With the rapid development of transcatheter techniques and instruments, transcatheter occlusion for patients with perimembranous ventricular septal defect (pVSD) and aortic valve prolapse (AVP) was constantly being tried, while the efficacy and safety of pVSD with AVP remain controversial.  The aim of this study was to evaluate long-term efficacy and safety of transcatheter occlusion of pVSD with AVP.  We retrospectively analyzed 164 children with pVSD and AVP who underwent transcatheter occlusion between January 2013 and November 2014. AVP was divided into 3 degrees according to right coronary leaflet morphology at end-diastole during aortic root angiography. Patient demographic and clinical data were collected. n = 63), moderate (n = 89), and severe (n = 12) AVP success rates were 93.7%, 89.9%, and 58.3%, respectively. Immediately after procedure, there was no new-onset aortic regurgitation (AR) above trivial degree, residual shunt above mild degree, or complications requiring medication or operation, except for 1 patient who developed transient complete atrioventricular block. During follow-up, 1 mild AVP patient aggravated from mild to moderate AR and 1 moderate AVP patient aggravated from trivial to moderate AR. The new-onset AR in mild, moderate, and severe AVP was 2%, 1.8%, and 20%, respectively. AR disappeared in 17 patients. Residual shunt occurred in 9 patients after procedure, 4 of which disappeared during the follow-up period. No serious complications occurred in any patient during follow-up. Five-year cardiovascular event-free survival rates for mild, moderate, and severe AVP were 89.6%, 94.5%, and 80.0%, respectively.  There were 97 males and 67 females  months; average weight, 16.94 ± 9.02 kg). Mild ( Transcatheter occlusion of pVSD with mild and moderate AVP has a high success rate and few complications, which is safe and effective in long-term follow-up. Transcatheter occlusion of pVSD with severe AVP has low success rates and high AR incidence. Therefore, transcatheter occlusion of pVSD with AVP is recommended for mild to moderate, but not severe, AVP. Ventricular septal defect (VSD) is the most common congenital heart disease excluding bicuspid aortic valve, accounting for approximately 40% of cases ; perimemWe retrospectively reviewed 164 patients with pVSD with AVP who underwent transcatheter occlusion of VSD between January 2013 and November 2014 at Guangdong Provincial People's Hospital. All patients fulfilled the following criteria: age ≥2 years and pVSD with AVP diagnosed by cardiac catheterization. Exclusion criteria were as follows: (i) other congenital heart diseases requiring transcatheter occlusion, such as atrial septal defect, patent ductus arteriosus, patent foramen ovale, etc.; (ii) other heart malformations requiring surgical treatment or residual shunt after VSD surgical repair; and (iii) bicuspid aortic valve. Patients' general demographic and clinical data were reviewed, including electrocardiogram, transthoracic echocardiography examinations, and catheterization procedure, etc.This study was approved by the research ethics committee of Guangdong Provincial People's Hospital (No. GDREC2016427A). Written informed consent was obtained from the guardians of all patients before the procedure.The transcatheter procedure was performed as previously described , 10. AorAVP was divided into 3 degrees according to the morphology of the right coronary leaflet at the end of diastole during angiography: mild (right coronary cusp buckling down the left ventricular outflow tract), moderate , and severe  , 11 Fig.All patients received oral aspirin (3–5 kg/kg/d) for 6 months after the procedure. Patients who had no complications after transthoracic echocardiography, electrocardiogram, and chest x-ray examination were discharged 48 hours post-procedure.Clinical evaluation, electrocardiogram, and transthoracic echocardiography were performed at 1, 3, 6, and 12 months after discharge, and thereafter yearly, to evaluate AVP, AR, position and shape of the occluder, residual shunt, tricuspid regurgitation, arrhythmia, etc. Cardiac adverse events were defined as more than moderate AR, new arrhythmias, residual shunts requiring surgical or transcatheter treatment, heart failure, cardiac arrest, and other complications requiring surgical treatment during follow-up.AR was classified as trivial , mild , moderate , and severe (AR jet reaching the left ventricular apex) by two-dimensional and color Doppler echocardiography in parasternal long-axis view –14.p < 0.05 was considered statistically significant.All statistical analyses were performed using SPSS Statistic version 25.0 . Categorical variables were expressed as numbers and percentages. Normally distributed data were expressed as mean ± standard deviation, and non-normally distributed data were expressed as median and interquartile range. Cumulative event-free survival was estimated using Kaplan–Meier analyses. A total of 164 patients with pVSD complicated with AVP were enrolled, including 97 male children (59.1%) and 67 female children (40.9%). The patients' demographic characteristics are described in Of the 164 patients who underwent transcatheter occlusion, the procedure was successful in 146. The success rates of mild, moderate, and severe AVP were 93.7%, 89.9%, and 58.3%, respectively, and the occluder sizes were 6.41 ± 1.40 mm, 6.52 ± 1.26 mm, and 6.00 ± 1.29 mm, respectively. The 18 patients in whom transcatheter occlusion failed ultimately underwent surgical repair of VSD.In the 59 mild cases of AVP, 44 symmetrical VSD occluders, 13 eccentric VSD occluders, and 2 ADO-II occluders were used for closure. Of the 80 patients with moderate AVP, 50 symmetrical VSD occluders, 28 eccentric VSD occluders, and 2 ADO-II occluders were used. For the 7 severe AVP patients, 1 symmetrical VSD occluder, 4 eccentric VSD occluders, and 2 ADO-II occluders were used.In the mild AVP group, for the 8 patients with trivial or mild pre-procedural AR, 4 patients decreased or disappeared and 4 patients remained unchanged after procedure. At the end of follow-up, the degree of AR was increased to moderate in 1 (1.69%) patient, who had a mild AR after procedure. In 51 patients with mild AVP without AR before procedure, 11 developed trivial post-procedural AR. At the end of follow-up, 1 patient aggravated to mild AR, and 9 patients had trivial AR.In the moderate AVP group, for the 25 patients with trivial or mild pre-procedural AR, 20 patients decreased or disappeared and 5 patients remained unchanged after procedure. At the end of follow-up, the degree of AR was increased to moderate in 1 (1.25%) patient, who had a trivial AR after procedure. In 55 patients with moderate AVP without AR before procedure, 13 developed trivial post-procedural AR. At the end of follow-up, 1 patient (1.8%) aggravated to mild AR.In the severe AVP group, for the 2 patients with trivial pre-procedural AR, 1 patient disappeared and 1 patient remained unchanged after procedure. At the end of follow-up, the degree of AR remained unchanged in both patients. In 5 patients with severe AVP without AR before procedure, 1 patient developed mild post-procedural AR. At the end of follow-up, the patient (20%) developed to mild AR and another patient developed trivial AR.As reported in Four patients (6.8%) developed post-procedural arrhythmia in mild AVP, including junctional tachycardia in 3 patients and complete right bundle branch block in 1 patient. Eight moderate AVP patients (10%) developed post-procedural arrhythmias, including junctional tachycardia in 2 patients, complete right bundle branch block in 1 patient, incomplete right bundle branch block in 3 patients, and left anterior bundle branch block in 2 patients. There was no post-procedural arrhythmia in patients with severe AVP. All patients with junctional tachycardia recovered after 2–6 days of treatment with methylprednisolone, and without recurrence at follow-up. More than half (57.1%) of the patients with various bundle branch blocks recovered at the 1-month follow-up.During the follow-up period, 1 patient developed ventricular extrasystole and 8 patients developed bundle branch block, all of which were asymptomatic, and half of which recovered at the end of the follow-up. None of these patients developed complete atrioventricular block or other severe arrhythmias requiring medication, pacemaker implantation, or surgical treatment.In the mild, moderate, and AVP groups, there were 4 (6.8%), 4 (5.0%), and 1 (14.3%) patients with residual shunt, respectively, after the procedure . At the The median follow-up time was 70 months . Follow-up data were available for 137 (93.8%) patients, as nine patients (6.2%) were lost to follow-up. Most patients lost to follow-up were due to changes in contact information.No hemolysis, cardiac arrest, shock, more than moderate AR, more than moderate residual shunt, surgical removal of device, implantation of pacemaker due to arrhythmia, or other complications requiring surgical treatment occurred during the follow-up period. Survival rates without cardiovascular adverse events for 6 years among three degrees of AVP are shown in The optimal time to close VSD with AVP is controversial. Due to the persistent existence of siphon of the VSD shunt, both the incidence of AVP and the possibility of AR increase with age. It is common belief that once AVP appears, VSD should be closed as soon as possible to avoid further aggravation of AVP and the occurrence of AR , 13, 14.AR is one of the most common complications after transcatheter occlusion and may be caused by aortic valve injury resulting from a guidewire or occluder-related injury –17. At tArrhythmia is another common complication, mainly including complete atrioventricular block, bundle branch block, ventricular extrasystole, and junctional tachycardia. The most serious arrhythmia is complete atrioventricular block, with an incidence of 1% to 5% –21. ThosAccurate pre-procedural evaluation of VSD and AVP and reasonable occluder selection are the key to a successful procedure. Pre-procedural transthoracic echocardiography and intra-procedural left ventriculography complement each other, which helps to avoid underestimating the true size of VSD due to partial occlusion of the prolapsed right coronary valve, resulting in inappropriate selection of the occluder. Aortic root angiography and intra-procedural transthoracic echocardiography should be performed before and after occlusion to help clarify AVP and the effect of the occluder on the aortic valve and the residual shunt. The occluders used in this study included symmetrical, eccentric, and ADO-II occluders. According to the experience of our center, when VSD is close to the aortic valve, an ADO-II occluder or eccentric occluder can be used. Symmetrical occluders are suitable for VSD with aneurysms. The softness of the ADO-II occluder can avoid injury to the aortic valve caused by the occluder . The uppWith the rapid development of transcatheter techniques and instruments, transcatheter occlusion of pVSD was constantly being tried , 8, whilAlthough transcatheter occlusion of pVSD with mild and moderate AVP is safe and efficient in long-term follow-up, this study was a single-center, retrospective study; these are some limitations of this study. Multicenter, large sample, prospective studies with longer follow-up results are still needed to determine the safety and efficacy of transcatheter occlusion. Furthermore, the selection of occluders in pVSD with AVP should be clarified in the future.pVSD with mild and moderate AVP has a high success rate and few complications; therefore, it is safe and efficient for transcatheter occlusion. However, pVSD with severe AVP has a low success rate and often leads to AR; transcatheter occlusion is not recommended.


