Blood-based tests for cancer detection are minimally invasive and could be useful for screening asymptomatic patients and high-risk populations. Since a single molecular biomarker is usually insufficient for an accurate diagnosis, we developed a multi-analyte liquid biopsy-based classification model to distinguish cancer patients from healthy subjects. The combination of cell-free DNA mutations, miRNAs, and cell-free DNA methylation markers improved the model’s performance. Moreover, we demonstrated that the androgen receptor mutation p.H875Y is not only relevant in prostate cancer but had a strong predictive value for colorectal, bladder, and breast cancer. Our results, although preliminary, showed that a single liquid biopsy test could detect multiple cancer types simultaneously.AR) was detected for the first time in bladder, lung, stomach, ovarian, brain, and pancreas cancer, all together in 51.3% of all cancer samples and in none of the healthy controls. A discriminant function model, comprising cfDNA mutations , cfDNA methylation markers  and miRNAs  could further classify healthy and tumor samples with 95.4% accuracy, 97.9% sensitivity, 80% specificity. This multi-analyte liquid biopsy-based test may help improve the simultaneous detection of several cancer types and underlines the importance of combining genetic and epigenetic biomarkers.Liquid biopsy-based tests emerge progressively as an important tool for cancer diagnostics and management. Currently, researchers focus on a single biomarker type and one tumor entity. This study aimed to create a multi-analyte liquid biopsy test for the simultaneous detection of several solid cancers. For this purpose, we analyzed cell-free DNA (cfDNA) mutations and methylation, as well as circulating miRNAs (miRNAs) in plasma samples from 97 patients with cancer  and 15 healthy controls via real-time qPCR. Androgen receptor p.H875Y mutation ( Cancer is mostly a manageable disease as long as it is diagnosed and treated before metastasis has begun. In most cases, higher-grade cancer evolves from lower-grade cancer. Thus, early tumor detection could increase the chances of successful treatment. In this way, carcinomas could be identified at an early stage when they can still be surgically removed and cured ,2. ThereLiquid biopsy is a rapidly developing tool for assessing biomarkers shed from difficult to access tissue in easily sampled bodily fluids, such as urine, blood, saliva, sweat, feces, and tears ,4. Such Cell-free DNA (cfDNA) and circulating miRNA from apoptotic, necrotic, or viable tumor cells are released into the bloodstream. Tumor-derived cfDNA harbors somatic mutations originating from the tumor and comprises tissue-specific DNA methylation patterns; thus, methylation can indicate tumor location ,8. HenceGiven that epigenetic alterations occur early in carcinogenesis, cfDNA methylation markers and miRNAs could be early cancer predictors. Genome-wide miRNA expression profiling by miRNA sequencing led to finding useful biomarkers for the early diagnosis of various cancers . MoreoveNevertheless, the heterogeneous phenotype of many diseases leads to variability in biomarker expression across individuals. Usually, a single molecular biomarker is not sufficient for an accurate diagnosis of cancer ,15,16. WThese methods’ huge amount of data needs automated data processing to deliver clinically relevant information. They range from simple approaches, such as logistic regression and support vector machines, to complex artificial neural networks with many hidden layers . Despite the numerous aforementioned advantages of liquid biopsy, this theoretically simpler approach to longitudinal disease monitoring, as opposed to tissue biopsy, is still not routinely applied in cancer management. The low amount of cell-free DNA, the limited sensitivity, and specificity remain a challenge, and the clinical applicability has yet to be established .Eventually, given the potential of this approach, an accurate, simple, and minimally invasive pan-cancer screening test could ensure wide use, especially in a high-risk population. This approach could reach more patients more rapidly since they would be screened for several cancer entities. Ideally, this test would be able to identify the tissue of origin if a malignancy is detected. In this study, our objective was to determine whether miRNAs, cfDNA mutations, and cfDNA methylation can be combined to differ samples from subjects with bladder, brain, breast, colorectal, lung, ovarian, prostate, stomach, and pancreatic cancers and samples from cancer-free subjects, consequently creating a multi-analyte liquid biopsy-based test. To our best knowledge, we are the first to combine mutations, miRNAs, and DNA methylation markers to test several tumor entities.n = 15) of subjects with no evidence of malignancies. Nonetheless, 7 subjects of the control group had one of the following conditions at the sample collection point: pulmonary fibrosis, renal cyst, hemorrhoidal disease, dyspepsia, peritonsillar phlegmon, or endometrial polyp. Cancer types included in the study were liver, lung, pancreas, colorectal cancer, prostate, ovarian, breast, stomach, bladder, and brain cancer. The clinical data of these study groups are summarized in The study was approved by the local ethics committees and carried out according to the current EU directives. All study subjects were recruited by Fidelis Research AD, Bulgaria, and included after written informed consent. Study participants had to be male or female above the age of 25 without a previously treated cancer. Plasma was obtained from a total of 205 patients with stage I, II, or III cancer prior to cancer therapy and a control group  on dry ice and stored temporarily at −20 °C upon arrival.Peripheral venous blood samples were collected prior to surgery and therapy in K-2 EDTA vacutainers. Subsequently, plasma was separated via double centrifugation as described previously . Whole bCell-free DNA (cfDNA) was isolated with MagMAX™ Cell-Free DNA Isolation Kit  using KingFisher™ Duo Prime Magnetic Particle Processor  according to the user guidelines. CfDNA was isolated from 4 mL plasma and eluted with elution solution in a final volume of 80 µL. The purified cfDNA samples were stored at −20 °C until further analysis. DNA was quantified using the dsDNA HS Assay Kit on Qubit 4 Fluorometer  according to the standard kit protocol. Prior quantitative real-time PCR (qPCR) analysis of cfDNA mutations, a blunt end ligation-mediated whole genome amplification (BL-WGA) was carried out as described previously . BrieflyAKT1, APC, AR, BRAF, CTNNB1, EGFR, ERBB2, ESR1, FBXW7, FGFR3, GNAS, HRAS, IDH1, KRAS, MED12, NRAS, PIK3CA, SMAD4, TERT, TP53, and VHL (The BL-WGA cfDNA (10–20 ng pro reaction) was then used for the mutational analysis with TaqMan™ Mutation Detection Assays (ThermoFisher). The array is designed to analyze 75 cancer-specific mutations in 21 genes and consists of a Reference Assay for the amplification of a mutation-free and polymorphism-free region of the target gene in addition to the Mutation Assay. Namely, the genes are  and VHL . ΔCt valThe presence of a mutation in a sample was determined upon an assay-specific cutoff point . The DNASEPT9, MLH1, MGMT, GATA5, GSTP1, SFN, MDR1, VIM, SHOX2, ALKBH3, APC, RASSF1A). Then, 2 µL of each fraction of cfDNA was amplified using a custom-designed primer  in a final volume of 10µL on QuantStudio 3 (ThermoFisher). The methylation level for each region was calculated using the following formula:For methylation analysis, 70 µL of the purified cfDNA was divided into two fractions—one containing methylated cfDNA and one containing unmethylated cfDNA, using MethylMiner™ Methylated DNA Enrichment Kit . This method is based on the binding of methylated DNA to MBD2 protein which is coupled to magnetic beads. The methylated fragments can then be eluted as a single enriched fraction with a high salt concentration solution (NaCl), thereby separating methylated (Me cfDNA) from unmethylated cfDNA (UnME cfDNA). Both fractions were subsequently quantified via real-time qPCR for 12 different cancer-relevant genetic regions  using KingFisher™ Duo Prime Magnetic Particle Processor  according to the user guidelines. Spike-in miRNA C. elegans spike-in control, After RNA purification, miRNA was transcribed into cDNA using the TaqMan™ Advanced miRNA cDNA Synthesis Kit (ThermoFisher). A 1:10 dilution of the cDNA was taken for the analysis of 48 miRNAs  was used to split the samples into subsets. The diagnostic potential of cfDNA mutations, cfDNA methylation markers, and miRNAs was analyzed in discriminant function analyses (DA) with a leave-one-out cross-validation. The performance of these DAs was further estimated by a receiver operating characteristic (ROC) analysis and area under the curve (AUC). Statistical analyses were carried out in IBM® SPSS® Statistics 20 Software. One-way ANOVA for continuous variables and χ2 test and cross-tabulation for categorical variables were used to analyze the characteristics of the subjects. A For the identification of the potential diagnostic markers, the correlation matrix of all variables was calculated . FirstlySubsequently, for each cancer type, the correlations of each cancer type with each biomarker were calculated and sorted by their absolute values . Since mThen, all combinations of these best biomarkers were tested regarding their importance to predict a particular tumor type versus the healthy control group. Variables with redundant information were eliminated based on a covariance matrix to further alleviate the effects of overfitting. Thus, superfluous biomarkers that yield no improvement concerning the classification performance of each cancer type were excluded. In order to do so, a score was defined, where false negatives are discouraged by a factor of two compared to false positives. All computations were carried out in R version 4.1.2.A total of 205 cancer and 15 cancer-free plasma samples were collected. One of the cancer samples was excluded since the patient was diagnosed with stage 4 ovarian cancer after plasma collection. We received only seven plasma samples from patients with liver cancer, making the size of this sample group too small to yield any meaningful results and thereby was excluded from the statistical data analysis. Hence, a total of 212 samples were analyzed for mutations, miRNAs, and DNA methylation. Patient characteristics are summarized in p < 0.001), CRC (p < 0.05), prostate cancer (p < 0.05), and stomach cancer (p < 0.005). The BMI of the subjects with ovarian cancer differed significantly from the BMI of the patients with CRC (p < 0.05) and stomach cancer (p < 0.001).One-way ANOVA test showed significant differences between the age of the healthy control group and bladder cancer (p = 0.439). However, a significant correlation between cfDNA concentration and cancer stage  was observed. Mean cfDNA plasma levels irrespective of cancer type were 0.435 ng/µL for stage I (n = 39), 1.091 ng/µL for stage II (n = 81), and 2.506 ng/µL for stage III (n = 77).Mean plasma cfDNA levels did not significantly differ between cancer patients and healthy controls , EGFR (COSM6224), FGFR3 (COSM718), KRAS (COSM517), PIK3CA (COSM760), TP53 . The six most frequently detected mutations among all samples were in AR , EGFR , TP53 , FGFR3 , TERT , APC  (CTNNB1), COSM6223 (EGFR), COSM22932 (FBXW7), COSM483 (HRAS), COSM499 (HRAS), COSM518 (KRAS), COSM10779 (TP53) were not detected in any sample and were therefore excluded from the analysis. All of the analyzed cfDNA alterations and their frequencies in this study population are listed in Targeted mutation analysis was