Phosphatidylinositol 3 kinase (PI3K)/AKT  signalling regulates various cellular processes, such as apoptosis, cell proliferation, the cell cycle, protein synthesis, glucose metabolism, and telomere activity. Corneal epithelial cells (CECs) are the outermost cells of the cornea; they maintain good optical performance and act as a physical and immune barrier. Various growth factors, including epidermal growth factor receptor (EGFR) ligands, insulin-like growth factor 1 (IGF1), neurokinin 1 (NK-1), and insulin activate the PI3K/AKT signalling pathway by binding their receptors and promote antiapoptotic, anti-inflammatory, proliferative, and migratory functions and wound healing in the corneal epithelium (CE). Reactive oxygen species (ROS) regulate apoptosis and inflammation in CECs in a concentration-dependent manner. Extreme environments induce excess ROS accumulation, inhibit PI3K/AKT, and cause apoptosis and inflammation in CECs. However, at low or moderate levels, ROS activate PI3K/AKT signalling, inhibiting apoptosis and stimulating proliferation of healthy CECs. Diabetes-associated hyperglycaemia directly inhibit PI3K/AKT signalling by increasing ROS and endoplasmic reticulum (ER) stress levels or suppressing the expression of growth factors receptors and cause diabetic keratopathy (DK) in CECs. Similarly, hyperosmolarity and ROS accumulation suppress PI3K/AKT signalling in dry eye disease (DED). However, significant overactivation of the PI3K/AKT signalling pathway, which mediates inflammation in CECs, is observed in both infectious and noninfectious keratitis. Overall, upon activation by growth factors and NK-1, PI3K/AKT signalling promotes the proliferation, migration, and anti-apoptosis of CECs, and these processes can be regulated by ROS in a concentration-dependent manner. Moreover, PI3K/AKT signalling pathway is inhibited in CECs from individuals with DK and DED, but is overactivated by keratitis. CECs are present in the outermost layer of the cornea and play an important role in maintaining CE homeostasis and optical function.The PI3K/AKT signalling pathway is widely involved in the proliferation, apoptosis, migration, and other functions of CECs.Many corneal diseases and wound healing are associated with the PI3K/AKT signalling and its interaction with ROS.How do CECs respond to different environmental conditions, such as health and disease states, through the PI3K/AKT signalling pathway?How does the PI3K/AKT signalling pathway interact with ROS in CECs to control the cellular response and influence disease progression?Can the PI3K/AKT signalling pathway be used as a major target for the treatment of corneal diseases in the future?AKT1 gene on human chromosome 14, band q32 [In 1987, Staal first discovered a proto-oncogene, i.e., the band q32 , 6. ThesThe eye is a complex structure that consists of the cornea, lens, vitreous, choroid, retina, optic nerve, and other accessory structures Fig. . The corDiabetic keratopathy (DK) is caused by diabetes-associated hyperglycaemia  and manifests as persistent corneal epithelial erosion, superficial punctate keratopathy, delayed epithelial regeneration, and reduced corneal sensitivity, which may cause impaired vision or permanent vision loss . Dry eyeThe PI3K/AKT signalling pathway plays an important role in the biological responses of CECs –19. Impohttps://pubmed.ncbi.nlm.nih.gov/) using the keywords “AKT AND corneal epithelial cells” and “AKT AND corneal epithelium”. Afterwards, we carefully read and categorized the results, removed some irrelevant articles, and then focused on recent studies and reviews (published between 2015 and 2022). Additional studies were discovered by consulting the reference lists of the selected articles. Most of the references cited in this review are experimental articles, although a few review articles, case reports, and book chapters are included; no other types of literature are cited. Our figures were edited with Adobe Illustrator CC 2018 software .We searched the PubMed database -dependent ectodomain shedding and transactivation of EGFR signalling (proto-oncogene tyrosine-protein kinase Src/PI3K/AKT), EGFR ligands, including epidermal growth factor (EGF), transforming growth factor-α, and heparin-binding EGF-like growth factor (HB-EGF), inhibit apoptosis and promote proliferation, migration in CECs, and healing of the CE .Eukaryotic translation initiation factor 5A (eIF5A), a nucleocytoplasmic shuttle protein, is the substrate of the EGF-induced EGFR/Src/PI3K/AKT/mTOR/p70S6K/rpS6 signalling pathway and induces the expression of MMP-9 in the extracellular matrix and proliferating cell nuclear antigen in vitro , 26. Fur2+ increases the activity of Src, and a disintegrin and metalloproteinase and transactivating the HB-EGF/EGFR/Src/PI3K/AKT cascade [The HB-EGF/EGFR/Src cascade is a target of various endogenous and exogenous molecules. Furthermore, the influx of extracellular Ca cascade , 28. In  cascade , 30. The cascade , 30.2+ influx-induced transactivation of EGFR/Src/PI3K/AKT signalling in hCECs and mouse CECs in vitro [The endocannabinoid system regulates the proliferation and migration of CECs partially by transactivating or inhibiting the EGFR/PI3K/AKT signalling pathway , 32. Twoin vitro . Howeverin vitro . This opIn hCECs, IGF1 binds to the IGF1 receptor to activate the insulin receptor substrate 2/PI3K/AKT signalling pathway and activate proliferation, but these effects are inhibited by IGF-binding protein 3 in CECs –38. FurtOther growth factors, including transforming growth factor-β (TGF-β), hepatocyte growth factor (HGF), keratinocyte growth factor, nerve growth factor, vascular endothelial growth factor B, and pigment epithelium-derived factor, also activate the PI3K/AKT signalling, which promotes the proliferation, migration of CECs, inhibits CEC apoptosis and promotes healing of the CE –44.The relationship between growth factors and DK is discussed in section 5.2.As a neuropeptide that is widely distributed in nerve fibres, NK-1  facilitate healing in the CE and the recovery of mitochondrial function in CECs , 46. By ROS are the byproducts of oxygen and are produced due to cellular metabolism, and regulate the redox balance, proliferation, cytotoxicity, and metabolic adaptation in a concentration-dependent manner , 53–56. Extreme environments including hyperglycaemic and hyperosmolar environments may cause excess ROS accumulation, which is considered an upstream suppressor of the EGFR-mediated PI3K/AKT signalling pathway, and delays healing of the CE . In vitrThe role of ROS in diabetes and hyperglycaemia is discussed in section 5.1.1.Interestingly, ROS production may also be induced by growth factors, and may function as a second messenger to activate PI3K/AKT signalling at low or moderate levels. These effects stimulate the proliferation and migration of healthy CECs, inhibit apoptosis of healthy CECs, and induce healing of the CE in vitro (in human and rabbit CECs) and ex vivo (in pig cornea) , 65. AltIn the healthy CE, the levels of phosphorylated PI3K and AKT are relatively low to maintain the homeostasis of CECs. When the CE is damaged, growth factors and NK-1 are secreted to activate the PI3K/AKT cascade, which promotes proliferation and migration and inhibits apoptosis and inflammation of CECs. These effects together enable the healing of the CE. However, under the influence of various factors , both the CE homeostasis and the normal expression of the PI3K/AKT signalling pathway are disrupted. These processes are associated with many diseases, including DK, DED, and keratitis Table , Fig. 3.Diabetes-associated hyperglycaemia stimulates apoptosis and inflammation but inhibits migration and tight junctions in CECs, thus causing DK .Hyperglycaemic conditions induce mitochondrial fragmentation, which leads to ROS overproduction that inactivates PI3K/AKT . Silent + and its precursors nicotinamide mononucleotide and nicotinamide riboside attenuate ROS accumulation, restore the mitochondrial membrane potential and facilitate the healing of the CE and regeneration of corneal nerves [Antioxidant therapy, which induces the clearance of excess ROS and activates the AKT pathway, is an effective drug treatment strategy for DK. For example, N-acetylcysteine reactivates the PI3K/AKT signalling pathway by inhibiting excess ROS production and restoring CEC migration and healing of the diabetic human and pig CE in vitro and ex vivo . Similarl nerves . Moreovel nerves , 78, 79.ER stress inhibits the PI3K/AKT signalling pathway and causes apoptosis and inflammation . In indiInterestingly, inhibition of ER stress activates the AKT pathway and promotes healing of the CE. As neuroprotective factors, most mesencephalic astrocyte-derived neurotrophic factors are retained in the ER and play an important role in maintaining ER stability , 84. MorHyperglycaemia inhibits the expression of growth factor receptors in CECs. In the context of diabetes, the expression of the c-met , which may directly inhibit HGF/c-met/PI3K/AKT signalling, is decreased in CECs in vitro . The expInactivation of the EGFR/PI3K/AKT signalling pathway leads to the overexpression of MMP-10 and cathepsin F. MMP-10 and cathepsin F then delay wound healing –91. TherClinically, growth factors, NK-1, and MMPs have been widely used to treat DK. We discussed that PI3K/AKT signalling is a target pathway related to DK, indicating that other drugs that reactivate PI3K/AKT signalling can be developed to treat DK.DED-induced tear film hyperosmolarity inhibits the PI3K/AKT signalling, which is partially responsible for the occurrence and progression of inflammation, autophagy, and apoptosis in CECs .Hyperosmolarity may increase the expression of high-mobility group box 1 (HMGB1), tumour necrosis factor α (TNF-α), and IL-1β in a dose-dependent manner by inhibiting PI3K/AKT signalling in CECs in vivo and in vitro . ActuallTherefore, reactivation of PI3K/AKT signalling may be an effective treatment for DED. As a type of carotene, astaxanthin restores the activity of the PI3K/AKT signalling pathway to promote HMGB1 expression and inflammation . In addiFurthermore, in an in vitro CEC model of DED, the transcription factor EB activates autophagy, and the PI3K/AKT signalling pathway inhibits transcription factor EB activity to exert an anti-autophagy effect . HoweverIn summary, CsA, astaxanthin, and trehalose inhibit inflammation, apoptosis, and/or autophagy in DED models by restoring PI3K/AKT signalling pathway activity. Clinically, we propose that PI3K/AKT signalling may be a crucial target for DED treatment.The PI3K/AKT signalling pathway is a key pathway that mediates keratitis and has potential as a therapeutic target for keratitis.Infectious keratitis is mostly caused by microbial infections, such as bacterial, fungal, and viral infections. Studies have shown an important role the PI3K/AKT signalling pathway plays in the infection of CECs with these pathogens and is a target of many drugs.Bacterial keratitis accounts for approximately 65–90% of all cases of microbial keratitis . BacteriFusarium, Aspergillus, and Candida. Previous studies have shown that extracellular polysaccharide EPS-II partially inhibits the adherence of Candida albicans to CECs, partially suppresses PI3K/AKT signalling, and decreases levels of inflammatory proteins (IL-6 and MMP-14) in fungal keratitis [C. albicans to hCECs and may activate cellular responses by binding with receptors, contains ligands similar to C. albicans in vitro [Fungal keratitis is caused by fungal infections, such as eratitis , 107. EPin vitro .Serratia marcescens, and a selective 3-phosphoinositide-dependent kinase 1 inhibitor called BX795 [Viral keratitis often causes infectious blindness, and herpes simplex virus type 1 (HSV-1) is one of the most common human pathogens . Most ofed BX795 , 109. Aned BX795 , 111. HeIn summary, inflammation and virus replication are mediated by PI3K/AKT signalling in infectious keratitis. C3G, asiatic acid, EPS-II, PD, BX795, and other drugs reverse overactivation of PI3K/AKT signalling to treat infectious keratitis.Noninfectious keratitis is associated with injury and other inflammatory agonists. The PI3K/AKT signalling pathway is closely related to noninfectious keratitis, but the specific mechanism must be further elucidated. For instance, exposure to nitrogen mustard, a vesicating agent, increases cyclooxygenase 2 (an inflammation mediator), MMP-9, and vascular endothelial growth factor levels in CECs potentially through the activation of the AKT-activator protein 1 pathway in vitro and ex vivo . MoreoveOverall, our review mainly summarizes the role of the PI3K/AKT signalling pathway in CECs. Growth factors , NK-1, and insulin activate the PI3K/AKT signalling pathway by binding to their receptors, which inhibit apoptosis and inflammation, promote the proliferation and migration of CECs, and accelerate healing of the CE. Extreme environments-induced excess accumulation of ROS inhibits PI3K/AKT signaling, thus inducing CEC apoptosis and inflammation, but low or moderate levels of ROS activate PI3K/AKT inhibit apoptosis, and promote the healthy CEC migration and proliferation.Diabetes-associated hyperglycaemia directly inhibit PI3K/AKT signalling by increasing ROS and ER stress levels or suppressing receptors of growth factors in CECs and partially induce DK. Similarly, in DED, the PI3K/AKT signalling pathway is suppressed by hyperosmolar conditions and ROS. However, in keratitis, overactivation of the PI3K/AKT signalling pathway is responsible for inflammation and virus replication in CECs Fig. .Fig. 4ThWe realized that a few studies have reported the status of PI3K/AKT signalling in different keratitis, but the specific mechanisms are not well known. Furthermore, the development of more effective drugs that target the PI3K/AKT signalling to treat related corneal diseases is needed.Main text and tables (marked)-R3 with Data availabilityTitle, running title and abstract-R3aj-checklist for CDDIS-22-0330REditing Certificate of AJE