Lipidomics is a lipid-targeted metabolomics approach that aims to the comprehensive analysis of lipids in biological systems in order to highlight the specific functions of lipid species in health and disease. Lipids play pivotal roles as they are major structural components of the cellular membranes and energy storage molecules but also, as most recently shown, they act as functional and regulatory components of intra- and intercellular signaling. Herein, emphasis is given to the recently highlighted roles of specific bioactive lipids species, as polyunsaturated fatty acids (PUFA)-derived mediators , endocannabinoids (eCBs), and lysophospholipids (LPLs), and their involvement in the mesenchymal stem cells (MSCs)-related inflammatory scenario. Indeed, MSCs are a heterogenous population of multipotent cells that have attracted much attention for their potential in regulating inflammation, immunomodulatory capabilities, and reparative roles. The lipidomics of the inflammatory disease osteoarthritis (OA) and the influence of MSCs-derived lipids have also been addressed.  The lipidome is defined as the complete set of lipids present within a cell, a tissue, or an organism ,2. In thMSCs are non-hematopoietic multipotent progenitor cells with the ability to differentiate into different mesodermal lineages including osteocytes, chondrocytes, and adipocytes ,16. The Furthermore, the therapeutic potential of MSCs, largely mediated by paracrine signaling , is currGenerally, MSCs can modulate both innate and adaptive immune responses in vitro and in vivo due to their ability to inhibit T-cell proliferation and dendritic cell maturation, recruit regulatory T-cells, and modulate B-cell functions ,27,28. EEndogenous bioactive lipids cover a pivotal role in very important biological phenomena, such as inflammation, immune regulation, and maintenance of homeostasis ,40. Inde2, PGE2, PGI2, and PGF2α), prostacyclins, and TXs [2), the bioconversion product of PGD2, increases during the resolution phase and acts as a brake on inflammation by inducing apoptosis of inflammatory cells [4 (lipoxin A4) is also increased during the resolution phase and acts as a stop signal for the acute response [The group of eicosanoids represents the widest family of bioactive lipids and includes several molecules characterized by the long carbon chain ω6 AA or ω3 EPA and DHA as common precursors. ω6 AA, released from membrane phospholipids firstly via phospholipase A2 and secondarily by phospholipase C, is the substrate for three different enzymes leading to the generation of pleiotropic and heterogenous compounds: (1) cyclooxygenases 1 and 2 (COX-1/2) drive the synthesis of PGs  P45 and TXs . ω3 PUFA and TXs , and are and TXs ,52,53, e and TXs , recruit and TXs ,56. LTs  and TXs . On the  and TXs ,58,59 an and TXs ,61. Two  and TXs , renal a and TXs . Moreove and TXs ,63. Howe and TXs . Indeed,ry cells . The conresponse . Finallyresponse .Cannabis sativa. The plant Cannabis sativa and its preparations, marijuana and hashish, are being used for many years for recreational and medical purposes [4, reducing the migration of inflammatory cells into areas of acute inflammation and inducing the death of inflammatory cells [eCBs are endogenous lipid compounds that can bind G-protein coupled cannabinoid receptors (CB1 and CB2) in the same way as tetrahydrocannabinol (THC), the major psychoactive component of purposes  because purposes . Thanks purposes . The compurposes , the firpurposes ,73, is apurposes . Moreovepurposes , whereaspurposes ,76. Thuspurposes . Moreovepurposes ,79, havepurposes . These ωpurposes  and adippurposes . In addipurposes ,84. In dpurposes ,86. Morepurposes , one of purposes ,89,90. Fpurposes ,92. Withpurposes ,94, vasopurposes  and seempurposes , and inhpurposes , the AEAry cells .O-acyl chain, generated from phospholipase-mediated hydrolyzation of membrane glycerophospholipids (GPLs) and sphingolipids (SLs). Consequently, LPLs are classified into two main categories: glyceryl-based LPLs (including LPA) and sphingosyl-based (including S1P) with a glycerol or a sphingosine backbone, respectively [LPLs are bioactive signaling lipids consisting of ectively ,99  proliferation through β-catenin-mediated c-Myc and vascular endothelial growth factor expression via exchange protein directly activated by cAMP (Epac1)/Ras-related protein 1 (Rap1)/Akt and PKA cooperation [2 and prostaglandin D2 (PGD2) effects on MSCs proliferation and osteogenic differentiation suggests that both their receptors are highly expressed in these cells and both prostaglandins seem to have a negative impact [2 firstly enhances the MSCs growth-rate, while longer stimulation leads to a growth-inhibitory effect. Contrarily, PGD2 inhibits MSCs growth regardless of the duration of the exposure. Moreover, their inhibitory effect on calcium deposition also suggests a negative impact on MSCs osteogenic differentiation [2 as potent modulator of ASCs migration and proliferation through ERK and p38 MAPK signaling mechanisms [2 appears to induce ASCs differentiation into smooth-muscle-like cells [Recently, profiles of glycerophopholipids (GPLs) present in human BMSCs were assessed from young and old donors and across passages during in vitro culture ,112,113.peration , and thrperation . On the e impact . In detantiation . Moreovechanisms . In addike cells ,120. Conke cells  describeke cells . ASCs seke cells . In addike cells .During MSCs differentiation, eCBS variation was also highlighted and the expression of CB1 and CB2 is considerably lower in undifferentiated cells and it increases during osteogenic ,126 and In addition, Pagano et al. found out that ASCs exposed to the synthetic cannabinoid WIN55,212–2 increase the glucose uptake, the calcium influx, and the expression of the adipogenesis regulator PPAR-γ; contrarily, these effects are inhibited by the specific CB1-antagonist Rimonabant . Finally2 secretion is increased upon incubation with the tumor necrosis factor alpha (TNF-α) and the interferon gamma (IFN-γ) [MSCs present anti-inflammatory properties and are being used with great success as treatment for inflammatory and autoimmune diseases. They have been shown to migrate towards injured tissues affected by inflammatory events, led by several growth factors, cytokines, and chemokines . Being p (IFN-γ) . To bett (IFN-γ)  have per (IFN-γ) ,136. The (IFN-γ) , with th (IFN-γ) , which h (IFN-γ) .2 was widely investigated given its key role in the immunosuppressive activity of MSCs [1, PGE2, PGE3, 6-keto PGF1α, PGF2α, and PGJ2 in the conditioned medium. Although PGE2 has been linked to the immunosuppressive effects of MSCs since their inhibitors production attenuate MSC-mediated immunomodulation [2 may have an immunostimulatory role by facilitating Th1 differentiation and expanding the Th17 T-cells population [Concerning the MSCs lipid secretion, PGE of MSCs . Masoodi of MSCs  have anadulation , PGs arepulation . Since ppulation ,140.2, also a higher expression of EP3, which is involved in the stimulation of angiogenesis, was obtained in MSCs suggesting a possible correlation with the early phases of inflammation [In the presence of PGEammation .4 (LXA4), the first identified anti-inflammatory and pro-resolving lipid mediator [Recent studies have evidenced the roles of LXs as regulators of the resolution phase of inflammation  and of Rmediator , signalimediator .2 after AA supplements intake. However, this possible harmful effect can be attenuated by the chain elongation on the less active precursor, ω6 22:4. The ω3 PUFAs precursor, the alfa-linolenic acid (18:3), shows a slight reduction of its GPL AA content, while the EPA (20:5) and DHA (22:6) acid supplements efficiently displace the AA, creating several pools of GPL species substrates that allow attenuation of inflammatory signaling [The ω3 fatty acids EPA and DHA, which are found mainly in marine oils, have long been thought to have anti-inflammatory properties, whereby they compete with AA, by reducing pro-inflammatory eicosanoids . The molignaling .2 and AA-derived oxylipin 15-HETE levels were related to knee OA [2, in OA synovial fluid, as well as oxylipins derived from ω3 and ω6 PUFA such as 15-HETE (derived from AA), 17-HDHA (derived from DHA), and 18-HEPE (derived from EPA). When the pro-inflammatory response occurs in the cartilage, some types of prostanoid enzymes, such as COX, will be produced and released in excessive amounts. COX activation will increase the production of MMP, inhibit the expression of PGE2 and collagen genes and will stimulate the apoptosis process. Studies conducted by Hardy et al. [2 in chondrocytes. The pro-inflammatory cytokine IL-1β stimulates the production of PGE2 in large quantities, and this could induce the degradation process of OA. At the molecular level, IL-1β will increase the expression of the COX-2 gene and the microsomal prostaglandin E synthase-1 at mRNA and protein levels. Therefore, an increase in PGE2 production is related to mPGES-1 and COX-2 derivatives from osteoarthritis chondrocytes stimulated by IL-1β. Another recent study has shown the beneficial effects of resolvin D1 on OA chondrocytes. RvD1 belongs to the family of D-series Rvs, which includes RvD2–RvD6 and share the common precursor 17-HDHA. In one study, RvD1 was found to inhibit the IL-1β-mediated upregulation of COX-2, PGE2, MMP13, and nitric oxide and to prevent chemically induced apoptosis in human osteoarthritis chondrocytes [2 gene expression was studied in the pathogenesis of OA. One study showed that MSCs could significantly (p < 0.05) reduce PGE2 expression in OA synoviocytes after 24 and 48 h co-culture compared to control cells [Osteoarthritis (OA) is a heterogeneous chronic joint disease characterized by the processes of degradation, repair, and inflammation that occur in the connective tissue, the vulnerable layer of joints, synovium, and subchondral bone . From a  knee OA , suggest knee OA  and sinc knee OA . All stu knee OA . These r knee OA  both in y et al.  and Shimy et al.  have anadrocytes . These edrocytes . Howeverdrocytes . MSC-secdrocytes ,159, anddrocytes , providiol cells . Moreoveol cells , and EVsol cells ,163. Lipol cells ,165. On ol cells . In detaIn the last few years, lipidomics has gathered the interest of the scientific community because of the recently confirmed role of lipids in several biochemical aspects as first actors. In detail, lipids are recognized as key players in cells membrane and signaling processes, such as inflammation and immunomodulation. Furthermore, cell lipidome changes according to different cell phases and microenvironment features. Therefore, by analyzing differences in profiles of specific lipid species, it is possible to obtain insights regarding lipids interference in cell signaling and other cellular mechanisms. Lipidomics has proved being successful in identifying viable and functional cell cultures, which could guarantee efficient and safe MSCs application. Despite the limited availability of data regarding MSC lipidomics, the pleiotropic biological actions of different lipid families indicate them as promising candidates for future therapeutic interventions.