implemented to investigate 75 alterations such as nucleotides insertions and substitutions , referre45, 21%) . The mutp < 0.001, mean difference = 4.819, std error = 0.463, 95% CI 3.896–5.741). Cancer patients had 6.15, while the control group had 1.33 mutations on average.A significant difference in the mutation burden of cfDNA between healthy subjects and cancer patients was prominent  (AR mutation-positive patients (AR+) for lung, stomach, ovarian, brain, and pancreas cancer were 48.28, 26.09, 20, 11.11, and 8.33, respectively. Interestingly, cancer samples with an AR mutation (n = 101) had an overall higher total mutational burden than samples without an AR mutation , 7.5 and 4.8 mutations on average, respectively (p < 0.001).A total of 101 (51.3%) of all cancer patients had a p.H875Y mutation in the androgen receptor gene (M238555) . This muSEPT9, MLH1, MGMT, GATA5, GSTP1, SFN, MDR1, VIM, SHOX2, ALKBH3, APC, RASSF1A) were analyzed for all 212 samples. The cfDNA methylation levels for MLH1, SFN, MDR1, VIM, and ALKBH3 of all groups are shown in SEPT9, MGMT, GATA5, GSTP1, SHOX2, APC, and RASSF1 between the different study groups were observed. The data for all analyzed genetic regions are provided in a heatmap in The methylation levels (m%) of 12 different cancer-relevant genetic regions  showed significantly lower levels of miRNAs 148a-3p, 148b-3p, 195-5p, 210-3p, 23a-3p, 25-3p when compared to samples without an AR mutation  ; therefore, they were excluded from the analysis. A heatmap was generated for the remaining 43 miRNAs . After c n = 96) L.A search algorithm for the most predictive cfDNA mutations, miRNAs, and cfDNA methylation markers for each cancer type was derived and implemented, and variables with redundant information were eliminated based on a score that discourages false positives. The correlations for each cancer type are shown in 2 14.688, p < 0.001): samples with an AR mutation  and samples without an AR mutation . The AR+ group consisted only of tumor samples since no AR mutation was detected in the control group. However, the AR– group contained the healthy controls (n = 15) and tumor samples (n = 96); therefore, no further classification of these groups was possible based only on AR mutation. In order to separate healthy from tumor samples in the AR– group, several discriminant function analyses with a leave-one-out cross-validation were carried out, including different sets of biomarkers, not including AR mutation. The sets of biomarkers were as follows: discriminant analysis 1 (DA1) incorporated all measured targets; discriminant analysis 2 (DA2) only cfDNA mutations; discriminant analysis 3 (DA3) only cfDNA methylation; discriminant analysis 4 (DA4) only miRNAs; discriminant analysis 5 (DA5) included the biomarkers with highest correlations identified through the correlation matrixes  and consequently classifying the AR– group in cancer patients and healthy subjects . The classification models, based solely on mutations, cfDNA methylation, or miRNAs, showed poor specificity . Combining all the analyzed biomarkers improved the specificity to some extent (DA1 57.3%); however, the sensitivity declined. The large number of biomarkers included in the DA1 model decreases the classifier’s performance since some contain redundant and superficial information. To alleviate the effects of this so-called “curse of dimensionality,” also known as the “Hughes phenomenon” [Here, we describe a model consisting of two steps, sorting samples in two groups—with and without an nomenon” ,33, the Interestingly, our results demonstrate that the combination of three different analytes could improve the performance of a classification model. Each analyte type provides distinct information and adds value to the classification model, highlighting the importance of a multi-analyte-based liquid biopsy test for cancer detection ,34. Although cfDNA concentration has been previously suggested as a biomarker for cancer detection , our resNonetheless, some genomic aberrations were detected among the control group . SomaticOur results suggest that plasma cfDNA could serve rather as a monitor for disease progression since cfDNA concentration correlated with cancer stage regardless of cancer type. However, mutational burden and miRNA expression and cfDNA methylation should be taken into account . As mentioned earlier, one key limitation of this study is the lack of follow-up in addition to the small number of healthy controls. Despite the small size of the control group, it sufficed to observe several statistically significant results since it was matched to reflect the average sample size of each cancer group. Another potential limitation is the clinical utility of this model, as the three different analytes require separate sample processing and analysis. Nevertheless, a blood-based test is a minimally invasive procedure in contrast to a tissue biopsy and is more frequently accepted by patients than other screening procedures such as colonoscopy  or a fecLiquid biopsy markers such as cfDNA mutations, cfDNA methylation, and CTCs are already successfully applied as prognostic and predictive tools for treatment response in several tumor types and monitoring tools for disease progression ,43,44,45In this study, we created a liquid biopsy-based classification model allowing the discrimination between healthy controls and patients with various solid tumors. We demonstrated that combining several analytes improves the performance of the test. Nevertheless, a bigger prospective cohort is required to confirm the clinical utility of this classification model and assess whether a subclassification of the different cancer types is possible.


Multiple endocrine neoplasia 2A (MEN2A) is a rare autosomal-dominant genetic syndrome, frequently misdiagnosed or neglected clinically, resulting in delayed therapy to patients.A 47-year-old Chinese male patient underwent laparoscopic right adrenal tumorectomy, and postoperative pathology confirmed the tumor as pheochromocytoma (PHEO). He was readmitted to the department of endocrinology and metabolism due to constant increase in carcinoembryonic antigen (CEA) at 5 months after the operation.The patient was confirmed with medullary thyroid carcinoma (MTC), multiple neck lymph node metastasis, and pituitary microadenoma. The p.Cys611Tyr  mutation was detected. Therefore, he was diagnosed with MEN2A.He underwent total thyroidectomy. The gene-sequencing analysis of his family was conducted, and the C611Y mutation was detected in his daughter.The level of carcinoembryonic antigen decreased significantly after thyroidectomy in this patient. Long-term follow-up management was conducted. Elevated serum calcitonin and bilateral thyroid nodules were found in his 13-year-old daughter. Thus, MEN2A was highly suspected and she was suggested to undergo total thyroidectomy.Patients with MEN2A should be screened regularly and managed by a multidisciplinary team. Four clinical variants of MEN2A have been recorded in the Revised American Thyroid Association Guidelines for the Management of Medullary Thyroid Carcinoma issued by the American Thyroid Association, including MEN2A, MEN2A with pruritic cutaneous lichen amyloidosis, MEN2A with Hirschsprung disease (HSCR), and familial thyroid medullary carcinoma. The main clinical manifestations of MEN2A are medullary thyroid carcinoma , pheochromocytoma , and primary hyperparathyroidism , with an incidence of about 1/30,000. Here, we report a case of MEN2A and conduct a literature review.Multiple endocrine neoplasia (MEN) is characterized by the occurrence of tumors involving ≥2 endocrine glands in a single patient. Up to now, 3 major types of MEN have been recognized and referred to as type 1 (MEN1), type 2A (MEN2A), and type 2B (MEN2B). Multiple endocrine neoplasia 2A (MEN2A) is a rare autosomal-dominant genetic syndrome,2The patient was a 47-year-old Chinese man, who underwent laparoscopic right adrenal tumorectomy in December 2018. Postoperative pathology confirmed the tumor as the PHEO. Five months after the operation, he was reexamined at the outpatient department and subsequently admitted to the department of endocrinology and metabolism, due to constant increase in carcinoembryonic antigen (CEA). The results of clinical biochemical tests at admission are shown in Table RET gene sequencing is the gold standard for the diagnosis of MEN, thus the gene-sequencing analyses of this patient and his family were conducted . The results showed that no RET gene mutation was detected in his brother or his son, but the p.Cys611Tyr  mutation was detected in his daughter and himself. Since it is one of the pathogenic mutations of MEN2A, we conducted the preliminary screening of his 13-year-old daughter and found bilateral thyroid nodules and elevation in the serum calcitonin level. Thus, she was highly suspected of MEN2A and suggested undergoing total thyroidectomy. Long-term follow-up management was conducted both on this patient and his daughter Fig. .–8 RET gene mutations have been found in almost all patients with MEN2. The most frequent mutations of the RET gene are point mutations in exons 10 and 11. The distributions of mutational hot spots probably vary across countries, regions, and ethnic populations. The 611 codon is located on exon 10 of the RET gene. The C611Y mutation (the sequence of TGC at the 611 site of the RET gene coding for Cysteine mutates to the sequence of TAC coding for Tyrosine) could cause the formation of ligand-independent RET dimer, automatically activate the downstream signal pathway without ligand, and induce excessive cell proliferation and carcinogenesis.RET proto-oncogene (rearranged during transfection proto-oncogene) with a total length of 60 kb is the only pathogenic mutation of MEN2A, which is located on the long arm of chromosome 10 (10q11.21) including 21 exons, encoding a RET transmembrane protein composed of 1100 amino acids. This mutation of the RET gene encodes abnormal RET protein, influencing the proliferation, apoptosis, invasion, and metastasis of different tumor cells through the signal pathway, leading to diseases such as MEN2A. which is mainly related to MEN2A, familial medullary thyroid carcinoma (FMTC), and MEN2A with HSCR.According to the risk of invasive MTC, the RET gene mutations were divided into 4 risk levels by the American Thyroid Association. The C611Y mutation belongs to ATA-b (moderate risk),33.1–13 One family was eliminated later due to incomplete data. The family data in the case presentation is also listed herein .Relevant articles from January 2009 to January 2020 were searched in the biomedical databases, including PUBMED, CNKI, WANFANG, and VIP, with the keywords of “Multiple endocrine neoplasia 2A,” “MEN2A,” “RET,” “p.Cys611Tyr,” “c.1832G>A,” and “C611Y.” Four Chinese MEN2A families with the C611Y mutation were retrieved (Table 44.14.1.1P < .05). Excluding 4 cases clinically suspected with MTC but not confirmed by pathological biopsy, the penetrance of MEN2A in women was 100% , while that in men was only 58.33% .In the 4 Chinese MEN2A families, 27 people were detected with the C611Y mutation, in which 22 people were diagnosed with MEN2A and the other 5 were mutation carriers, with a total penetrance of 81.48% (22/27). It was noted that there was a significant penetrance difference in sex (4.1.245.45% (10/22) of them were initially diagnosed with MTC, with an average age of 46.10 years (23–74 years). 18.18% (4/22) of the patients had PHEO as the initial symptom, with an average age of 39.67 years (35–47 years). 