Osteoblasts are discovered to secrete hormones with endocrine effects on metabolism, and osteocalcin (OC) is the most abundant non-collagenous protein in bone. We investigate the relationship between serum OC levels and glycolipid metabolism and muscle function in children with osteogenesis imperfecta (OI).A total of 225 children with OI and 80 healthy controls matched in age and gender were included in this single center study. Serum levels of fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), low- and high-density lipoprotein cholesterol  were measured by automated analyzers. Serum levels of fasting insulin (FINS) were measured using an automated electrochemiluminescence system. Serum levels of OC and undercarboxylated osteocalcin (ucOC) were measured by enzyme-linked immunosorbent assay. Grip strength and timed-up-and-go (TUG) test were measured. Bone mineral density (BMD) and body composition were measured using dual-energy X-ray absorptiometry.P<0.05). Serum OC, ucOC levels, and ucOC/OC in OI type III patients were significantly lower than those in OI patients with type I and IV. Serum levels of OC, ucOC, and ucOC/OC were negatively correlated to BMI, FBG, insulin levels, and HOMA-IR . The ratio of ucOC/OC was positively correlated to grip strength , lean mass percentage (%LM) of the total body and limbs, and negatively correlated to fat mass percentage (%FM) of the total body, %FM and fat mass index (FMI) of the trunk .OI patients had significantly higher body mass index (BMI), FBG, and HOMA-IR, but lower HDL-C levels, lower grip strength and longer TUG than control group (all Obesity, glucolipid metabolic abnormalities, and reduced grip strength were common in children with OI. Circulating osteocalcin and ucOC may play an important role in the regulation of glucose metabolism, as well as the muscle function of children with OI. Mutations in unctions , 8. Receunctions , which wTherefore, the objective of this study is to evaluate glycolipid metabolism and muscle function of children with OI and to investigate their relationship to serum levels of OC and ucOC.This was a single-center cross-sectional study conducted in endocrinology department of Peking Union Medical College Hospital (PUMCH) from January 2018 to April 2021. Patients with OI less than 18 years old and age- and gender- matched normal controls were included. The patients were diagnosed as OI if they met the criteria: Patients had a history of more than one fracture under minor trauma, and with an age- and sex-adjusted BMD Z-score less than or equal to -1.0 at lumbar spine (LS) or femoral neck (FN); or patients with BMD Z-scores ≤ -2.0 at LS or FN; and then the pathogenic mutation was found in these patients , 11. PatPatients with OI were classified into subtypes according to Sillence classification and clinical characters: type I, mild phenotype; type II, perinatally lethal; type III, a severe form with progressive deformity; type IV, moderate severity; and type V, characterized by calcification of the hyperplastic callus formation, forearm interosseous membrane and radial head dislocation.The study was approved by the Scientific Ethics Committee of PUMCH. The legal guardians of OI patients and normal controls provided written informed consents before they participated in this study.2). Overweight and obesity were defined as BMI higher than the 85th and 95th percentile of the reference of Chinese normal children , and the age- and gender-specific Z scores were calculated according to the normal reference of Chinese children . Body machildren .Blood samples were collected after an overnight fast. Serum levels of OC and ucOC were measured by enzyme-linked immunosorbent assay  following the manufacturer’s instructions, with the detection range of OC and ucOC as 0.5-16.0 ng/ml and 0.25-8.0 ng/ml, respectively. The intra-assay coefficients of variation (CV) were 3.0%-4.8% and 4.4%-6.7% for OC and ucOC measurement, respectively. The inter-assay CVs were 0.7%-2.4% and 5.7%-9.9% for OC and ucOC detection, respectively.Serum levels of fasting blood glucose, triglyceride, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, calcium (Ca), phosphate (P), alkaline phosphatase , alanine aminotransferase (ALT) and creatinine (Cr) were measured by automated analyzers . Serum levels of fasting insulin (FINS), beta cross-linked carboxy-terminal telopeptide of type I collagen , 25-hydroxyvitamin D (25OHD), and intact parathyroid hormone (PTH) were measured using an automated electrochemiluminescence system . Values of insulin sensitivity/resistance were calculated as Homeostasis model assessment insulin resistance (HOMA-IR), which equaled to FBG (mmol/L) × FINS (μU/mL)/22.5. Homeostasis model assessment islet beta-cell function (HOMA-β) was equal to 20×FINS (μU/mL)/-3.5) (%). As renal function was a possible influence factor of OC , we used2). Appendicular mass was calculated as the sum of upper and lower limb mass. For males, slight-moderate or severe increased body fat was defined as body fat mass percentage more than 20% or more than 25%, respectively. For females, slight-moderate or severe increased body fat was defined as body fat mass percentage more than 30% or more than 35%, respectively , femoral neck (FN) and total hip of OI patients were measured by dual-energy X-ray absorptiometry . BMD Z scores of LS and femoral neck were calculated based on reference data of BMD in Chinese and Asian children , 17. Eacectively .Grip strength of the dominant hand was measured by a handheld dynamometer , and the highest of three attempts was recorded. Physical function was measured by the timed-up-and-go (TUG) test, of which patients were timed to stand from the chair, walk 3 meters, turn, and then return to the seated position in the chair .COL1A1, COL1A2, IFITM5, SERPINF1, CRTAP, SERPINH1, FKBP10, SP7, BMP1, TMEM38B, PLOD2, P3H1, P4HB, PPIB, SEC24D, SPARC, WNT1, PLS3, CREB3L1 and MBTPS2 (Genetic mutations of OI were identified by a targeted next-generation sequencing (NGS) panel  which was previously described in detail  were expressed as mean ± standard deviation (SD). Abnormal distribution  were presented as median (quartiles). Categorical data were expressed as the number and percentage (%). The independent sample t-test and the analysis of variance (ANOVA) were utilized to compare continuous data of normal distribution among different clinical or genetic subgroups. Continuous data of abnormal distribution for two groups and more than two groups were analyzed by the Mann-Whitney U-test and Kruskal-Wallis test, respectively. The Chi-squared test and the Fisher test were used to analyze categorical variables. Relationships between OC or ucOC levels and glycolipid metabolic parameters or muscle function were analyzed using the Spearman correlation. Multiple linear regression models were used for adjustments of age, gender, 25OHD, eGFR, ambulatory status, and clinical classifications.P value was less than 0.05.The SPSS software version 20.0  was used to perform all statistical analyses. Statistical significance was considered if the two-tailed 2, which were higher than that of control group(P=0.003). There were 45 (20.0%) OI patients with obesity, and the percentage was higher than the control group (P=0.043). OI patients had significantly higher FBG levels and HOMA-IR, but lower HDL-C levels than the control group . The serum levels of OC in OI patients were (24.56 ± 11.51) ng/ml, which were lower than the control group (P=0.040). The serum of ucOC and the ratio of ucOC/OC had no significant difference between the OI patients and control group . Overweight was found in 8 (8.3%), 3 (7.5%), and 1 (1.2%) patients with type I, type III and type IV OI, and obesity existed in 16 (16.7%), 11 (27.5%) and 14 (17.3%) of the above subgroups patients with OI, respectively. Serum levels of FBG were significantly higher in OI type III patients than in OI type I and type IV patients, but HOMA-IR, HOMA-β, TC, LDL-C, HDL-C, and TG were similar among OI type I, III and IV groups. Serum OC, ucOC levels, and ucOC/OC in OI type III patients were significantly lower than those in OI patients with type I and IV , COL1A2 , IFITM5 , FKBP10 , WNT1 , PLS3 , TMEM38B , PLOD2 , SERPINF1 , BMP1 , SERPINH1 , CRTAP , and P3H1 .The mutated genes identified in OI patients included COL1A1, COL1A2, IFITM5 and the autosomal recessive (AR) gene mutations group. Patients with IFITM5 mutation had significantly lower BMI than AR group. There was no significant difference in the percentage of overweight or obesity, serum levels of FBG, insulin, HOMA-IR, HOMA-β, TG, TC, LDL-C, HDL-C, OC, ucOC and ucOC/OC among the four subgroups of genotypes , and trunk/limb fat mass ratio of the COL1A2 mutant group was higher than COL1A1 mutant group , but negatively correlated with total body and trunk %FM . The ratio of ucOC/OC was positively correlated with total body and appendicular %LM (P=0.046). After adjusting for age, gender, 25OHD level, eGFR, ambulatory status and clinical classifications, the correlation between ucOC and body composition still existed (P=0.033).Body composition was measured in 25 OI patients. 14 patients had moderately or severely increased body fat percentage, and 4 patients had slightly increased body fat percentage. The total body LMI of OI type III was significantly lower than that of OI type IV patients .Grip strength and TUG test were measured in 23 and 15 OI patients, as well as 20 healthy controls, respectively. OI patients had significantly lower grip strength and longer TUG than control group ’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.W-BZ carried out the biochemical measurement, collected the clinical data from the patients, analyzed the data and wrote the manuscript. JH, B-NZ and D-CZ contributed to collection of clinical data and blood sample. OW, YJ, W-BX and X-PX contributed to review the manuscript. ML contributed to the conception and design of the research, acquisition and interpretation of the data, and revised the manuscript. All authors contributed to the article and approved the submitted version.This work is supported by National Key R&D Program of China (2018YFA0800801), National Natural Science Foundation of China , and Beijing Natural Science Foundation (7202153).We appreciated the OI patients and normal controls for their participation.