Signal transducer and activator of transcription 3 (STAT3) is a transcription factor and regulates tumorigenesis. However, the functions of STAT3 in immune and drug response in cancer remain elusive. Hence, we aim to reveal the impact of STAT3 in immune infiltration and drug response comprehensively by bioinformatics analysis. The expression of STAT3 and its relationship with tumor stage were explored by Tumor Immune Estimation Resource (TIMER), Human Protein Altas (HPA), and UALCAN databases. The correlations between STAT3 and immune infiltration, gene markers of immune cells were analyzed by TIMER. Moreover, the association between STAT3 and drug response was evaluated by the Cancer Cell Line Encyclopedia (CCLE) and Cancer Therapeutics Response Portal (CTRP). The results suggested that the mRNA transcriptional level of STAT3 was lower in tumors than normal tissues and mostly unrelated to tumor stage. Besides, the protein expression of STAT3 decreased in colorectal and renal cancer compared with normal tissues. Importantly, STAT3 was correlated with immune infiltration and particularly regulated tumor-associated macrophage (TAM), M2 macrophage, T-helper 1 (Th1), follicular helper T (Treg), and exhausted T-cells. Remarkably, STAT3 was closely correlated with the response to specified inhibitors and natural compounds in cancer. Furthermore, the association between STAT3 and drug response was highly cell line type dependent. Significantly, the study provides thorough insight that STAT3 is associated with immunosuppression, as well as drug response in clinical treatment. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor transmitting signals from numerous receptors. Besides, it can also regulate the expression of various genes contributing to tumorigenesis ,2,3. STAApart from tumor cells, STAT3 is also implicated in many other non-malignant cells in the tumor microenvironment . The tumGrowing evidence points to the aberrant activity of STAT3 in cancer providing a potential therapeutic option . It has Owing to the recent advancement in bioinformatics, we aim to reveal the impact of STAT3 in tumor immune microenvironment and drug response comprehensively by bioinformatics analysis. Our results shed light on the potential role of STAT3 in mediating the immune response in the tumor microenvironment. Moreover, the study have also indicated the association between STAT3 expression and drug response in different cancer cell lines. Importantly, the data provide a new insight into the combination of immunotherapy and STAT3 related inhibitors in clinical cancer treatment.https://cistrome.shinyapps.io/timer/) includes more than 10,000 samples across 23 cancer types from The Cancer Genome Atlas (TCGA) [+ T-cells, CD4+ T-cells, macrophages, neutrophils, and dendritic cells. Some candidate marker genes of certain immune cells were selected from previous studies [x-axis with gene symbols, and related marker genes were represented on the y-axis as gene symbols. The transcriptional level was displayed with log2 RSEM. The correlation was exhibited by the purity-corrected partial Spearman method .The database Tumor Immune Estimation Resource  . The dats (TCGA) . The gens (TCGA) . Based o studies ,30, thenhttp://ualcan.path.uab.edu/) was used to analyze the correlation between STAT3 mRNA transcriptional level and pathological stage in cancer. The pathological stage in cancer was divided into Stages 1, 2, 3, and 4. The platform UALCAN is a web-based tool to analyze gene expression data deeply using genomics data from TCGA and clinical data from 31 cancer types [The database UALCAN (https://www.proteinatlas.org/). The antibody used in the database was HPA001671. The HPA database represents the protein expression in 44 major human tissues and some cancer tissues by immunohistochemistry [p-value was determined using the Mann–Whitney U-test.The protein expressions of STAT3 in human normal tissues and tumor tissues were validated via the Human Protein Altas . The drug response was measured by the area under the curves (AUCs) for 545 compounds across cancer cell lines, and the datasets were obtained from the Cancer Therapeutics Response Portal , including CTRPv2.0 2015 ctd2 Expanded Dataset, CTRPv2.1 2016 pub Nat Chem Biol 12 109, and CTRPv2.2 2015 pub Cancer Disc 5 1210. The compounds contain FDA-approved drugs, clinical candidates, and small molecule probes. Then, the correlation between drug sensitivity and gene expression level was evaluated by Pearson correlation coefficients [p-value of less than 0.05. The data analysis was conducted in RStudio Version 1.2.5033.The mRNA profiles for 823 cancer cell lines were downloaded from the Cancer Cell Line Encyclopedia , breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and prostate adenocarcinoma (PRAD). Besides, the data also showed that STAT3 expression was aberrantly higher in HPV-positive HNSC than HPV-negative HNSC A.Subsequently, to have a better understanding of the relationship between STAT3 and clinicopathological features, we analyzed the STAT3 expression level in different pathological stages by UALCAN. The results showed that STAT3 expression was lower in different pathological stages of BLCA, COAD, KICH, KIRC, LUAD, and LUSC compared with normal tissues. Meanwhile, STAT3 in KIRC with Stage 3 was significantly lower than that with Stage 1. However, there was no significant difference in STAT3 expression level among Stages 1–4 in BLCA, COAD, KICH, LUAD, and LUSC B, suggesAdditionally, the protein expression level of STAT3 was found by using clinical specimens from the HPA database. The STAT3 protein expression data was shown for each of the 44 normal tissues A, and itr = −0.383), KICH (r = −0.436), and LIHC (r = −0.315) , COAD, esophageal carcinoma (ESCA), KICH, KIRC, LIHC, PRAD, and rectum adenocarcinoma (READ). The results suggested that STAT3 expression level was inversely correlated with tumor purity, indicating that STAT3 was highly correlated with non-tumor cells, especially in BLCA ( −0.315) .+ T-cells in 5 cancer types , CD4+ T-cells in 4 cancer types , macrophage in 4 cancer types , neutrophils in 7 cancer types , and DCs in 7 cancer types . Additionally, the moderate correlations between STAT3 and neutrophils (r = 0.51), DCs (r = 0.427) were identified, respectively, in BLCA. Similarly, the moderate or strong correlations between STAT3 and infiltrating levels were shown, involving neutrophils (r = 0.47) and DC (r = 0.476) in COAD; B-cell (r = 0.534) and DC (r = 0.742) in KICH; neutrophils (r = 0.411) in KIRC; CD8+ T-cells (r = 0.567), macrophages (r = 0.497), neutrophils (r = 0.505) and DCs (r = 0.502) in PRAD; DCs (r = 0.463) in READ , CD8 in READ .The results implied that STAT3 was associated with immune infiltration in BLCA, CESC, COAD, ESCA, KICH, KIRC, LIHC, PRAD, and READ. Although the correlation between STAT3 and abundance of TIICs was verified in different cancer types, STAT3 was particularly correlated with the infiltrating level of neutrophil and DC.+ T-cells, monocytes, TAM, M1 macrophages, M2 macrophages, neutrophils, DCs, as well as various subsets of T-cells, to be specific, T-cells , T-helper 1 (Th1) cells, regulatory T (Tregs) cells, follicular helper T (Tfh) cells, and exhausted T-cells. Considering the correlation between STAT3 and tumor purity, STAT3 and abundance of TIICs, we focused on BLCA, KICH, and PRAD to study the correlation between STAT3 and immune cells. The results showed that STAT3 was positively correlated with most gene markers of immune cells in BLCA, KICH, and PRAD. Specifically, STAT3 showed significant correlation with CD+8 T-cell marker genes CD8A and CD8B; monocyte marker genes CD86 and CD115; TAM marker genes CCL2, CD68, and IL10; M1 macrophage marker genes inducible nitric oxide synthase (INOS), interferon regulator factor 5 (IRF5), and cytochrome c oxidase subunit II (COX2); M2 macrophage marker genes CD163, V-set and immunoglobulin domain containing 4 (VSIG4), and membrane-spanning 4-domains A4A (MS4A4A)  . The datAt the same time, TAM markers CD68 and IL10, M2 macrophage markers CD163 and VSIG4, were closely correlated with STAT3 expression . TAMs arIn addition, we also revealed the significant positive correlation between STAT3 and marker genes of different subsets of T-cells, including T-cell  marker genes CD3E and CD2; Th1 marker genes, T-bet, signal transducer and activator of transcription 4 (STAT4), signal transducer and activator of transcription 1 (STAT1), and tumor necrosis factor alpha-like (TNF-α); Treg marker genes forkhead box P3 (FOXP3) and signal transducer and activator of transcription 5B (STAT5B); Tfh marker genes BCL6 and interleukin 21 (IL21); exhausted T-cell marker genes PD-1, cytotoxic T-lymphocyte associated protein 4 (CTLA4), and TIM-3 . FurtherA combination of immunotherapy and small molecule inhibitors was an effective choice and targeting STAT3 directly or indirectly was also the potential approach for cancer therapy. So, we evaluated the association between STAT3 expression and the response to 545 drugs in 823 cancer cell lines by CCLE and CTRP. Additionally, the results showed that only two drugs, VX-680 and GSK-J4, had a correlation coefficient greater than 0.2  to the greatest extent compared with other compounds. In other words, STAT3 expression was correlated with sensitivity to abiraterone in breast cancer cell lines. Abiraterone not only was approved by the FDA for the treatment of prostate cancer but also displayed the potential benefit for breast cancer [r = −0.999) in skin cancer. Bafilomycin A1 is a promising candidate drug targeting growth, apoptosis, and metastasis in cancer cell lines [r = 0.555). As an inhibitor of fatty acid synthase, cerulenin could affect the proliferation and metastasis of cancer cells [In breast cancer, abiraterone, targeting CYP17A1, was negatively correlated with STAT3 (t cancer ,41. Addill lines ,43, and ll lines . The datll lines , and STAll lines . The strer cells . Hence, er cells .To further investigate the potential mechanism of phytochemical approaches to cancer treatment, we summarized the correlation between transcriptional expression level of STAT3 and sensitivity to natural compounds in cancer cell lines by CCLE and CTRP databases . We founBesides, some reports revealed that betulinic acid could regulate STAT3 in many cancer types ,52. In cBioinformatics has been rapidly applied to cancer research in recent years. Using some powerful bioinformatics tools like software packages and databases, profiles of gene expression in cancer can be analyzed. With the aid of some statistical methods, bioinformatics can provide a platform to discover the underlying molecular mechanisms in cancer biology and development . Here, uApart from the known role of STAT3 in promoting cancer cell proliferation, invasion, and angiogenesis, STAT3 also showed the crucial role in immunosuppression . In thisκB and STAT3 pathways in HT-29 colorectal cancer cell [More and more studies have shown that combination of immunotherapy and small molecule inhibitors is more effective than immunotherapy alone. Apart from the role of STAT3 in immune suppression, this study also demonstrates the significant association between STAT3 expression and drug response. The data was interesting that the significant association between STAT3 expression and VX-680, GSK-J4 treatment response in 823 cell lines. VX-680 might mediate cell death by acting on P53/Bax/caspase-3 dependent pathway in cholangiocarcinoma cell . Additiocer cell . Additiocer cell . Overallcer cell , and shocer cell ,82. The To be honest, there were some limitations in this study. First, the maximum sample size of cancer patients was 12 and normal was 3 in the HPA dataset, which made the protein expression of STAT3 in cancer less objective. Moreover, the sample size of KICH was relatively small and the correlation with immune infiltration in KICH was less reliable compared with BLCA and PRAD. Therefore, succeeding studies are still needed to address these problems. Although there were limitations in our study, some of the results in our study were pretty meaningful and could offer directions for future study. For instance, we comprehensively analyzed the association between STAT3 and TIICs and our results showed that the correlation between STAT3 and M2 macrophage, Tregs, and T-cell exhaustion was higher compared with other immune cell types. As we know, M2 macrophages, Tregs, and T-cell exhaustion play an important role in immune suppression. Based on the above findings, we made the conclusion that STAT3 was particularly correlated with immune suppression in the tumor microenvironment. Furthermore, we systematically investigated the association between STAT3 expression and drug response in various cancer cell lines. Interestingly, our results revealed that VX-680 and GSK-J4 showed a positive response to STAT3 in cancer. Besides, the response of FASN inhibitor was closely correlated with STAT3, which provided insight into STAT3 might regulate lipid homeostasis. In addition, our results also revealed that some natural compounds might mediate STAT3 in cancer.In summary, our study suggests that STAT3 is related to immune cell infiltration and closely associated with immune suppression. Additionally, STAT3 expression is tightly correlated with specified inhibitors and natural compounds treatment response in cancer. Therefore, STAT3 may play an important role in immune infiltration and cancer clinical therapy.