18.18% (4/22) of them had both PHEO and MTC initially, and their average age at diagnosis was 51.00 years (42–69 years). There was no statistical difference in sex among the above 3 groups.4.1.3P < .05).Among the 22 patients with MEN2A, 17 were confirmed with MTC, and 8 were confirmed with PHEO, but no one with PHPT (Table 4.24.2.1P > .05). Nine patients had lymph node metastasis of varying severity, accounting for 52.94% (9/17), and there was no significant difference in sex as well (P > .05). In addition, there was no adverse prognosis report related to PHEO or PHPT.Five out of 17 patients pathologically diagnosed with MTC underwent postoperative recurrence, with an incidence of 29.41% (5/17), and there was no statistically significant difference in sex .6.2In the literature review, there was a significant sex difference in the clinical characteristics of the 4 Chinese MEN2A families with the C611Y mutation, with a penetrance of 58.33% and 100% in male and female patients, respectively. Therefore, regular MTC screening and long-term follow-up management are particularly important for people with C611Y mutation, especially for women. Only MTC-related adverse prognosis reports were found in the literature and it was more prone to postoperative recurrence or lymph node metastasis. So, the selection of operation and treatment of cervical lymph nodes were worth further study. All patients had a recurrence of MTC after subtotal thyroidectomy in the 4 Chinese MEN2A families. Could total thyroidectomy be more beneficial for MTC patients with C611Y mutation? More high-level evidence-based medical studies are expected. The effect of MTC lymph node metastasis or recurrence on the survival rate of MEN2A patients with C611Y mutation, the incidence of surgical complications and adverse outcomes associated with PHEO, and the morbidity of PHPT. In-depth studies are required as unsettled issues still existed.There are certain limitations in this study. The small number of research subjects may affect our preliminary conclusions. In the future, it is necessary to enlarge the sample size.6.3–24It was difficult to make an accurate diagnosis of MEN2A in clinical since its incidence in population was rather low and its clinical symptom was not representative. As a result, the clinical data of the proband (Table  In conclusion, MEN2A patients should be screened regularly and managed by a multidisciplinary team.Furthermore, MEN2A should be highly suspected in his daughter (Table Conceptualization: Yan Li, Ya-qin Tan, Yu hua Chen.Data curation: Yan Li, Ya-qin Tan, Zhi-xiang Tang, Qing-hui Liao, Zhong-qiu Guo, Kang-bao Lai, Rong Wang, Yu hua Chen.Formal analysis: Yan Li, Yu hua Chen.Funding acquisition: Yu hua Chen.Investigation: Yan Li, Ya-qin Tan, Zhi-xiang Tang, Qing-hui Liao, Zhong-qiu Guo, Kang-bao Lai, Rong Wang, Yu hua Chen.Methodology: Yan Li, Ya-qin Tan, Zhi-xiang Tang, Qing-hui Liao, Zhong-qiu Guo, Kang-bao Lai, Rong Wang, Yu hua Chen.Project administration: Yan Li, Ya-qin Tan, Yu hua Chen.Resources: Yan Li, Ya-qin Tan, Zhi-xiang Tang, Yu hua Chen.Software: Qing-hui Liao, Zhong-qiu Guo, Kang-bao Lai, Rong Wang.Supervision: Qing-hui Liao, Zhong-qiu Guo, Kang-bao Lai, Rong Wang.Validation: Yan Li, Ya-qin Tan, Yu hua Chen.Visualization: Zhi-xiang Tang, Qing-hui Liao, Zhong-qiu Guo, Kang-bao Lai, Rong Wang.Writing – original draft: Yan Li.Writing – review & editing: Yan Li, Ya-qin Tan, Zhi-xiang Tang, Qing-hui Liao, Zhong-qiu Guo, Kang-bao Lai, Rong Wang, Yu hua Chen.


The mortality rate of young female COVID-19 patients is reported to be lower than that of young males but no significant difference in mortality was found between female and male COVID-19 patients aged over 65 years, and the underlying mechanism is unknown. We retrospectively analyzed clinical characteristics and outcomes of severely ill pre- and post-menopausal COVID-19 patients and compared with age-matched males. Of the 459 patients included, 141 aged ≤55, among whom 19 died . While for patients >55 years (n=318), 115 died . In patients ≤55 years old, the levels of NLR, median LDH, median c-reactive protein and procalcitonin were significantly higher while the median lymphocyte count and LCR were lower in male than in female . In patients over 55, these biochemical parameters were far away from related normal/reference values in the vast majority of these patients in both genders which were in contrast to that seen in the young group. It is concluded that the mortality of severely ill pre-menopausal but not post-menopausal COVID-19 female patients is lower than age-matched male. Our findings support the notion that estrogen plays a beneficial role in combating COVID-19. In December 2019, a series of unexplained cases of pneumonia were reported in Wuhan, Hubei, China, with clinical manifestations very similar to viral pneumonia ), followed by diabetes (78 [17.0%]) and cardiovascular disease (72 [15.7%]). Fever (425 [92.6%]) is the most common symptom on admission, followed by dry cough (275 [59.9%]), dyspnea (270 [58.8%]), fatigue (194 [42.0%]), expectoration (154 [33.6%]), anorexia (94 [20%]), diarrhea (65 [14.2%]), abdominal pain (42 [9.2%]), sore throat (35 [7.6%]), dizziness (27 [5.9%]), headache (24 [5.2%]), nausea (23 [5.0%]), and vomiting (14 [3.1%]). Hemoptysis (6 [1.3%]) and abdominal pain (6 [1.3%]) were rare . Of thesThe laboratory values on admission are summarized in In patients aged ≤ 55 years old, the levels of lymphocyte and T lymphocyte counts in males were significantly lower, while the levels of alanine aminotransferase and aspartate aminotransferase, total bilirubin, urea nitrogen, creatinine, CRP, procalcitonin, LDH, Myoglobin, Creatine kinase were significantly higher than those in the female patients . For patOf the 459 patients, the vast majority (410 [89.3%]) of patients used antiviral drugs , followed by antibiotics (365 [79.5%]) and glucocorticoids (168 [36.6%]). Only 6 patients (1.7%) received continuous renal replacement therapy . 308 patUnivariate logistic regression analysis showed that gender, hypertension, chronic kidney disease, mechanical ventilation, ARDS, sepsis, septic shock were associated with nosocomial death in severe COVID-19 patients aged ≤55 years old. Hypertension, cardiovascular disease, cerebrovascular disease, glucocorticoid therapy, renal replacement therapy, mechanical ventilation, ARDS, sepsis, septic shock were associated with nosocomial death in severe COVID-19 patients aged > 55 years old. Multivariate logistic regression analysis results further showed that gender, hypertension and septic shock were associated with the incidence of nosocomial death in severe COVID-19 patients aged ≤55 years old. Cardiovascular disease, glucocorticoid therapy, ARDS, sepsis, and septic shock were associated with the incidence of nosocomial death in severe COVID-19 patients aged > 55 years old; .Studies have shown that the risk factors for adverse outcomes caused by COVID-19 include advanced age and the presence of comorbidities  , 4, 19. It has been well known that COVID-19 not only causes pneumonia, but also damages other organs, such as the heart, liver and kidneys, as well as organ systems such as the blood and immune system . The patAmong patients who aged ≤55 years old in this cohort, female patients had a much lower incidence of developing complications than male patients, especially in terms of lung injury . However, this difference in incidence between males and females was not manifested among the patients aged more than 55 years old. The most possible explanation for this phenomenon is that the levels of estrogen in females after menopause were decreased, and thus the beneficial effects of estrogen seen in females younger than 55 no longer exist.Estradiol-mediated lung inflammation control has been shown to be attributable to down-regulated NF-κB signaling. NF-κB is a transcription factor that directly regulates the expression of various pro-inflammatory cytokines (including TNF-α and IL-1β) . It is kThe main target of SARS-CoV-2 invading the human body is the angiotensin converting enzyme 2 (ACE2) receptor. ACE2 is widely distributed in the human body, and is expressed in the heart, kidney, lung, liver, testis, and intestine , 34. TheHuman endocrine pancreas also express ACE2, so coronavirus may damage islets and cause acute hyperglycemia . TherefoIn short, estrogen, especially E2, provides a protective effect on the regulation of inflammatory response and immune cell function, the influence of the cardiovascular system, insulin sensitivity and the improvement of monocyte function, thus most female patients with COVID-19 was saved from death. However, this protective effect gradually weakens in female who have passed menopause. In addition, the WHO classified estrogens as carcinogenic in humans and one of the most important risk factors of breast cancer . Breast This cohort study may have the following limitations. First, the study was conducted in only two hospitals with a limited sample size, which may affect our interpretation of the study results. Second, there may be selection bias in determining protective factors that affect clinical outcomes. Third, lack of or insufficient adherence to standard supportive care during difficult times may lead to poor clinical outcomes in some patients. A larger cohort study of patients with multiple centers or multiple classifications is also needed to validate our conclusions.Supplementary Table 1Supplementary Table 2


In fact, much evidence has suggested that hydrogen sulfide plays a significant role in many physio-pathological processes, such as inflammation, oxidation, neurophysiology, ion channels regulation, cardiovascular protection, endocrine regulation, and tumor progression. Considering the plethora of physiological effects of this gasotransmitter, the protective role of H2S donors in different disease models has been extensively studied. Based on the growing interest in H2S-releasing compounds and their importance as tools for biological and pharmacological studies, this review is an exploration of currently available H2S donors, classifying them by the H2S-releasing-triggered mechanism and highlighting those potentially useful as promising drugs in the treatment of cardiovascular diseases.Hydrogen sulfide (H T. T119]. ammation ,121 , and cad AP-39) .Indeed, Rossoni and co-workers demonstrated that ACS14 exerts strong protective effects against buthionine sulfoximine (BSO)-induced cardiovascular diseases, by increasing systolic blood pressure and lowering heart rates in rats . Additio2S donor, named AP-39, resulting from the reaction of triphenylphosphonium with ADT-OH, was developed [2S-releasing properties and potential pharmacological effects. More recently, another hybrid Heveloped  and studAP-39 significantly inhibits oxidative stress-induced toxicity and protects against acute cardiac arrest, renal, and myocardial I/R injury, by inhibiting the mitochondrial permeability transition pore ,132,133.Starting from GYY4137 structure, Kang and coworkers developed a series of phosphonamidothioates, named JK donors . To synthesize these compounds, the phenylphosphonothioic dichloride was treated with 3-hydroxypropionitrile and, consequently, with a selected C-protected amino acid, such as glycine, phenylalanine, valine, alanine, and proline. These steps provided the intermediate, which underwent LiOH-mediated hydrolysis, furnishing the final compounds .2S donors. Indeed, in aqueous media under acidic conditions (pH = 2.0–6.0), JK donors had higher H2S release rates, whereas at neutral and mildly basic pH (7.4–8.0), they caused slower and less H2S release. These donors represent the first class of pH-controllable H2S  of the series  showed p4)2MoS4, was identified as a pH-dependent H2S donor [2S can be generated from ATTM under strong acidic conditions (5% H2SO4) , an excellent copper chelator with the formula  .2S donor [Later, Xu and co-workers described ATTM as a water-soluble and a pH-sensitive slow-releasing H2S donor . More re2S donor . 