmental and management stressors due to reduced muco-ciliary clearance and impaired immune-inflammatory function. PEN spreads progressively with high morbidity rate, causing major economic losses to pig producers worldwide, associated to diminished production indexes and increased control costs, based on vaccination and antimicrobial treatments, used to mitigate those detrimental effects. Vaccination is commonly used for Mhy prophylaxis. While vaccines improve growth performance and reduce clinical signs and lung lesions, the results are often variable. In response to multiple stimuli, cytokines -molecular signals produced by a variety of immuno-modulatory cells- are involved in numerous protective biological activities. To investigate the role of cytokines in vaccinated and non-vaccinated pigs, an experimental model of infection was developed, by combining trans-tracheal and aerosol route, using a mixture of Mhy-field strains. Comparison of clinical, microbiological, pathological, and immunohistochemical parameters revealed significant differences depending upon the profile of cytokines expressed. These results indicate the importance to enhance Th1-type cytokine response—cell-mediated immunity -, to improve the effectiveness of Mhy-immunization, and to develop more efficient vaccines against PEN.Mycoplasma hyopneumoniae (Mhy) is the causative agent of enzootic pneumonia, characterized by high morbidity and low mortality rates in intensive swine production systems. To better understand the mechanisms underlying the protection of an inactivated whole cell vaccine, we investigated the immunohistochemical differences in the cytokine expression in vaccinated and non-vaccinated pigs experimentally infected with Mhy. Four-week-old Mhy-negative pigs (n = 24) were allocated to negative control (n = 8), or one of two Mhy-infected groups: vaccinated (n = 8) and non-vaccinated (n = 8). Infection was carried out by a combination of trans-tracheal and aerosol route. Lung samples were processed for histopathological and immunohistochemical studies, by using antibodies against Mhy, IL1-α, IL1-β, IL-2, IL-4, IL-5, IL-6, Il-8, IL-10, IL-12p35, IL-13, IL-17A, TNF-α, IFN-γ, and CD-4 lymphocytes. Although all cytokines increased in both infected groups, IL-2, IL-12, and IFN-γ were significantly overexpressed in vaccinated pigs. These findings, in conjunction with the decrease of macroscopic and histological lesions in vaccinated animals, indicate the importance to enhance Th1 response in the immunization strategies to control Mhy infection. Mycoplasma hyopneumoniae (Mhy) is the etiologic agent of porcine enzootic pneumonia (PEN) [ia (PEN) . The disia (PEN) . Colonizia (PEN) . Histoloia (PEN) ,5. The cia (PEN) .On-farm control and eradication plans are frequently carried out to combat Mhy infection in porcine production worldwide. Improved management strategies, vaccine-based prophylaxis, and antibiotic therapy is used, alone or in combination, to reduce the detrimental outcomes of Mhy infection . Some ofMhy induces the production of several cytokines by macrophages and other immune or non-immune cells, detected in bronchoalveolar lavage fluid and lung tissue of infected pigs, which have been implicated in the pathogenesis of PEN, and in the development of lesions in the lung and respiratory tract of infected animals ,11,12. IOur study intended to establish the potential association between vaccination, clinical and pathological findings, and cytokine expression in the lungs of pigs experimentally infected with Mhy.2 area with filtered air. Animals were kept in a 15 h light/9 h dark regimen, they had unlimited access to water, and were fed with feed from a local supplier. Nasal swabs were taken on arrival from all piglets to demonstrate the absence of Mhy in the upper respiratory tract by qPCR, following the protocol described by Marois et al. [®, Louvain-la-Neuve, Belgium, 0.025 mL/Kg, intramuscular (IM)) was performed on arrival.Twenty-four Large White pigs of 30 days of age, males and females, and originating from a certified Mhy-free farm were utilized as part of this study. Animals were identified with ear tags and randomly allocated in 3 groups  with 8 piglets each. Each group was kept in separated identical boxes, to prevent the risk of pathogen spread between groups during the experiment, provided with 16 ms et al.  using a ® Hurricane Ultra) with a 20 μm diameter droplet, during 30 min in 3 consecutive days. Control group (C0) received 10 mL of sterile modified Friis liquid medium by transtracheal route. All Mhy cultures used for inoculation of the piglets were applied at a concentration of 105 colour changing units (CCU).Piglets in group C1 were vaccinated, by the intramuscular route in the neck, with 2 mL of Mhy chemically-inactivated saponin-adjuvated whole bacteria vaccine containing two local strains. Piglets in group C2 were non-vaccinated and kept as infection controls. Twenty-three days post-vaccination pigs of groups C1 and C2 were inoculated, by transtracheal route using 10 mL of a pool of cultures including seven Mhy field strains, filtered through a 0.22 μm pore size membrane. All strains were grown in modified Friis liquid medium and collected for inoculation in the logarithmic phase of growth. In addition, a Mhy aerosol was administered using an electric portable aerosol applicator . Lungs were taken and examined in order to determine the extension of gross lesions, and the results expressed, as previously described [All pigs were euthanized 29 days after infection, by means intramuscular deep sedation with Zoletil™ , followed by intracardiac injection of pentobarbital , following the semiquantitative criteria described by Livingston et al. [M. hyorhinis, M. hyosynoviae, and M. flocculare) in culture samples. In addition, lung samples were also processed for general bacteriology using standard methods. Prior to culturing, lung tissue samples were processed for direct DNA extraction using E.Z.N.A.