Mucosal-associated invariant T (MAIT) cells are innate-like T cells present at considerable frequencies in human blood and barrier tissues, armed with an expanding array of effector functions in response to homeostatic perturbations. Analogous to other barrier immune cells, their phenotype and function is driven by crosstalk with host and dynamic environmental factors, most pertinently the microbiome. Given their distribution, they must function in diverse extracellular milieus. Tissue-specific and adapted functions of barrier immune cells are shaped by transcriptional programs and regulated through a blend of local cellular, inflammatory, physiological, and metabolic mediators unique to each microenvironment. This review compares the phenotype and function of MAIT cells with other barrier immune cells, highlighting potential areas for future exploration. Appreciation of MAIT cell biology within tissues is crucial to understanding their niche in health and disease. Abunnd fungi –10. All nd fungi , thus beBarrier surfaces are sites of cross-talk between the host and diverse external environments. MAIT cells are found in the intestine, skin, respiratory, oral and female genital mucosa, which all house microbial communities adapted to the local environment and in symbiosis with the host  Table , 35, allThe range of MAIT cell effector functions is only just being explored. It is increasingly appreciated that resident macrophages and lymphocytes are in constant cross-talk with tissues, integrating cues from the local microbiome, cellular, environmental and metabolic milieus for their development and function –44. In tLactobacillus sp.-derived lactic acid and hydrogen peroxide, with additional structural and immunological changes with the menstrual cycle, pregnancy and age , regulatory T (Treg), and CD8+ tissue-resident memory (TRM) and Tc17 cells. MAIT cells and other unconventional T cells, such as invariant natural killer T (iNKT) and gamma-delta T (γδT) cells, share features with both innate and adaptive cells. They can be activated in a TCR-dependent or independent manner  challenge similar frequencies of murine skin MAIT, γδT, and iNKT cells are host-derived and thus tissue-resident  and common, but not universal, among MAIT cells within the oral and gastric mucosa  associated with CD8c mucosa , 15, skic mucosa , 57, andc mucosa –29  cells are derived from the host long term, donor-derived cells can be found over a year after transplantation  are features shared with other barrier tissue-resident cells, including TH17, peripherally derived Treg (pTreg), ILC3, iNKT and γδT cells  driven by the transcription factor promyelocytic leukemia zinc finger protein (PLZF) (TBX21) and RORγt, whereas naïve SPF mice have two almost mutually exclusive populations  , 55. RORn (PLZF) , 68. Unuulations , 70: neaculation . Followi species .+ cells biased towards type-17 function   . It is uE. coli) , and botE. coli) . Ultimat+ T cells (and Tregs) in mouse skin  on resolution of infection from Legionella longbeachae and with re-infection, suggesting significant functional parallels  necessary for long term maintenance . MDR1 (ABCB1) is an ATP-binding cassette B1 drug resistance transporter expressed on IL-17 producing CD4eostasis ; a subse of MDR1 , 45, 1082O2). H2O2 can be transported by the plasma membrane water channel, aquaporin 3 (AQP3), which is part of a core transcriptional signature shared among type-17 secreting NKT17, ILC3, γδT, and TH17 cells in mice  released after cell lysis and has cell type specific effects -lactate; and both lactate and Lactobacilli-derived factors dampen Staphylococcus aureus (S. aureus)-induced MAIT cell activation in whole PBMC  , can directly modulate barrier RORγtesponses . Acetateesponses . These S GF mice  and are xpansion –141 and xpansion . Barriereptor 2) , which i T cells . As SCFAin vitro , it woulIn addition to the microbiome, mucosal immune cells are capable of directly sensing chemicals, including nutrients in the mammalian diet . Dietaryall-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (RARA), RARβ (RARB), and RARγ (RARG)  . In miceependent –148, witathology . Similarathology , plasticathology , and IL-athology . In humae repair . Vitamin−γδT cell IL-17 production is inhibited by RA through reduction in IL-1R, IL-23R, and pSTAT3 expression  is a conserved ligand activated transcription factor highly expressed by cell types at barrier surfaces, in keeping with its role as an environmental sensor promoting mucosal integrity. Physiological AhR ligands include: indole-derived ligands from dietary cruciferous vegetables; host and microbe-derived tryptophan-metabolites ; and exogenous chemicals . Initialand Treg , 170, Ahand Treg , 172. Sentegrity . Ahr-defsent IEL , with re tissues , 176, wh tissues , 177.Ahr is dispensable for MAIT cell thymic development in mice   . It woul+ T cells share functionality and a conserved transcriptional signature by bulk microarray, which includes enrichment for two candidate GPCR proton sensors, GPR65 and GPR68  modulates ILC2-mediated tissue protection , 223; vaH17 and ILC3  signaling, which promotes STAT3 signaling and aerobic glycolysis to increase type-17 effector functionality of Tand ILC3 . IGFbp4,ionality . Indeed ionality , which mUV-light can regulate the immune system , RORA, PER1, and CRY1 enriched among MAIT, iNKT, and Vδ2+ γδT cells . PK is funded by the Wellcome Trust [WT109965MA]; the Medical Research Council (STOP-HCV); a National Institute of Health Research Senior Fellowship, the National Institute of Health Research Oxford Biomedical Research Centre, and the National Institutes of Health (U19 I082360).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.


Corylus spp.) are economically important nut-producing species in which ovule development determines seed plumpness, one of the key parameters reflecting nut quality. microRNAs (miRNAs) play important roles in RNA silencing and the post-transcriptional regulation of gene expression. However, very little is currently known regarding the miRNAs involved in regulating ovule growth and development.Hazels , early ovule growth (Ov2), rapid ovule growth (Ov3), and ovule maturity (Ov4). On the basis of small RNA and mRNA sequencing using the Illumina sequencing platform, we identified 970 miRNAs in hazel, of which 766 and 204 were known and novel miRNAs, respectively. In Ov1-vs-Ov2, Ov1-vs-Ov3, Ov1-vs-Ov4, Ov2-vs-Ov3, Ov2-vs-Ov4, and Ov3-vs-Ov4 paired comparisons, 471 differentially expressed microRNAs (DEmiRNAs) and their 3117 target differentially expressed messenger RNAs (DEmRNAs) formed 11,199 DEmiRNA/DEmRNA pairs, with each DEmiRNA changing the expression of an average of 6.62 target mRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of all DEmRNAs revealed 29 significantly enriched KEGG pathways in the six paired comparisons, including protein export (ko03060), fatty acid elongation (ko00062), starch and sucrose metabolism (ko00500), fatty acid biosynthesis (ko00061), and amino sugar and nucleotide sugar metabolism (ko00520). Our results indicate that DEmiRNA/DEmRNA pairs showing opposite change trends were related to stress tolerance, embryo and seed development, cell proliferation, auxin transduction, and the biosynthesis of proteins, starch, and fats may participate in ovule growth and development.These findings contribute to a better understanding of ovule development at the level of post-transcriptional regulation, and lay the foundation for further functional analyses of hazelnut ovule growth and development. Corylus spp.) are important nut-producing species in the Betulaceae family , the nut kernel of which is an important raw material in food processing industries [Hazels , miR168 regulates phase transition, leaf epinasty, and fruit development via regulation of its target gene SlAGO1 [ARF8, thereby producing the parthenocarpy trait in both Arabidopsis and tomato [microRNAs (miRNAs) are small non-coding RNA molecules ranging from 18 to 24 nucleotides in length that occur extensively in plants, animals, and certain viruses, and play important roles in mRNA cleavage, RNA silencing and the post-transcriptional regulation of gene expression –10. In pd of 25% . Similar in rice –14. Furte SlAGO1 , whereasd tomato . Hence, Genome-wide analysis of miRNAs and their target genes can provide valuable insights into miRNA-based regulatory mechanisms in plants . To dateIn total, we obtained 201.1 million raw reads through Illumina sequencing of 12 small RNA libraries for ovules at four successive developmental stages  Fig. , and theIn order to discover novel miRNAs, we used the miRDeep2 software package with modifications for plant miRNAs to analyze the unannotated reads . We accoDue to the specificity of the Dicer enzyme and the DICER-LIKE (DCL) enzyme, the final length of mature miRNA is mainly in the range of 20 nt to 24 nt, of which 21 nt or 24 nt are the main miRNAs in plants . In totaDicer enzyme or DCL is necessary for the formation of mature microRNA (miRNA), and it cleaves pre-miRNA correctly to generate mature miRNA with the correct seed regions . When DiDEmiRNAs among the six paired sample groups were identified. For the paired comparisons Ov1-vs-Ov2, Ov1-vs-Ov3, Ov1-vs-Ov4, Ov2-vs-Ov3, Ov2-vs-Ov4, and Ov3-vs-Ov4, we identified 203, 258, 283, 281, 320, and 35 DEmiRNAs, respectively Fig. , indicatSimilarly, we used DESeq2 to identify differentially expressed (DE) mRNAs among the six paired sample groups, with threshold criteria of | log2 (Fold Change) | > 1 and FDR < 0.05. We accordingly detected 8940, 10,689, 12,525, 8007, 9807, and 3004 DEmRNAs in the Ov1-vs-Ov2, Ov1-vs-Ov3, Ov1-vs-Ov4, Ov2-vs-Ov3, Ov2-vs-Ov4, and Ov3-vs-Ov4 comparisons, respectively, indicating a greater abundance than the corresponding DEmiRNAs Fig. . As withWe used Target Finder to screen hazel mRNA transcripts having high complementary sequence with both the predicted known and novel miRNAs, and accordingly identified 18,137 mRNA targets of the predicted miRNAs. In order to obtain annotation information for the target genes, we used BLAST software to compare the predicted target gene sequences with sequences in the NR, Swiss-Prot, GO, COG, KEGG, KOG, and Pfam databases. We accordingly succeeded in annotating 15,412 of the targeted genes (Table S6). To identify the most biologically relevant miRNA/target pairs, we selected all interacting pairs of DEmiRNAs and target DEmRNAs, resulting in subsets of 1042, 1325, 1653, 894, 947, and 54 DEmiRNA/target DEmRNA pairs for the Ov1-vs-Ov2, Ov1-vs-Ov3, Ov1-vs-Ov4, Ov2-vs-Ov3, Ov2-vs-Ov4, and Ov3-vs-Ov4 comparisons, respectively Fig. . These DChanges in the expression of each DEmiRNA and its target DEmRNAs are shown in Fig. We found that a large number of DEmRNAs targeted by DEmiRNAs encode important transcription factors involved in developmental regulation, including, but not limited to, members of the families bHLH, WRKY, ARF, MYB, and MADS. In order to assess the biological function of the DEmRNA targets of DEmiRNAs, we performed gene ontology (GO) classification analysis using the BMKCloud platform. All 3117 target DEmRNAs with GO annotation identified from the six paired comparisons were assigned to one of three