2S is responsible for protective effects in doxorubicin- and adriamycin-induced cardiomyopathy during cancer therapies [Considering that some preclinical data suggested that Hherapies ,139,140 herapies , the res2S-releasing compounds were some of the first synthetic donors to be reported. They can release H2S by reacting with endogenous thiol-containing molecules, such as GSH, which are relatively abundant in mammals. Some of these H2S donors were found to exhibit protective effects in cardiovascular diseases benzamides were synthesized by Zhao et al. [Among the first nucleophile-triggered Ho et al.  with the2S only following a nucleophilic attack by thiols. The structure–activity relationship studies showed that electron withdrawing groups cause a faster H2S release, unlike electron donating groups. In addition, H2S release from N-(benzoylthio)benzamides was also detected in the plasma, revealing that N-SH compounds have efficacy in complex systems. The resulting compounds were proven to be stable in aqueous buffers and to be able to release HThese compounds protect human keratinocytes against methylglyoxal (MGO)-induced cell damage and dysfunction, which is prevalent among patients with diabetes mellitus . Moreove2S donors starts with a thioester exchange between the donor and a first molecule of cysteine generating S-benzoyl cysteine and N-mercaptobenzamide, an N-SH intermediate , confirming their ability to release. Moreover, the selected donors were evaluated into a murine model of myocardial I/R injury. The results proved that they exhibit cardioprotective activity due to their ability to reduce the infarct size.HAnalogous perthiol-based donors are the dithioperoxyanhydrides (RC(O)–S–SC(O)R’) , which aBoth alkyl and aromatic dithioperoxyanhydride donors were obtained by two possible synthetic strategies: iodine oxidation of the thiocarboxylates  or react2S release from these compounds was measured amperometrically and found in both buffers and cellular lysates, confirming H2S-releasing properties of dithioperoxyanhydrides. When treated with thiols, these donors formed a key intermediate for H2S production, the acyl-persulfides, which, reacting with the excess of thiols, could directly release H2S and RSSAc  and 3-pyridyl-isothiocyanate  exhibite2S release from these compounds has already been described above  , which a2Cl2, using both the conventional synthetic strategy, previously described in literature [2S release by using a combination of HPLC, MS, and NMR to isolate and characterize the intermediates and the final products. These compounds were obtained through oxidative condensation of isothiocyanates with isocyanates in the presence of SOterature , and theterature . The sec2, and H2S. As depicted in It was also found that these donors exerted vasodilating activity in a concentration-dependent manner on isolated aorta rings. 2S prodrug is a water-insoluble microcrystalline material, mostly consisting of α-sulfur (about 99% S8) hydrophilized with traces of ionic substances, such as sodium sulfate, sodium thiosulfate, and sodium polythionates, which enhance its bioavailability [Furthermore, a sodium polysulthionate, named SG-1002 , was devlability . 2S-releasing from S8 is activated by thiols and this reaction with GSH is depicted in SG-1002 was obtained via comproportionation of sulfur atoms in the -2 and +4 oxidation states in a strongly acidic medium of high ionic strength. This synthetic route, together with properties and some therapeutic applications of SG1002 are described in a patent . H2S-rel2S and sulfane sulfur levels in models of pressure overload heart failure. Moreover, it improved coronary artery vascular reactivity and attenuated high-fat diet-induced cardiac hypertrophy and dysfunction [SG-1002, when administered orally, produced a more sustained and consistent increase in Hfunction ,158,159.2S-donating ability in patients with congestive heart failure was investigated [Importantly, SG-1002 has completed the phase I clinical trial where its Hstigated . The resstigated . To date, it is the only sulfide-based therapy clinically tested in patients with cardiovascular diseases.2S donors have been received increasing interest due to their many advantages over the other triggers already mentioned above. Among these, a series of esterase-sensitive prodrugs of H2S, based on a lactonization reaction, also known as ‘‘trimethyl lock” (TML) [In recent years, enzyme-triggered Hk” (TML) , was devk” (TML)  -releasing compounds. Indeed, COS is a substrate of carbonic anhydrase (CA), which catalyzes its conversion into H2S [Another class of enzyme-triggered Hinto H2S ,167.2S donors are N-thiocarboxyanhydrides (NTAs)   [168,169The sarcosine NTA derivative (NTA1)  was synt2S production from NTA1 consists of ring-opening by a biological nucleophile, such as an amine. The resulting dipeptide byproducts were confirmed by GC-MS and LC-MS, respectively. After COS release, its conversion into H2S occurs through the rapid enzymatic action of CA  were developed [Among the class of COS/Heveloped , using aeveloped ,174. The2S in the presence of an oxidant, such as H2O2. In this mechanism, the boronate ester was converted to a phenol by ROS, releasing COS, which was quickly converted into H2S by CA [The obtained compounds were found to release H2S by CA   in dichloromethane furnished the desired compounds -3-prop-2-ynylsulfanylpropionic acid, named ZYZ803, was developed [Recently, a novel Heveloped  by coupleveloped ,182.2S concentration in plasma through the activation of CSE enzyme [As already described above, SPRC could enhance HE enzyme  while fuE enzyme .2S and NO, respectively , releasing Hectively . These gAs reported by Hu et al., ZYZ803 significantly promoted endothelial cell angiogenesis both in vitro and in vivo .2S and NO releasing from ZYZ803 [Indeed, the oral administration of ZYZ803 in a mouse model of hindlimb ischemia stimulated angiogenesis more effectively than individually administered SPRC and furoxan, confirming the additive protective actions of Hm ZYZ803 .Additionally, ZYZ803 regulated the vascular tone in isolated rat aortic rings , attenua2S donor with activity in the cardiovascular system is zofenopril, an angiotensin-converting enzyme (ACE) inhibitor. Undergoing hydrolysis in the liver, it forms zofenoprilat, its active metabolite containing a thiol group  have completed the phase I clinical trials and registered on clinicaltrials.gov for cardiovascular disease and breast cancer (Among them, at least two Ht cancer .2S-releasing compounds have yet to be more tested and fully characterized.Nevertheless, there are still too few available donors with oral bioavailability, which is crucial for clinical translation, and being available for testing in in vivo pharmacokinetic studies. Furthermore, the safety and long-term effects of many H2S donors exhibiting cardioprotective activity and thus providing an insightful knowledge of their chemical nature and mechanisms of release, could enhance the chances to design novel H2S-donating molecules useful to help further reduce the risks of cardiovascular disease in the coming years.Bearing in mind the above considerations, this work, by reviewing the available H


To explore the potential function of methyltransferase-like 5 (METTL5) in uterine corpus endometrial carcinoma (UCEC) and verify the relationship between deficient DNA mismatch repair (MMR) and METTL5. We used bioinformatics to predict the possible role of METTL5 and molecular biology methods to analyze METTL5 expression. We observed UCEC proliferation, development, and apoptosis using a METTL5 knockdown lentivirus and, coupled with METTL5 bioinformatics and Western blot analysis, detected microsatellite instability (MSI) and MMR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Finally, some METTL5-associated gene mutations in UCECs were detected. Results show that METTL5 expression in UCEC tumor tissue was increased, and UCEC patients with high METTL5 expression had worse prognostic outcomes. We also observed the highest METTL5 expression level in KLE cells. Furthermore, knocking down METTL5 weakened the proliferation, reduced tumor volume and biomarkers, and increased apoptosis. Moreover, METTL5 knockdown induced the MSH2, MSH6 and PMS2 expression in MMR. METTL5 was negatively correlated with gene silencing, mRNA binding, olfactory receptor activity, antigen processing and presentation, cytosolic DNA sensing, olfactory transduction, and RIG-1-like and Toll-like receptor signaling pathways. METTL5 may regulate MMR protein levels in UCECs, thus enhancing UCEC proliferation, development, and prognosis.  Uterine corpus endometrial carcinoma (UCEC) is the most common and threatening gynecological malignant tumor that leads to death due to its high recurrence rate . EndometDeficient DNA mismatch repair (MMR), a strong mutator phenotype of microsatellite instability (MSI), and has recently been described in ECs ,5. In adAs previously reported, regulating MMR may be a prognostic factor for tumor recurrence and adjuvant chemoradiotherapy in patients with advanced UCECs ,8. ThereMethyltransferase-like 5 (METTL5) is a protein-coding gene associated with intellectual developmental disorder, autosomal recessive 72 and primary autosomal recessive microcephaly. It is related to nucleic acid binding and protein methyltransferase activity . Recent We aimed to investigate the potential role of METTL5 in UCECs, then used a METTL5 knockdown lentivirus to regulate MMR and UCEC development in vivo and in vitro. The present findings indicate that METTL5 may regulate MMR in UCECs. These results may provide novel strategies for UCEC treatment via MMR-associated mechanisms.We selected 16 patients from the Department of Pathology in our hospital and pathologically diagnosed them with UCEC from May 2019 to May 2021. We obtained adjacent para-carcinoma tissues as controls. All patients provided informed consent, and the Ethics Committee of The First Affiliated Hospital of Hebei North University approved the experimental design.http://timer.cistrome.org/). The survival assay of the UCEC patients in the METTL5 low and METTL5 high expression groups was performed using the Kaplan-Meier plotter website , and the cutoff of expression was 50%. We downloaded the Pan-Cancer Assay from The Cancer Genome Atlas (TCGA) database, as described previously [https://www.mutarget.com/analysis?type=target.We determined METTL5 expression using the TIMER2.0 website , KLE cells, RL952 cells, Ishikawa cells, and ECC-1 cells. They were maintained and cultured as previously described . The celThe following antibodies were used: Anti-METTL5 , anti-Ki-67 , anti-CEA , anti-CA199 , anti-CA153 , anti-HE4 , anti-MLH1 , anti-MSH2 , anti-MSH6 , anti-PMS2 , and anti-β-actin .Chongqing Tengxin Biotechnology Co., Ltd.  supplied four-week-old healthy male Balb/c mice. We subjected the mice to specific pathogen-free (SPF) conditions and housed them under the Guide for the Care and Use of Laboratory Animals. The Ethics Committee of the First Affiliated Hospital of Hebei North University approved the study design (Protocol No.: 2020ECHNU033).We designed and chemically synthesized METTL5 knockdown lentiviruses (sh-METTL5) and control lentiviruses  and stored them at −80°C. Lentivirus cell administration has been well-established . Table 1The subjects were randomly divided into four groups (n = 6 in each group) : Con groThe mice were subcutaneously injected