On this account, L1CAM expression in the MELF component may stratify the prognosis and management in patients with uterine-confined, low-grade carcinomas.L1CAM overexpression (≥10%) and the microcystic, elongated, and fragmented (MELF) pattern of invasion have previously been assessed as prognostic factors in endometrial carcinoma. We aimed to assess the relationship between L1CAM expression, MELF glands, and lymph node involvement in endometrial carcinoma, as all these factors are related to epithelial-to-mesenchymal transition. We evaluated L1CAM expression in 58 cases of uterine-confined, low-grade endometrioid carcinomas. We found that most cases (65.5%) expressed L1CAM in a limited manner to MELF glands. Cases with L1CAM expression in ≥10% of the MELF component showed a significantly higher tendency to lymph node spread  and substantial lymphovascular space invasion . All cases were p53-wild-type; 17 (29.3%) were mismatch repair-deficient. Twenty cases (34.5%) had positive nodes. No cases showed L1CAM positivity in ≥10% of the whole tumor. MELF glands expressed L1CAM at least focally in 38 cases (65.5%). L1CAM positivity in ≥10% of the MELF component was found in 24 cases (41.4%) and was the only significant predictor of lymph node involvement in both univariate (p < 0.001) and multivariate analysis (p < 0.001). In conclusion, L1CAM might be involved in the development of the MELF pattern. In uterine-confined, low-grade endometrioid carcinomas, L1CAM overexpression in MELF glands may predict lymph node involvement.In endometrial carcinoma, both L1CAM overexpression and microcystic, elongated and fragmented (MELF) patterns of invasion have been related to epithelial-to-mesenchymal transition and metastatic spread. We aimed to assess the association between L1CAM expression, the MELF pattern, and lymph node status in endometrial carcinoma. Consecutive cases of endometrial carcinoma with MELF pattern were immunohistochemically assessed for L1CAM. Inclusion criteria were endometrioid-type, low-grade, stage T1, and known lymph node status. Uni- and multivariate logistic regression were used to assess the association of L1CAM expression with lymph node status. Fifty-eight cases were included. Most cases showed deep myometrial invasion ( Endometrial carcinoma is the most common gynecological malignancy in developed countries and the second most common worldwide after cervical carcinoma . EndometPOLE mutated/ultramutated, microsatellite unstable/hypermutated, copy number high, and copy number low. This classification allows the stratifying prognosis in endometrial carcinoma [POLE mutation, which requires molecular analysis; molecular testing of POLE gene is commonly based in the search for hotspot pathogenic mutations [POLE mutated group (surrogate of the ultramutated group) [POLE mutated group show good prognosis regardless of clinicopathological features, which not uncommonly are worrisome; for instance, about half of POLE-mutated endometrial carcinomas are high-grade [In 2013, The Cancer Genome Atlas (TCGA) Research Network  found foarcinoma . The TCGarcinoma ,8,9. Thearcinoma . In recearcinoma ,8,10. Suarcinoma . This prarcinoma ,8. Two larcinoma ,13 and tarcinoma ,15, haveutations ,12,15. Td group) ,12. Integh-grade . POLE-mutated endometrial carcinomas up to FIGO stage II are included in the low-risk prognostic group, regardless of other clinicopathological features, such as tumor grade, histotype, LVSI, and depth of myometrial invasion. On the other hand, all p53 abnormal carcinomas are included in the high-risk prognostic group, except for cases with no myometrial invasion, which are considered at intermediate risk instead; in this way, p53 abnormal endometrioid carcinomas are considered prognostically analogous to serous carcinomas. For the NSMP and MMR-deficient groups, the risk group is based on traditional clinicopathological factors. In particular, cases at FIGO stage IA, endometrioid histotype, and low grade, with no or focal LVSI, are included in the low-risk group; this means that these tumors do not need adjuvant treatment. FIGO IA high-grade endometrioid carcinomas, as well as FIGO IB low-grade endometrioid carcinoma, fall into the intermediate risk group in the case of no or focal LVSI. Endometrioid carcinomas with substantial LVSI  fall into the high-intermediate risk group, which also includes endometrioid carcinomas at FIGO stage II [In the 2020 ESGO-ESTRO-ESP guidelines, all stage II ,11. SuchCTNNB1 exon 3 mutations [However, there is still a paucity of prospective data from randomized controlled trials in this field.  Furthermutations . Among these factors, L1CAM has emerged as one of most promising . L1CAM iThe MELF pattern of invasion was first described by Lee, Vacek, and Belinson  as an “eAs both L1CAM expression and MELF pattern are related to epithelial to mesenchymal transition and, likely, to metastatic spread, we aimed to assess (i) the relationship between these two factors in endometrial carcinoma and (ii) their association with lymph node metastasis.n = 58). The study outcomes were (i) the expression of L1CAM in MELF and (ii) its association with lymph node status.Patients were selected from a larger study which assessed L1CAM in a consecutive series of endometrial carcinomas; all patients underwent hysterectomy at the Gynecologic Oncology Unit, Fondazione Policlinico Universitario A. Gemelli, Rome, Italy, with a diagnosis of presumed uterine-confined endometrial carcinoma . Among these patients, we selected all consecutive cases (from January 2018 to October 2021) of low-grade (FIGO G1-G2) endometrioid carcinoma with a MELF pattern of invasion and who underwent pelvic lymphadenectomy or sentinel lymph node biopsy  with lymph node involvement. A p < 0.001) and multivariate analysis (p < 0.001) with a negative predictive value of 81.6% and a positive predictive value of 80%. Because the significance of ITCs is undefined, we repeated the analysis after considering ITC cases as negative. Again, we found that an expression of L1CAM in ≥10% of MELF glands was the only significant predictor of lymph node micro-/macrometastasis on both univariate (p = 0.001) and multivariate analysis (p = 0.004) . Forty-one cases (72.4%) showed deep myometrial invasion; 14 cases (24.1%) showed focal LVSI, and 34 cases (58.6%) showed substantial LVSI. P53 immunohistochemical expression was wild-type in all cases; 17 cases (29.3%) showed MMR deficiency. Positive lymph nodes were found in 20 cases (34.5%), including 6 cases of ITC (10.3%), 6 cases of micrometastasis (10.3%) and 8 cases of macrometastasis (13.8%) . None of= 0.004) .In our study, we aimed to assess the relationship between L1CAM expression and the MELF pattern of invasion in endometrial carcinoma, as they are both related to epithelial to mesenchymal transition and aggressiveness. Interestingly, none of the included cases showed L1CAM positivity in more than 10% of the whole tumoral area. However, almost two thirds of the tumors showed L1CAM expression in the MELF glands, which ranged from weak and focal to strong and diffuse. This first result suggests that L1CAM may be involved in the epithelial to mesenchymal transition process which underlies the MELF pattern of invasion, potentially being useful as a marker of MELF glands. Then, we used the threshold of 10% to define L1CAM overexpression in MELF glands. We found that L1CAM overexpression in MELF glands was significantly associated with lymph node involvement. In particular, L1CAM was the strongest predictor of lymph node involvement and was independent from LVSI, deep myometrial invasion, and MMR status. This finding suggests that the tendency of the MELF glands to spread to lymph nodes may depend on the degree of L1CAM expression. In the previous study by Stelloo et al., L1CAM showed unfavorable prognostic value in a cohort of endometrioid carcinomas including low-grade tumors with deep myometrial invasion and high-grade tumors with superficial myometrial invasion. Although a 10% threshold was adopted, such a percentage was assessed on the whole tumoral area and did not consider where L1CAM was expressed . Our resTP53 mutations [Previous studies have shown that L1CAM is often associated with p53 abnormalities . In our utations . Indeed,utations ) may regutations .Noticeably, L1CAM overexpression in MELF glands predicted lymph node involvement with a positive predictive value of 80% and a negative predictive value of 81.6%; such data support a possible role in directing patient management. The evaluation of L1CAM expression in MELF glands may be integrated in the prognostic stratification of endometrial carcinoma, regardless of the overall expression in the whole tumor. For instance, a L1CAM expression ≥10% in MELF glands might have a prognostic significance similar to LVSI; uterine-confined, low-grade endometrioid carcinoma with L1CAM positive glands might be included in the high-intermediate risk category according to the ESGO-ESTRO-ESP risk classifier . FurtherRemarkably, all cases in our series were low-grade endometrioid carcinoma with no direct extension beyond uterus. Such homogeneity in our sample is a strength of our study. Furthermore, all cases in our series fell into the NSMP or the MMR deficient group, which are considered groups at intermediate prognosis, as discussed above ,8,10,11.In conclusion, L1CAM is often expressed in MELF glands of endometrioid carcinoma, suggesting its involvement in the development of the MELF pattern. L1CAM positivity in ≥10% of cells in the MELF component, regardless of the overall expression in the whole tumor, appears as the strongest independent predictor of lymph node involvement in MELF cases. This may suggest that the prognostic value of MELF pattern depends on L1CAM expression. On this account, quantifying L1CAM expression in MELF glands might be prognostically relevant and might potentially impact the patient management. Further studies are warranted in this regard.