categories, namely, biological process, cellular component, and molecular function  values obtained based on Illumina sequencing were consistent with those acquired from qRT-PCR  [By mining genomic data in silico, 57 putative miRNAs were predicted and identified in 703.007) –24. In t703.007) . These f703.007) . ClearlyArabidopsis, overexpression of miR858 promotes susceptibility to pathogen infection, whereas the inhibition of miR858 activity via target mimics (MIM858 plants) can confer disease resistance [MYB41, MYB3, and MYB108, respectively, were upregulated by 5.01-, 1.41-, and 1.08-fold, respectively. In Arabidopsis, AtMYB41 is known to regulate transcriptional and metabolic responses to osmotic stress [AtMYB101 plays a role in controlling pollen tube–synergid interactions during fertilization [PtrMYB3 participates in the regulation of secondary wall biosynthesis [MYB108 is involved in the defense response against Verticillium dahliae infection [MIR858 suppressed development due to nutrient competition, resulting in the highly upregulated expression of stress tolerance target genes at the Ov3 and Ov4 stage, including the expression of transcription factors MYB41, MYB3, MYB108, which may be beneficial in promoting tolerance adaption in developing ovules. Similarly, MIR403, and MIR482 may also are involved in ovule development regulation though their target mRNA, including zinc finger CCCH domain-containing protein 2 gene (ZFP2) [In plants, endogenous miRNAs play important roles as regulators of gene expression, and are undoubtedly implicated in the regulation of important biological processes, including growth, reproduction, development, differentiation, signal transduction, and defense responses , 26. In sistance . In pairc stress , whereaslization . In poplynthesis , and in nfection . The diae (ZFP2)  and disee (ZFP2) .Arabidopsis, agamous-like MADS-box protein AGL62 is expressed exclusively in the endosperm, and encodes a Type I MADS domain protein that functions as a transcription factor. In agl62 mutants, there is a premature formation of cell walls in the endosperm, indicating that AGL62 is required for suppression of cellularization during the syncytial phase, and thereby plays an important regulatory role in seed development [AGL62-encoding genes (g15301 and g34233) were highly upregulated during ovule development, whereas the miRNAs targeting these genes  showed a decreasing expression pattern. APETALA 2 (AP2) is known to regulate the expression of several floral-specific homeotic genes, including AGAMOUS [AP2-encoding genes (g4294 and g15260) upregulated concomitant with the downregulation of targeting DEmiRNAs . Hence, high and increasing expression pattern of AGL62 and AP2 in ovules is suggestive of their important roles in ovule development. Furthermore, we found that their expression is regulated by MIR159, MIR168, and MIR172; however, further molecular analyses will be necessary to determine whether AP2 regulate the expression of AGL62.According to the floral quartet model (FQM), the identity of the different floral organs is specified during development by quaternary protein complexes comprising MADS-domain proteins . In Arabelopment . Somewha AGAMOUS . In the GAMYB-like genes [MIR166 genes are dynamically controlled in regulation of the shoot apical meristem (SAM) and floral development in parallel with the WUSCHEL (WUS)-CLAVATA (CLV) pathway [MIR166 contributes to the induction of somatic embryogenesis via targeting PHABULOSA, a positive regulator of LEAFY COTYLEDON2 that controls embryogenic induction via activation of the auxin biosynthesis pathway [grf mutants have been found to be characterized by embryogenic defects such as cotyledon fusion [myb domain protein 101 , ABSCISIC ACID INSENSITIVE 5 , GROWTH REGULATING FACTOR5 , GRF12 (g10975), GRF1 (g13479), polyadenylate-binding protein C1 , and T-complex protein 1 . The DEmiRNAs also regulate important biological processes, including seed development, embryogenesis, ovule growth, and cell proliferation. The aforementioned five miRNA families include a number of the DEmiRNAs identified in the present study, each of which is predicted to cleave several differentially expressed target genes, thus forming hundreds of DEmRNAs/DEmiRNA pairs. Collectively, these DEmRNAs/DEmiRNA pairs constitute a complex regulatory network that function to coordinate development of the hazelnut ovule.Other important developmental regulators are known to be regulated by MiR159, MIR166, MIR396, and MIR319. MiR159 is a highly conserved miRNA that plays roles in short-day flowering, anther development, and seed germination via repression of  pathway . It is c pathway . The MIRn fusion . miR319 n fusion . These DAUX1, encoded by g30973, was identified to be significantly unregulated in the Ov1-vs-Ov2 pairwise comparison, indicating that auxin influx into the ovule is beneficial for embryo development at stage Ov2.Our GO and KEGG pathway classification of all target DEmRNAs revealed that multiple DEmRNAs are involved in plant hormone signal transduction Figs.  and 8. AAux/IAA proteins are important repressors in auxin signaling pathway. Auxin response factors (AFRs) are responsible for the expression regulation of early auxin response genes, and Aux/IAA can form heterodimer with ARFs and inhibit their transcriptional regulation activity subsequently . We founArabidopsis, ARF2/MNT (MEGAINTEGUMENTA) is a repressor of cell division and organ growth [Camellia oleifera, silencing of ARF2 using amiRARF234 was found to promote identity defects in cells at the micropylar pole of embryos and subsequent seed abortion [ARF6 is known to regulate gynoecium maturation, and the flowers of arf6 arf8 double mutants are characterized by immature gynoecia in Arabidopsis [ARF6, ARF2, ARF17, and ARF9, respectively. Among these, ARF6 and ARF2 are predicted to be cleaved by dozens of DEmiRNAs belonging to the MIR167_1, miR159, and miR319 families. The expression abundance of ARF6 and ARF2 deceased and increased along with development, respectively. ARF6 is targeted by DEmiRNAs of the MIR167_1 family , the expression of which tended to increase with ovule development. Conversely, the expression of miR159 and miR319 family DEmiRNAs targeting ARF2  was observed to decrease with ovule development. Taken together, our results indicate that the relatively high expression of DEmiR159 and DEmiR319 and low expression of their target ARF2 at stage Ov3 and Ov4 contribute to ovule growth and filling, whereas a decrease and increase in the expression of MIR167_1 and its target ARF6, respectively, at all four developmental stages indicated their regulatory role in embryo sac maturation and subsequent ovule growth and filling.In n growth , and arfn growth , 47. In abortion . Furtherbidopsis , 49. In AUX1 expression was relatively higher at stage Ov2 than stage Ov3 and Ov4. As active repressors, the expression of Aux/IAA proteins were relatively lower at stages Ov1 and Ov2, whereas expression of ARF2, a repressor of cell division and organ growth, increased concomitant with development, and that of ARF6 decreased with development. These results accordingly indicate that auxin signal transduction tends to be active at stages Ov1 and Ov2, which may facilitate the development and differentiation of the embryo sac and embryo at these stages, whereas subsequent inactivation of auxin signal transduction may contribute to promoting the growth and filling of ovules at stages Ov3 and Ov4. Moreover, we found that the expression of this set of important regulators was strictly regulated by multiple miRNA families, including MIR164, MIR 319, MIR167_1, miR159, and miR319.Results obtained from histochemical analysis of auxin indicated that auxin is enriched in the growth center of pistillate inflorescences and young ovaries . In thisGlgB (g8185) was upregulated by 1.82- and 1.79-fold, respectively, indicating a pattern of increasing expression with development. GlgB is the target gene of the novel miR_278, which was found to be downregulated by 1.59-fold in the Ov1-vs-Ov3 pairwise comparison. Sucrose-phosphate synthase (SPS) plays a major role in photosynthetic sucrose synthesis by catalyzing the rate-limiting step of sucrose biosynthesis from UDP-glucose and fructose-6-phosphate, and has been demonstrated to play a role in regulating carbon partitioning in plant leaves [GlgB and SPS may promote the biosynthesis of starch and carbon partitioning in hazelnut, and that novel miR_278 and cme-miR166i may be involved in these biological process via their regulation of GlgB and SPS.GlgB, a 1, 4-alpha-glucan branching enzyme, is a key enzyme in the starch synthesis pathway, in which it catalyzes the formation of the alpha-1, 6-glucosidic linkages in glycogen by cleaving 1, 4-alpha-linked oligosaccharides from growing alpha-1, 4-glucan chains and subsequently attaching these oligosaccharide to the alpha-1, 6 position . In our t leaves . In our KEGG pathway enrichment analysis indicated that DEmRNAs were significantly enriched in several amino acid- and protein-related pathways (Table S10), including protein export (ko03060), arginine and proline metabolism (ko00330), alanine, aspartate and glutamate metabolism (ko00250), and tryptophan metabolism tryptophan metabolism (ko00380). Glutamate synthase (NADH) (GLT1), encoded by g3579, is a key enzyme in ammonia assimilation processes, generating l-glutamate from l-glutamine and 2-oxoglutarate, and represents an alternative pathway to l-glutamate dehydrogenase for the biosynthesis of l-glutamate . In the g121, was upregulated by 2.41-fold, whereas its targeting miRNA (novel miR_278) was downregulated by 2.17-fold. Furthermore, we found that g20632 and g8996, which encode ACSL, were upregulated by 1.09- and 2.90-fold, respectively, whereas the miR159 family miRNA bdi-miR159a-3p, which targets both these genes, showed a decrease in expression with ovule development, being downregulated by 1.81-fold in the Ov2-vs-Ov4 comparison. Taken together, ACCase and ACSL are key enzymes in the initial stages of fat biosynthesis and the final step prior to fatty acid elongation, respectively, and all the three DEmRNAs were upregulated in the Ov2-vs-Ov4 comparison, concomitant with a downregulation of their targeted DEmRNAs. These DEmRNA/DEmiRNA pairs may be responsible for the activated lipid biosynthesis at stage Ov3 and Ov4.The conversion of acetyl CoA to malonyl CoA, catalyzed by acetyl-CoA carboxylase (ACCase), is an initial step in fat synthesis . The conMYB41, MYB3, MYB108, and ZFP2. We also deduced that DEmiRNAs within the MIR159, MIR166, MIR167_1, MIR396, and MIR319 family play important roles in seed development, embryogenesis, ovule growth, and cell proliferation via their regulation of MYB101, ABSCISIC ACID-INSENSITIVE 5, GRF5, GRF12, GRF1, polyadenylate-binding protein gene, TCP-1, and AP2. Similarly, we suggest that auxin signal transduction may be regulated by DEmiRNAs such as miR159 and miR319 in the MIR164, MIR 319, MIR167_1 families, whereas novel miR_278 and cme-miR166i may be involved in starch and sucrose metabolism pathways via their regulatory roles related to GlgB and SPS expression. Moreover, novel miR_278 and bdi-miR159a-3p may