with KLE cells with or without sh-METTL5 lentivirus administration. After five days, we observed their vital signs and inoculation sites, as described previously . The micA total of 50 μg of protein was transferred to nitrocellulose membranes, as described previously . These wSections were incubated with METTL5 and Ki-67 primary antibodies at 4°C overnight, as previously described . ImmunohCells from each group were analyzed using a PI-Hoechst assay , as described previously .Tissue fractions from each group were analyzed using a TUNEL assay , as described previously .Cell viability was studied using the MTT assay, and absorbance was measured at 490 nm, as described previously .GraphPad Prism 6.0 and SPSS 18.0 were used to analyze the data, which are expressed as the mean ± standard deviation (SD). One-way or two-way ANOVAs were performed and multiple comparisons are analyzed using a Holm-Sidak test. A P-value < 0.05 was considered statistically significant.To explore the potential function of METTL5 UCEC and verify the relationship between MMR and METTL5. We used bioinformatics to predict the possible role of METTL5 and molecular biology methods to analyze METTL5 expression. We observed that the proliferation, development, and apoptosis using a METTL5 knockdown lentivirus and coupled with METTL5 bioinformatics and Western blot analysis, detected MSI and MMR.Bioinformatic analysis results suggest that METTL5 may be a gene related to UCECs, as we detected varying METTL5 levels in different tissues and cells . METTL5 As the previous results indicated that METTL5 might be correlated with UCEC progression, we designed a method to block METTL5 levels: sh-METTL5 . We obseAs knocking down METTL5 administration regulated UCEC development, we repeated the process in vivo. First, the tumor volumes at 21 and 28 days were significantly decreased in sh-METTL5 mice (P < 0.05, We used Pan-Cancer and correlation analyses to detect the role of METTL5 in MSI and MMR in UCECs. As METTL5 was predicted to be a gene related to MSI and MMR in UCECs based on previous results, we detected MMR marker levels in vivo. In the GO assay, we found that METTL5 was negatively correlated with gene silencing, gene silencing by RNA, mRNA binding, olfactory receptor activity, and RNA binding involved in post-transcriptional gene silencing . In the EC is a threatening gynecological malignant tumor associated with a 20% mortality rate in Western and developing countries ,24. EC mIn short repetitive DNA sequences, MSI refers to the hypermutator phenotype second to frequent polymorphisms and single nucleotide substitutions. It is a consequence of DNA MMR deficiency and vital to ECs ,26.Liu J, et al investigated a hotspot R298P mutation in UCEC was present in a key component of METTL14, exhibiting reductions in m6A methylation that are likely due to either this METTL14 mutation or reduced expression of METTL3 . Qin L, Apoptosis is a programmed cell death process without any inflammatory responses . We knocThrough Pan-Cancer and correlation analyses, we found that METTL5 was positively correlated with MSI and negatively correlated with MRR proteins, especially MSH2, MSH6, and PMS2. There is no research on METTL5 with MRR proteins. Hence, we have only explored this process according to our bioinformatics results. We used Western blotting and IHC to prove our bioinformatic outcomes related METTL5 to MMR. Our results showed that sh-METTL5 could improve MSH2, MSH6, and PMS2 expression but not MLH1 expression.First, we will report the results. However, some methodological deficiencies due to our limited lab conditions must be taken into consideration. Furthermore, more rigorous and advanced experiments by other scholars are required.In further bioinformatics analysis of METTL5 in UCEC patients using Pan-Cancer and the target gene system, we found that METTL5 was negatively correlated with gene silencing, mRNA binding, olfactory receptor activity, antigen processing and presentation, cytosolic DNA sensing in GO, antigen processing and presentation, cytosolic DNA sensing pathways, olfactory transduction, and RIG-1-like and Toll-like receptor signaling pathways in KEGG.Finally, METTL5 may be involved in UCEC prognosis and related to ZFP59, JPH4, OR52K1, PSG11, TMPRSS4, SEPT7, SLC2A7, OR10C1, NDUFA9, DCTD, RFPL4A, IL10, RDH5, CLK3, TPCN2, ISL2, NCR3LG1, RAB27B, TMED8, SLC25A31, OAS1, SHOX2, RAB11A, and AGTR1 mutations. Several studies are needed to verify this data, which will be the focus of our subsequent experiments.A limitation of this study is that METTL5 mRNA levels should be detected in endometrial cells to explain whether differences in METTL5 expression are due to transcription or translation levels.Our study found that knocking down METTL5 could significantly activate apoptosis and inhibit EC development via MMR administration. Hypothetically, our findings may offer a new approach for UCEC treatment by regulating MMR.


Store-operated calciumentry (SOCE) is a pivotal mechanism in calciumhomeostasis, and, despite still being under investigation, its dysregulationis known to be associated with severe human disorders. SOCE modulatorsare therefore needed both as chemical probes and as therapeutic agents.While many small molecules have been described so far, their poorproperties in terms of drug-likeness have limited their translationinto the clinical practice. In this work, we describe the bioisostericreplacement of the ester moiety in pyrazole derivatives with a 1,2,4-oxadiazolering as a means to afford a class of modulators with high metabolicstability. Moreover, among our derivatives, a compound able to increasethe calcium entry was identified, further enriching the library ofavailable SOCE activators. STIM1 and its homologue STIM2are single-span membrane proteins residing on the ER membrane andact as sensors of calcium levels. Orai are a family of proteins  that form the ion channels on the plasma membrane.6 When a depletion of calcium from the ER stores occurs, STIM undergoesa conformational change leading to self-association into puncta andmigration near the plasma membrane. There, interaction with Orai promotesthe opening of the channel and the subsequent influx of calcium. Theempty ER stores are then refilled by the sarco-endoplasmic reticulumcalcium ATPase (SERCA) pump.8 Due to its prominentrole in encoding calcium signals, SOCE is implicated in several humandisorders, including cancer,10 inflammatory boweldisease,11 allergy,12 and acute pancreatitis.14 Therefore,it is not surprising that this cellular pathway has become a promisingtarget for the therapeutic treatment of several pathological conditions.The ubiquitous second messengercalcium is a central regulator of a plethora of physiological processes,spanning from fertilization of oocytes to cell death, mobility, secretion,and gene expression. Infinitesimal oscillations of this ion concentrationin the different cell compartments control a multitude of processes.This is the reason why every cell maintains a huge gradient betweenthe plasma membrane or the intracellular calcium repository and thecytoplasm, with various pumps and exchangers in charge of finely regulatingits levels.18 able to modulate SOCE have beenreported. The earliest modulators are SKF-96365,20 2-APB and its derivatives,22 and bis(trifluoromethyl)pyrazolescompounds, initially named BTP (BTP1 (1), BTP2 (2), 23 followed by Pyr’s (Pyr3 (3), Pyr6 (4), Pyr10 (5), 25 Among others, GSK-7975A ,29 whose structurehas not been disclosed. These are not the only SOCE inhibitors thatare in human use. Indeed, a virtual screening performed on FDA-approveddrugs recently unveiled that two approved drugs are also SOCE inhibitorswith relevant activity at therapeutic doses,30 the prodrug leflunomide inhibitor, while leflunomide is devoid of in vitro activity on this enzyme.Over the years, many small molecules7975A 6, 1,26 RO29O2959 7, 1,27 and nta66 8, 111,12 haM4620 9, 1 for acumide 10, 131,32 anmide 11, 1.33,34 T12, 35Our group of research has contributedto the development of druglikeSOCE modulators by reporting pyrtriazoles, among which the most promisingcandidate is CIC-37 12, 1.3535 However, the molecule is affected by poor metabolic stability, andthis is attributable to the ester group, which is shared by pyrtriazolesand some of the Pyr’s , and CIC-37 . Moreover, 15 additional coupling reactions were performed study was undertaken. To this aim, eight coupling reactionswere performed  in the presence of the compounds at 10 μMconcentration. After 600 s, Ca2+ was added and intracellularlevels were measured in fluorescence microscopy with the calcium dyeFura-2. As shown in 22, % of SOCE residual activity: 38.6%), BTP1, leflunomide , Pyr2, and Pyr10 . Also,compounds 27 (40.8%) and 29 (49.6%), displayingan electron-withdrawing group in para position on the aromatic ring,or 31 (47.4%) in which the aromatic ring is fused withan imidazole, afforded a good inhibitory activity. For these compounds,the cell viability was therefore evaluated. An MTT assay was performedand the compounds that affected cell viability by more than 25% at10 μM were discarded . Notably, the remaining five compounds  were not cytotoxic under these conditions. According to both activityand cytotoxicity, for the selected five compounds, the representativetraces are depicted in With this approach, 24 oxadiazole-bearingpyrazoles were synthesizedand tested in human embryonic kidney (HEK) cells for SOCE inhibition.To this aim, intracellular Ca50 valueswere calculated and are reported in 22, with an IC50 value of 3.1 μM.Moreover,for these selected compounds, IC37, in which the side chainis represented by a linear aliphatic substructure, afforded an increasein calcium entry, with a percentage of SOCE activity of 146.2% comparedto the control. More in detail, the compound, tested at the concentrationof 10 μM, significantly increased the area under the curve (AUC)of calcium entry and the peak amplitude, without affecting the slope and 1 μM (116.7%) but was not detected at concentrationsabove 10. In particular, at 30 and 100 μM, 37 actsas weak SOCE inhibitor, with a percentage of residual activity ofabout 70%.Surprisingly,compound he slope4. We als43), NM-3G (44), 35 and biphenyltriazoles , 40 A similarbehavior is also shared by 2-APB (47), a well-known inhibitorof IP3 receptors and TRP channels. The compound is a SOCE modifierin Orai1- and Orai3-expressing cells, acting as SOCE enhancer at lowconcentrations, while high concentrations induce a transient increasefollowed by complete inhibition.41 Althougha precise rationale can not be extrapolated from the scaffolds ofthe enhancers, in our experience it often occurs that a minimal structuralmodification in a class of compounds designed to negatively modulateSOCE is capable of turning an inhibitor into a molecule able to increasecalcium entry.The identification of a SOCE enhancer among a class of inhibitorshas been reported in three previously published classes of modulators,with two of them described by us and represented by pyrtriazoles  and the identifiedSOCE enhancer (37) were then evaluated for their in vitro hepatic metabolic stability. To this aim, the candidateswere incubated in MLS9 fraction supplied with NADPH and the residualsubstrate was measured after 1 h. Under these conditions, the oxadiazole-bearingpyrazoles resulted overall metabolically stable, with only two compoundsaffording a residual substrate lower than 80% and the remaining moleculeshigher than 90% after incubation (Supporting Information). In contrast, Pyr3 and CIC-37, which suffered from hydrolysis ofthe ester function, provided a residual substrate of 43 and 74%, respectively-like disorders.