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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The sinonasal cavities harbor a wide variety of rare cancer types. Histopathological classification can be challenging, especially for poorly differentiated tumors. Despite advances in surgery and radio-chemotherapy, the 5-year survival rate is still very low. Thus, there is an unmet clinical need for new therapeutic options. We retrospectively evaluated poorly differentiated tumors of 9 different histological subtypes from 69 patients who had received conventional treatments for the presence of CD8+ tumor-infiltrating lymphocytes (TILs), as well as the expression of PD-L1 and microsatellite instability (MSI) markers MLH1, MSH2, MSH6 and PMS2, as biomarkers for immunotherapy. CD8+ TILs were present in 23/69 (33%) cases, PD-L1 expression was observed in 23/69 (33%), and markers for MSI positivity in 5/69 (7%) cases. CD8+ TILs correlated with PD-L1 positivity, while both were mutually exclusive with MSI markers. None of the biomarkers were associated with clinical features as age, gender or tumor stage. Cases with CD8+ TILs and PD-L1 positivity showed a tendency toward worse disease-specific survival. Immune checkpoint inhibitors are emerging as new options for treatment of many tumor types. Our results indicate that also a substantial subset of patients with poorly differentiated sinonasal tumors may be a candidate to be treated with this promising new therapy. Approximately 5% of all head and neck cancers arise in the sinonasal cavities . They reA growing number of these tumors can now be diagnosed by molecular genetic analysis, such as NUT carcinoma by chromosomal translocation t 15;19) NUT-BRD4, HPV-related multiphenotypic sinonasal carcinoma by high-risk HPV in the absence of the t 6;9) MYB-NFIB rearrangement, and subsets of SNSCC by EGFR exon 20 mutation and by DEK::AFF2 fusions [;19 NUT-B;9 MYB-NFA common factor of sinonasal tumors is their very low incidence, making it difficult for the clinician not only to reach a correct diagnosis, but also to develop and test treatment options. Despite advances in endoscopic surgery, precision radiotherapy, heavy ion radiotherapy, and induction chemotherapy, the 5-year survival rate remains less than 50%. For SNEC, SNUC, and NUT carcinoma prognosis is counted in months ,14,15. LTo improve these outcomes, new treatment options for neoadjuvant, concomitant or adjuvant therapy are necessary, and immunotherapy may be such an option. Immune checkpoint inhibitors have improved outcomes for different types of solid tumors, including head and neck squamous cell carcinoma, non-small cell lung cancer and oesophageal squamous cell carcinoma ,20,21. OPrevious studies on sinonasal tumors have indicated PD-L1 expression in 26% of ITAC and 30–46% PD-L1 expression in SNSCC ,24,25,26MSI is due to defects in the genes involved in the MMR (mismatch repair) system, such as MLH1, MSH2, MSH6 and PMS2. Although it was initially described in patients with sporadic colon cancer and hereditary non-polyposis colorectal cancer, it has now been demonstrated in many types of tumors. In two recent studies using MLH1, MSH2, MSH6 and PMS2 staining as surrogate markers of MSI, 3.2% (4/125 cases) and 2.3% (3/131 cases) were found to be MSI positive, indicated by loss of or reduced PMS2 and MLH1 ,29. In aTo our knowledge, apart from SNSCC, ITAC and ONB, no other sinonasal tumor types have been studied with regard to biomarkers for immunotherapy. The aim of this study was to evaluate the presence of CD8+ TILs, PD-L1 expression and the expression of MLH1, MSH2, MSH6 and PMS2 as surrogate markers of MSI, in a series of poorly differentiated sinonasal carcinomas who had received conventional treatments, as possible indicators for treatment with immunotherapy. In addition, results were correlated with clinico-pathological and follow-up data.Samples from poorly differentiated sinonasal carcinomas of 69 patients who had received conventional treatments were collected from the Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain; from the VU Medical Center (VUmc), Amsterdam, the Netherlands, and from University Medical Center Utrecht (UMC), Utrecht, the Netherlands. All experimental protocols were approved by the Institutional Ethics Committee of the Hospital Universitario Central de Asturias and by the Regional CEIC from Principado de Asturias . All methods were carried out in accordance with the guidelines of the Institutional Ethics Committee of the Hospital Universitario Central de Asturias. Informed consent was obtained from all patients.Our cohort included 14 olfactory neuroblastoma (ONB), 6 neuroendocrine carcinoma (SNEC), 25 (SNUC), 6 poorly differentiated squamous cell carcinoma (PD-SNSCC), 5 HG-non-ITAC, 10 solid-type ITAC, 1 SMARCB1-deficient carcinoma, 1 SMARCA4-deficient carcinoma and 1 NUT carcinoma. Representative photomicrographs of hematoxylin and eosin staining of the tumor types are given in The initial diagnoses of all cases were revised by an experienced pathologist (AF). All the available slides were reviewed in each case, and tumors were diagnosed using the diagnostic criteria described in the fourth edition of the World Health Organization Classification . AdditioTumor samples which were obtained from biopsy or surgery were fixed with formalin and embedded in paraffin. When several paraffin blocks were available for the same tumor, the most representative block was selected. Tissue microarray blocks were prepared using the Beecher Tissue Microarrayer . In total, 4 blocks were constructed, containing three 1 mm cores from different areas of 69 tumors .Immunohistochemistry was performed on an automatic staining workstation  with antigen retrieval by EnVision FLEX + Mouse  over 20 minutes. The following antibodies were applied: anti-CD8 clone C8/144B, IR623 , anti-PD-L1 clone E1L3N , and MSI markers anti-MLH1 clone M1, anti-PMS2 clone A16-4, anti-MSH2 clone G219–1129 and anti-MSH6 clone SP93 . The slides were evaluated in a double-blind manner by three observers , and discrepancies between the observers were resolved by a consensus review after simultaneous reevaluation.CD8+ TIL scoring was adapted from Fuchs et al. , who defp-values < 0.05 were considered to indicate statistically significant. Statistical analysis was carried out with the use of SPSS Base, version 15.0 and SPSS Advanced models, version 15.0  software. Pearson’s chi-squared test and Fisher’s exact test were used to test possible associations between CD8+ TILs and PD-L1 expression and MSI presence and various clinicopathological factors. Univariate Kaplan–Meier curves were plotted to assess the relations of CD8+ TILs, PDL-1 expression and MSI to overall, disease-specific and disease-free survival using the log-rank-test. p = 0.006), while there was no age difference regarding disease-free survival . During this period, 22 (67%) patients developed a recurrence or metastasis. At the time of writing, 17 (52%) patients remained alive, 13 (39%) died of disease and 3 (9%) died of other causes. The 5-year overall survival was 43% and the 5-year disease-free survival was 34%. Tumor stage was not correlated with overall or disease-free survival. Patients of 60 years of age and older showed worse overall survival . The presence of CD8+ TILs was not related to age, tumor stage or clinical outcome, although there was a tendency toward worse overall survival  tumors showed a high presence of CD8+ TILs, either in the tumor or stromal compartment. In 21/23 cases, CD8+ TILs were present in both compartments. CD8+ TILs were most frequent in solid-type ITAC and HG-non-ITAC (60% of tumors), followed by SNUC (34%), PDSCC (33%), ONB (21%) and SNEC (17%). SMARCB1-deficient carcinoma, SMARCA4-deficient carcinoma and NUT carcinoma were devoid of CD8+ TILs . Examplesurvival .p = 0.006). We found no correlation between PD-L1 expression and age or tumor stage. PD-L1 positive cases did demonstrate both worse overall  tumors showed membranous PD-L1 staining in >5% of tumor cells. The staining pattern was diffuse in the majority of cases, whereas HG-non-ITAC mostly demonstrated focal expression . PD-L1 e overall  and diseWe applied PMS2, MLH1, MSH2, and MSH6 staining as surrogate markers of MSI and found 5/69 (7%) cases positive, two were SNEC, two SNUC and one was HG-non-ITAC. Four tumors demonstrated complete absence and one reduced expression of PMS2, accompanied by complete loss of MLH1 in two cases and reduced MLH1 in two cases, while in one case PMS2 was the only lost of the four MSI markers. One case showed complete absence of all four markers, and in one case shown in Standard treatment of poorly differentiated sinonasal carcinomas consists of surgery and radiotherapy, in some cases complemented with histo-type driven chemotherapy ,17. In sConsidering the whole cohort of 69 tumors, we found 33% to have CD8+ TILs, 33% with PD-L1 expression and 7% as MSI-positive. In nearly all cases, CD8+ TILs were present both in the tumor and the stromal compartment and correlated with PD-L1 positivity, which is in agreement with many previous studies. In contrast with previous studies on MSI-positive tumors, we found MSI positivity to be mutually exclusive with CD8+ TILs and nearly also with PD-L1 expression ,39,40. TComparing the different tumor types, CD8+ TILs were observed most frequently (60% of cases) in the adenocarcinoma subtypes HG-non-ITAC and solid-type ITAC, which is higher than previous studies on sinonasal tumors reporting 19–50% in SNSCC ,26,41,42Although follow-up information in our study was very limited and the statistical power of the survival analysis low, our results indicated a tendency toward worse clinical outcome for tumors with CD8+ TILs. This finding contrast with a number of studies on ITAC, ONB and SNSCC have indicated CD8+ TILS as indicators of better survival ,27,42,43However, there are also reports that associated CD8+ TILs with worse survival in SNSCC , and in Our data also suggest a possible association between PD-L1 expression and worse clinical outcome. This is in accordance with a similar study on SNSCC by Hongo et al. , althougWe found 5/69 tumors with an indication for MSI positivity, four with complete loss of PMS2 together with lost/reduced MLH1 expression. In general, MSI positivity is considered a predictor of favorable clinical outcome. With follow-up information of only two of the MSI-positive cases in our cohort, no meaningful assessment of prognostic vale was possible; however, these patients had a very poor survival of 2 and 6 months. Our cohort included one case each of SMARCB1-deficient carcinoma, SMARCA4-deficient carcinoma and NUT carcinoma, unfortunately without follow-up information. The FDA has approved MSI status, analyzed by immunohistochemistry or by multiplex PCR, as predictor for the efficacy of immunotherapy of unresectable or metastatic cancer, irrespective of the primary site of origin . More spOur results on poorly differentiated sinonasal carcinomas revealed that at least one of the immunotherapeutic biomarkers >10% CD8+ TILs, >5% PD-L1 expression or MSI-positivity occurred in 39% (27/69) of cases. For immunotherapy to be most effective, tumors ideally should express PD-L1 and have tumor-activated CD8+ TILs , and thiThe three cases SMARCB1-deficient carcinoma, SMARCA4-deficient carcinoma and NUT carcinoma may be given special attention. Recent studies suggest that SMARCB1 and SMARCA4 deficiencies lead to enhanced levels of TILs and PD-L1, despite low TMB ,59,60. OOur study has several limitations. First, the series of cases is not homogeneous. Although all tumors are considered poorly differentiated, they do belong to distinct sinonasal cancer subtypes. Second, due to the fact that these are rare tumors, we could only analyze a relatively low number of cases, and thus more studies are necessary. Finally, the patients in our retrospective cohort had received conventional treatments that did not include immunotherapy, so it was not possible to correlate response to therapy with CD8+ TILs and PD-L1 expression status or to find an explanation as to why cases with CD8+ TILs had worse clinical outcomes. However, this negative correlation does not preclude that these patients would be less responsive to immune checkpoint inhibitors.To conclude, immune checkpoint inhibitors represent new options for treatment for an increasing number of tumor types. Our results on CD8+ TILS, PD-L1 expression and MSI indicate that also a substantial subset of patients with poorly differentiated sinonasal tumors may be candidates to be treated with this promising new therapy.