Most gastric cancer (GC) patients have already benefited from immune checkpoint inhibitors, but some of them may terminate immunotherapy due to immune-related adverse events (irAEs). Extracellular vesicles have been shown to carry proteins, nucleic acids and other biomacromolecules to recipient cells, which is very important for exploring the potential of biomarkers of irAEs via EV-derived proteins. In 62 GC patients, EV-ICOS and EV-IDO1 were screened from 42 vital proteins as biomarkers of irAEs, and then confirmed in a validating cohort of 40 GC patients. In summary, EV-ICOS and EV-IDO1 can perfectly predict irAEs of ICI treated GC patients.Immune checkpoint inhibitors (ICIs) initiate a new stage for gastric cancer (GC) therapeutics, and plenty of patients have already benefited from ICIs. Liquid biopsy promotes the development of precision medicine of GC. However, due to the lack of precision biomarkers of immune-related adverse events (irAEs), the safety of ICIs-treated GC patients cannot be guaranteed. In our study, GC patients treated with ICIs were included for investigating the correlation between irAEs of ICIs and corresponding outcomes. We also explored the potential of biomarkers of irAEs via EV-derived proteins. Dynamic plasma was taken from 102 ICIs-treated GC patients generated retrospectively or prospectively, who were divided into discovery and validating cohorts. Plasma EV-derived protein profiles were described, and two EV-proteins, inducible T-cell co-stimulator (EV-ICOS) and indoleamine 2,3-dioxygenase 1(EV-IDO1), from 42 vital proteins were screened to predict the prognosis of ICIs with irAEs. Our work is the first to propose that EV-proteins can predict ICIs-corresponding irAEs, which can be conducive to the diagnosis and treatment of GC patients, and to facilitate the screening of beneficiaries. Gastric cancer (GC) is the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide . With thExtracellular vesicles (EVs), defined as nano-size particles with a small diameter (40–160 nm), are secreted by almost all types of cells. Containing various bioactive molecules including proteins, lipids, and nucleic acids, EVs not only mediate signal transduction in cellular communication but also participate in the physiological process including immune regulation and cancer progression . Due to In this study, a total of 102 GC patients who received ICI-based therapy were enrolled in our study and were divided into two independent cohorts. EVs derived from patient plasma were analyzed by protein microarray array paneled with 42 key proteins, and 2 vital members were screened from them to predict the prognosis and monitor the irAEs of GC patients treated with ICIs. Our study first described the potential of EV-derived proteins as novel biomarkers in predicting and monitoring irAEs, which offers reference value in the clinical practice.Recruitment, treatment, management, follow-up and blood samples of all patients were executed by the Department of Gastrointestinal Oncology, Peking University Cancer Hospital & Institute. A total of 102 GC patients diagnosed with unresectable or advanced GC  and non-GEJ types) and administrated with ICI-based regimens from August 2016 to April 2021 were enrolled. All patients had normal function of the heart, liver, lung, kidney and had been ruled out bacterial and viral infections. All patients’ irAEs were assessed following Management of Immune Checkpoint Inhibitor-Related Toxicity that was published in 2020 and updated to the NCCN Guidelines , and theg, 10 min for separating plasma and blood cells, then centrifugated at 25 °C, 1800× g, 8 min for obtaining lymphocytes after lysis red blood cells. Extracted blood components were stored at −80 °C for future experiments. EV was isolated from plasma samples by using the SEC-based qEV column , and the centrifugation method  was also applied in this work for EVs extracting. Finally, EV eluates were filtrated with an Amicon Ultra-4 10 kDa centrifugal filter device .Approximately 10 mL peripheral blood was collected with EDTA tube, centrifugated at 4 °C, 3000× w/v) diluted in PBS for 1 h at room temperature, then incubated with 10 μL unpurified plasma sample that diluted (1:10) in wash buffer (0.05% Tween20 in PBS) sequentially at room temperature on a shake flask at 30 rpm for 2 h, and then incubated overnight at 4 °C. The next day, the slides were washed and incubated with biotinylated detection antibodies  diluted 1:1500 in wash buffer. After that, Cy3-labelled streptavidin  was added in the slides for incubation 1 h at room temperature. After washing, the slides were scanned using the GenePix 4000A microarray scanner  and GenePix Pro image analysis software  was used to analyze the signal intensity of fluorescent images.In our study, 42 candidate marker proteins, including 19 representative cellular biomarkers in tumor-immune microenvironment, 11 immune checkpoint modulators, 12 therapeutic biomarkers or targets of gastrointestinal cancer, and 4 canonical EV markers, were embraced in the EV expression array. Then the 46 key EV-proteins were printed onto a 3D modified slide surface  using Arrayjet microarrayer  to construct a protein chip spectrum. For EV-proteins capture experiment, the protein chip spectrum was initially blocked with 3% BSA (https://www.ncbi.nlm.nih.gov/geo/ (accessed on 2 November 2021)) and The Cancer Genome Atlas (TCGA) ), as well as gastric cancer cohort (n = 102) previously published by our group.Databases were downloaded from the Gene Expression Omnibus (GEO)  received ICI monotherapy, 62.5% (25/40) received ICI plus chemotherapy and 7.5% (3/40) received double-ICI therapy, while irAEs were observed in 32.5% (13/40) patients. These irAEs were composed of 20% (8/40) grade 1, 10% (4/10) grade 2 and 2.5% (1/40) grade 3, while for all irAEs cases, 46.1 (6/13) were related with skin, 15.4% (2/13) with digestive system, 15.4% (2/13) with hematopoietic system, 15.4% (2/13) with endocrine system, and 7.7% (1/13) with bone and muscle . When coIn the validating cohort, patients with irAEs achieved higher DCR than patients without irAEs , while pSimilar to discovery cohort, there is no evident correlation between the two EV-proteins and ORR/DCR a, while Since EV-derived ICOS and IDO1 exhibited similar predictive roles for GC irAEs, we assessed their expressional profiles and found that these two EV-derived proteins were tightly linked in both discovery and validating cohorts a. The inContemporarily, immunotherapy has become a central pillar of cancer treatment in various tumor types, including gastric cancer. Although a great many GC patients benefit from immunotherapy, the accompanying irAEs also disturb clinicians for they impede patients from deriving durable responses. IrAEs are a spectrum of diseases featured with unrestrained autoimmunity and inflammations affecting multiple organs. Though generally manageable, certain varieties of irAEs could be life-threatening. Therefore, it remains challenging to seek predictive biomarkers identifying patients at stake in irAEs to facilitate timely clinical intervention.A series of studies have reported candidate biomarkers correlated with IrAEs. Gene expression profiles, C-reactive proteins, human leukocyte antigens (HLAs) and gut microbiome were found to be capable of predicting irAEs ,21,22,23+ Tregs mediates allergic airway diseases, and targeting the ICOS/ICOS-L pathway may disrupt T follicular helper cell responses and ameliorate allergic asthma by disrupting the disease [ICOS and IDO1 are proteins related to immunotherapy; relevant target drugs have been produced ,31, and  disease ,34. In a disease ,36. As a disease . Moreove disease ,39. HoweImmunotherapy innovates cancer therapy while unpredictable irAEs restrict its clinical practice. In our study, a protein chip spectrum that contains 46 key tumor immune microenvironment molecules and classic targets was invented to screen irAEs related markers. Considering that plasma samples are easily obtained, less consumed, and minimally invasive, our detecting procedure was very convenient and accurate to predict irAEs. In conclusion, our results indicate that EV-ICOS and EV-IDO1 are efficient predictors of irAEs, which could assist the decision-making for clinicians and could reduce unnecessary suffering for GC patients treated with ICIs.


Chemokines constitute a group of small, secreted proteins that regulate leukocyte migration and contribute to their activation. Chemokines are crucial inflammatory mediators that play a key role in managing viral infections, during which the profile of chemokine expression helps shape the immune response and regulate viral clearance, improving clinical outcome. In particular, the chemokine ligand CXCL10 and its receptor CXCR3 were explored in a plethora of RNA and DNA viral infections. In this review, we highlight the expression profile and role of the CXCL10/CXCR3 axis in the host defense against a variety of RNA and DNA viral infections. We also discuss the interactions among viruses and host cells that trigger CXCL10 expression, as well as the signaling cascades induced in CXCR3 positive cells. Chemokines are small, secreted molecules that enhance cell interaction via G-protein-coupled receptors . The cheFurther, chemokine receptors can be categorized into conventional (cCKRs) and atypical . ConventSeveral physiological processes, including cell proliferation and angiogenesis, as well as pathological conditions, including inflammation, neoplasia, autoimmunity, and infections , are controlled by binding chemokines to their cognate receptors . During Three chemokines are known to be interferon (IFN)-induced angiostatic CXC chemokines. These include monokine induced by interferon (MIG/CXCL9), interferon gamma-induced protein 10 (IP-10/CXCL10), and interferon-inducible T-cell chemoattractant (I-TAC/CXCL11). The chemokine CXCL10 binds CXCR3, which is present in T helper lymphocytes, natural killer (NK) cells, dendritic cells (DCs), macrophages, and B cells ,25,26,272+ mobilization [CXCL10 possesses an upstream regulatory sequence that contains several critical regulatory elements for nuclear factor-κB (NF-κB) and interferon-stimulated response element (ISRE), as well as sites for the binding of proteins such as heat shock (HS) factors . Cytokinlization ,46. Sinclization ,48,49,50lization . Moreovelization . CXCL10 could be protective or pathogenic in viral infections depending on the host’s immune status and the type of viral infection . CXCL10 + T cells in the liver of adenovirus infected mice [The absence of CXCL10 using antibodies or knockout mouse models resulted in increased viral titers and reduced T cell infiltration within the brains of mice infected with a murine hepatitis virus (MHV), leading to increased mortality ,68. CXCLted mice ,70. Thested mice ,72,73,74ted mice . Moreoveted mice ,76. For ted mice . Additioted mice .