participate in fat biosynthesis via their regulatory roles related to ACCase and ACSL expression. We believe that the findings of this study provide valuable insights that will contribute to gaining a better understanding of regulatory roles of miRNAs in ovule development in hazelnut.We identified a total of 970 miRNAs in hazel, 471 of which were found to be differentially expressed in six paired stage-wise comparisons. Most of the highly expressed DEmiRNAs were found to contribute to stress tolerance, embryo and seed development, and cell proliferation. Among these, we assume that MIR858, MIR403, and MIR482 function to depress development due to nutrient competition, resulting in the high upregulated expression of stress tolerance target genes, including Corylus heterophylla × C. avellana ‘Dawei’, and C. heterophylla × C. avellana ‘Bokehong’ was used as a pollination cultivar. Pollen of ‘Bokehong’ was collected and air-dried using the male flower branch culture method, as described previously [In 2018, the plant samples used in the present study were collected from a hazel orchard in Siping, Jilin Province, China, in which the major hazelnut cultivar was eviously . These heviously , 5. In thttps://www.ncbi.nlm.nih.gov/sra/?term=PRJNA591492). The raw sequencing reads were filtered to generate clean reads using Trimmomatic software [https://www.cavellanagenomeportal.com).Total RNA was extracted using a Plant Total RNA Kit  according to the product instructions. Purity and concentration of RNA were determined by a spectrophotometer , and integrity of total RNA was assessed on a 1% denaturing formaldehyde-agarose gel. Good quality RNA was used to construct RNA libraries using an NEB Next Ultra small RNA Sample Library Prep Kit for Illumina . Initially, 3′ and 5′ adaptor sequences were ligated to the total RNA, and cDNA was obtained after reverse transcription. Thereafter, PCR amplification and gel purification were performed to generate the small RNA libraries, and single-end 50 nt (SE50) sequencing were performed using a HiSeq 4000 sequencing platform . Raw small RNA sequencing and mRNA data of 12 samples at four developmental stages were deposited in the Sequence Read Archive  software package (version v2.0.5)  to deterPutative precursor sequences were obtained by comparing the positional information in the hazel genome with the mapped reads. On the basis of read distribution information regarding the precursor sequences  and the precursor structural energy information (RNA fold randfold), we used a Bayesian model to identify novel microRNAs .2(FC) | > 1 and a false discovery rate (FDR) < 0.05 were used as discrimination criteria. Fold change (FC) represents the ratio of expression between two sample groups. Comparisons were performed between each pair of sample libraries , in which the former was used as the control and the latter as the experimental group in each paired comparison. The six pairs of miRNA profiles were compared in order to determine the changes in miRNA expression during ovule development and growth in hazel.Expression data were obtained for the miRNAs in each sample, and the miRNA expression levels were normalized as tags per million (TPM) using following formula : TPM = rOn the basis of known and newly predicted miRNAs and gene sequence information for corresponding species, we used Target Finder software (version v1.6)  to prediA paired design of mRNA extraction, mRNA library construction, and sequencing of the aforementioned 12 samples was carried out as described previously , 5, sequhttp://www.biocloud.net), GO and KEGG classification and enrichment analysis of all identified miRNA target gene and DEmiRNA-targeted mRNA with GO terms was conducted. The threshold for significant enrichment was set as P < 0.05.Using the online Gene Ontology (GO) and KEGG tools of BMKCloud (http://downloads.fyxm.net/Primer-Premier-101178.html) and synthesized by Sangon Biotech Co, Ltd. . The real-time quantitative RT-PCR analysis of mRNA was performed as described previously [−ΔΔCT method was applied to calculate the relative changes in gene expression [The total RNA samples used in sequencing were also subjected to real-time quantitative RT-PCR of miRNAs and predicted target genes. The primer pairs of several target genes used for real-time quantitative RT-PCR were designed using Primer 5.0 (eviously , 5. We spression .Additional file 1 Figure S1 Sample correlation diagramAdditional file 2 Table S1 All microRNAs and their expressionAdditional file 3 Table S2 Analysis of miRNA family of all identified miRNAAdditional file 4 Table S3 Target mRNA of all identified miRNAAdditional file 5 Table S4 All miRNA count at four developmental stagesAdditional file 6 Table S5 All DEmiRNA expression at four developmental stagesAdditional file 7 Table S6 Annotation of all target mRNAAdditional file 8 Table S7 DEmRNAs of DEmiRNAsAdditional file 9 Table S8 Target DEmRNA of DEmiRNA showing opposite changing trendAdditional file 10 Table S9 GO enrichment of DEmRNA of DEmiRNAAdditional file 11 Table S10 KEGG enrichment of DEmRNA of DEmiRNAAdditional file 12 Table S11 Primers for qRT-PCR analysis


Hepatocellular carcinoma (HCC), a leading cause of cancer-related death, is initiated and promoted by chronic inflammation. Inflammatory mediators are transcriptionally regulated by several inflammatory signaling pathways, including nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK). cJun N-terminal kinase (JNK), a member of the MAPK family, plays a central role in HCC pathogenesis. Pathogen-associated molecular patterns (PAMPs) activate JNK and other MAPK upon recognition by toll-like receptors (TLRs). Apart from TLRs, PAMPs are sensed by several other pattern recognition receptors, including cytosolic NOD-like receptors (NLRs). In a recent study, we demonstrated that the NLR member NLRP12 plays a critical role in suppressing HCC via negative regulation of the JNK pathway. This article briefly reviews the crosstalk between NLRP12 and JNK that occurs during HCC. CTCNB1, WNT, AXIN, TP53, CCND1, and CDKN2A are commonly found in HCC [Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer ,2. With d in HCC ,5. The pd in HCC . Irresped in HCC ,8.Nlrp12-/- mice are highly susceptible to hepatocellular carcinoma [Nlrp12-/- mice was associated with increased activation of the c-Jun N-terminal kinase (JNK) signaling pathway. Here, we will review our findings focusing on the link between NLRP12 and JNK in the regulation of HCC.The major pathways regulating inflammation in the liver include nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) ,11,12,13arcinoma . IncreasThe Ras/Raf/MAPK pathway is one of the major pathways associated with HCC ,29. ThreSince the JNK pathway plays a central role in maintaining homeostasis in the liver, its dysregulation is associated with inflammatory liver disorders, including steatosis, fibrosis, cirrhosis, and HCC ,33,34. JBecause of its tumor-inducing potential, activation of JNK in the liver needs to be maintained tightly, and such a regulation involves other inflammatory pathways and molecules. Mice deficient in IκB kinase β (IKKβ) in hepatocytes developed increased HCC following the administration of DEN . This fiJnk1-/- mice, but not Jnk2-/- mice [Among three isoforms of JNK , liver expresses JNK1 and JNK2. However, DEN-induced HCC development was significantly reduced in -/- mice ,47. Furt-/- mice . In agre-/- mice . While i-/- mice . Given t-/- mice ,49,50,51Although most in vitro and in vivo studies depicted JNK as a promotor for HCC, it is surprising that mice double-deficient in Jnk1 and Jnk2 in hepatocytes are susceptible to HCC . In contThe innate immune response against pathogens is initiated with germline-encoded PPRs ,54,55. AYersinia pestis [Nlrp12-/- mice are associated with higher inflammatory responses and activation of NF-κB and ERK pathways [Nlrp12-/- mice are hyper-responsive to TLR ligands, such as LPS, Pam3, and Poly I:C [Nlrp12-/- mice during Salmonella Typhimurium infection helped resolve the infection [NLRP12 belongs to the NLRP subfamily of NLRs. Like other NLRP family proteins, NLRP12 is composed of an N-terminal pyrin domain (PYD), central nucleotide binding domain (NBD), and a C-terminal leucine-rich repeat (LRR). NLRP12 is abundantly expressed in myeloid cell lineage, including macrophages, dendritic cells, monocytes, and neutrophils ,66. An ia pestis . Most ota pestis ,70,71. Ma pestis ,73,74. Ma pestis . Increaspathways . SupportPoly I:C . Cytokinnfection , suggestNlrp12-/- mice are highly responsive to antigen immunization. Thus, Nlrp12-/- mice develop atypical experimental autoimmune encephalomyelitis (EAE) due to increased production of Th2 cytokine IL-4 into the central nervous system [Nlrp12-/- T cells into immunodeficient Rag-/- mice also elicited exacerbated colitis [Nlrp12-/- mice [Nlrp12-/- mice should be validated by independent studies from other laboratories.While most studies found NLRP12 to inhibit NF-κB and MAPK pathways in myeloid cells, increasing evidence suggests that NLRP12 regulates these pathways in other cell types as well. T cells of s system . Transfe colitis . The rol colitis . Deficie colitis ,76,77. I colitis ,79. Rece-/- mice ,79. WhilNlrp12-/- mice developed significantly higher numbers of and larger tumors compared to WT mice [NLRP12 is altered in about 2% of HCC patients [NLRP12 is not a major cancer suppressor gene, its expression and activation status may regulate HCC pathogenesis.Given that chronic inflammation is a major driver for HCC and NLRP12 negatively regulates inflammatory responses, it is intriguing whether NLRP12 plays any role in HCC. In a recent study, we investigated the role of NLRP12 in HCC using mouse models in which tumors were induced by DEN or DEN plus carbon tetrachloride. In both experimental settings,  WT mice . This obpatients . AlthougNlrp12-/- mice was associated with higher expressions of the HCC marker Afp, inflammatory cytokines, and chemokines, including IL-6, TNFα, Cxcl1, Cxcl2, and Ccl2, protooncogene cJun, cMyc, and Cyclin D1, and reduced expression of p21 [Nlrp12-/- mouse livers, therefore, reflect an overall hyperinflammatory response [Nlrp12-/- livers [Increased HCC pathology in n of p21 . IL-6 ann of p21 ,80,81,82n of p21 ,84. In an of p21 ,8,82. Inn of p21 ,86. Highresponse . Notably- livers , indicatNlrp12-/- livers. Interestingly, Nlrp12-/- HCC showed higher JNK activation, but not NF-κB and ERK [Nlrp12-/- hepatocytes; there was no major difference in JNK activation in Kupffer cells and hepatic stellate cells isolated from wild-type and Nlrp12-/- mouse HCC [Nlrp12-/- mice exhibited increased activation of JNK and expression of cJun, cMyc, and Ccnd1 upon stimulation with LPS and other TLR ligands, e.g., Pam3 and PGN [As discussed above, inflammatory signaling pathways, including NF-κB, ERK, P38, JNK, and STAT3, regulate inflammatory responses and tumorigenesis. Since NLRP12 has been shown to downregulate the activation of NF-κB and ERK, these pathways were expected to be hyperactivated in  and ERK . This obouse HCC . The hep and PGN . Knockdo and PGN . Corrobo and PGN . OverallNlrp12-/- liver upon DEN-induced HCC induction [Nlrp12-/- livers did not show any difference