Piper species are being considered as possible medicinal plants for the development of new sources of antioxidants. Several studies have been carried out starting from the extract levels, fractions, and compounds of the Piper species, which showed good antioxidant activity. Currently, some of these plants are being used as ingredients in traditional medicines to treat allergies, toothaches, and coughs. This review examines the distribution, botanical data, pharmacology, especially antioxidant activity, and the compounds contained in five Piper species, namely Piper amalago L., Piper betle L., Piper hispidum Sw., Piper longum L., and Piper umbellatum L.Antioxidants are compounds that are able to inhibit the negative effects that come from free radicals. The phenomenon of imbalanced antioxidant production and the accumulation of free radicals in cells and tissues can cause oxidative stress. Excessive free radicals that enter the body cannot be warded off by endogenous antioxidant compounds so that the required antioxidant compounds can come from the outside, which helps in the performance of endogenous antioxidants. Antioxidants that come from outside consist of synthetic and natural antioxidants; however, synthetic antioxidants are not an option because they have toxic and carcinogenic effects. Therefore, the use of natural ingredients is an alternative method that is needed to create a new natural antioxidant compound.  Piperaceae have good antioxidant activity  pyrrolidine (158) [P. hispidum, namely piperamine (159) and piperine (160). The chalcones group of flavonoids was isolated from P. hispidum. 2′-hydroxy-3,4,4′,6′-tetramethoxychalcone (161), 2′-hydroxy-4,4′,6′-trimethoxychalcone (162), and 2′,3-dihydroxy-4,4′,6′-trimethoxychalcone (163) were successfully isolated from the same part of P. hispidum [164) from the leaves extract of P. hispidum. Meanwhile, a new compound belonging to the butenolide group that was isolated from P. hispidum leaves was 9,10-methylenedioxy-5,6-Z-fadyenolide (165) [Several secondary metabolites contained in ne (158) . In anotne (158)  succeedehispidum . Ruiz ethispidum  successfde (165) .P. hispidum belong to the monoterpenes and sesquiterpenes groups. P. hispidum essential oils also presented phenylpropanoids and alcohols. Most of the essential oils identified from the leaves of P. longum. The plant parts that have been studied to date are fruits, roots, leaves, and seeds [P. longum. In addition, several compounds were isolated, including asarinine (191), pellitorine (192), retrofractamide A (193), piperlongumine (194), brachystamide A (195), and longamide A (196) [P. longum.Alkaloids, terpenoids, sterols, and essential oils are chemical compounds isolated from nd seeds . Piperin A (196) ,119. TabP. longum are shown in P. longum. This consisted of monoterpenes, aromatic ester, and sesquiterpenes.The essential oils isolated from P. umbellatum are alkaloids, flavonoids, sterols, and terpenoids . Piperumbellactam A–D (226–229) are each classified as an alkaloid compound, which is a pure compound that has recently been discovered in the branch of plants [P. umbellatum are shown in P. umbellatum.In general, the bioactive compounds contained in f plants . Other b−9 s, respectively, which are two molecules that are highly reactive and can attack nearby molecules quickly and cause severe damage [2 which activates NADPH oxidase to produce superoxide anion radicals and is further converted to O2 and H2O2 by superoxide dismutase (SOD). However, the presence of increased ROS or excessive pro-oxidants in the body can cause oxidative stress. This also happens due to a lack of antioxidant production in the body [The chain of chemical reactions can take place well in life because there is oxygen from the air for the oxidation process, which provides energy in the form of ATP . Furthere damage ,128. Brithe body ,130. Oxithe body . TherefoAntioxidants are molecules that can neutralize free radicals and can inhibit the oxidation process of other molecules or can be used as reducing agents. The main function of this antioxidant is to end the chain reaction of ROS formation . Antioxi2O2). In mammalian tissue, SOD is divided into three types, namely superoxide dismutase 1 (SOD1), which is found in the cytosol in the form of copper, superoxide dismutase 2 (SOD2), which is found in the mitochondria in the form of manganese, and extracellular superoxide dismutase. Recently, SOD1 is a major antioxidant enzyme in dealing with oxidative stress [2O2 in the water and protects cells against oxidative stress. CAT can transfer oxygen bound with H2O2 to other molecules. The decomposition of H2O2 used the peroxidative activity method (H2O2 + AH2 A + 2H2O) and the catalytic activity mode (2H2O2 + O2 + 2H2O). The breakdown of H2O2 by enzymes depends on the concentration of H2O2 and follows the first-order reaction [2O2 to water or alcohol. The mechanism of action of this enzyme starts from reduced glutathione, which functions as an effective electron donor because the thiol dioxide group becomes a sulfide bond (H2O2 + 2GSH → GS-SG + 2H2O) [Enzymatic antioxidants can protect the body against oxidative attack because of their ability to decompose ROS. Superoxide dismutase (SOD), catalase (CAT), and glutathione Peroxidases (GSHPx) are the main enzymes involved in the defense against ROS . SOD is e stress ,137. CATreaction ,138,139. + 2H2O) ,141.Exogenous antioxidants are further divided into synthetic antioxidants and natural antioxidants. These antioxidant compounds react with ROS to inhibit or eliminate the activity of these ROS . Synthet50 lower than 1000 mg/kg body weight, and must pass mutagenic, teratogenic, and carcinogenic tests. Currently, the development of natural antioxidants continues to be carried out because they have the same benefits as synthetic antioxidants but have no side effects [Natural antioxidants can strengthen endogenous antioxidant defenses in neutralizing and inhibiting the performance of ROS . The mai effects ,155. Est effects  reported effects , garlic  effects , and the effects .The determination of antioxidant activity in one sample can be performed with several models of antioxidant activity test methods. Currently, a test procedure has been developed to evaluate antioxidant activity in vitro. Antioxidant activity test methods cannot be compared with each other because they have different target mechanisms; therefore, each has advantages and disadvantages . The met2+) reducing antioxidant power (CUPRAC), nitric oxide (NO) scavenging, superoxide anion (SA) radical scavenging and hydrogen peroxide (H2O2) scavenging assays [SET-based methods are widely used for the in vitro testing of antioxidants. Several SET-based methods are 2,2-Diphenyl-1-picrylhydrazyl radical scavenging (DPPH), ferric ion reducing antioxidant power (FRAP),2,2-Azinobis 3-ethylbenzthiazoline-6-sulfonic acid radical scavenging (ABTS), cupric ions , one of the compounds isolated from P. betle, has good antioxidant activity, with an IC50 value of 114.34 ± 0.46 g/mL when using the nitric oxide radical method [At the compound level, Tabopda et al.  succeedel method .Piper amalago L., Piper betle L., Piper hispidum Sw., Piper longum L., and Piper umbellatum L. are Piper species that have tremendous potential as a new source of natural-based medicines. This can be seen from the traditional use of this plant, which is used for dental and oral care. In addition, the content of extracts, fractions, and secondary metabolites has several biological activities, especially, in several methods, antioxidants. Based on this review, it is hoped that it can become a reference and guide in the development of several activities for the discovery of new drugs based on natural ingredients from this Piper species.