+ T cell and NK cell migration towards the airway epithelial milieu was significantly affected by the ability of the airway epithelial cells to dramatically upregulate CXCL10 [Previous studies reported a synergy among double-stranded RNA (dsRNA) and IFN-γ-induced signaling in regulating CXCL10 production. As mentioned earlier, pro-inflammatory mediators and IFN-γ increased CXCL10 expression by a number of cell types, including keratinocytes, macrophages, endothelial cells, smooth muscle cells, and epithelial cell fibroblasts and astrocytes ,86,87,88e CXCL10 . Other de CXCL10 ,92,93. + T cells. CXCL10 regulates the persistence and heterogeneity of CD8+ T cells in the spleens of mice infected with lymphocytic choriomeningitis virus (LCMV). Moreover, functional CD8+ T cell responses were found to be greater in Cxcl10−/− mice and were associated with a lower viral count [CXCL10 was found to have a protective function during certain RNA viral infections such as the severe acute respiratory syndrome coronavirus (SARS-CoV) and Epstein-Barr virus (EBV) ,95,96. Oal count . + T cells and CD4+ T helper cells into HCV-infected livers [Among various chemokines, CXCR3 has been studied in viral hepatitis as it plays a significant role in the recruitment of T cells into the peripheral inflammatory sites ,106. Thed livers ,111,112,d livers ,115,116.d livers ,119,120.d livers .+ T cells. CXCL10 plasma levels were significantly higher in HCV-infected patients with advanced fibrosis [In chronic HCV infection, CXCL10 contributed to the development of necroinflammation and liver fibrosis. Moreover, the intrahepatic production of IFN-γ promoted CXCL10 expression by sinusoidal endothelium and hepatocytes, thereby triggering the recruitment of CXCR3fibrosis . Additiofibrosis . Additiofibrosis . This thfibrosis .+ T cells into the central nervous system, which subsequently aids in limiting viral replication [+ T cells that amplify neuroinflammation through the IFN induction of chemokine release. Therefore, the CXCL10/CXCR3 signaling pathway may influence glial biology and may repair virally induced neurologic diseases [CXCL10 is also known to play a critical role in the host defense against infection caused by the John Howard Mueller (JHM) strain of mouse hepatitis virus (JHMV). Early expression of CXCL10 was observed to promote the attraction of CXCR3lication . On the diseases .CXCL10 is a key player in respiratory syndromes. Its level was higher in the plasma and bronchial alveolar lavage fluid (BALF), which correlated with the disease severity ,126,127.Similar to other respiratory diseases, high CXCL10 levels in COVID-19 patients showed a strong correlation with disease severity ,135. As Intriguingly, across all three coronaviruses , CXCL10 was identified as a crucial contributor to pulmonary pathogenesis. CXCL10 was associated with lung injury and the activation of the toll-like receptor 4 (TLR4) signaling pathway ,149. Alt+ macrophages, the main producers of IL-6. These observations highlight the vicious cycle between CXCL10, IL-6, and macrophages in the lungs of COVID-19 patients, which could regulate the onset, maintenance, and progression of cytokine storms during COVID-19 infection [IL-6 is one of the cytokines released during COVID-19-mediated cytokine storm ,155. Durnfection .+ DCs, triggering myeloid DC maturation [As in respiratory coronavirus infections, CXCL10 showed a significant increase during measles virus (MeV) infection, which may distinguish MeV from other diseases such as rubella virus (RuV), parvovirus B19 (B19V), human herpesvirus 6 (HHV6), EBV, and human cytomegalovirus (HCMV). Another common factor was the high level of CXCL10 in hospitalized MeV patients compared with non-hospitalized ones, and higher CXCL10 level was observed in cases of primary infection compared with re-infected cases ,158. Thituration . CXCL10 expression was higher in Zika virus (ZIKV)-infected individuals. This expression could be mediated through the induction of IFN-γ signaling pathway by the ZIKV NS5 protein. Previously, CXCL10 was discovered to be a biomarker of several bacterial infections . Similar+ cells towards the infected dorsal root ganglia. Consequently, CXCL10 could be a distinct chemokine that attracts inflammatory cells, specifically CXCR3+ cells causing ganglionitis [As illustrated in lionitis .+ cells towards the liver that could cause liver fibrosis [According to several studies, CXCL10 is implicated in hepatitis B virus (HBV) infection, including a study where the polymorphism G-201A in the promoter of the CXCL10 gene predicted susceptibility to chronic HBV infection . During fibrosis . + effector memory (TEM) and CD8+ residual memory (TRM) cells within latently infected trigeminal ganglia following HSV-1 reactivation [+ TEM and CD8+ TRM cells associated with protection against recurrent ocular herpes. These findings demonstrate that CXCL10/CXCR3 axis is crucial for CD8+ T cell immunity, which protects against recurrent herpesvirus infection [+, CD8+ T cells, and NK cells at the sites of HSV-1 replication [−/−) and CXCL10-deficient (CXCL10−/−) animals compared with wild types [−/− mice exhibited impaired NK and HSV-1-specific CD8+ T cell activation, which increased their susceptibility to HSV-1 infection [CXCL10 is crucial in host defense against multiple neurotropic viruses, including herpes simplex virus-1 (HSV-1) . It was tivation . In contnfection . The chelication ,179,180.ld types ,177. In nfection . Unlike HSV-1, CXCL10 was found to promote the infection caused by stimulating virus replication in immune cells during herpes simplex virus type 2 (HSV-2) infection . The exp+ T cells into the colon tumor microenvironment corroborated with increasing the efficacy of PD-1 antibody [Multiple strains of oncolytic viruses such as vesicular stomatitis virus (VSV), HSV-2, maraba virus, chikungunya virus, and reolysin have been reported to drive the high expression of chemokines in infected tumors ,189,190.antibody . These rantibody ,194. An impaired immune response is a critical hallmark in the development and progression of viral infections. Notably, the role of chemokines during viral infections has been explored and analyzed during the context of many RNA and DNA viral infections. CXCL10 was found to mediate its inflammatory activity through CXCR3, which is mainly expressed on T cells, NK cells, macrophages, and dendritic cells. Several studies report the significance of CXCL10 and its chemokine receptor CXCR3 in host defense and viral clearance, while other reports demonstrate its role in disease pathogenesis. Moreover, CXCL10 was suggested to be a potential diagnostic and prognostic marker in several viral infections such as COVID-19. Further, CXCL10 has a potential role as an anti-tumor agent mediated by oncolytic viruses. Therefore, targeting CXCL10 could be a possible therapeutic modality against many viral infections, as well as against tumor development. Further in-depth understanding of the chemokine system may allow the development of such therapeutics against viral infections, which could be beneficial in preventing opportunistic microbes.


This study aimed to validate the efficacy of NTP42:KVA4, a novel oral formulation of NTP42 in clinical development, in preclinical models of PAH while also, critically, investigating its direct effects on RV dysfunction.Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary artery pressure leading to right ventricular (RV) failure. While current PAH therapies improve patient outlook, they show limited benefit in attenuating RV dysfunction. Recent investigations demonstrated that the thromboxane (TX) ANTP42:KVA4 were evaluated in the monocrotaline (MCT) and pulmonary artery banding (PAB) models of PAH and RV dysfunction, respectively, and when compared with leading standard-of-care (SOC) PAH drugs. In addition, the expression of the TP, the target for NTP42, was investigated in cardiac tissue from several other related disease models, and from subjects with PAH and dilated cardiomyopathy (DCM).The effects of NTP42:KVA4 alleviated disease-induced changes in cardiopulmonary hemodynamics, pulmonary vascular remodeling, inflammation, and fibrosis, to a similar or greater extent than the PAH SOCs tested. In the PAB model, NTP42:KVA4 improved RV geometries and contractility, normalized RV stiffness, and significantly increased RV ejection fraction. In both models, NTP42:KVA4 promoted beneficial RV adaptation, decreasing cellular hypertrophy, and increasing vascularization. Notably, elevated expression of the TP target was observed both in RV tissue from these and related disease models, and in clinical RV specimens of PAH and DCM.In the MCT-PAH model, NTP42:KVA4 attenuates experimental PAH pathophysiology, not only alleviating pulmonary pathologies but also reducing RV remodeling, promoting beneficial hypertrophy, and improving cardiac function. The findings suggest a direct cardioprotective effect for NTP42:KVA4, and its potential to be a disease-modifying therapy in PAH and other cardiac conditions.This study shows that, through antagonism of TP signaling,  Pulmonary arterial hypertension (PAH) is a rare yet devastating disease with progressively debilitating symptoms and high mortality. The underlying etiology of PAH is characterized by excessive vasoconstriction and remodeling of the pulmonary vasculature leading to increased pulmonary vascular resistance (PVR). However, the ultimate determinant of survival in PAH patients is the response of the right ventricle (RV) , 2.Upon elevated afterload due to increased PVR, the RV in PAH patients initially responds through compensatory mechanisms termed adaptive hypertrophy. These physiological responses aim to preserve systolic and diastolic right heart function and are characterized by an increased RV wall thickness facilitated by hypertrophic remodeling, increased angiogenesis, altered sarcomere organization and increased intrinsic cardiomyocyte contractility , 3, 4. HWhile considerable advances have been made in the clinical management of PAH, patient mortality remains high. Current PAH standard-of-care (SOC) therapies include the phosphodiesterase type-5 inhibitors (PDE5is), endothelin receptor antagonists (ERAs), prostacyclin analogs (PCAs) or prostacyclin receptor agonists (PRAs) and soluble guanylate cyclase (sGC) stimulators, with various other pipeline compounds in clinical development. The key focus of the SOCs and those in clinical development is to reduce PVR, either by alleviating excessive pulmonary vasoconstriction or enhancing pulmonary vasodilation, and/or reduce pulmonary vascular remodeling. However, while it is recognized that RV function is the main determinant of prognosis in PAH, current SOC therapies have limited cardiac-specific effects , 9. Furt2 receptor, or TP, primarily mediates signaling of the