American Heart Association’s (AHA) Life’s Simple 7 (LS7), an index of cardiovascular health risks, has been associated with worse brain outcomes but few examined this relationship in midlife. We examined whether LS7 scores at midlife were associated with brain morphometry in early old age. Participants were 471 men who participated in the Vietnam Era Twin Study of Aging. The LS7 index was assessed at mean age 62 (range 55-66) and 68 (range 61-71) and included smoking, physical activity, diet, body mass index, cholesterol, glucose, and blood pressure. Each factor was coded, per AHA criteria, on a 3-point scale  and summed to create a composite score (0-14). At mean age 68, participants underwent structural magnetic resonance imaging, which was used to create the previously validated brain measures. Scores included: the ratio of abnormal white matter to white matter, and two Alzheimer’s disease brain signatures  signature). Analyses controlled for age, education, income, ethnicity, and APOE genotype. Concurrently at mean age 68, the LS7 was associated with cortical thickness/volume , MD  signatures and abnormal white matter ratio . Prospectively, the LS7 at mean 62 was significantly associated with age 68 cortical thickness/volume  and MD  signatures but not with abnormal white matter ratio. These results suggest that prevention strategies that promote heart healthy behaviors could have implications for healthy brain aging.


KCNQ1_rs2237892, ZPR1_rs2075291, APOA5_rs662799, APOA1_rs5072, and SIK3_rs151139277, influenced 3GO risk, and the minor alleles of their haplotype had a 3GO risk 3.23 times higher than the major alleles. For subjects with a high energy intake, the 3GO risk of the minor alleles was significantly higher than that of the major alleles  = 2.062~5.061, p < 0.001). BMI, HbA1c, SBP, and serum concentrations of glucose, HDL, and triglyceride were significantly higher for the minor allele than the major alleles (p < 0.001). The haplotype interacted with the intakes of protein (p = 0.033), digestible carbohydrate (p = 0.012), fat (p = 0.008), and undigestible carbohydrates (p = 0.015) to increase 3GO risk. An interaction was also observed between smoking and the haplotype (p = 0.007). The minor allele effects on 3GO incidence were higher in the high digestible carbohydrate intake and smoking groups. By contrast, the minor allele impacts on 3GO frequencies were much higher in the low intake of undigestible carbohydrates, protein, and fat. In conclusion, people who carry a minor allele of the 11q23 locus haplotype should avoid smoking and replace digestible carbohydrate intake with consuming high-quality protein, healthy fat, and undigestible carbohydrates.3GO is a condition in which hypertension, hyperglycemia, and dyslipidemia co-occur, and these conditions are related to each other and genetic and environmental factors. We hypothesized that common genetic variants and their interactions with lifestyles influenced 3GO risk. We aimed to explore common genetic variants to affect 3GO risk and their haplotype interaction with lifestyles in a city hospital-based cohort in 58,701 Koreans > 40 years. 3GO was defined as SBP ≥ 140 mmHg and DBP ≥ 90 mmHg for hypertension, fasting blood glucose ≥ 126 mg/dL for hyperglycemia, and LDL ≥ 160 mg/dL or HDL ≤ 40 mg/dL, or triglyceride ≥ 200 mg/dL for dyslipidemia. Haplotypes were generated by genetic variants selected from genome-wide association study ((GWAS) an observational study of the genetic variation of the whole genome in different individuals, used to see if any variation is related to traits) after adjusting for age, sex, area of residence, and body mass index (BMI). Nutrient intakes were assessed using food frequency questionnaires. Interactions between haplotype and lifestyles and 3GO risk were investigated. Parameters related to metabolic syndrome were significantly different in the 0GO, 1–2GO, and 3GO groups, that is, groups of individuals with none, one to two, or all three of the components of 3GO. At the 11q23 locus,  The World Health Organization has stated that reducing metabolic syndrome (MetS) development should be considered the first defense against cardiovascular diseases . The morThe other three components of MetS  are closely interrelated and share risk factors, such as aging, menopause, obesity, inflammation, and oxidative stress . HyperteRelatively non-obese Asians with MetS are susceptible to progress to T2DM, dyslipidemia, and hypertension. 3GO is a newly coined word for a syndrome comprised of hypertension, hyperglycemia, and dyslipidemia, and it is well-recognized in China since these diseases often simultaneously occur in one person ,12. GO cThe epidemiological data used to represent the Korean urban population was obtained from the Korean Genomics and Epidemiology Study (KoGES) conducted by the Korean Center for Disease Control and Prevention. The Institutional Review Board of the Korean National Institute of Health (KBP-2015-055) approved the study protocol. All participants volunteered to take part in the study and provided written informed consent. Before conducting the research, we also obtained approval from the Institutional Review Board of Hoseo University (1041231-150811-HR-034-01). KoGES was initiated in 2004 and terminated in 2013 and was a population-based, prospective cohort study that recruited 58,701 Korean adults aged 40–77 years . Follow-up surveys are conducted every two years, and study participants were followed up to 5 times during the 10-year study period. Information about lifestyles, current medications, and sociodemographic characteristics was obtained by a questionnaire-based survey.Questionnaires provided details of residence area, gender, age, smoking, drinking, and exercise status. Physical activity was estimated by multiplying rated physical activity intensities by times per week. Physical activity was classified as light, moderate, and heavy, based on moderate physical activity for <30, 30–120, and >120 min/week. Participants were classified as current, past, and never smokers. Never smokers had a lifetime smoking history of <100 cigarettes, and past-smokers had not smoked during the previous six months. Alcohol intake was assessed by multiplying the amount of drinking per occasion by the number of occasions per week, and daily alcohol intakes were classified as none (0 g), light (<2 g), moderate (2–20 g), or heavy (>20 g).2), and obesity was defined as a BMI ≥ 25 kg/m2.After at least 10 min rest upon arrival to the hospital between 7:00 a.m. and 9:00 a.m., blood pressure was measured at the upper right arm in a sitting position three times using a mercury sphygmomanometer  using a standard protocol ,15. TherAccording to the International Hypertension Federation , the diaUsual food intakes were assessed using a semi-quantitative food frequency questionnaire (SQFFQ) composed of 103 food items, and frequencies and average serving sizes were obtained for each food item. Serving sizes were assessed as 0.5, 1, or 2 times a reference serving size. Average intakes were calculated for all food items by multiplying frequencies and average daily serving sizes. The validity and reproducibility of the SQFFQ were confirmed using a 3-day food record as previously described . Dietaryhttp://www.cdc.go.kr/CDC/eng/mobile/CdcKrContentView.jsp?cid=74266&menuIds=HOME001-NU1130-MNU1890 (accessed on 18 March 2020).). After genotyping, genotyping accuracy was assessed by Bayesian Robust Linear Modeling with the Mahalanobis Distance genotyping algorithm [p < 1 × 10−5), and a minor allele frequency (MAF) of <1% [According to standard protocols, genomic DNA was extracted from peripheral blood monocytes of all participants using the QuickGene DNA whole-blood kit with QuickGene-610 L equipment . Single nucleotide polymorphisms (SNP) were identified using a Korea Biobank array (K-CHIP), which contained 830,000 SNPs customized for Koreans  and designed by the Center for Genome Science of the Korea National Institute of Health  of <1% .p < 5 × 10−6 since genetic effects were determined by haplotype. Subsequently, SNP-related genes were identified by searching the SCAN annotation database site (http://scandb.org/newinterface/ (accessed on 22 July 2020).). Linkage disequilibria (LD) were determined to check for correlations between SNPs in the selected locus of the chromosome using Locus Zoom .Genetic variants were selected from genome-wide association study (GWAS) results for the 3GO risk, where GWAS was conducted using EditPlus software after adjusting for age, gender, residence area, and BMI. Genetic variants affecting 3GO risk were selected using The haplotype was generated from the selected 5 SNPs in the 11q23 locus, and polygenetic scores (PRS) of the haplotype were estimated by adding the number of risk alleles in each genetic variant. PRS of the haplotype was divided into three groups: 0–3 , 4–6 , and ≥7 . Haplotype alleles were used to estimate genetic and genetic × lifetime impacts on 3GO.The impacts of interactions between genetic variants and lifestyles on 3GO risk were analyzed. Daily energy, protein, fat, carbohydrate, Ca, coffee and alcohol intake, smoking status, and physical activity were included in the interaction analysis as lifestyle variables. Dietary intakes, smoking statuses, and physical activities were dichotomized as ‘high’ or ‘low’ about median values. Residence area, age, gender, BMI, smoking, drinking status, coffee, energy, fat, carbohydrate, cholesterol intakes, physical activity, and plasma total cholesterol concentration were included as covariates in the interaction regression model.p-values < 0.05.Statistical analysis was performed using SPSS Ver. 24.0 (IBM SPSS Statistics). Descriptive statistics of categorical variables, such as gender and dietary frequency distribution, were obtained using the chi-square test. Descriptive statistics of continuous variables are presented as means and standard deviations (SDs). Covariates for adjustment were as follows: age, gender, area of residence, BMI, daily energy intake, cholesterol intake, fat intake percentages, physical activity, smoking status, and alcohol consumption. Five SNPs in the identified chromosome 11 (the 11q23 locus) were used for haplotype analysis. The major, heterozygous, and minor alleles of the haplotype were scored as 0, 1, and 2, respectively. The Chi-square test was used to analyze the frequency distributions of categorical variables. One-way analysis of variance (ANOVA) was used to determine the significances of differences between the control, 1–2GO, and 3GO groups after covariate adjustment for age, gender, area of residence, and BMI. One-way ANOVA was used to determine the differences between haplotype groups after adjusting for age, gender, residence area, and BMI. Duncan’s test was used to determine multiple comparisons among the groups if ANOVA was significantly different among the groups of each parameter. Multivariate logistic regression analysis controlled for covariates was used to