in cell death and proliferation [Nlrp12-/- mice. The proliferation rate of hepatocytes was significantly higher in Nlrp12-/- mice, although no major difference in cell death was observed between WT and Nlrp12-/- hepatocytes [Nlrp12-/- liver was due to extrinsic factors, such as higher abundance of reactive oxygen and nitrogen species, cytotoxic cytokines , and proapoptotic molecules in the tumor microenvironment. Higher proliferation of Nlrp12-/- hepatocytes was associated with increased expression of pro-proliferative molecules, including cMyc, cJun, and Ccnd1. The JNK inhibitor dramatically reduced proliferation rate of hepatocytes and expression of cJun, cMyc, and Ccnd1, suggesting the key role of JNK in the hyperproliferative nature of Nlrp12-/- hepatocytes [A unique feature of the liver is its regeneration capacity. Liver that is subjected to partial hepatectomy regains its complete mass within 7 to 10 days ,44. Livenduction . Notablyferation . The rolatocytes . Notablyatocytes . Thus, hatocytes .Nlrp12-/- tumors is due to increased activation of TLR pathways by LPS or other PAMPs. Consistently, Nlrp12-/- mice treated with antibiotics did not develop visible tumors [JNK can be activated by PAMPs, as well as host-derived factors, such as TNFα, ROS, and stress ,82,89,90e tumors . In factGut microbiota-derived PAMPs play a central role in HCC pathogenesis. Both parenchymal and non-parenchymal cells express TLRs, and therefore, may respond to PAMPs, leading to the activation of JNK and other inflammatory pathways. Therefore, suppression of this gut-liver axis of JNK pathway may constitute an effective means for inhibiting HCC pathogenesis. Our recent study points to NLRP12 as an important molecular brake of this signaling axis and HCC pathogenesis. It is interesting that NLRP12 regulates JNK but not NF-κB and ERK in hepatocytes. Whether NLRP12 negatively regulates the JNK pathway in a manner similar to its inhibition of NF-κB and ERK in myeloid cells remains unknown. Previous studies have documented that NLRP12 interacts with IRAK1 to suppress canonical NF-κB, and NIK and TRAF3 to inhibit non-canonical NF-κB in myeloid cells ,91,92. I


TP53 gene in favor of their survival and propagation. Some of the mutant p53 proteins not only lose the wild-type activity, but also acquire oncogenic function, namely “gain-of-function”, to promote cancer development. Growing evidence has revealed that various E3 ubiquitin ligases are able to target both wild-type and mutant p53 for degradation or inactivation, and thus play divergent roles leading to cancer cell survival or death in the context of different p53 status. In this essay, we reviewed the recent progress in our understanding of the p53-targeting E3 ubiquitin ligases, and discussed the potential clinical implications of these E3 ubiquitin ligases in cancer therapy.The tumor suppressor p53 prevents tumorigenesis and cancer progression by maintaining genomic stability and inducing cell growth arrest and apoptosis. Because of the extremely detrimental nature of wild-type p53, cancer cells usually mutate the  The carcinogenesis is a complex event involving mutation or dysregulation of tumor suppressor genes, and amplification or activation of oncogenes. Wild-type p53 is the most influential tumor suppressor in human cancer. In response to a variety of stress signals, p53 is activated to inhibit malignant transformation, tumor cell growth, proliferation and motility, and cancer-fostering metabolism through different mechanisms.TP53 encodes a transcription factor, p53, consisting of three functional protein motifs, the transactivation domain (TAD), DNA-binding domain (DBD), and tetramerization domain (TD), critical for its tumor-suppressive activity. It has been well documented that p53 harnesses tumor growth and propagation through various mechanisms [The tumor suppressor gene chanisms ,2. Whilechanisms . In addichanisms ,7,8,9. Achanisms . In somechanisms . In addichanisms . Recentlchanisms ,14 by orchanisms ,16, cholchanisms , cardiolchanisms , polyamichanisms , α-ketogchanisms , and esschanisms ,22. AltoBesides acting as a transcription factor, the cytoplasmic p53 has also been found to play roles in tumor suppression . The cytAs the cytotoxic effect of p53 is so detrimental to cancer cells, a plethora of mechanisms have been evolved to inhibit p53 activity in order for tumors to survive and propagate . For instance, the E3 ubiquitin ligase MDM2 and its homologous partner, MDMX, which are often amplified or overexpressed in cancer ,29, haveHowever, it has been found that p53 can be modulated and activated via diverse post-translational modifications (PTMs) in response to various stress signals, including DNA damage, nucleolar stress, hypoxia, oxidative stress, telomere erosion, and oncogene activation ,2,48,49.Over the past decade, the nucleolus has emerged as a considerable cellular compartment monitoring the p53 signaling pathway ,57. The Taken together, p53 can execute its tumor-suppressive function through both transcription-dependent and -independent manners. Although p53 usually maintains a restricted level due to overexpression of a great many oncogenic inhibitors in cancer cells, it can be activated in response to various stress signals, which is critical for the development of strategies for cancer treatment.TP53 is the most frequently mutated gene in human cancers. For instance, it is mutated in over 95% of high-grade serous ovarian cancer [TP53 mutation across all cancer types is around 50% [n cancer ,65, 74% n cancer , 50–80% n cancer . The oveound 50% . The canound 50% .TP53 can repress the activity of the remaining wild-type allele in cancer cells. Two possible mechanisms have been described to account for this ‘dominant-negative’ effect. First, mtp53 may form cotetramers with its wild-type counterpart leading to the abolishment of its transcriptional activity [The emergence of missense mutations can abrogate the tumor-suppressive function of wild-type p53 (wtp53), because the mutations in the DBD prevent mutant p53 (mtp53) from binding to the p53-responsive DNA elements on the target gene promoter. In addition, mutation of one allele of activity . Moreoveactivity . Remarkaactivity . p53 mutactivity ,73. mtp5activity . It has activity ,76. Likeactivity  and caspactivity , and inhactivity  through activity . The mtpactivity ,82.Although the vast majority of p53 mutants lose the wild-type function or exert a ‘dominant-negative’ effect on the remaining wild-type allele in cancer cells, many of them, particularly the missense and hotspot mutations, promote cancer development by acquiring the oncogenic GOF, and are therefore potential targets for anti-cancer therapy.Although missense mutations mostly occur in the DBD and may locally or conformationally affect the structure and function of the p53 proteins, these mutants have been shown to share a multitude of binding partners with wtp53. Accumulating evidence has revealed that numerous E3 ubiquitin ligases can target both wt and mtp53 for degradation or inactivation . It is tThe RING-finger E3 ligase MDM2, encoded by a wtp53 target gene, interacts with and induces ubiquitination and proteolytic degradation of wtp53, as thus forming a negative feedback circuit, which plays a central role in the surveillance of wtp53 stability and activity ,52. ConsRNF128 encodes two isoforms of the ring finger protein RNF128 that was characterized as a crucial regulator of T cell function, cytokine production, and nutrient metabolism through in vitro and in vivo studies [ studies ,122,123. studies . Interes studies , reveali studies . The oth studies . TogetheTwo more members of the RING-finger E3 ubiquitin ligase family, PIRH2 and COP1 that are also encoded by wtp53 target genes, were found to negatively regulate wtp53 stability by promoting its proteolytic degradation ,88,90. TThe tripartite motif protein TRIM71 that also possesses a RING-finger motif with E3 ligase activity was found to interact with and degrade wtp53 through the ubiquitin-proteasome system leading to restricted apoptosis during neural stem cell differentiation and early brain development . It had Another tripartite motif protein, TRIM24, was initially described as a liver-specific tumor suppressor. In Trim24-null mice, hepatocytes were prone to undergo malignant transformation and progress to metastatic hepatocellular carcinoma by upregulating the retinoic acid signaling . HoweverAn additional case is the tumor suppressor TRIM19, better known as PML, that was found to induce wtp53 stabilization and activation through multiple mechanisms. Although having a RING-finger motif, TRIM19 has not been reported to possess E3 ligase activity. Instead, it enhances wtp53 acetylation through CBP  and MOZ The tripartite motif protein family comprises over 70 members, most of which contain a RING-finger motif displaying E3 ubiquitin ligase activity. Besides the above three TRIM proteins, a multitude of family members have been shown to modulate wtp53 activity . For insThe U-box E3 ligase CHIP (C-terminus of HSP70-interacting protein) was reported to induce degradation of wt and mtp53 involving UbcH5b as an E2 and the chaperone protein HSP70 as a bridge between the E3 ligase and p53s . CHIP apIn our recent work, by screening mtp53-interacting partners in ovarian cancer tissues harboring mtp53 , severalAlthough it is now evident that p53 is an essential determinant for the role of its associated E3 ubiquitin ligases, they do show some preference to regulate different cancer-related signaling pathways in certain cancer types. For example, TRIM24 was able to boost the ER, AR, and STAT3 pathways in breast, prostate and brain cancers, respectively ,129,130,Interestingly, it is noticed that many of the E3 ubiquitin ligases, including MDM2, RNF128, PIRH2, COP1, TRIM19 and TRIM24, are encoded by wtp53-inducible genes. This may partially explain why these E3 ligases are overexpressed in wtp53-harboring cancer as tumor promoters (genomic amplification should be another reason), whereas underexpressed in mtp53-harboring cancer as tumor suppressors. Chemotherapy or radiotherapy-induced DNA damage stress has been shown to induce wtp53 activation and, thus, the expression of these E3 ubiquitin ligases as negative feedback regulators for wtp53. Under such circumstances, it is therefore rewarding to investigate if targeting these E3 ligases can untie the auto-regulatory loops and potentiate the efficacy of the traditional cancer therapies.In conclusion, the p53 signaling pathway constitutes an overwhelming intricate regulatory network, and the cancer-associated mutations, particularly the GOF mutants, make it even more complex. Although the missense mutations cause the local or conformational structural alterations to generate a variety of p53 mutants, they do share some common binding partners with wtp53. The diverse functions of the E3 ubiquitin ligases, by targeting both wtp53 and mtp53, not only achieve distinct cellular outcomes leading to cancer cell survival or death, but also open new opportunities for potential targeting therapy. To thoroughly understand the underlying mechanisms is certainly instructive for precision medicine for cancer.