prostanoid TXA2 and of the free-radical derived isoprostane 8-iso-prostaglandin F2α (8-iso-PGF2α), as well as other endogenous ligands, regulating processes including platelet aggregation, and constriction and proliferation of vascular and pulmonary smooth muscle. TP-mediated signaling also mediates potent pro-inflammatory, pro-mitogenic, and pro-fibrotic effects, and levels of TXA2, 8-iso-PGF2α, and TP expression are elevated in many cardiovascular and pulmonary diseases, inflammatory disorders and in certain cancers  A cancers , 12. Mul cancers –19. TXA2l models . TP exprl models , 19. In rtension , 25. Whirtension . Taken tNTP42 is currently in clinical development for PAH and other cardiopulmonary indications. Previous efficacy evaluations demonstrated that NTP42 attenuates preclinical PAH in both the monocrotaline (MCT)-and Sugen/Hypoxia (SuHx)-induced animal models of PAH  in 79 healthy male volunteers where it was confirmed as safe, well-tolerated, with good pharmacokinetic and pharmacodynamic profiles following single and repeat oral dosing. To specifically assess its potential to impart direct cardioprotective effects on the RV, the aim of this study was to validate the efficacy of NTP42, delivered as the orally formulated NTP42:KVA4, in the MCT-PAH model while also exploring its effect in the pulmonary artery banding (PAB) preclinical model of RV dysfunction and pressure overload. Furthermore, in this study, we also examined expression levels of the TP, the target receptor for NTP42, in RV tissues from several highly relevant disease models as well as in clinical specimens from subjects with PAH and dilated cardiomyopathy (DCM), where the data further supports the hypothesis that the TP is a bona fide target for treatment of PAH and certain other cardiac dysfunctions.The TP antagonist s of PAH , 28. As NTP42:KVA4 (1 mg/kg), the PDE5i Sildenafil (50 mg/kg), the ERA Macitentan (30 mg/kg), the PRA Selexipag (1 mg/kg), or the sGC stimulator Riociguat (5 mg/kg) was commenced on Day 7 post-MCT and continued to Day 28 (NTP42:KVA4 (1 mg/kg), or the sGC stimulator Riociguat (5 mg/kg) was started on Day 2 post-PAB and continued to Day 27  were used in all models. MCT-PAH was induced using a single injection of 60 mg/kg MCT, where twice-daily oral treatment with placebo, o Day 28 . The PABo Day 27 .Human tissues, following autopsy, were obtained from the Institute of Cardiometabolism and Nutrition BioCollection , detailed in P-values < 0.05 were considered to indicate significant differences.A detailed description of the Materials and methods is presented in NTP42 alleviates pulmonary pathologies and cardiopulmonary hemodynamics in both the MCT- and SuHx-induced PAH animal models  following its dissolution in an organic-based drug vehicle unsuited and not approved for use in man. Hence, for use in the clinical setting, NTP42 has since been uniquely formulated with the widely used pharmaceutical polymer Kollidon® VA 64 yielding the investigational medicinal product (IMP) referred to as NTP42:KVA4. Herein, it was first necessary to confirm or validate the oral efficacy of NTP42 administered as NTP42:KVA4 in preclinical PAH. Thereafter, the study specifically aimed to investigate the preclinical efficacy of NTP42:KVA4 in the PAB model of RV pressure overload to assess the potential of NTP42 to impart direct cardiac benefits.We previously reported that l models , 28. In NTP42:KVA4 was first evaluated in a delayed interventional MCT-PAH model in rodents, where disease was allowed to develop for 7 days prior to initiating treatment, where efficacy was also compared with drugs from each of the four clinical SOC PAH therapies.Hence, efficacy of NTP42:KVA4 significantly alleviated the MCT-induced increases in RVSP and Fulton’s Index parameters and alleviated the MCT-induced increase in cardiomyocyte size , and significantly attenuated pulmonary pathologies, including vessel occlusion and muscularization, CD68+ macrophage infiltration, perivascular fibrosis, and edema (MCT led to increased RV systolic pressure (RVSP) and RV hypertrophy, as measured by Fulton’s Index . Quantifyte size . While Non Index . Moreovefibrosis . In linefibrosis  treatmennd edema .NTP42:KVA4, none of these SOCs significantly reduced cardiomyocyte size  or heart rate (HR) per se, animals treated with Riociguat or Selexipag displayed either increased mAP or decreased HR, respectively  or Riociguat  was initiated 2 days after PAB surgery, where Riociguat was chosen as an appropriate comparator compound as, in previous preclinical investigations, it has been shown to prevent deterioration of RV function induced by PAB  and RV dilation as evidenced by increased RV end-diastolic dimension (RVEDD) and RV end-diastolic area (RVEDA) . In addi reduced . Notablynchanged . In addiargement . Comparaargement .P = 0.1998, Detailed pressure-volume (PV) loop analyses showed that PAB animals demonstrated profound RV overload with marked signs of RV dysfunction . In PAB NTP42:KVA4 significantly improved RV function. In line with ECHO data  in diastolic function, as measured by Eed  was significantly increased across all sarcomere lengths in PAB animals, indicative of increased cardiomyocyte stiffness . ConsistP42:KVA4 , PT was  animals  and was  animals , while r4 groups . Similar4 groups .+/Ca2+ exchanger 1 (NCX1) or sequestration into internal cellular stores by sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a)  . AlteratSERCA2a) –40, and SERCA2a) . FurtherSERCA2a) –44. HereSERCA2a) . MoreoveSERCA2a) . SpecifiSERCA2a) , where tSERCA2a) , 44. Tre evident .per se, expression of the TP was examined in RV tissues from experimental PAH and cardiac disease models, as well as in clinical specimens of PAH and dilated cardiomyopathy (DCM).While signaling through the TP is implicated in pathological cardiac conditions, few studies have examined TP expression levels in RVs of subjects with PAH. Thus, as a rationale for the therapeutic potential and utility of TP antagonists NTP42:KVA4 in both MCT-PAH and PAB models led to a non-significant trend toward reduction in TP expression -induced PAH model , and notIn clinical specimens from healthy human donors , TP exprRight heart function is widely viewed as the most important determinant of clinical outcome in PAH and indeed in various other forms of pulmonary hypertension (PH) . InsuffiWhile PAH SOCs demonstrate robust pulmonary vasodilatory enhancing effects and lead to meaningful reductions in PVR, they show limited evidence of directly targeting right heart adaptation and function. In PAH, there is an unmet need to not only alleviate pulmonary pathology and PVR, but to also directly target mechanisms underlying RV dysfunction to enhance patient quality-of-life and improve survival. Current PAH SOCs have variable effects on RV function in both experimental and clinical settings . In prec2/TP pathway in preclinical PAH demonstrated conflicting results  in alleviating pulmonary pathologies in the MCT-PAH model was confirmed to be in line with previous findings using the NTP42 API. Moreover, this MCT-PAH study demonstrated that NTP42:KVA4 attenuates RV hypertrophy and promotes a more beneficial pattern of RV adaptation. Thereafter, we aimed to investigate the potential for NTP42:KVA4 to directly target the compromised RV using the PAB model of right heart pressure overload. With an absence of confounding pulmonary pathologies, the PAB model allows for a direct assessment of therapeutic intervention on RV structure and function  in man than in rat. Consistent with this proposition, in the recent Phase I clinical trial of the IMP NTP42:KVA4 in healthy subjects (NCT04919863), the IC50 of NTP42 for inhibition of U46619-induced aggregation of human platelets ex vivo was confirmed to be 9.9 nM. Extending this translation of preclinical to predicted clinical data for the PAH SOCs, based on their relative IC50 in rats and humans, both Sildenafil    . As an important regulatory-compliant safety parameter required by both the European and US EMA and FDA agencies before proceeding to FIH Phase I clinical trials, the in vivo effect of NTP42:KVA4 on the cardiovascular system was also investigated in conscious telemetered dogs, where no inotropic or chronotropic effects were observed at doses up to 450 mg/kg NTP42:KVA4 PO  , 65 and n, 1 nM)  are expe and man , 68, the and man . With rectively) , 70, it P42:KVA4 , 5. In a:KVA4 PO .2/TP pathway on RV dysfunction, TP antagonism was protective against mild RV pressure overload in a mouse PAB model, where pressures and cardiac output were improved  inhibitor was evaluated in a Phase II clinical trial, but this study was prematurely terminated during enrolment due to the development of acute leg pain in trial participants  D2 (DP1), PGE2 , PGF2α (FP) and PGI2/prostacyclin (IP) receptors, with no agonist activity at the TP itself  in many cell types. Specifically, in cardiomyocytes, basal and peak Ca2+ concentrations as well as width of Ca2+ transients are increased following treatment with the TXA2 mimetic U46619, and prolonged stimulation results in irregular Ca2+ oscillations and a marked increase in cytosolic-free Ca2+ concentrations (3) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP2) cellular stores (3 release leads to rapid mobilization of intracellular Ca2+ from the sarco/endoplasmic reticulum (SR) via activation of ligand-gated IP3 receptors (IP3Rs). In this regard, treatment of isolated cardiomyocytes with U46619 increased intracellular Ca2+ in a dose-dependent manner, where these increases were blocked by the TP antagonist SQ29548 or inhibitors of the IP3 pathway , triggers a larger Ca2+ release from the SR via ryanodine receptors (RyRs), leading to cardiac contraction. A further mechanism through which the TP may lead to increased intracellular Ca2+ is following protein kinase (PK) C activation due to second-messenger DAG liberation from PIP2. It has been shown by multiple groups that TP stimulation in vascular smooth muscle cells leads to dysregulation of resting membrane potential and subsequent activation of LTCCs  transcriptional mechanisms . All patients/participants, or their surrogates, provided written informed consent for the use of these biomaterials for research purposes. Animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of IPS Therapeutique , or by the Ethical Committee of the University of Porto , certified by the Portuguese National Authority for Animal Health.EM, FR, RA, ED, LB, VS, HR, GC, JG, AL-V, J-BM, CB-S, CL, LH, DM, MH, AV, FP, PM-F, and BK: substantial contributions to the conception or experimental design of the work or the acquisition, analysis, and interpretation of data for the work. EM, LH, DM, MH, AV, FP, PM-F, and BK: drafting the manuscript or revising it critically for important intellectual content. All authors have read and approved the final manuscript.