Rheumatoid arthritis (RA) is a chronic, progressive, autoimmune disease caused by a malfunction of the immune system. The aim of this study was to examine the anti-arthritic effects and suggest the mechanisms of actions of diosmin and trolox in male Wistar rats. Complete Freund’s adjuvant (CFA) was used to establish RA in the animals by subcutaneous injection of 100 µL CFA/rat into plantar region of right hind leg in two consecutive days. Diosmin and/or trolox were administered orally at a dosage of 20 mg/kg/day to CFA-induced arthritic rats for 2 weeks. The normal and arthritic control groups were orally given the same equivalent volume of a vehicle (1% carboxymethyl cellulose) in which treatment agents were dissolved. At the end of the experiment, blood samples were collected from the jugular vein for the detection of the total leukocyte count (TLC) and differential leukocyte count (DLC) in blood and the detection of rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPA), tumor necrosis factor-α (TNF-α), interleukin-13 (IL-13), and interleukin-17 (IL-17) levels by enzyme-linked immunosorbent assay (ELISA), as well as markers of oxidative stress and the antioxidant defense system in serum. The right hind ankle regions of three rats from each group were dissected out and fixed in 10% neutral-buffered formalin for histological examination and the other three were kept at −30 °C for Western blot analysis of nuclear factor-kappa B (NF-κB) protein 50 (NF-κB p50), NF-κB p65, inducible nitric oxide synthase (iNOS), nuclear factor erythroid-2-related factor 2 (Nrf2), and matrix metalloproteinase (MMP)-1 (MMP-1), MMP-3, and MMP-9. The CFA injection was deleterious to the ankle joint’s histological architecture, manifesting as infiltration of inflammatory cells into the articular cartilage, hyperplasia of the synovium, and erosion of the cartilage. All these effects were ameliorated by diosmin and/or trolox, with the combined dose being the most effective. The two compounds significantly lowered the elevated serum levels of RF, ACPA, TNF-α, and IL-17, as well as other pro-inflammatory mediators, such as NF-κB p50, NF-κB p65, iNOS, MMP-1, MMP-3 and MMP-9. They also increased the levels of the anti-inflammatory cytokine, IL-13, and the cytoprotective transcription factor Nrf2. The compounds stimulated higher activities of antioxidants, such as glutathione, glutathione-S-transferase, catalase, and superoxide dismutase, and reduced lipid peroxidation in the serum of arthritic rats. In conclusion, diosmin, trolox, and their combination, which was the most potent, exerted anti-arthritic, anti-inflammatory and antioxidant effects by suppressing NF-κB signaling, inhibiting matrix metalloproteinases, and activating Nrf2. Rheumatoid arthritis (RA) is a debilitating, inflammatory, and autoimmune disease , which cComplete Freund’s adjuvant (CFA)-induced arthritis exhibits pathophysiological features similar to human RA , making Currently, conventional drug therapies prescribed for RA have side effects, prompting a global search for new therapeutics from natural sources ,16,17,18Diosmin (diosmetin-7-O-rutinoside) is an unsaturated glycoside distributed in various citrus fruits , obtaineThe aim of this study was to scrutinize the effects of diosmin and trolox on CFA-induced arthritis in male Wistar rats, with a focus on their mechanisms of action and the roles of NF-κB, iNOS, Nrf2 and MMPs.Between January 2019 and January 2020, 40 male Wistar rats weighing 100–120 g and aged 6–7 weeks were studied at Beni-Suef University’s Faculty of Science in Egypt. The rats were obtained from the Egyptian Organization for Biological Products and Vaccines (VACSERA) animal house in Helwan, Cairo, Egypt. Animals were monitored for around 10 days before starting to rule out any concomitant infections. The animals were kept in polypropylene cages with well-ventilated stainless steel covers and were provided access to standard balanced food and water at their discretion at room temperature (25 °C ± 5 °C). The animals were weighed weekly during the experimental period. All animal procedures followed the IACUC rules at Beni-Suef University in Egypt . Every attempt was made to limit the number of animals as well as their misery and suffering.Mycobacterium tuberculosis, diosmin, and trolox . Sigma Chemical Company provided CFA, a mineral oil (1 mg/mL) suspension of heat-killed A subcutaneous injection of 100 µL CFA/rat/day was administered into the plantar region of the rat’s right hind paw for two successive days to induce arthritis .After inducing RA by injecting CFA, Wistar rats were segregated into five groups  designed as follows :Group 1 : This group comprised normal rats orally administered the equivalent volume of the vehicle  for 15 days.Group 2 (Arthritic control group): This group consisted of arthritic rats who, similar to the normal control group, were orally administered the equivalent volume of the vehicle  for 15 days. Group 3 (Arthritic group treated with diosmin): This group comprised arthritic rats administered a safe oral therapeutic dose of diosmin (20 mg/kg/day)  dissolveGroup 4 (Arthritic group treated with trolox): This group comprised arthritic rats treated with a safe oral therapeutic dose of trolox (20 mg/kg/day)  dissolveGroup 5 (Arthritic group treated with diosmin and trolox): This group comprised arthritic rats administered a safe oral therapeutic dose of diosmin and trolox (each dissolved in 1% CMC) for 15 days. At the end of the experiment, rats were euthanized by a cervical dislocation under diethyl ether inhalation anesthesia, and blood samples were obtained from the jugular vein. Part of the blood was collected in an anti-coagulated tube for the total leukocyte count (TLC) and differential leukocyte count (DLC). The other part of the blood was collected in a coagulating tube, centrifuged at 3000 rpm for 15 min, the clear non-hemolyzed supernatant sera were rapidly extracted, split into three aliquots for each individual animal, and stored at −20°C until required for further analyses. Three rats from each group had their right hind ankle region excised and fixed in 10% NBF  for histological examination. The right hind ankles of three other rats from each group were stored at −30°C until they were used for Western blotting investigations.The swelling rate and paw edema in the different groups were measured by wrapping a thread around the circumference of the right paw just above the tarsal surface, as an indicator. A ruler was used to determine the length of the thread. Measurements were taken once a week after the adjuvant injection (on the 7th and 14th days) ,18.Following sacrifice and dissection, the right ankles were put into 10 percent neutral-buffered formalin (NBF) for 48 h for fixation before being transferred to the National Cancer Institute’s pathology department in Cairo, Egypt, for paraffin section preparation and hematoxylin and eosin staining (H&E).Decalcification of the ankle was performed using a 10% formic acid solution. The ultimate point of decalcification was physically assessed with a surgical blade over the course of two weeks, with the formic acid solution being refilled twice weekly. Complete decalcification was followed by a graded ethanol series of dehydration before the specimen was embedded in paraffin wax. H&E was used to stain sections that were 5 µm thick.TLC and DLC were determined from a portion of the blood from the jugular vein collected in tubes containing ethylenediaminetetraacetic acid (EDTA) solution (50 μL of 15% EDTA for 2.5 mL of blood). For TLC, the cells were loaded onto a Neubauer hemocytometer slide using diluted gentian violet solution while the Giemsa stain was used to estimate DLC .® . TNF-α and ACPA were measured using ELISA kits provided by MyBioSource, Southern California, San Diego, CA, USA.Serum levels of rheumatoid factor (RF), interleukin-17 (IL-17), interleukin-13 (IL-13), TNF-α, and anti-citrullinated protein antibodies (ACPA) were measured with specific ELISA kits, following the manufacturer’s instructions. RF and IL-17 were detected using ELISA kits purchased from CUSABIO . IL-13 was measured using an ELISA kit acquired from PicoKineLPO, detected as malondialdehyde (MDA) levels and reduced glutathione (GSH) levels were estimated using the methods documented by Yagi  and Beutry antibodies targeting the proteins of interest and β-actin . The membranes were washed thrice using Tris-buffered saline with Tween 20 at room temperature. The blots were incubated with horseradish peroxidase-conjugated 2ry antibodies  at a dilution of 1:5000 for 30 min. Finally, the blots were washed again and the chemiluminescent signal was detected on an X-ray film.The expressions of NF-κB protein 50 (NF-κB p50), NF-κB p65, iNOS, nuclear factor erythroid-2-related factor 2 (Nrf2), matrix metalloproteinase 1 (MMP-1), MMP-3, and MMP-9 were assayed in rat ankles by Western blotting, according to the method described by Harlow and Lane . An ice-p value less than 0.05.The data are presented as mean ± standard error. The Statistical Package for Social Sciences  was used to examine the data and the Duncan’s test was used to compare the results of different groups. For right hind paw circumference, a two-way analysis of variance (ANOVA) as well as a one-way ANOVA were performed. Statistical significance was defined as a Swelling, edema, and redness, which are external indicators for assessing the severity of arthritis, were observed in the right hind paws and ankles of CFA-induced arthritic rats. These gross lesions were reduced in the arthritic groups treated with diosmin and/or trolox, with the combined treatment being the most effective .The effects of diosmin, trolox, and their combination on the circumference of the right hind paw in CFA-induced arthritic rats is depicted in p < 0.05) throughout the experiment. The two-way ANOVA, on the other hand, depicted that the effect of treatment, time, and treatment–time interaction was significant at p < 0.05 . When arthritic rats were given diosmin, trolox, or both, their elevated serum levels of RF, TNF-α, ACPA, and IL-17 were significantly downregulated, while the depleted level of IL-13 was significantly upregulated (p < 0.05). The combinatory effect was the most potent on RF, ACPA, TNF-α, IL-13 and IL-17 serum levels, recording percentages of −77, −67, −65, 116 and −72%, respectively. p < 0.05) than normal rats. Upon treatment with diosmin, trolox, or both, serum LPO levels decreased by 38, 33, and 38%, respectively, when compared with arthritic rats. p < 0.05) the serum activities of all these antioxidant enzymes compared with arthritic rats. In CFA-induced arthritic rats, the serum GSH, GST, SOD, and CAT activity reduced by 53, 38, 77, and 75%, respectively, in comparison to normal rats. Treatment with diosmin, trolox, or both significantly increased (p < 0.05) with considerable increases in the counts of