ORF1, ORF2, and ORF17 genes, either individually or in combination. Mutant viruses were analyzed for their replication properties in cultured equine dermal cells, PBMC infection efficiency, virus-induced changes in the PBMC proteome, and cytokine and chemokine expression profiles. ORF1, ORF2, and ORF17 are not essential for virus replication, but ORF17 deletion resulted in a significant reduction in plaque size. Deletion of ORF2 and ORF17 gene significantly reduced cell-to-cell virus transfer from virus-infected PBMC to EC. EHV-1 infection of PBMC resulted in upregulation of several pathways such as Ras signaling, oxidative phosphorylation, platelet activation and leukocyte transendothelial migration. In contrast, chemokine signaling, RNA degradation and apoptotic pathways were downregulated. Deletion of ORF1, ORF2 and ORF17 modulated chemokine signaling and MAPK pathways in infected PBMC, which may explain the impairment of virus spread between PBMC and EC. The proteomic results were further confirmed by chemokine assays, which showed that virus infection dramatically reduced the cytokine/chemokine release in infected PBMC. This study uncovers cellular proteins and pathways influenced by EHV-1 after PBMC infection and provide an important resource for EHV-1 pathogenesis. EHV-1-immunomodulatory genes could be potential targets for the development of live attenuated vaccines or therapeutics against virus infection.Equine herpesvirus type 1 (EHV-1) causes encephalomyelopathy and abortion, for which cell-associated viremia and subsequent virus transfer to and replication in endothelial cells (EC) are responsible and prerequisites. Viral and cellular molecules responsible for efficient cell-to-cell spread of EHV-1 between peripheral blood mononuclear cells (PBMC) and EC remain unclear. We have generated EHV-1 mutants lacking  Alphaherpesviruses are ubiquitous pathogens affecting human and animal populations ,2,3,4. HVaricellovirus in the subfamily Alphaherpesvirinae and are important pathogens that infect horses . Ho. Ho32]. ORF1 did not affect virus transfer at any stage.Following inhalation of EHV-1, the virus primarily replicates in respiratory tract epithelial cells, subsequently passes through the basement membrane, establishes cell-associated viremia, and, finally, is transferred to the endothelial epithelium . To mimip-values ≤0.05, as calculated by Perseus software. In total, 63 cellular proteins were upregulated and 78 proteins were downregulated. KEGG-pathway enrichment analysis of differentially expressed proteins (p ≤ 0.05) was calculated using Benjamini-corrected Fisher’s exact test. In Ab4-wt-infected PBMC, upregulation of several pathways including platelet activation, Ras signaling, leukocyte transendothelial migration, endocytosis, lysosome, oxidative phosphorylation pathways, and cAMP signaling pathways was detected. On the other hand, downregulation of proteins associated with herpesvirus infection, chemokine signaling, spliceosome, RNA degradation, and apoptotic pathways was observed  out of 23 cytokines tested in the panel. All standards and quality controls were within the specified range. In general, the release of most cytokines and chemokines was strongly inhibited upon Ab4-wt infection of PBMC. Only FGF-2 was expressed in infected PBMC at 6 hpi  . Infectiytokines . All mutytokines C. IFNγ l6) were infected with Ab4-wt at an MOI of 1. At 24 hpi, infected PBMC were applied over EC, and supernatants were collected at 3 and 6 h for cytokine estimation. In PBMC-EC co-cultures, non-infected PBMC released more cytokines than virus-infected PBMC. Further, Ab4-wt infected PBMC in the presence of EC did not show much difference in terms of release of additional cytokines and chemokines, except release of IL-6  clearly altered the cytokine expression profile and restored cytokine expression partially at different time points. ORF2 gene deletion expressed higher levels of IL-1β cytokine at all time points.Taken together, infection of PBMCs with Ab4-wt mostly resulted in downregulation of cytokine expression and deletion of different viral genes ; however, they had comparable viremia to Ab4-wt [ORF2 deletion mutant was attenuated; however, it had strong immunogenicity without altering viremia in Icelandic horses [ORF1, ORF2, and ORF17) are essential for virus pathogenesis and play an important role in virus spread from PBMC to EC.EHV-1 infection of PBMC is a critical step in deciding EHV-1 pathogenesis; thereby the virus can spread from respiratory epithelium to the endothelium without being captured by the host immune system. As described earlier, the process of virus transfer is complex, requires coordinated action of viral proteins, adhesion molecules expressed by both cells, and cytokines and chemokines for facilitating inter-cellular adhesion, intra-cellular trafficking, and cellular polarity ,72. Seven ponies . Experimo Ab4-wt . In anotc horses ,76. Failc horses ,49,50. AORF1, ORF2, and ORF17 genes of EHV-1 are dispensable for virus replication in equine cells as confirmed by growth kinetics and plaques size assays. Double and triple gene deletion mutants also replicated normally as with the parental virus. Only ORF17 deletion mutant showed a significant, but slight, reduction in plaque size, ranging between 10–15%. Albeit the plaque size reductions were less, similar plaque size reductions of UL43 (ORF17 homolog) deletion mutants were observed in HSV-1 and PrV [ and PrV ,78. DespORF17 and ORF2 as single-gene deletion mutant significantly reduced virus transfer between PBMC and EC under static and flow conditions. Furthermore, ORF17 and ORF2 mutants (as double and triple gene deletions mutants in combination with ORF1) also reduced virus transfer to EC. Double and triple gene deletions showed an additive reduction in virus transfer to EC in a flow condition compared to static condition, which can be attributed to a synergetic effect on the functional dynamic rolling of infected PBMC over EC. The flow chamber system creates homogenous fluid shear stress in the endothelium similar to that observed in the blood vessel environment in vivo. Fluid flow over the endothelium results in ranges of ion fluxes, modulation of several pathways including biochemical pathways and gene and protein expression in both in vitro and in vivo. Specifically, shear forces modulate cell surface transmembrane adhesion molecules via cytoskeleton by integrin-dependent activation of MAP kinases via Ras GTPase and RhoA activation [ORF2 and ORF17 modulated the MAP kinase and Ras GTPase pathways, which might explain the reduction of virus spread.We show here that PBMC can be infected, to similar levels, with all mutant viruses without any significant differences. Co-cultivation and flow chamber assays with parental EHV-1 virus showed efficient virus spread from infected PBMC to EC as reported previously . Howevertivation . Furthertivation , which wORF2 and ORF17) are involved in modulating several pathways in PBMC, including cell signaling, adhesion, and immune pathways, thereby ORF2 and ORF17 gene deletions resulted in reduced virus transfer to EC. Our findings clearly correlate with previous in vivo findings where ORF2, ORF1/ORF71, and ORF1/ORF2 deletion mutants showed respiratory infection, comparable viremia, but no noticeable endothelial cell infection in four different experimental studies in horses [In the epithelium-PBMC contact assay, all mutant viruses transferred from infected epithelial cells to PBMC similar to the parental virus. However, subsequent flow chamber assay with infected PBMC showed reduced virus transfer to EC. It is very clear that gene deletions affected only the virus transfer step to EC, but not from the epithelium to the PBMC. The process of cell-to-cell virus transfer from infected epithelial cells to PBMC is relatively simple. EHV-1 undergoes full replication cycles in the epithelium and the released infectious virus particles result in dendritic cell infection ,82,83. Sn horses ,49,50,76It was shown before that all three PBMC subpopulations could be infected with EHV-1 ,20,85,86EHV-1 undergoes limited virus replication in PBMC ,85, but Parental EHV-1 infection upregulated proteins associated with several host pathways including MAPK, Ras signaling, endocytosis, oxidative phosphorylation, lysosomal pathways but downregulated herpesvirus and spliceosome pathways. MAPK pathway is involved in the manipulation of cellular functions such as signal transduction, cell adhesion, virus replication in target cells, and cell survival ,91. Ras-ORF1, ORF2, and ORF17 gene deletions have modulated several pathways, mainly chemokine signaling, MAPK, herpesvirus infection, and oxidative phosphorylation pathways. Ab4-wt infection downregulated herpesvirus infection pathway, while Ab4∆ORF1/ORF2/ORF17 mutant virus upregulated this pathway. Similar to the single gene deletion (Ab4∆ORF1 and Ab4∆ORF2), triple gene deletions modulated several pathways including chemokine signaling pathways. A previous in vitro study showed the potential role of MAPK pathway in EHV-1-infected PBMC. Inhibition of this pathway with a chemical inhibitor reduced virus transfer from infected monocytes to EC [ORF17 and ORF2 gene deletions downregulated the proteins associated with MAPK pathway and showed reduced virus transfer to EC. We surmise that ORF17 and ORF2 viral proteins are involved in upregulating the MAPK pathway thereby facilitating virus transfer to EC. As described earlier, MAPK is essentially involved in transmitting various extracellular signals that induce cellular proliferation, differentiation, and survival [ORF17 and ORF2 in modulating MAPK pathway will be the scope of our future studies.While comparing the proteomic profile of Ab4-wt and mutants infected PBMC, es to EC . In our survival . It has survival ,106. StuORF1 deletion showed release of more cytokines in infected PBMC. Similar findings were confirmed by mRNA expression, cytokine quantification, and migration assay [ORF1 deletion mutants. IFNγ is produced by antigen-activated leukocytes. Secreted IFNγ activates antigen-specific immunity, innate-cell mediated immunity, and cytokines/chemokines release. Ab4-wt infection blocks IFNγ release from PBMC and deleting ORF1 gene resulted in release of more cytokines/chemokines which shows the immune-modulating potential of ORF1 in Ab4 strain. ORF2 gene deletion also showed release of more cytokines in both in vitro and in vivo [ORF2 gene deletion resulted in expression of higher levels of IL-1β cytokine, which is considered as a host defense against virus infection. It is a well-known immune evasion strategy employed by HSV-1 to retain IL-1β in the intracellular space of infected macrophages without being released to the extracellular space by blocking the function of caspase-1, which blocks the pro-inflammatory activity of IL-1β [ORF2 of EHV-1 also may have a similar function, thereby Ab4-wt infection in PBMC results in decreased release of IL-1β, while ORF2 deletion restores expression. The role of ORF2 in modulating of IL-1β secretion could be the potential area for further investigation. ORF17 gene deletion did not restore cytokine/chemokine release completely, however, released more cytokines than parental EHV-1.Parental EHV-1 infection greatly reduced cytokine and chemokine release. Higher levels of FGF-2 in Ab4-wt infected PBMC can be correlated with activation of MAPK pathway as confirmed by proteomic analysis. FGF-2 results in the activation of Ras-MAPK pathway, which is essential for virus spread ,108. Preon assay . Further in vivo ,76. ORF2of IL-1β ,112. We ORF1 and ORF2 in its genome in comparison to Ab4-wt and showed release of more cytokines upon infecting PBMC [ORF1 and ORF2 genes in Ab4 restored expression of more cytokines, which clearly demonstrates that ORF1 and ORF2 have a major role in cytokine production.RacL11 strain, which is poorly spread to EC, naturally lacks ing PBMC . SimilarORF1 probably plays a major role in Ras-signaling, chemokine signaling, and cell adhesion pathways. ORF2 and ORF17 are implicated in chemokine signaling and MAPK signaling pathways thereby facilitating cell-to-cell virus spread. Furthermore, ORF1, ORF2, and ORF17 deletion mutants  were capable of stimulating strong cytokine responses in vitro. These genes could be potential targets for the development of live attenuated vaccine therapeutics against EHV-1 infection in equines. In addition, identifying possible interactions and finding interaction partners for ORF1, ORF2, and ORF17 proteins including viral and cellular proteins are the potential area of future studies.In conclusion, we have identified and confirmed immune evading and cytokine modulating properties of different viral proteins of herpesviruses. Based on our flow chamber, proteomics, and cytokine/chemokine assay, all three targeted genes were multifunctional in nature. Nevertheless, these viral proteins are involved in more than one pathway in PBMC. We presume that