Head and neck squamous cell carcinoma (HNSCC) is a frequently lethal malignancy, and the mortality is considerably high. The tumor microenvironment (TME) has been identified as a critical participation in cancer development, treatment, and prognosis. However, competing endogenous RNA (ceRNA) networks grouping with immune/stromal scores of HNSCC patients need to be further illustrated. Therefore, our study aimed to provide clues for searching promising prognostic markers of TME in HNSCC.  ESTIMATE algorithm was used to calculate immune scores and stromal scores of the enrolled HNSCC patients. Differentially expressed genes (DEGs), lncRNAs (DELs), and miRNAs (DEMs) were identified by comparing the expression difference between high and low immune/stromal scores. Then, a ceRNA network and protein-protein interaction (PPI) network were constructed for selecting hub regulators. In addition, survival analysis was performed to access the association between immune scores, stromal scores, and differentially expressed RNAs in the ceRNA network and the overall survival (OS) of HNSCC patients. Then, the GSE65858 datasets from Gene Expression Omnibus (GEO) database was used for verification. At last, the difference between the clinical characteristics and immune cell infiltration in different expression groups of IL10RA, PRF1, and IL2RA was analyzed.  Survival analysis showed a better OS in the high immune score group, and then we constructed a ceRNA network composed of 97 DEGs, 79 DELs and 22 DEMs. Within the ceRNA network, FOXP3, IL10RA, STAT5A, PRF1, IL2RA, miR-148a-3p, miR-3065-3p, and lncRNAs, including CXCR2P1, HNRNPA1P21, CTA-384D8.36, and IGHV1OR15-2, were closely correlated with the OS of HNSCC patients. Especially, using the data from GSE65858, we successfully verified that IL10RA, PRF1, and IL2RA were not only significantly upregulated in patients high immune scores, but also their high expressions were associated with longer survival time. In addition, stratified analysis showed that PRF1 and IL2RA might be involved in the mechanism of tumor progress.  In conclusion, we constructed a ceRNA network related to the TME of HNSCC, which provides candidates for therapeutic intervention and prognosis evaluation. Head and neck cancer is a frequently lethal malignancy, with approximately 800,000 new cases every year , 2. The Currently, the tumor microenvironment (TME), consisting of extracellular matrix, stromal cells, and tumor-infiltrating immune cells, is known to be involved in cancer development, distant metastasis, and immune escape . BidirecIn this study, we firstly divided HNSCC patients into two groups according to the immune/stromal scores with the ESTIMATE algorithm. Differentially expressed genes (DEGs), lncRNAs (DELs), and miRNAs (DEMs) were identified between the high- and low-score groups. Then, Kaplan-Meier survival analysis was performed to explore the relationship between immune/stromal scores and overall survival (OS). In light of the better OS of patients with high immune scores, a ceRNA network was constructed using the DEGs, DELs, and DEMs from the high and low immune score groups. In addition, a PPI network of DEGs was constructed to select hub genes, and survival analysis was performed to evaluate the prognostic roles of these RNAs included in the ceRNA network. Furthermore, the different expression and prognostic value of the survival-related RNAs were verified using the GSE65858 dataset from Gene Expression Omnibus (GEO) database. Finally, analysis of the clinical relevance and immune cell infiltration for IL10RA, PRF1, and IL2RA were conducted.https://tcga-data.nci.nih.gov/tcga/). Patients with other malignant tumors were excluded from our study, and samples that possessed the mRNA, miRNA, and lncRNA expression data simultaneously were included. One HNSCC cohort of GEO database (GSE65858) with 270 HNSCC patients was used for validation.The RNA-sequencing (FPKM) and clinical characteristics of 468 HNSCC patients were obtained from the TCGA Database  . AccordiP < 0.05, false discovery rate (FDR) < 0.05, and log2|fold change (FC)| > 1.5. When determining the DELs, P < 0.05, FDR < 0.05, and log2|FC| > 1.2 were used as cutoff values because there were so few candidate lncRNAs. Furthermore, the heatmap packages were applied to generate the heatmaps of DEGs, DELs, and DEMs.The DEGs, DELs, and DEMs between the two groups were determined with the limma package of R. The DEGs and DEMs were selected with P values less than 0.05 were considered significantly enriched.Gene ontology (GO) categories by molecular function (MF) and cellular component (CC) and biological process (BP), as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs were conducted by using ggplot2, enrichplot, and clusterProfiler package of R. P value for further analysis. MiRanda, TargetScan, and miRWalk were used to predict miRNA-mRNA interactions, and miRanda and PITA were used to predict miRNA-lncRNA interactions. Then, the intersection was taken between the target mRNAs/lncRNAs and the previously identified DEGs/DELs. Furthermore, DEMs that negatively regulated the expression of DEL and DEGs were retained to construct the ceRNA network and visualized via Cytoscape v3.8.0.Considering the prognostic relevance of immune/stromal scores in HNSCC patients, we selected the groups with a better P < 0.05 was recognized as a statistically significant difference.By using the STRING database, a PPI network of DEGs included in the ceRNA network was constructed and then visualized with Cytoscape. And Kaplan-Meier analysis was performed to investigate the relationship between the expression of DEGs, DELs, and DEMs in the ceRNA network and OS of HNSCC patients. https://icbi.i-med.ac.at/software/quantiseq/doc/) was employed to access difference in immune cells infiltration.Based on clinical characteristics , HNSCC patients were stratified into distinct subgroups. A Chi-square test was performed to determine the difference in clinical characteristics between different expression groups of IL10RA, PRF1, and IL2RA. Besides, QUANTISEQ   activity” in MF, “apical part of cell” in CC, and “cellular ketone metabolic process” in BP. The enriched KEGG pathways of those upregulated DEGs were mainly involved in “allograft rejection” and “viral protein interaction with cytokine and cytokine receptor,” and the main KEGG term enriched by the downregulated DEGs was “metabolic pathways” Figures .In addition, the top GO terms of the upregulated DEGs in the stromal score groups included “extracellular matrix structural constituent,” “collagen-containing extracellular matrix,” and “external encapsulating structure organization” in MF, CC, and BP, respectively. For the downregulated DEGs, the top GO terms included “enzyme inhibitor activity” in MF, “cornified envelope” in CC, and “epidermis development” in BP. The KEGG pathways associated with the upregulated DEGs mainly involved pathways related to “cornification,” and the KEGG terms associated with the downregulated DEGs included “estrogen signaling pathway” Figures .P = 0.0639 and P = 0.8799), while it did not mean that the DEGs, DELs, and DEMs between the two groups had no prognostic values. Thus, we chose DEGs, DELs, and DEMs of immune score groups, which had a relatively better survival for the construction of ceRNA network  were prominent for having many connections with other genes . FurtherTo verify whether the eleven prognostic biomarkers above were differentially expressed and of prognostic significance in another independent HNSCC cohort, we downloaded GSE65858 from the GEO database for validation. However, for lacking sufficient RNA sequencing data of miRNAs and lncRNAs, we only successfully performed the differential expression and survival analysis of the five DEGs  between high and low immune score groups. As shown in The distribution of clinical variables with corresponding expression subgroups was visualized Figures . The resIn addition, the immune infiltrating analysis showed that patients with high IL10RA, PRF1, and IL2RA expression exhibited high immune cells infiltration, such as CD8+ T cell, cytotoxic lymphocytes, NK cell, B cell, monocyte, and myeloid dendritic cell Figures . This wa6A methylation, alternative splicing, increased tumor mutation burden, and prognosis [In this study, 468 HNSCC patients were divided into two groups based on immune/stromal scores using the ESTIMATE algorithm. ESTIMATE, a method that infers the fraction of immune and stromal cells in tumor samples based on gene expression , enablesrognosis –25. TherP = 0.0296; The ceRNA network composed of 97 DEGs, 79 DELs, and 22 DEMs were constructed, and hsa-miR-149-5p, has-miR-17-5p, hsa-miR-3065-3p, hsa-miR-767-5p, and hsa-miR-96-5p were the top 5 nodes. Then, the PPI network identified 12 hub genes , which might play important roles in the network. Among these genes, only PTGS2 was downregulated in the high immune score patients. KEGG analysis showed that PTGS2 was significantly involved in the terms “arachidonic acid metabolism,” “metabolic pathway,” and “chemical carcinogenesis.” PTGS2, also known as COX-2, is an enzyme critical for PGE2 that is associated with the enhancement of cancer cell survival, growth, migration, and invasion , 27. PTGOf the upregulated hub genes in the PPI network, increased levels of IL10RA, PRF1, and IL2RA were significantly associated with longer survival time of HNSCC patients in both TCGA and GEO database. GO and KEGG analyses showed that IL2RA was significantly associated with “cytokine-cytokine receptor interaction” pathway. IL-2 interacts with IL2RA, which stimulates Tregs to express the transcription factors STAT5 and Foxp3, which play an essential role in Treg development and homeostasis , 31. TreInterestingly, in the ceRNA network, miR-3065-3p and the lncRNAs CXCR2P1, HNRNPA1P21, CTA-384D8.36, and IGHV1OR15-2 were significantly correlated with OS in HNSCC patients in the TCGA database; the relevant interactions within the network include the following: miR-3065-3p/IL2RA; CXCR2P1/miR-210-3p/FOXP3; HNRNPA1P21/miR-767-5p/IL10RA; CTA-384D8.36/miR-149-5p/STAT5A; CTA-384D8.36/miR-149-5p/PRF1; and IGHV1OR15-2/miR-744-3p/IL2RA. MiR-3065-3p was downregulated in high immune score patients, and patients with low miR-3065-3p expression had improved overall survival. The overexpression of the lncRNAs CXCR2P1, HNRNPA1P21, CTA-384D8.36, and IGHV1OR15-2 were correlated with longer overall survival for HNSCC patients. Among these, CXCR2P1 has been speculated to be related to immune checkpoints PD-1, PD-L1, and CTLA4, which are crucial for successful cancer immunotherapy that contribute to the immune response , 41, butIn conclusion, we estimated the level of infiltrating stromal and immune cells in the TME of HNSCC patients and constructed a ceRNA network, in which FOXP3, IL10RA, STAT5A, PRF1, IL2RA, miR-148a-3p, miR-3065-3p, CXCR2P1, HNRNPA1P21, CTA-384D8.36, and IGHV1OR15-2 were significantly correlated with the OS of HNSCC patients. Besides, the expression and survival roles of IL10RA, PRF1, and IL2RA were verified in another GEO cohort. This might provide novel targets in the TME of HNSCC and contribute to therapeutic intervention and prognosis evaluation.