neutrophils (156%), lymphocytes (163%), monocytes (147%), and eosinophils (117%). After treatment with diosmin, trolox, or both, the TLC dropped by 22, 25, and 20%, respectively, relative to arthritic rats. The expression of NF-κB p50 and NF-κB p65 increased in arthritic rats by 298 and 361%, respectively, compared with control rats, while treatment with diosmin, trolox, and their combination reduced NF-κB p50 levels by 45, 48, and 76%, NF-κB p65 levels by 38, 42, and 72%, compared with arthritic rats.When compared with the control group, the expression of iNOS increased by 409% in arthritic rats. Treatment with diosmin, trolox, or their combination decreased iNOS levels by 46, 47, and 73%, respectively, compared with arthritic rats.Nrf2 expression was 86% lower in arthritic rats relative to control rats, while diosmin, trolox, or their combination therapy raised Nrf2 levels by 224, 298, and 601%, respectively, compared with arthritic rats.p < 0.05).The expression of MMP-1, MMP-3, and MMP-9 was higher in arthritic rats than in control rats by 576, 388, and 364%, respectively. Diosmin, trolox, or their combination, on the other hand, significantly reduced the levels of these matrix metalloproteinases (MMPs) when compared with arthritic rats  and T helper 17 (Th17) cytokines prevail over Th2 cytokines ,49,54. TIn the current study, arthritic rats showed reduced levels of IL-13, which is consistent with Ahmed et al. , who shoNF-κB, a heterodimer of the p65 and p50 subunits of the Rel protein family, is found in the cytoplasm of cells and maintained in an inactive state by binding to the IκBα inhibitory protein. IκBα is phosphorylated by IκB kinase in response to distress stimuli, such as ROS, inflammatory cytokines, and adjuvants like CFA  64], wh, wh64], In accordance with previous studies, our present investigation showed that CFA-induced arthritic rats had elevated levels of MMP-1, MMP-3, MMP-9, p65/p50 NF-κB complexes, and iNOS ,67,68,69Mycobacterium tuberculosis antigens and adjuvant, meant to promote an immune response in the host, could be the reason for this leukocytosis. Once the immune system has been stimulated, adjuvants cause inflammation due to leukocytic infiltration; as a result, the leukocyte count may rise, which correlates with arthritis severity. Treatment with diosmin and/or trolox significantly reduced the elevated TLC as well as the individual lymphocyte, monocyte, and neutrophil counts, proving that these therapies have an anti-inflammatory effect on arthritic rats [The white blood cells (leukocytes) play a crucial part in the body’s defense system. During inflammation, inflammatory cells, such as macrophages and neutrophils, as well as leukocytes and plasma components, travel to the site of infection or injury to eliminate threats . In accotic rats ,73.Besides inflammation, oxidative stress is the other hallmark of RA. Multiple studies have indicated that people with RA have elevated oxidative stress along with increased inflammation . In pathIn our present investigation, CFA-induced arthritic rats revealed a marked elevation in LPO accompanied with a reduction in the antioxidants GST, SOD, CAT, and GSH, which is consistent with Ahmed et al.  and AhmeNrf2 is a key modulator of cellular defense that counteracts the damage caused by oxidative stress . It actiAlong the lines of results reported by Xie et al. , the expHistopathological examinations revealed that arthritic rats showed an infiltration of inflammatory cells, hyperplastic synovial membranes, bone erosion, and degenerative degradation of cartilage. Diosmin treatment, on the other hand, considerably reduced joint damage and exhibited only a mild stage of these lesions, including an intact articular surface, sound meniscus, mild synovial membrane projection, and mild congestion and edema of the periarticular tissue. trolox-treated arthritic rats exhibited a nearly normal articular surface, focal mild hyperplasia in synovial membrane, and no congestion or infiltration of inflammatory cells. Finally, the combination treatment of diosmin and trolox resulted in a nearly normal articular surface and synovial membrane, without any features of inflammation. As a result, this study clearly demonstrated the effectiveness of the test compounds in suppressing inflammation inside tissues and inhibiting the paw swelling characteristic of arthritis.The current investigation demonstrated that an anti-arthritic therapy comprising diosmin and/or trolox protected against oxidative stress and inflammation while ameliorating the histological effects on the articular joints of CFA-induced arthritic rats. The combined dose was the most effective. The anti-arthritic effects of these two compounds may be mediated by the suppression of NF-κB signaling, the activation of Nrf2 and antioxidant defense systems, and the repression of MMPs and pro-inflammatory cytokines. Clinical trials, however, are required to assess the efficacy and safety of this combination before it can be approved for use in humans.


BMP4, LEF1, IGF1R, TGFβ3, TGFα, and KRT17. These DEGs were enriched in HF growth- and development-related pathways. We further verified the function of LEF1 and showed that overexpression of LEF1 increased the expression of HF development-related genes and proteins, enhanced HFSC proliferation, and reduced HFSC apoptosis, while knockdown of LEF1 reversed these effects. DPC-Exos could also rescue the siRNA-LEF1 effect in HFSCs. In conclusion, this study demonstrates that DPC-Exos mediated cell-to-cell communication can regulate HFSCs proliferation by stimulating LEF1 and provide novel insights into HF growth and development regulatory mechanisms.Hair follicle (HF) growth and development are controlled by various cell types, including hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs). Exosomes are nanostructures that participate in many biological processes. Accumulating evidence indicates that DPC-derived exosomes (DPC-Exos) mediate HFSC proliferation and differentiation during the cyclical growth of hair follicles. In this study, we found that DPC-Exos increase ki67 expression and CCK8 cell viability readouts in HFSCs but reduce annexin staining of apoptotic cells. RNA sequencing of DPC-Exos-treated HFSCs identified 3702 significantly differentially expressed genes (DEGs), including  Both animal and human hair have functions in defense, protection, communication, and aesthetics ,2. Hair Extracellular vesicles with a diameter of 30–150 nm, also known as exosomes, are critical carriers of intercellular communication and play essential biological functions by transferring nanoscale molecules, such as mRNAs, miRNAs, and proteins, between cells . There iHair follicle morphogenesis is regulated by many signaling pathways, including Wnt, hedgehog, bone morphogenetic protein (BMP), transforming growth factor beta (TGF-β), and Notch ,33,34,35Yan H et al. used miRNA sequencing between DPCs and DPC-Exos and found that the miR-22-5p–LEF1 axis regulates HFSC proliferation . HoweverDPC-Exos were first characterized at the structural and protein levels by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and protein level detection, respectively. The ultrastructure of DPC-Exos showed the double-layered membrane typical of exosomes A. NTA shIn order to explore the functions of DPC-Exos in HFSCs, HFSCs were incubated with DPC-Exos for 48 h. The HFSC-control group was increased by 100 µL PBS for negative control. DPC-Exos-treated HFSCs (HFSCs + Exos) showed significantly increased ki67 staining A. CCK8 cp < 0.05, including LAMC1, BMP4, LEF1, FGF5, Wnt10b, FOSL1, and KRT17 , where the distance was calculated using the Euclidean formula A. A totand KRT17 B. To verelopment D. In theelopment E. Table LEF1 expression in DPC-Exos-treated HFSCs, and previous studies revealed that LEF1 promotes HF growth and regulates HF cells [LEF1 expression in HFSCs. HFSCs were transfected with the expression vector pcDNA3.1-LEF1, which elevated LEF1 in HFSCs and increased the expression of HF growth-related genes such as BCL2, BMP2, CCND1, CTNNB1, and EGF . For DPC and HFSC isolation, dorsal skins of 6-month-old Angora rabbits were harvested under Zoteil-50 anesthesia. Wounds were cleaned with iodine solution after surgery, and the animals were allowed to recover under controlled housing conditions .DPCs and HFSCs were isolated as previously described . DPCs weg for exosome harvesting. For DiI labeling, 10 mg of DiI was added to the culture medium for 10 min. The staining was terminated by adding 10 mL PBS, and the exosomes were extracted as above.Exosomes were isolated from the DPC culture medium using the Total Exosome Isolation Kit . Briefly, DPC supernatant was centrifuged to remove cell debris, followed by concentration with Ultra-15 centrifugal filter units  . ExosomeA 20 μL sample of diluted exosomes was dropped onto a copper mesh, fixed with uranyl acetate solution, and air-dried at room temperature. Exosomes were imaged with transmission electron microscopy  at 80 kV.Exosomes were diluted in PBS and analyzed on a Flow NanoAnalyzer instrument  to detect particle size and concentration of exosomes.HFSCs were washed with PBS, fixed with 4% paraformaldehyde, and incubated with Ki67 primary antibody  overnight. Detection was performed with an antirabbit secondary antibody . Cell nuclei were stained with DAPI. Pictures were quantified with ImageJ software.Oryctolagus cuniculus) genome from Ensemble OryCun2.0 , and the cDNA was synthesized with HiScript III RT SuperMix for qPCR  as instructed. RT-qPCR was performed with ChamQ SYBR qPCR Master Mix . The primers used are listed in TM automatic protein expression analysis system  [Cellular protein levels were detected with the Wesose, CA) . ProteinThe mRNA sequence of rabbit LEF1 (GenBank accession no. XM_002717171.4) was used for the primers designed from the LEF1 CDS sequence, and the PCR was performed using Phanta Max Super-Fidelity DNA Polymerase (Vazyme) and subcloned into EcoR I and Xba I  digested pcDNA 3.1(+) vector, which was subsequently called pcDNA3.1-LEF1. The small interfering RNA (siRNA)-LEF1 was designed and purchased from Shanghai GenePharma Co., Ltd.  The primers for the overexpression vector and siRNA sequence are listed in Cells were replated into 96-well plates 8 hours after transfection to detect cell proliferation. CCK8  reagent was added at 0, 24, 48, and 72 h and incubated for 2 h, and the absorbance was measured at 450 nm using Infinite 200 Pro .Cells were harvested two days after transfection, washed twice with precooled PBS, stained with FITC and PI , and detected by flow cytometry  within 1 h to detect apoptosis.All statistical analyses were performed using SPSS 22.0 . Paired sample t-test was used to examine the OD value, apoptosis rate, relative gene expression, and protein expression. Each analysis comprises three or more biological replicates. All data are presented as mean ± SD.