determine the adjusted odds ratio (OR) and 95% confidence interval (CI) of the haplotype for the risk of 3GO and its components. Multivariate interaction regression models were used to evaluate the impacts of interactions between haplotype alleles and lifestyles on 3GO risk, after adjusting for covariates of age, gender, residence area, and BMI (model 1), and those for model 1, physical activity, and energy, alcohol, and coffee intake (model 2). When a significant interaction was found, the lifestyle variable concerned was dichotomized into low and high groups, and in each group, adjusted OR and CI between the haplotype and 3GO risk were estimated. Statistical significance was accepted for Of the 58,701 individuals that participated in KoGES, 1454 (2.48%) had 3GO. In the 2GO group, 2695 (4.59%) had hypertension and hyperglycemia, 6413 (10.9%) had hypertension and dyslipidemia, and 2969 (0.51%) had dyslipidemia and hyperglycemia . A totalTotal physical activity and coffee intake were not significantly different in the three groups, whereas alcohol drinking and smoking were. Interestingly, energy intake, which was calculated as a percentage of estimated energy requirement, was lower in the 3GO group than the other two groups . In termp > 0.05) and MAF (>0.1). Four of the 5 SNPs, except rs2237892, were positively associated  with 3 GO risk (APOA5 (apolipoprotein A5) rs662799, APOA1 (apolipoprotein A1)_rs5072, ZPR1 (zinc finger protein)_rs2075291, and SIK3 (SIK family kinase 3)_rs151139277. No LD was observed among the SNPs (KCNQ1 (potassium voltage-gated channel subfamily Q member 1)_rs2237892 exhibited a negative relationship with 3GO risk , strong , moderate , weak , and weakest  between the designated SNP and rs2237892. Five SNPs had a week or weakest relation with each other and had no LD relation.KCNQ1_rs2237892, ZPR1_rs2075291, APOA5_rs662799, APOA1_rs5072, and SIK3_rs151139277 in 11q23. It showed that 14,317, 13,118, and 1005 in the control participants belonged in the major, heterozygous, or minor alleles of the haplotype . We also analyzed the associations between the haplotype containing KCNQ1_rs2237892, ZPR1_rs2075291, APOA5_rs662799, APOA1_rs5072, and SIK3_rs151139277 and anthropometric and biochemical parameters. In model 2, the haplotype’s minor alleles were positively associated with SBP, HbA1c, serum glucose, total cholesterol HDL, and triglyceride concentration by 2.135-, 2.688-, 2.138-, 3.095-, 3.196-, and 3.568-fold respectively, as compared with the major alleles of haplotype (p < 0.001)  . However< 0.001) . Althoug< 0.001) .p = 0.033), carbohydrates (p = 0.012), fat (p = 0.008), and undigested carbohydrates . As regards lifestyle factors, haplotype interacted with smoking status to influence 3GO risk (p = 0.007). However, alcohol intake, coffee intake, and physical activity did not interact with haplotype to modify the risk of 3GO.After dichotomizing subjects into low- and high-intake groups, haplotype was found to interact with nutrient intake and 3GO risk. For those with the minor haplotype allele, 3GO risk was increased by 3.3- and 3.2-fold in low-protein and low-fat intakes respectively , and forAlthough BMI is low, Asians are more inclined to 3GO than whites ,25. ThisFurthermore, Asians and Caucasians also consume different proportions of carbohydrates, fats, and proteins . A very KCNQ1 is a voltage and lipid-gated potassium channel expressed in various tissues, including the myocardium, inner ear, kidney, lung, stomach, and intestines. KCNQ1 affects insulin secretion, and its mutation is related to the prevalence of T2DM [ZPR1 is a cell proliferation and signal transduction regulator protein with various physiological functions [ZPR1 has been associated with T2DM hypertriglyceridemia in Asians [ZPR1 rs2075291, influences MetS risk with serum triglyceride and HDL levels [ZPR1 rs2075291 is associated with hypo-LDL cholesterolemia, hypertriglyceridemia, and hyperglycemia. of T2DM ,29. Its  of T2DM . The rs2 of T2DM ,31. The unctions , and rs9n Asians . The 11qAPOA5, which has been implicated in regulating plasma triglyceride levels and the prevalence of MetS either independently or via complex gene–gene and/or gene–environment interactions. Consistent with the result of the present study, rs662799 of APOA5 has been reported to increase the risk of MetS in Caucasians [APOA1 is a primary lipoprotein associated with serum HDL concentrations in T2DM [APOA1 is related to dyslipidemia in a gender-specific manner, as shown in the present study [SIK3 plays crucial mediating roles in the three essential cancer signalings: cell proliferation, inflammation, and metastasis [SILK3 can be associated with MetS risk. The present study shows that SIK3 rs151139277 is related to 3GO, which suggests that inflammation may be associated with MetS. Summarizing these results, it appears that the 11q23 locus haplotype may be associated with hypertriglyceridemia, hypo-HDL-cholesterolemia, hyperglycemia, and inflammation, and contribute to blood flow disturbances, leading to hypertension.The 11q23 locus also encodes ucasians , Japanesucasians , Koreansucasians , and Chiucasians ,36. APOA in T2DM , and rs5nt study . SIK3 pltastasis , and hastastasis  and chrotastasis , SILK3 cIn this present study, a high carbohydrate intake but low protein, fat, and undigested carbohydrate intakes were associated with significant risks of 3GO, especially in participants with the minor alleles of haplotype. Protein, carbohydrate, fat, and undigested carbohydrate intakes and smoking status interacted significantly with the 11q23 locus haplotype to modulate 3GO risk. Some Asians, including Koreans, consume a white rice-based diet high in carbohydrates but low in protein and fat, and previous studies have shown that Asian adults on a rice-based diet have a high incidence of MetS ,42,43. D3GO risk is known to be reduced by an undigested carbohydrates-containing diet , as was Tobacco seriously harms cardiovascular health, and thus, helping cardiovascular patients quit smoking and avoid secondhand smoke exposure can improve prognosis, and interestingly, the effect of smoking cessation can be equal or better than single drug treatments for metabolic diseases . Furthern = 58,701). (3) Nutrient intakes were calculated using self-estimates of food intakes determined by the SQFFQ, and thus, some daily nutrient intakes may have been inaccurate. However, the SQFFQ included 103 foods that Koreans consume frequently and demonstrated good validity and reproducibility compared with 12-day food records [The present study was undertaken to explore common genetic variants associated with 3GO risk and the impacts of interactions between their haplotype at the 11q23 locus and intakes of carbohydrates, protein, fats, and undigested carbohydrate, and smoking status for the risk of 3GO and its components. This study shows that the increased prevalence of 3GO in Asian countries might be linked to genetic factors and their interactions with nutrient intake and smoking status. However, this study has several limitations. (1) The study was conducted using a case-control design, and thus, its results cannot establish causal relationships. (2) The study was conducted on an urban hospital-based cohort recruited from several cities without stratification, which means our results may not represent the Korean population, despite the large number of participants recruited  in the 11q23 locus had a 3GO risk. Interactions of haplotype with carbohydrate, fat, protein, and undigested carbohydrate intakes and smoking status influenced 3GO risk. Low intakes of fat, protein, and undigested carbohydrate were positively associated with 3GO risk in individuals with the haplotype’s minor allele. Higher carbohydrate intake and smoking by individuals with the haplotype’s minor allele were also associated with an elevated risk of 3GO. We conclude that individuals having the minor allele of the 11q23 locus haplotype need to prohibit smoking and replace digestible carbohydrates intake into the consumption of undigested carbohydrates (mainly dietary fiber), high-quality protein, healthy fat, and calcium. The results of this study could be applied to prevent 3GO in those at genetically elevated 3GO risk by modifying lifestyles.3GO was found to be associated with high BMIs, waist circumference, elevated fasting serum glucose, total cholesterol, triglyceride levels, systolic and diastolic pressures, and low serum HDL levels in our adult Asian cohort. We also found the haplotype generated from 5 SNPs (


MAGI1 and MAGI2 genetic loci generate a series of long non-coding RNAs (lncRNAs) that act as promoters or suppressors of tumors in a tissue-dependent manner by sponging some sets of miRNAs or by regulating epigenetic processes. This review details current knowledge of paths followed by the three MAGIs to control carcinogenesis.MAGI1, 2, and 3 belong to a subgroup of the MAGUK family of scaffolding proteins, and are comprised of 6 PDZ domains, 2 WW domains, and 1 GUK domain. MAGIs associate with cell surface receptors, junctional complexes, and interact selectively with a wide range of effectors, including the PTEN tumor suppressor, the β-catenin, and YAP1 proto-oncogenes. The regulation of the PI3K/AKT, the Wnt, and the Hippo signaling pathways, on the one hand, the downmodulation of MAGIs in various types of cancers, and its physiopathological significance, on the other, make these scaffolding proteins considered to be tumor suppressors. Interestingly, MAGI1 and MAGI2 genetic loci generate a series of long non-coding RNAs that act as a tumor promoter or suppressor in a tissue-dependent manner, by selectively sponging some miRNAs or by regulating epigenetic processes. Here, we discuss the different paths followed by the three MAGIs to control carcinogenesis.Scaffolding molecules exert a critical role in orchestrating cellular response through the spatiotemporal assembly of effector proteins as signalosomes. By increasing the efficiency and selectivity of intracellular signaling, these molecules can exert (anti/pro)oncogenic activities. As an archetype of scaffolding proteins with tumor suppressor property, the present review focuses on MAGI1, 2, and 3 (membrane-associated guanylate kinase inverted), a subgroup of the MAGUK protein family, that mediate networks involving receptors, junctional complexes, signaling molecules, and the cytoskeleton. MAGI1, 2, and 3 are comprised of 6 PDZ domains, 2 WW domains, and 1 GUK domain. These 9 protein binding modules allow selective interactions with a wide range of effectors, including the PTEN tumor suppressor, the β-catenin and YAP1 proto-oncogenes, and the regulation of the PI3K/AKT, the Wnt, and the Hippo signaling pathways. The frequent downmodulation of MAGIs in various human malignancies makes these scaffolding molecules and their ligands putative therapeutic targets. Interestingly,  The cellular response to external stimuli e.g., hormones, growth factor, cell–cell contact, stress requires the spatio-temporal