Diabetes is a metabolic disease that raises the risk of microvascular and neurological disorders. Insensitivity to insulin is a characteristic of type II diabetes, which accounts for 85-90 percent of all diabetic patients. The fundamental molecular factor of insulin resistance may be impaired cell signal transduction mediated by the insulin receptor (IR). Several cell-signaling proteins, including IR, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase (PI3K), have been recognized as being important in the impaired insulin signaling pathway since they are associated with a large number of proteins that are strictly regulated and interact with other signaling pathways. Many studies have found a correlation between IR alternative splicing, IRS gene polymorphism, the complicated regulatory function of IRS serine/threonine phosphorylation, and the negative regulatory role of p85 in insulin resistance and diabetes mellitus. This review brings up-to-date knowledge of the roles of signaling proteins in insulin resistance in order to aid in the discovery of prospective targets for insulin resistance treatment. Diabetes is a complex metabolic disorder associated with increased incidence of cardiovascular and neurological complications. The global prevalence of diabetes has increased rapidly from 4.7% 1980) to 8.7% 2014) . IR. IRINSR;nd liver . There hnd liver . Alternae levels . The abnlinaemia , 20. As nd SFRS7 . HNRNPA1nclusion . Increasnclusion .PTP1Bgene knockout can increase insulin sensitivity by enhancing IR signaling [The level of tyrosine phosphorylation of IR is crucial for controlling insulin signaling, while activation of IR can be inhibited by various proteins for posttranslational modifications, such as tyrosine phosphatase, the Grb protein family (growth-factor-receptor bound protein), SOCS , and PC-1 or ENPP1 . PTP1B (protein tyrosine phosphatase 1B) is the most studied among the tyrosine phosphatases, which interacts with IR and dephosphorylate key tyrosine residues to limit its activity. ignaling . Other rignaling , 26. Grbignaling , 28.SOCS expression has been found to be upregulated in insulin resistance, implying that it plays a role in the feedback control of insulin signaling and the development of diabetes . In the PC-1 expression was found to be higher in T2D patients' muscle, fibroblasts, and adipose tissues, while overexpression of PC-1 in established cell lines inhibited IR autophosphorylation and resulted in insulin resistance , 33. MorThe 121st amino acid of PC-1 is critical for its interaction with IR, and a functional missense nucleotide polymorphism resulting in a lysine to glutamine amino acid change has a stronger interaction with IR and is more effective in lowering IR autophosphorylation . AlthougIRS is a crucial mediator of insulin action and serves as a major site for both positive and negative control of insulin signaling transduction. IRS is made up of six members, from IRS1 to IRS6, all of which have relatively similar gene sequences and three-dimensional structures.The pleckstrin-homology (PH) domain, the adjacent phosphotyrosine-binding (PTB) domain, and the C-terminal domain of them are all extremely similar , 52. TheIRS1 gene knockout causes cell differentiation abnormalities in preadipocytes, whereas IRS2 gene knockout has no effect on cell differentiation but causes nonresponsiveness to insulin-stimulated glucose transport [IRS1 gene knockout mice exhibit insulin deficiency in muscle tissue, whereas IRS2 gene knockout animals have insulin deficiency mostly in the liver and generate growth abnormalities in a few tissues, including neurons and pancreatic cells [As a key node of insulin signaling pathways, the loss of each isoform of IRS leads to varied physiologic results. The distribution and function of the IRS isoforms are summarized in ransport . IRS1 geic cells , 56. IRSThe downregulation of the IRS1 gene causes the expression of genes involved in gluconeogenesis to increase, whereas the downregulation of the IRS2 gene causes the expression of genes involved in abiogenesis to increase . IRS1 coThe prevalent polymorphism of IRS1 is a glycine to arginine substitution in codon 972 (Gly972Arg), which is located between two potential tyrosine phosphorylation sites involved in binding with p85. This polymorphism has been linked to the development of T2D in obese Caucasian children , EgyptiaIRS1 Gly972Arg GDM has been observed. The Gly1057Asp variation of the IRS2 gene has been widely documented, and it is thought to be linked to insulin resistance, T2D, and obesity. Gly1057Asp inside IRS2 has been linked to greater insulin resistance and T2D in obese Caucasian youngsters, Kurdish ethnics, Iranians, and Asian Indians [Gestational diabetes mellitus (GDM), which affects 2-22 percent of all pregnancies, has a genetic background that is similar toT2D, including decreased insulin production and insulin resistance. As a result, similar genetic variations linked to T2D could be applied to predict the risk of GDM. In Saudi , Egyptia Indians , 76–78,  Indians . However Indians , while n Indians , implyinSix novel IRS4 SNPs have been reported to be linked with BMI in schizophrenic patients , while aα (TNF-α) [There are more than 70 potential serine/threonine phosphorylation sites in IRS protein, in addition to tyrosine phosphorylation sites, which can be induced by a variety of factors, including tumor necrosis factor- (TNF-α) , 91, c-J (TNF-α) , protein (TNF-α) , glycoge (TNF-α) , SOCS-3  (TNF-α) , and mit (TNF-α) . Serine  (TNF-α) , 98. Inc (TNF-α) –101. IRS (TNF-α) , 103.hSer1223/m1214 (human and rat IRS1) interferes with IRS1's interaction with SHP-2, a negative modulator of IRS1 tyrosine phosphorylation, most likely due to steric hindrance, implying a positive regulatory function [In fact, serine phosphorylation of IRS1 has been shown to have a positive regulatory role, promoting tyrosine phosphorylation while inhibiting serine/threonine phosphorylation at other sites. For example, phosphorylation of IRS1 at Ser629 causes phosphorylation at Ser636 to decrease, adversely regulating IRS1 and increasing insulin activity . While afunction . Basal pfunction , 107.Serine/threonine phosphorylation at specific sites on IRS provides negative charge, altering protein interactions and reducing downstream cell signaling. By inducing conformational changes in IRS, phosphorylation at certain sites promotes protein interaction and enhances downstream signaling. Furthermore, depending on the time course of insulin action, the interaction status, and the implications of temporal variations in IRS phosphorylation, phosphorylation at specific serine/threonine residues may have varied effects . PhosphoPI3K inhibitors inhibit practically all of insulin's metabolic activities, including glucose transport, glycogen synthesis, lipid synthesis, and adipocyte differentiation, demonstrating that PI3K plays an important role in insulin signaling . Class IJNK1 gene knockout mice fed a high fat diet (HFD), suggesting that it may have a protective role in the occurrence of impaired glucose tolerance and insulin resistance [Insulin sensitivity was increased by a mutation in p85, suggesting that p85 plays a negative function in insulin signal transduction , 114. Thsistance .PI3KR1, the coding gene of the PI3K p85 subunit, may be associated with GDM [PI3KR1 and GDM, although the sample size was small, with only 38 pregnant women and 240 controls included [PI3KR1 was involved in abnormal glucose metabolism [Gene polymorphism of with GDM , T2D [12with GDM , insulinincluded . Anothertabolism . A similtabolism .Tyrosine residues of IR and IRS are dephosphorylated by PTP1B, which is one of the most well-studied tyrosine phosphatases, acting as a crucial negative regulator of the insulin signaling pathways . PTP1B mSince the prevalence of diabetes has increased considerably around the world, understanding the molecular basis of insulin resistance is both theoretically and practically important. More research into the molecular processes of insulin signal transduction, particularly aberrant IR splicing, IRS gene polymorphism, regulatory effects of IRS phosphorylation, and the regulatory function of p85 on insulin activities, is valuable and still needed.


Nuclear factor erythroid 2-related factor 2 (Nrf2) may harbor endogenous neuroprotective role. We strived to ascertain the prognostic significance of serum Nrf2 in severe traumatic brain injury (sTBI).This prospective cohort study included 105 controls and 105 sTBI patients, whose serum Nrf2 levels were quantified. Its relations to traumatic severity and 180-day overall survival, mortality, and poor prognosis  were discerned using multivariate analysis.P < 0.001), as compared to controls. Serum Nrf2 levels were independently correlative to Rotterdam computed tomography (CT) scores  and Glasgow Coma Scale (GCS) scores . Serum Nrf2 levels were significantly higher in non-survivors than in survivors  and in poor prognosis patients than in good prognosis patients . Patients with serum Nrf2 levels > median value (10.9 ng/ml) had markedly shorter 180-day overall survival time than the other remainders . Serum Nrf2 levels were independently predictive of 180-day mortality , overall survival , and poor prognosis . Serum Nrf2 levels distinguished the risks of 180-day mortality and poor prognosis with areas under receiver operating characteristic curve (AUCs) at 0.768 and 0.793, respectively. Serum Nrf2 levels > 10.3 ng/ml and 10.8 ng/ml discriminated patients at risk of 180-day mortality and poor prognosis with the maximum Youden indices of 0.404 and 0.455, respectively. Serum Nrf2 levels combined with GCS scores and Rotterdam CT scores for death prediction  had significantly higher AUC than GCS scores (P = 0.028), Rotterdam CT scores (P = 0.007), or serum Nrf2 levels (P = 0.006) alone, and the combination for poor outcome prediction  displayed significantly higher AUC than GCS scores (P = 0.035), Rotterdam CT scores (P = 0.006), or serum Nrf2 levels (P = 0.008) alone.There was a substantial enhancement of serum Nrf1 levels of patients (median, 10.9 vs. 3.3 ng/ml; Increased serum Nrf2 levels are tightly associated with traumatic severity and prognosis, supporting the considerable prognostic role of serum Nrf2 in sTBI. Severe traumatic brain injury (sTBI), one of the most severe traumas, is characterized by high morbidity and mortality . Its patvia regulating the expression of genes coding antioxidant, anti-inflammatory, and detoxifying proteins (Nuclear factor erythroid 2-related factor 2 (Nrf2) is a kind of recently discovered nuclear factor for antioxidant response element (ARE)-regulated genes, which is also a key regulator of endogenous inducible defense systems in the body . Acute oproteins –13. ThusPatients diagnosed with sTBI (GCS scores < 9) were enrolled into this prospective observational study, which was performed at our hospital from February 2017 to May 2021. The inclusion criteria were as follows: (1) age ≥ 18 years, (2) injury severity score < 9 in non-cranial aspects, (3) blunt trauma, and (4) hospital admission within 12 h after trauma. The exclusion criteria were as follows: (1) previous neurological diseases, such as stroke, intracranial tumors, cerebral aneurysms, vascular malformations, and severe head trauma, and (2) other specific conditions or diseases, e.g., severe infections within recent a month, autoimmune diseases, pregnancies, severe hepatic, renal or cardiac diseases, and malignancies. In addition, controls were a group of healthy non-patient volunteers recruited from May 2020 to May 2021 at our hospital, who were free of some chronic diseases, such as hypertension, diabetes mellitus, and coronary heart disease, and were normal in routine tests, e.g., blood leucocyte count, blood hemoglobin levels, and blood platelet count. This study followed the ethical guidelines of the Declaration of Helsinki, and the approval for the protocol of this study was acquired from the institutional review committee at our hospital. All