However, mechanism underlying seed morphology variation has not been systematically investigated yet. Thus, using the near isogenic lines (NILs) harboring one, two or three of the fruit shape loci, histological, physiological and transcriptional bases of seed morphology change have been studied. sun and ovate showed potential abilities in decreasing seed size, whereas, fs8.1 had a potential ability in increasing this parameter. Interactions between two loci and the interaction among three loci all led to significant decrease of seed size. All the loci significantly down-regulated seed shape index (SSI), except for sun/fs8.1 double NIL, which resulted in the reductions in both seed length and width and finally led to a decreased trend of SSI. Histologically, seed morphological changes were mainly attributed to the cell number variations. Transcriptional and physiological analyses discovered that phytohormone-, cytoskeleton- as well as sugar transportation- and degradation-related genes were involved in the regulation of seed morphology by the fruit shape loci.Seed size and shape are not only critical for plant reproduction and dispersal, but also important agronomic traits. Tomato fruit shape loci  Seed morphology, which mainly refers to the seed size and shape, is an important factor that impacts the above-mentioned processes. Seed size affects seed environmental adaptability as well as seed dispersal efficiency. Larger seeds accumulate more nutrients for germination and are more tolerant to abiotic stresses. Whereas, plants carrying smaller seeds tend to produce more seeds, which is conducive to the seed dispersal . Seed shSeed morphology is controlled by the coordinated growth of the maternal (seed coat) and the zygote tissues (embryo and endosperm), which is regulated by multiple factors . Among tTGW6, the gene underlying a major grain weight quantitative trait loci (QTL), increased grain length and weight by delaying endosperm cellularization through affecting IAA (indole-3-acetic acid) production (AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) and AUXIN RESPONSE FACTOR (ARF) which encode transcriptional repressor and transcription factor, respectively, are key components of the auxin signaling pathway  changed the basipetal distribution of auxin and enhanced cell division and expansion in the spikelet hulls, which finally led to the enlargement of rice grain , was associated with grain weight through association mapping  to take the advantage of the DEFL1’s promoter and to express at a much higher level. The sun locus as well as overexpression of SUN all led to the elongation of fruit, cotyledon and leaflet, which could be attributed to the increased cell number in proximal-distal direction and the decreased cell number in medio-lateral direction. Members of IQD family have been confirmed to act as Ca2+-regulated scaffolds and are able to impact the arrangement of microtubules through recruiting CaM, KLCR1 (KINESIN LIGHT CHAIN-RELATED PROTEIN-1) and SPR2 to the microtubules  and SlOFP20 (Solyc10g076180) all belong to the OFP gene family. ovate, a null mutation of OVATE gene, is resulted from a SNP which leads to the emergence of a pre-mature stop-codon. ovate changes the round-shaped fruit into elongated pear-shaped fruit by promoting cell division along the proximal-distal axis but simultaneously repressing cell proliferation along the medial-lateral direction at the proximal end. Overexpression of OVATE not only affected the shape of the reproductive organs, but also decreased plant height and leaf size (Sov1 (suppressor of ovate) is attributed to a ~31-Kb deletion in the upstream of SlOFP20, which increases the gene’s expression and eventually leads to ovary cell number variation by influencing cell division pattern . At the histological level, seed size variations were attributed to the changes of the seed coat area and embryo area, which were mainly caused by the cell number changes. Transcriptomic and physiological analyses disclosed that phytohormones and their related genes, sugar transportation and degradation-related genes as well as cytoskeleton-related genes were impacted by the fruit shape loci, which may be the reasons for the seed size and shape changes.Near-isogenic lines (NILs) are a powerful genetic tool not only for mapping QTLs but also for clarifying their functions . Fruit sit shape . In our 22.1Solanum pimpinellifolium accession LA1589 background, of which the interval regions harbor one, two or three of the tomato fruit shape loci , were investigated in this study. Even though the mutant alleles of the fruit shape genes were not caused by the loss-of-function mutations, the three loci were still represented by lowercase italics. Details about the interval regions and the morphological characteristics of the NILs were described in sun, ovate, fs8.1, sun+ovate (s/o), sun+fs8.1 (s/f), ovate+fs8.1 (o/f), sun+ovate+fs8.1 (s/o/f), were grown in the greenhouse with standard fertilizer application and water supply at China Agricultural University in year 2021 and 2022. In each repeat, at least eight plants of each genotype were grown and all the investigated plants were placed randomly in the greenhouse.NILs in 2.22.2.1http://oardc.osu.edu/vanderknaap/tomato_analyzer.php) and seed area, length, width and shape index were measured. The thousand seed weight was calculated from the weight of 200 shade-dried seeds. In each repeat, 1000 seeds were collected from at least 25 fruits which were harvested from at least 8 plants of each NIL.Mature seeds were collected from red-ripe fruits of the NILs. In order to eliminate the effect of pollination efficiency on seed number and development, fruits developed from hand-pollinated flowers were selected for seed collection. Mature seeds were first treated with 9% HCl for 30 min and then were rinsed with tap-water for 10 min. After that, seeds were placed on paper towel and dried in room temperature for 2 weeks. This experiment was repeated three times, where seeds of replicates 1 and 2 were fixed on petri dishes using double-sides taps, whereas, seeds of replicate 3 were fixed on petri dishes using 1% agarose. After that, the petri dishes were scanned with the EPSON V600 scanner in 1200 dpi. Images were analyzed by Tomato Analyzer 4.0 software  and Turkey’s mean separation tests were performed in this paper using SPSS software. Significant effects and interactions were shown by the p-values computed from the F ratio in ANOVA. Visualization of the results were conducted using GraphPad.2.4www.biocloud.net). Since very few genes were statistically significant when using DESeq2 method, genes that met the following conditions were considered as the differentially expressed genes (DEGs) and selected for the Venn diagram analysis. (i) the average value of a gene’s FPKM must be ≥2 in any of the eight NILs; (ii) the ratio of FPKM value of a gene (any of the seven mutant NILs/WT) must be ≥1.50 or ≤0.67. Venn diagram analyses were conducted using Venny 2.0.2 software (https://bioinfogp.cnb.csic.es/tools/venny/index2.0.2.html). Heatmaps were drawn via using the TBtools software.Sixty to eighty developing seeds were dissected from at least three fruits of each NIL at 8 dpa and immediately frozen in liquid nitrogen. Total RNA extract, library construction and sequencing were performed by Biomarker Technologies Corporation  on a GAII platform (Illumina). Preliminary data analysis, including short sequences (reads) filtering, alignment, and FPKM calculation, was performed in BMKCloud (2.5DWF1 (Solyc02g069490), 2ODD (Solyc02g062490), GRP13 (Solyc06g007920), GA2ox2 (Solyc10g007560), NCED3 (Solyc07g056570), VI2 (Solyc08g079080), SWEET14 (Solyc03g097560) and WLIM1 (Solyc03g114000). Total RNA used for RNA-seq analysis was reverse-transcribed into cDNA using a PrimeScript RT reagent kit . Primers were designed using Primer5 software and listed in SAND (Solyc03g115810) was selected as the internal control gene according to Expositó-Rodríguez et al. (2008). 2-ΔΔCt method was used to calculate relative expression values. For each gene, three technical repeats were performed.For verifying the RNA-seq results, quantitative real-time PCR (qRT-PCR) was performed to clarify the expression patterns of some of the interested genes, including 2.6http://www.metware.cn/) based on the AB Sciex QTRAP 6500 LC-MS/MS platform. The hormones analyzed were: indole-3-acetic acid (IAA), abscisic acid (ABA), cytokinin (CK), gibberellins (GAs), brassinosteroid (BR) and ethylene (ETH). The significance of the effect of a locus on the accumulation of a certain hormone and interactions between loci were calculated by ANOVA. Paired Student’s t-tests were performed to compare the group means when the effect was significant. The effects of each locus were tested in the WT and mutant backgrounds.Phytohormones were detected by MetWare  to length) were investigated  areas in s/f NILs, but was not changed in the proximal area  was significantly decreased in mal area Table 3.s/f NIL was opposite to variation trends of seed length and width  =124/131), 174 (u/d=69/105), 559 (u/d=274/285), 311 (u/d=133/178), 707 (u/d=329/378), 603 (u/d=298/305) and 1051 (u/d=617/434) DEGs, respectively , GRP13 (encoding a gibberellin-regulated protein), GASA4 (encoding a gibberellin-regulated protein), DCR , CML19 , NCED3 (encoding a 9-cis-epoxycarotenoid dioxygenase which plays a part in ABA biosynthesis) and MAP70-5  were highly expressed in fs8.1 NIL; meanwhile, IQD10/21, CYP724A1 (encoding a cytochrome P450 enzyme which plays a role in brassinosteroid biosynthesis), IAA19/29 , PIN3 (encoding an auxin polar transporter), NCED1, CYP704A2  and CKX7 (encoding a cytokinin oxidase which is involved in cytokinin degradation) were up-regulated in NILs harboring fs8.1; in contrast, expression of NR2 (encoding a nitrate reductase which takes part in nitrogen assimilation), NRT1.1 , IAA36, CKI7 (encoding a cyclin-dependent kinase inhibitor 7 which is involved in cell cycle control), GIF3 (encoding a GRF1-interacting factor whose homolog controls seed size in Arabidopsis), GA2ox2 (encoding a gibberellin 2-oxidase which plays a role in gibberellin degradation), WLIM1 , CYP85A1 (encoding a BR6ox enzyme which is involved in brassinosteroid biosynthesis), IAA1, CYP707A1 , they highly expressed in NILs s/f, o/f and s/o/f; expression of SWEET14 , 2ODD (encoding a 2-oxoglutarate-dependent dioxygenases which is involved in gibberellin biosynthesis), DWF1 (encoding a steroid synthetase which plays a role in brassinosteroid biosynthesis), NRT1.11, GA2ox2, OFP10, WLIM1, 2ODD was obviously higher in WT NIL , STP1 (encoding a sugar transporter protein) and NRT1.1 clearly highly expressed in o/f NIL , BEH4 ( in rice ), CEPR2 modeling ) was redsun, ovate and s/o, 135, 75 and 192 DEGs were unique to NILs sun, ovate and s/o, respectively; meanwhile, 31, 51 and 30 DEGs were only shared between NILs sun and ovate, NILs sun and s/o, and NILs ovate and s/o, respectively; in addition, 38 DEGs were shared by the three NILs  and NRT1.1/2/2.4 were obviously up-regulated in the s/o NIL; meanwhile, an abscisic acid receptor gene PYR1-L was clearly down-regulated in s/o NIL but expressed higher in NILs WT, sun, ovate and s/o/f; as to CKX3, it highly expressed in NILs s/f, o/f and s/o/f; expression of SCL3, a gene involved in GA signaling, was higher in NILs WT and sun; as to GA20ox1, it was obviously up-regulated in fs8.1 NIL, but was clearly down-regulated in s/o NIL; in regard to the expression of IAA1 and 2, the highest levels were recorded in NILs s/o/f and s/o, respectively , GH3-10, GA2ox2, CKX1/3/7, IPT1 (encoding an isopentenyl transferase which is involved in trans-zeatin