® MicroElute® Genomic DNA extraction Kit  following the manufacturer’s instructions, and the nucleic acid obtained used for Mhy bacterial load quantification by qPCR [t-test with SPSS Inc. 26.0 for Windows . Differences were considered significant when p < 0.05.Lung tissue samples were sampled using sterile viscose swabs. After that, the tip of the swabs was placed in Friis modified liquid broth and incubated for 24 h at 37 °C in constant stirring. Following the initial incubation, cultures were filtered using 0.45 μm membranes  and incubated until a color change evidencing a pH shift was observed. Subcultures were performed on solid Friis medium, incubated at 37 °C in a humid chamber. Species-specific PCR  was used by qPCR . Statist2O2 in methanol for 30 min at room temperature. Tissues were subjected to heat induced antigen retrieval (water bath at 98 °C) with antigen retrieval solution pH 6.0  for 15 min. All sections were incubated with 10% rabbit or mouse normal serum  for 30 min at room temperature. The primary monoclonal or polyclonal reagents, diluted 1:150, which included rabbit polyclonal anti-Mhy [2O2 0.1%. After rinsing in tap water, slides were lightly counterstained with Harris’s hematoxylin and mounted under DPX Mountant  for microscopy. Substitution of normal sera for the primary antibodies served as negative controls. Control tissues were processed alongside the test slides. Positive cells were counted in 40 non-overlapping and randomly selected high magnification fields of 0.237 mm2 from each sample and antibody. Results were expressed as means ± SD of positive cells per mm2, and analysis of variance for mean comparisons assessed by a nonparametric one-way ANOVA on ranks  with Dunn´s post-hoc test for multiple pairwise comparisons between groups, with the SPSS Inc. 26.0 for Windows . For parametric data, different groups were compared using two-tailed ANOVA and multiple comparisons were performed using Tukey test. Differences were considered significant when p < 0.05. The evaluation of the histological lesions, density of alveolar macrophages, and counting of cells positive for the different antibodies was carried out by one observed (FR), who was blinded to the group of animals to which each histological slide belonged.For immunohistochemistry, sections were dewaxed and rehydrated, and endogenous peroxidase activity was blocked by incubation with 3% HClinical signs of non-productive spontaneous and frequent dry cough, characteristic of Mhy infection, was consistently present from 9 dpi until the end of the experiment in all animals of group C2 . In addiM. hyorhinis, M. hyosynoviae, and M. flocculare was negative in all samples tested. Mhy was detected in lung tissue samples from all animals included in group C2. Mhy specific DNA was also detected in lung tissue samples from animals 12, 13, 14, and 15 belonging to group C1. All animals in group C0 were negative for the presence of Mhy DNA in lung tissue samples. Mhy detection was confirmed with both conventional [5) was significantly higher than group C1 (mean relative copy number = 1.09 × 102) (p = 0.038).General bacteriological examination of the lung samples post-mortem did not demonstrate the presence of any common microorganism in the tissue samples analyzed. Likewise, molecular identification of entional  and specentional . Mhy PCR2) was observed in all pigs of group C2. Two pigs of group C0 (nos. 3 and 6) and four of group C1  showed between 20–40 cells/mm2. Significant differences (p < 0.05) between vaccinated and un-vaccinated groups were detected in the scores for gross and microscopic lesions.All pigs of group C2 showed variable but typical well demarcated areas of dark red to purple, consolidated areas in the cranial, cardiac, and intermediate lobes of the lungs. The percentage of affected areas is presented in Numerous Mhy cells were immunolabeled, as a granular reaction, at the luminal surface of bronchial and bronchiolar epithelial cells of all pigs of group C2 A, and, wMhy is considered one of the main pathogens in the porcine respiratory disease complex, by producing major economic losses associated to diminished production indexes and increased treatment and control costs, based on antimicrobial and vaccination treatments ,23. One The precise mechanisms of vaccine-associated protection are not yet fully understood, although both mucosal antibodies and cell-mediated immunity may play a role . After cNumerous studies have demonstrated the increase of IL-1, IL-8, and TNF-α in the bronchoalveolar exudate of infected animals ,14,15,17INF-γ is a significant activator of phagocytic and bactericidal activities of macrophages in injury sites ,17,24, aBALT hyperplasia is a characteristic histological finding during Mhy infection ,4,5. MhyMacrophages are critical in the production of various pro-inflammatory cytokines. These cytokines are vital in the resolution of the infection but are similarly responsible for tissue damage . IL-12 aIL-10 is a down-regulating cytokine, mainly produced by Th2 cells and macrophages, that inhibits antigen-presenting cells, suppresses the secretion of IL-1, TNF-α, IL-12 by mononuclear cells, and exhibits inhibitory effects on the production of IFN-γ by Th1 cells ,13,16. TApart from the fact that Mhy adhesion to host cells is of paramount significance in pathogenesis, the exact mechanisms underlying the immunopathogenesis and cytokine induction during PEN are still unclear. Alveolar macrophages phagocytose and neutralize foreign organisms that reach lower airways, and generate oxygen radicals, cytokines, lysozyme, complement, and other products. The production of IL-12 by alveolar macrophages influences neighboring dendritic cells activation and Th1/Th2 differentiation ,29. IL-2IL-5 and IL-3 are two relevant cytokines produced by Th2 lymphocytes which have been associated with epithelial hypertrophy and metaplasia of mucin-producing cells, stimulation of mucin MU-C5AC secretion, cellular infiltration and hyperplasia of the BALT during Mhy infection . In addiThacker et al.  demonstrIn conclusion, the results of this study suggest that Mhy induces a mixed Th1/Th2 immunity in non-vaccinated animals, and promotes Th1-response, by means the overexpression of IL-2, IL-12, and IFN-γ in vaccinated animals, which was related with protection against the infection. These outcomes, combined with the decrease of macroscopic and histological lesions observed in vaccinated animals, suggest the importance to improve Th1 response in the immunization strategies aimed at controlling Mhy infection.