the relatives of patients and controls themselves gave informed written consent prior to inclusion in the study.We gathered demographic information, past medical history, cigarette smoking history, and alcohol drinking history. Baseline GCS scores were recorded to reflect clinical severity of head trauma. The radiological parameters were determined by research personnel blinded to clinical data using the first available head CT scan. Positive radiological appearances included abnormal cisterns, midline shift, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, intraventricular hemorrhage, intracerebral hematoma, brain contusion, and pneumocephalus. Rotterdam CT scores were calculated to assess radiological severity of head trauma. A functional outcome was evaluated using eight-grade extended Glasgow Outcome Scale (GOSE) at 180 days following trauma. GOSE was dichotomized into poor outcome (1–4 points) and good outcome (4–6 points) .Blood samples were collected from all study participants, including patients and controls. The blood was centrifuged, and then, serum samples were immediately preserved at −80°C until required for further analysis. The same technician, who was blinded to clinical data, performed measurement of serum Nrf2 levels using a commercially available DNA-binding enzyme-linked immunosorbent assay (ELISA)  as per the manufacturer's protocol. In brief, the samples were incubated for 1 h in a pre-coated 96-well-plate, followed by primary antibody incubation for 1 h. After incubation, all the wells were washed and incubated with horseradish peroxidase-conjugated antibody for 1 h, followed by trimethylbenzene substrate for 15 min in the dark. The reaction was stopped by the addition of stop solution, and the plate was read at 450 nm on a microplate reader . Each sample was in duplicate determined, and the results were averaged for further analysis.t-test for normally distributed data and the Mann–Whitney test for non-normally distributed data. Using the Kruskal–Wallis H test, serum Nrf2 levels were compared among multiple groups divided by GCS scores and Rotterdam CT scores. Serum Nrf2 levels were dichotomized based on its median value, and 180-day overall survival time was compared using the log-rank test. Under ROC curve, the prognostic predictive capability was assessed, and area under ROC curve (AUC) and the corresponding 95% confidence interval (CI) were yielded. 180-day death and poor prognosis were regarded as two dependent variables. The two multivariate binary logistic regression models were established, and significant variables in univariate analysis were forced to identify independent predictors of 180-death and poor prognosis. Associations were reported as odds ratio (ORs) and 95% CIs. Univariate Cox's proportional hazard analysis was performed to screen variables, which were substantially related to 180-day overall survival, and afterward, multivariate Cox's proportional hazard analysis was performed to discern independent predictors. Hazard ratio (HRs) and 95% CIs were reported for showing associations. Bivariate correlations were analyzed using the Spearman test, and thereafter, multivariate linear regression model was configured to ascertain variables, which were independently correlated with serum Nrf2 levels. For all tests, the two-sided P < 0.05 were considered statistically significant.Receiver operating characteristic (ROC) curve analysis was carried out using MedCalc statistical software version 17.4 , and other data analyses were performed utilizing the SPSS 20.0 statistical package . Qualitative variables were shown as frequencies (proportions). Categorical data were compared between two groups using Pearson's Chi-square test or Fisher's exact test as appropriate. Quantitative data, which were tested for normal distribution, were presented as means  if normally distributed or medians (percentages 25th−75th) if non-normally distributed. Statistical methods of intergroup comparisons of quantitative data included the independent P > 0.05).In 9/l .Among this cohort of patients, 32 were cigarette smokers, 33 consumed alcohol, 17 suffered from hypertension, 12 presented with diabetes mellitus, and 17 were inflicted with hyperlipidemia. Hospital admission time ranged from 0.5 to 12.0 h after trauma . Blood-sampling time ranged from 1.0 to 14.5 h  following trauma. Traumas of 52 cases were caused by automobile/motorcycle; those of 44 cases, by fall/jump; and those of 9 cases, by others. GCS score ranged from 3 to 8 . Rotterdam CT score ranged from 3 to 6 . The mean systolic and diastolic arterial pressures were 125.7 mmHg  and 74.7 mmHg , respectively. Blood glucose levels ranged from 5.2 to 20.7 mmol/l . Blood leucocyte count ranged from 3.2 to 16.1 × 10P < 0.001). Just as listed in P < 0.001) and Rotterdam CT scores . Alternatively, 14, 25, 29, 15, 10, and 12 patients had GCS scores 3, 4, 5, 6, 7, and 8, respectively. Moreover, 11, 50, 25, and 19 patients had Rotterdam CT scores 3, 4, 5, and 6, respectively. In P < 0.001). Similarly, there were markedly highest serum Nrf2 levels in patients with GCS score 3, followed by GCS scores 4, 5, 6, and 7, and pronouncedly lowest in those with GCS score 8  and Rotterdam CT scores . Serum Nrf2 levels combined with GCS scores and Rotterdam CT scores  had significantly higher AUC than GCS scores (P = 0.028), Rotterdam CT scores (P = 0.007), or serum Nrf2 levels (P = 0.006) alone.In this group of sTBI patients, 180-day mortality was 24.8% (26/105). Non-survivors were significantly older than survivors, GCS scores were substantially lower, and Rotterdam CT scores were markedly higher in the dead than in the alive, and serum Nrf2 levels and blood glucose levels were profoundly raised in non-surviving patients, as compared to surviving ones . Just as displayed in P < 0.05). Furthermore, the afore-mentioned significant pertinent variables were incorporated in multivariate Cox's proportional hazard model, and sequentially, it was confirmed that GCS score, Rotterdam CT score, and serum Nrf2 levels were independently associated with 180-day overall survival after head trauma  during 180-day follow-up after head trauma. In accordance with the median value of serum Nrf2 levels , all patients were dichotomized. In P < 0.001), and patients with GOSE 1 had significantly highest serum Nrf2 levels and those with GOSE 8 displayed substantially lowest serum Nrf2 levels (P < 0.001). In aggregate, 49 patients had the development of a poor outcome (46.7%) at 180 days after trauma. In P < 0.05, P = 0.299) and Rotterdam CT scores . Serum Nrf2 levels combined with GCS scores and Rotterdam CT scores  had significantly higher AUC than GCS scores (P = 0.035), Rotterdam CT scores (P = 0.006), or serum Nrf2 levels (P = 0.008) alone.At post-injury 180 days, GOSE scores of patients ranged from 1 to 8 , and a total of 26, 9, 6, 8, 23, 16, 10, and 7 patients showed GOSE scores 1, 2, 3, 4, 5, 6, 7, and 8, respectively. In To the best of our knowledge, this study, for the first time, measured Nrf2 levels in the peripheral blood of patients with acute brain injury and further discerned the relationship between serum Nrf2 levels and illness severity plus functional outcome after sTBI. The main findings of our study are that (1) sTBI patients had significantly higher serum Nrf2 levels than healthy controls; (2) serum Nrf2 levels were in independent correlation with GCS scores and Rotterdam CT scores; (3) serum Nrf2 was an independent predictor of 180-day death, overall survival, and poor outcome; and (4) serum Nrf2 levels were in possession of significant prognostic predictive ability. Assumably, serum Nrf2 levels may be highly correlated with trauma severity and be tightly associated with a long-term functional outcome after sTBI, indicating that serum Nrf2 should represent a potential prognostic biochemical marker of sTBI.via reducing oxidative damage.Oxidative stress harbors an important role in some pathophysiologic processes that emerge following acute brain injury, including TBI . As a faNuclear factor erythroid 2-related factor 2 was greatly expressed in mouse brain after transient middle cerebral artery occlusion  and in bIn the current study, in order to assess relationship between serum Nrf2 levels and trauma severity in addition to long-term prognosis, GCS and Rotterdam CT classification were selected as the two indicators of trauma severity, and 180-day GOSE scores and death were regarded as prognostic parameters. As regards statistical methods, multiple multivariate models were configured. Our data showed that serum Nrf2 levels were in independent correlation with trauma severity reflected by GCS scores and Rotterdam CT scores and were independently predictive of death, overall survival, and poor prognosis at 180 days after head trauma. Meanwhile, its prognostic predictive capability was confirmed under ROC curve. Intriguingly, serum Nrf2 levels combined with GCS scores and Rotterdam CT scores for death or poor outcome prediction displayed significantly higher AUC than GCS scores, Rotterdam CT scores, or serum Nrf2 levels alone. In summary, serum Nrf2 may serve as a promising prognostic biomarker.in vitro (There are several limitations in this study. First, the blood of sTBI patients was collected at a median value of 6.3 h after trauma. No treatments for glycemic control had been done, and serum Nrf2 levels were not significantly correlated with blood glucose levels in this study. However, Nrf2 signaling pathways regulation was associated with the hypoglycemic effect of specific agents and hypoglycemia-induced blood–brain barrier endothelial dysfunction in vitro . Thus, iin vitro  and therin vitro , 25. Lenin vitro , 27. Henin vitro , 29 and in vitro  and is din vitro , 29. So,in vitro , 29 may To the best of my knowledge, our study, for the first time, showed that increased serum Nrf2 levels of sTBI patients are independently correlated with GCS scores and Rotterdam CT scores and were independently predictive of 180-day death, overall survival, and poor outcome. Moreover, serum Nrf2 levels combined with GCS scores and Rotterdam CT scores for death or poor outcome prediction had significantly higher discriminatory efficiency than any one of them. Thus, it is hypothesized that serum Nrf2 may represent a potential prognostic biomarker of sTBI.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Quzhou Affiliated Hospital of Wenzhou Medical University. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TP53 is associated with a worse prognosis due to the occurrence of metastases, increased chemoresistance, and tumor growth. Currently, numerous compounds capable of p53 reactivation or the destabilization of mutant p53 are being investigated. Several of them, APR-246, COTI-2, SAHA, and PEITC, were approved for clinical trials. This review focuses on these novel therapeutic opportunities, their mechanisms of action, and their significance for potential medical application.Continuous development of personalized treatments is undoubtedly beneficial for oncogenic patients’ comfort and survival rate. Mutant  TP53 gene and known as “the guardian of the genome” [TP53 is the most frequently mutated gene in cancers. It has been recognized that 50% of cancer patients acquire certain types of TP53 gene alterations [TP53 mutations are labeled as loss of function (LOF) or gain of function (GOF). The presence of GOF TP53 mutants increases the malignancy of tumors in various ways. Occurring metastases, greater chemoresistance, invasiveness, and shorter survival are typical traits of GOF. These mutations account for 30% of all missense mutations in the TP53 gene. Widespread screening of patients allowed for the recognition of the hotspots R175, G245, R248, R249, R273, and R282, which are present in the DNA-binding domain (DBD) or near this interface of p53 [The tumor suppressor p53, encoded by the  genome” , perform genome” . The p53 genome” ,5,6,7. R genome” . TP53 iserations ,10. The e of p53 .Kip1  and Pai1 (phosphoribosylanthranilate isomerase 1), while TIGAR (TP53 induced glycolysis regulatory phosphatase) and glutaminase 2 (encoded by GLS2) are responsible for the arrangement of metabolic changes [HSP70 or HSP90, which stabilize MUTp53, therefore allowing its accumulation in the cell [Non-altered p53 acts as a transcription regulator in charge of the cellular responses to stress factors, hypoxia, nutritional stress, differentiation signals, and DNA damage. Under stress conditions, an affected cell can respond in numerous ways. p53 induces cell cycle arrest, apoptosis, or senescence. Each of those processes is regulated by p53 and its target genes. Cell cycle arrest is induced by p21 and p57; apoptosis is activated via Puma, also known as BCL2 binding component 3, Bax (BCL2-associated X), and Noxa (phorbol-12-myristate-13-acetate-induced protein 1); and senescence is induced via p27 changes ,16. Addi changes . The neg changes ,19 Howev changes . It is a changes . Severalthe cell . Howeverthe cell .Missense mutations occurring in the six hot spots consist of eight mutants, which account for nearly 30% of all missense mutations. They consist of R175H, G245S, R248Q, R248W, R249S, R273C, R273H, and R282W. The gain of function characteristics are designated to all of them, although the mechanisms behind their novel functionalities are different. This group can be further divided, based on the dysfunctionality that is present in the final protein. Mutation in the contact mutants, R248Q, R248W, R273H, and R273C, occurs in their DNA-binding domain, directly affecting their ability to control the transcription of targeted genes. Conformational mutants such as R175H, G245S, R249S, and R282H, on the other hand, are unable to fold properly, leading to the loss of the zinc coordinates and, thus, general DNA-binding activity ,30. ContSLC7A11  gene, and its overexpression is observed in various types of cancer cells, especially in cancer stem cells (CSC). xCT alters metabolic pathways via participation in glutathione biosynthesis and, in this way, protects cancer cells from oxidative stress conditions and ferroptosis [SLC7A11 and sensitize cells to ferroptosis. It was also shown that cells with non-functional p53 are highly resistant to chemotherapeutics and radiotherapy; however, the knockout of the SLC7A11 gene results in the restoration of sensitivity to applied therapy. These observations are related to GSH depletion and, consequently, to the reduced protection from oxidative stress upon xCT inhibition. This suggests that, in the resistance to oxidative stress, the regulation of the xCT-glutathione axis plays an important role, which allows the tumor to survive in unsuitable conditions. Therefore, the inhibition of the xCT-glutathione axis may represent a promising approach to overcoming resistance associated with MUTp53 [TP53 knockdown [R273H and R175H. Interestingly, the mutant D281G did not cause such effects [MDR1 (multidrug resistance gene 1) [CDK1 (cyclin-dependent kinase 1) and CCNB1 (cyclin B1) expression [R248Q mutant’s resistance to cisplatin could be modulated by fibroblast growth factor-inducible 14 (Fn14). High-grade serous ovarian cancer cells became sensitive to cisplatin, due to p53R248Q degradation, which was possible when expression of Fn14 was restored [Cancer cells’ ability to manage oxidative stress is possible due to the presence of the xCT , a functional subunit of the cystine/glutamate antiporter system xc-. xCT is coded by the roptosis . Generalh MUTp53 ,37. Thisnockdown . Etoposi effects . Cisplat effects . The und gene 1) . Each ty gene 1) . The respression , while Rpression . Long nopression . Anotherrestored .Novel strategies in the therapies targeting MUTp53 have been presented over the past decade. These strategies include the elimination of mutant p53 and restoration or reactivation of WTp53, destabilization of MUTp53, or inhibition of the downstream signaling resulting from mutant p53 gain of function and, thus, initiation of synthetic lethality in the cells expressing mutant p53. Numerous compounds targeting MUTp53 have already been discovered, and various brilliant reviews have already described them in a comprehensive manner ,49,50,51PUMA, NOXA, and BAX, which, in consequence, promotes apoptosis [APR-246 was discovered by cell-based screening in 2002 by Bykov and collaborators . This mopoptosis ,57.SLC7A11 by targeting NRF2 (nuclear factor erythroid 2-related factor 2), which prevents the formation of the antioxidant-glutathione (GSH). MQ, additionally, leads to ROS abundance by binding to the thiol groups of GSH [R175H or R273H. The authors revealed that this mechanism disrupts the functioning of the R175H mutant but not of R273H. APR-246 demonstrates higher toxicity, when SLC7A11 is downregulated, but only for the R175H mutant and not for R273H [TP53 mutation status may not be the best predictive factor for APR-246 sensitivity. Recently it was concluded that the SLC7A11 expression is a significantly more precise factor. Additionally, the SLC7A11 genetic regulators, such as ATF4, Mdm2, WTp53, and c-Myc, modulate the cancer resistance to APR-246 [TP53 mutations, such as OVCAR-3R248Q (human ovarian cancer), NSCLCR248W, and R273H , to become sensitive to doxorubicin and cisplatin [TP53 (R248W cell line), it was discovered that introducing WTp53 increased the sensitivity to APR-246 [APR-246 is transformed into a reactive compound methylene quinuclidinone (MQ), which is capable of covalent binding to the thiol groups in MUTp53 and WTp53 . This prs of GSH ,60,61. Tor R273H . This inor R273H ,64,65. D APR-246 . Neverthisplatin . Differe APR-246 .APR-246 is currently registered for 13 clinical trials in patients suffering from various cancers . Within COTI-2 is a third generation of thiosemicarbazone that targets p53 mutants and restores their native conformation. The molecule was registered for clinical trials and further studied in 2016 by Salim and co-workers via machine learning in silico screening  . COTI-2 TP53 mutations, HT-29 , HCT-15 , OVCAR-3 , K562 , SF-268 , SNB-19 , T47D , MDA-MB-231 , KRAS mutations , PIK3CA mutations , APC mutations , and PTEN mutations , which showed its therapeutic potential by inhibiting the growth of all the above mentioned cell lines. The results of the study conducted by Salim and co-workers offered several promising discoveries. COTI-2 was also compared to approved therapeutics such as cetuximab, erlotinib, cisplatin, and carmustine on various colon cancer and glioblastoma cell lines, in which it was proven to be more effective [TP53 status [The efficacy of this therapeutic agent has been tested on multiple cell lines and mice xenografts. Mostly, it demonstrated antiproliferative activity, greater than the one observed with the use of cetuximab or erlotinib, both of which were already approved for cancer treatments. COTI-2 was tested on human cell lines with different kinds of 3 status . Another3 status .TP53 mutations; however, the recruitment status is currently unknown.There is one registered clinical trial for COTI-2. This is a phase I study of COTI-2, as a monotherapy or in combination with cisplatin . The patSuberoylanilide hydroxamic acid  is a histone deacetylase inhibitor (HDACi). Originally it was used for cutaneous T-cell lymphoma treatment. SAHA was already approved by the FDA; however, more possibilities for its use in cancer therapy are continuously under development. Recently, it was shown to target MUTp53 specifically and induce its degradation . PrimariR280K and DLD1S241F ; however, SAHA-induced cell death was only present in the MDA-MB-231 cells and not in DLD1 [SAHA induces cell senescence, independently of MUTp53 status. The mechanism is still under examination. Some studies suggest that it promotes MUTp53 degradation selectively, in this case in MDA-MB-231WAF1/CIP1 elevation via the inhibition of Mdm2, but only in the LNCaP (human prostate adenocarcinoma) cell line and not in MCF-7, despite elevated levels of p53 and p27Kip1 [TP53 gene (U266A161T and RPMI8226E285K), although it had a greater impact on RPMI226 cells [Additionally, SAHA is capable of inducing apoptosis in cells that lack p53 mutations in a mechanism completely independent from p53. The mechanism consists of p21 p27Kip1 ,82. A di26 cells .R280K, BT-474E285K, and prostate adenocarcinoma PC3p.K139fs*31, where the antiproliferative effect was present due to the increased expression of CDKN1A (cyclin-dependent kinase inhibitor 1A encoding p21), while, at the same time, CCND1 (cyclin D1) and TP53 expression levels decreased [The study conducted by Huang and co-workers suggests a direct reaction between SAHA and p53, in cases where apoptosis was a result of p53 activation via phosphorylation. This research was carried out on an in vitro model, where nasopharyngeal carcinoma cells showed that SAHA activates tumor suppressors such as p53 and Rb1 (retinoblastoma protein), while, at the same time, inactivating AMPK (5′ AMP-activated protein kinase) signaling, which leads to apoptosis . SAHA isSuccessful in a variety of cancers in in vitro research  and well-tolerated by patients diagnosed with cutaneous T-cell lymphoma, SAHA has established its multipurpose use . In the Selected clinical trials of SAHA, whether used alone or in combination with other drugs, cover a variety of cancers, and the range is summarized in β-phenylethyl isothiocyanate (PEITC) is a phytochemical that can be found in cruciferous vegetables. This compound acts as a Zn2+ chelator, which inhibits the misfolding of MUTp53. Its therapeutic properties targeting cancer diseases were proposed by Aggarwal et al., who found this compound through cell-based screening . VariousR175H hotspot mutant and successfully restored its native conformation. In consequence, this mutant was exposed to proteasome-mediated degradation [Studies conducted in the mouse breast cancer xenograft model (the SK-BR-3 xenograft mouse) have shown that PEITC selectively targets p53 mutants. This compound acted preferentially towards the p53radation . Moreoveradation . In vitrradation . PEITC iradation . PEITC aradation ; thereforadation . Generalradation . The mecradation . PEITC iradation . Currentradation .Targeted, personalized therapy still has a long way to go, but, with each study, that goal becomes closer. A key question to ask at this stage, of any cutting-edge therapeutic work, is what benefit it may bring to the patient.E177R variant is considered. However, other mutants have not been studied yet. Researchers point out that when even a partial loss in activity of p53 is introduced, the cancer risk is increased. However, when there is minimal to no activity at all, the phenomena known as p53 addiction [TP53 mutation. Some recent reviews argue that targeting MUTp53 will provide effective treatment in the future. The development of machine learning technology may come with an easier answer to the future of cancer treatment. Recently a study using such technology reported new genes  that may be associated with p53 functions, which seem to be a better fit for the platinum-based therapies for patients than their TP53 status [TP53, should be pursued or not has yet to be unraveled [The results of the in vitro and in vivo studies of new potential drugs often show the benefits of their use. Unfortunately, at the stage of clinical trials, the expected benefits of treatment often turn out to be far from ideal, especially in oncological patients. When cancer occurs, the changes in p53 are most notably related to its antitumor functions. Not only do mutations of p53 appear but also other abnormalities; thus, specific approaches for such a broad group of patients might be almost impossible. Taking into account variables such as interactions with other genes and metabolites and the dominant negative effects, regardless of whether the mutation is germinal or somatic, the application of personalized therapy in the clinic seems almost impossible . Neverthddiction ,101,102 ddiction . The useddiction . Other tddiction  should a3 status . The ongnraveled ,108,109.