biosynthesis), SWEET1, SUS4 (encoding a sucrose synthase protein), CYCD3.1/6.1, CYP78A10, IQD6 and OFP13 were up-regulated in NILs s/f, o/f and s/o/f; in contrast, CID1L2 and CAR4 were down-regulated in the above-mentioned NILs; expression of SAUR50, encoding a SAUR-like auxin-responsive protein, was increased in NILs harboring fs8.1, while, expression of WLIM1-L was decreased in the same NILs; expression of GH3.2, GAST1-L and IQD19 was enhanced in sun NIL, but was repressed in s/f NIL; as to CYCB2.4 and ATN (encoding a microtubule-binding protein), their expression levels peaked in s/f NIL, and meanwhile, the expression of SAUR71 peaked in fs8.1 NIL; in regard to 2ODD, its expression was up-regulated in NILs WT and s/o  was increased in NILs sun, fs8.1, s/f, o/f and s/o/f; as to the CML3/8/15/22/24, they all highly expressed in NILs o/f and s/o/f , GH3.1/3.6, GA2ox8-L, DWF2, CYP85A1, CYCA3.2, GRF3-L, WRKY31, dUTPase  and OFP10, the lowest expression levels were all discovered in the s/o/f NIL (In order to clarify the epistatic interaction among  in rice ), NRT1.1fs8.1; only in fs8.1 NIL; in NILs s/f and s/o/f; in NILs s/f, o/f and s/o/f; only in s/o/f NIL; in NILs WT, ovate and fs8.1; in NILs WT and sun; in NILs WT and fs8.1; in NILs WT, ovate, fs8.1 and s/o; in NILs WT, sun and fs8.1; in NILs WT and ovate; in NILs WT, ovate and s/o; in NILs WT, sun, ovate and s/o; in NILs WT, sun, ovate, fs8.1 and s/o; only in sun; only in s/o; and DEGs that lowly expressed in fs8.1 NIL, respectively  as well as our developing seeds. In the WT NIL, SUN barely expressed in anthesis ovules, 4-dpa embryos, 4-dpa endosperm, 4-dpa seed coat and 4-dpa funiculus but highly expressed in 8-dpa seeds  and JA-Ile (isoleucine conjugated-JA) levels in the absence of the fs8.1; ovate had an impact on iP  accumulation; fs8.1 not only tend to reduce IAA and increase ABA in 9-dba flower buds but also inhibited TRA  accumulation in anthesis ovaries and 5-dpa fruits  gene family and plays a key role in maintaining source/sink characteristics  and is capable of catalyzing the first degradative step in sucrose utilization   but increased the expression of MAP70-5  . Interesd by sun , suggest5 , expression of another WLIM1 (Solyc12g088320) was also reduced in the s/f NIL, and meanwhile, expression of ATN, a gene involved in the identification of the division plane during mitosis, was up-regulated  was still down-regulated, however, TUB3 was up-regulated by the interaction. In contrast, interaction between sun and ovate eliminated the effect of single loci on cytoskeleton. As to the interaction of sun×ovate×fs8.1, it eliminated the effects of single loci, but at the same, established a new regulatory pathway which consisted of MAP65-6 and ROP9.Interactions among different loci also impacted the cytoskeleton-related pathways. The impacts of SUN and OVATE in WT NIL (sun could be directly attributed to effects of this locus; whereas, the physiological and transcriptomic variations in NILs carrying ovate were probably ascribed to the after effects of the locus or influence of nearby tissue, such as funiculus, since OVATE still highly expressed in this tissue at 4 dpa (Besides what is mentioned above, based on the expression patterns of sun impacted tomato seed morphology mainly through reducing BR biosynthesis, inhibiting sugar transport and degradation as well as repressing WLIM1-related cytoskeleton arrangement; meanwhile, ovate regulated seed morphology mainly by inhibiting auxin polar transportation as well as GA biosynthesis, sugar transport and degradation, and WLIM1-related cytoskeleton arrangement; as to fs8.1, although a decreased CK level and an increased ABA level were observed in the developing seeds of this NIL, the increased GA and BR levels as well as the enhanced source-sink relationship were still able to result in an increased trend of seed size. Interaction between sun and fs8.1 changed seed morphology mainly through decreasing the IAA, GA and CK contents as well as further repressing the WLIM-related cytoskeleton pathway; interaction between ovate and fs8.1 impacted seed morphology mainly through down-regulation of the IAA and CK contents, and repressing WLIM1-related cytoskeleton arrangement; interaction between sun and ovate changed seed morphology mainly through reducing GA and CK levels; in regard to the interaction of sun×ovate×fs8.1, the effects of the decreased IAA, GA and CK levels may overcome that of increased BR level and finally down-regulated the seed size and shape index together with the rearrangement of cytoskeleton which was caused by the different expression of MAP65-6 and ROP9.Taking together, 5sun and ovate showed potential abilities in decreasing tomato seed size, while, fs8.1 had a potential ability in increasing this parameter. Interactions between two loci as well as the interaction among three loci all led to significant decrease of seed size. All three fruit shape loci significantly down-regulated SSI, except for s/f, which resulted in the reductions in both seed length and width and finally led to a decreased trend of SSI. Histologically, the seed morphological changes were mainly depended on the cell number variations. Transcriptional and physiological analyses discovered that effects of fruit shape loci on seed morphology were high-likely mainly due to the changes of phytohormone-, cytoskeleton- as well as sugar transportation- and degradation-related genes.Based on the data obtained in this study, it can be presumed that https://www.ncbi.nlm.nih.gov/bioproject, PRJNA902156.The datasets presented in this study can be found in online repositories. The names of the repository and accession number can be found below: LS, HS, and JC designed the experiments; JC, BP, YX, XC, and JJ carried out the morphological, histological and physiological experiments; JC and ZL performed the RNA-seq analysis; JC wrote the first draft of the manuscript; LS revised the manuscript and draw figures. All the authors are contributed to the modification of the manuscript.


Ramucirumab, as a vascular endothelial growth factor receptor-2 inhibitor, was first approved in 2014 for treated advanced or metastatic gastric/gastroesophageal junction (GEJ) adenocarcinoma. This study deeply analyzed the efficacy and safety of advanced or metastatic cancer treated with ramucirumab, which included 11 global, double-blind, phase 3 randomized controlled trials with a total of 7410 patients. Subgroup analysis based on different cancer types showed that standard regimens plus ramucirumab significantly increased progression-free survival and overall survival compared with placebo groups in patients with advanced non-small-cell lung cancer (NSCLC), hepatocellular carcinoma, gastric cancer, or GEJ adenocarcinoma. Although a higher proportion of patients achieved overall response and disease control than those treated with placebo, the overall response was not statistically significant between the two groups in advanced NSCLC. Grade 3 or worse treatment-emergent adverse events (TEAEs) that occurred in at least 5% of patients were neutropenia (30.5% in the ramucirumab group vs. 23.5% in the placebo group), leucopenia (14.8% vs. 9.2%), weight decreased (14.2% vs. 8.0%), myalgia (11.7% vs. 7.7%), fatigue (10.9% vs. 7.7%), hypertension (9.2% vs. 2.3%), and anaemia (6.2% vs. 7.7%). In the TEAEs of special interest, the ramucirumab group had a significantly higher incidence of bleeding , hypertension, proteinuria, liver injury/failure (grade 1-2), venous thromboembolism (grade 1-2), and gastrointestinal perforation (grade ≧3) than the control group. Cancer is a serious global public health problem and is expected to become the leading cause of death in countries around the world in the 21st century, which may bring great harm to human health .3 requires new blood vessels to promote, and gave some evidence to support this view in subsequent studies , but there was no statistically significant difference at gra de≧3 .Epistaxis is a type of bleeding/haemorrhage, and it was analyzed separately in 4 RCTs, included 393 (25.0%) patients in the ramucirumab group and 149 (10.2%) in the control group with grade 1-2 events, 3 (0.2%) in the ramucirumab group and 1 (0.0%) in the control group with grade≧3. The incidence of epistaxis in the ramucirumab group was higher than the control group at grade 1–2 , GI haemorrhage events , and gra de≧3 GI perforation events  in the ramucirumab group.Congestive heart failure (CHF), infusion-related reaction (IRR), venous thromboembolic (VTE), arterial thromboembolic (ATE), gastrointestinal (GI) perforation, GI haemorrhage, renal failure, fistula, healing complication, and pulmonary hemorrhage were adverse events with low probability (<5%). There was no significant difference between the two groups except for the higher incidence of grade 1-2 VTE events , nausea (grade 1–2), decreased appetite, and stomatitis were some of the most frequently reported nonhaematological events. Neutropenia, leucopenia (grade 1–2), and thrombocytopenia were the most frequently reported haematological TEAEs. Moreover, the incidence of TEAEs reported above was higher in the ramucirumab group. In addition, the TEAEs of special interest such as bleeding/haemorrhage , hypertension, liver injury/failure (grade 1–2), proteinuria, VTE (grade 1–2), and GI perforation (grade 3) were also more common in the ramucirumab group, as expected, consistent with the incidence of adverse events associated with most antiangiogenic therapies (38–40). Most of grade 4 or 5 TEAEs of special interest occurred with a very low incidence in both groups. There were only 0.07% of patients (3/4040) reported grade 4 hypertensive crisis, and 0.03% of patients (1/3782) reported grade 4 proteinuria in the ramucirumab group (0% in control), with no grade 5 events. Grade 4 and grade 5 GI perforation events were 0.36% (10/2783) and 0.22% (6/2783) in the ramucirumab group (only 1 case in each control group). Fortunately, in the reported cases of RCTs, these events were all within the safe range that could be controlled by medical treatment, with no unexpected safety events reported.VEGF has been proved to be essential for the occurrence, development, and metastasis of cancer, and its mediated carcinogenesis is mainly completed by affecting the formation of new blood vessels and vascular permeability. In the past few decades, many anticancer drugs that target VEGF receptors or subtypes or signaling pathways have been approved by the United States Food and Drug Administration. These targeted drugs can be divided into the following categories according to the mechanism of action: (1) block the binding of ligands to receptors, such as bevacizumab, which targets VEGF-A, (2) block signaling through VEGFR, such as ramucirumab, which targets VEGFR2, and (3) tyrosine kinase inhibitors that block VEGFR1, VEGFR2, and VEGFR3 kinase activity, such as pazopanib and lenvatinib , 42. TheIn conclusion, the meta-analysis showed that standard regimens combined with ramucirumab resulted in more beneficial PFS and OS outcomes in advanced NSCLC, HCC, gastric cancer, or GEJ adenocarcinoma. The importance of the results for clinical practice was that the incidence of hypertension, proteinuria, bleeding, liver injury/failure, VTE, and GI perforation were consistent with reported TEAEs of special interest associated with the angiogenesis inhibitor class. However, the authors do not observe an increased incidence associated with ramucirumab for high-grade bleeding, ATE, IRR, renal failure, fistula, or healing complication across these RCTs. Also, these adverse events were largely controllable after dose adjustment and supportive treatment.