integration of the several signaling pathways to produce an adapted biological response in terms of metabolism, proliferation, differentiation, cell–cell interaction, migration, or cell death. These processes are orchestrated at specific sites by scaffolding proteins that serve as the backbone for supramolecular signaling complexes termed signalosomes ,2,3,4. Shttp://pawsonlab.mshri.on.ca/index13a5.html accessed on 12 April 2021). Some of these domains are highly selective and have few targets, whereas others might interact with motifs present in a broad range of proteins, e.g., sequences rich in proline amino residues for SH3 and WW domains, the last 4–7 amino acids at the C-terminus of target proteins for PDZ domains (see below), a phosphorylated tyrosine residue for SH2 and PTB domains, phosphorylated serine or threonine residues for 14-3-3 domain. It is worth noting that the sequence inside or surrounding these recognition motifs contributes to the relative selectivity towards the interaction with these domains. Thus, each member of these families might selectively interact with a subset of target proteins. Scaffolding molecules are constituted by the layout of distinct protein interaction modules, e.g., SH3, PDZ for MAGUK family of proteins, or by the multiplicity of the same interaction domain, e.g., 13 PDZ domains for MUP1.At the molecular level, scaffolding proteins can be considered as the modular assembly of domains able to interact with other proteins, nucleic acids , or lipids at the plasma membrane. More than 80 distinct protein interaction modules for intracellular proteins were referenced in Tony Pawson’s website in 2016 . One single PDZ domain may interact with many different PDZ binding motifs. Conversely, one PDZ binding motif may be targeted by distinct PDZ domains. For instance, the C-terminus of β-catenin can interact with more than 25 proteins with the PDZ domain, among them NHERF1, DVL1, SAP97, and TIAM1, and MAGI1B via its domain PDZ5 ,21,22. OIn line with their ability to regulate various transduction pathways, many scaffolding proteins with PDZ domains, including syntenin, disheveled, TIAM1, GIPC, NHERF2, SCRIB, DLG, and MAGIs have been implicated positively or negatively in different steps of cancer development and dissemination. In the present review, we focused on the three MAGIs, as an archetype of scaffolding molecule with antitumor activity.Among the scaffolding molecules with PDZ domains, the membrane-associated guanylate kinases (MAGUKs) are a superfamily of proteins characterized by the inclusion of one or three PDZ domains at the N-terminus, followed by one SH3 domain and then the guanylate kinase (GUK) domain. This GUK domain is catalytically inactive due to the absence of the P-loop that binds ATP. Nevertheless, it allows protein interaction, mainly with cytoskeleton proteins and effectors of signal transduction. Many MAGUK proteins contain other modules, e.g., homologous of CaMKII, WW, or L27 domains . PSD95, The three membrane-associated guanylate kinase inverted (MAGI) proteins constitute a subgroup of MAGUK proteins. They are characterized by an inverted arrangement: the GuK domain is at the NH2 terminus, the SH3 domain is replaced by two WW domains, and follows five PDZ domains –numbered 1 to 5- ,30. A moTwo MAGI2 variants termed (MAGI2α and MAGI2β) depleted of PDZ0 and part of the GUK domain as the result of alternative translational initiation sites have been also identified in the rat brain . Such vaMAGI1 and MAGI3 transcripts have been evidenced in human small intestinal neuroendocrine tumors and breast cancers, respectively . Th. Th26]. Different levels of MAGI tumor suppressor activities are displayed in MAGI gene loci are involved in the control of carcinogenesis, independently of the transcription of the corresponding MAGI scaffolding molecules, their splice variants, and alternative polyA truncated isoforms, but through transcripts that act as competing endogenous (ceRNA) RNAs. CeRNA appears as a novel regulatory mechanism between non-coding miRNA and coding RNA. These long non-coding RNAs (lncRNAs) can function as miRNA sponges, thus inhibiting the degradation of miRNAs target transcripts.Interestingly, it was recently evidenced that MAGI1 gene [On the search of lncRNAs involved in cardiac hypertrophy, Song et al. identified MAGI1-IT1, an intronic transcript 1 of GI1 gene  . Knock-down of the lncRNA in the human gastric cancer AGS and MGC-803 cell lines decreases proliferation in vitro and tumor growth in nude mice .A MAGI1 antisense RNA has been identified, but to our knowledge, no physiological role has been described .Three MAGI2 antisense RNAs have been identified . If no fcell adhesion molecule 2 (CADM2) and PTEN transcripts, leading to upregulation of CADM2, PTEN, caspase 3, and Bax, and reduced level of Bcl-2 [In non-small cell lung carcinomas (NSCLCs), MAGI2-AS3 is downregulated and associated with tumor size, TNM stage, distant metastasis, and shorter overall survival ,214,230.of Bcl-2 . MAGI2-Aof Bcl-2 . InteresIn breast cancer, MAGI2-AS3 is downregulated compared to adjacent control tissues, and this accumulation is negatively correlated with histological grade, TNM stage, ER expression, PR expression, and HER-2 expression . Among tSMG1 knockdown reverses the suppressive function of MAGI2-AS3 in HCC cells [Interestingly, in hepatocellular carcinoma (HCC), MAGI2-AS3 targets miR-374b-5p, and inhibits the proliferation and migration of HepG2, Hep3B, and MHCC-97H HCC cell lines in vitro, and the growth of HepG2 xenografted in nude mice . In thisCC cells . WhetherCC cells  is also CC cells .As observed in NSCLC and breast cancer, downregulation of MAGI2-AS3 in HCC is strongly related to tumor size, lymph node metastasis, TNM stage, and shorter overall survival ,221. FurLikewise, MAGI2-AS3 proved also to be downregulated in gliomas and associated with shorter overall survival . CCDC19 restored the proliferation, migration, and invasion of bladder cancer cells suppressed by MAGI2-AS3 overexpression [An in sillico analysis of expression profiles of lncRNA, miRNA, and mRNA, along with the clinical information of bladder carcinoma patients allowed us to identify MAGI2-AS3 as a key node encompassing 17 miRNA nodes and 46 mRNA nodes. Among the putative miRNA targets, miR-200 and miR-143 are considered tumor suppressors in human bladder cancer . Neverthpression . Tang etpression .PTEN, RECK, Metastasis suppressor protein 1 (MTSS1), Homeobox -A5 (HOXA5) [In the context of epithelial ovarian cancer, specifically high-grade serous ovarian carcinoma, MAGI2-AS3 acts as a tumor suppressor by sponging miR-15b-5p, miR-374a-5p, and miR-374b-5p, and may regulate the expression of their target mRNAs  (HOXA5) . MAGI2-A (HOXA5) .MAGI2-AS3 promoter, leading to lower MAGI2-AS3 accumulation [A polymorphism in MAGI2-AS3 (rs7783388 GG genotype) seems to enhance colorectal cancer risk as a result of decreased binding affinity of glucocorticoid receptor to the mulation . In thismulation . In thismulation .RACGAP1 promoter in an RNA-DNA manner. The demethylation of histone H3 at lysine 4 (H3K4me2) enriched in the RACGAP1 promoter region downmodulates RacGAP, inhibits proliferation, and promotes apoptosis of HEPG2 cells in vitro, and decreases tumor growth in nude mice [LGIR1 promoter. The enhanced expression of LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) might negatively regulate the EGFR, MET, and RET receptor tyrosine kinases [HOXB7 promoter region. The ensuing decreased transcription of HOXB7 inhibits proliferation and radio-resistance of esophageal cancer cell lines in vitro and in vivo [ACY1 (aminoacylase 1) promoter region [Another mechanism for the tumor suppressor activity of MAGI2-AS3 independent of miRNA sponging has also been evidenced in HCC cells . MAGI2-Aude mice . Likewis kinases . The low in vivo . Both MAr region . Restorar region .MAGI2 promoter methylation through TET1 recruitment. Restoration of MAGI2-AS3 expression in the MCF-7 mammary cell line induces MAGI2 expression, leading to the inactivation of AKT and Wnt/β-catenin pathways and impaired cell proliferation and migration [Interestingly in breast cancer cells, MAGI2-AS3 proves to act as a cis-acting regulatory element, decreasing igration .All these studies report the downregulation of MAGI2-AS3 during carcinogenesis and support a role of this lncRNA as a tumor suppressor in adrenal, brain, breast, bladder, liver, lung, colorectal, prostate, pancreatic, ovarian, and renal cancers ,238,239.MAGI2-AS3 [In contrast, MAGI2-AS3 proved to be overexpressed in gastric cancers and nasopharyngeal carcinomas ,228,240.AGI2-AS3 . In the AGI2-AS3 . This efIn cervical cancer, conflicting results have been reported. Wang et al. showed that the expression of lncRNA MAGI2-AS3 was reduced in tumors compared to control tissues and that the promoter region of the lncRNA was hypermethylated . In thisThese dual effects of MAGI2-AS3 as a tumor promoter or suppressor might be related to the cellular context, including tissue origin, the patterns of mRNA/ miRNA expression, the genetic defects, the pathways dysregulated but also the existence of different variants of this lncRNA, -due to different translation initiation sites, and alternative splicing- that might target distinct miRNAs. It appears therefore of major importance to perform the fine analysis of the pattern of expression of these variants in relation to physiopathology.https://clinicaltrials.gov, accessed on 6 July 2021). Such compounds might reactivate MAGI expression silenced by promoter methylation. The recent success of the RNA vaccine demonstrates the technological feasibility of RNA delivery in humans that could be applied to selectively exhaust some oncomiRs which sponge transcripts encoding tumor suppressors, including MAGIs. Decreased methylation in MAGI3 large exon is correlated with the accumulation of the MAGI3pPA pro-oncogenic variant in breast cancers. Thus, targeting RNA m6A demethylases, such as ALKBH5 which is overexpressed in breast cancer and promotes stem cell maintenance [MAGI1 and MAGI2 genomic loci also generate lncRNAs that modulate neoplastic progression and might serve as non-invasive biomarkers of cancer progression [Scaffolding molecules exert a critical role in orchestrating cellular response by increasing the efficiency and selectivity of signal transduction. The emerging role of PDZ domain molecules in carcinogenesis might be related to some redundancy between these different protein families. In this concern, PTEN does interact with the three MAGIs, but also with other scaffolding molecules such as DLG1 and SCRIB. This further highlights the essential role of these scaffolds in the control of signal transduction. Nevertheless, we report here strong evidence concerning the downmodulation of MAGIs in various types of cancers and their physiopathological implications . Biochemntenance ,249,250 ntenance . As far ntenance . Growingntenance ,8. Such ntenance . These pgression ,230. The