IA5 bearing a p-quinoid structure reduced the cytoplasmic level of phosphorylated YAP and the YAP–TEAD complex transcriptional activity and reduced cancer cell growth. IA5 is a promising hit compound for TEAD activity modulator development.The transcriptional regulators YAP (Yes-associated protein) and TAZ  are the major downstream effectors in the Hippo pathway and are involved in cancer progression through modulation of the activity of TEAD  transcription factors. To exploit the advantages of drug repurposing in the search of new drugs, we developed a similar approach for the identification of new hits interfering with TEAD target gene expression. In our study, a 27-member in-house library was assembled, characterized, and screened for its cancer cell growth inhibition effect. In a secondary luciferase-based assay, only seven compounds confirmed their specific involvement in TEAD activity.  Yes1-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are two oncogenic transcriptional co-activators that bind the transcriptional enhanced associate domain (TEAD) transcription factors and are, thus, involved in cell proliferation. In fact, the YAP and TAZ functional activity is implicated in the tumorigenesis of various cancers, including breast , colon , and lunCurrent inhibitors of YAP–TEAD transcriptional activity may act by directly altering the YAP–TEAD interaction or by indirectly interfering with the complex transcriptional activity through modulation of the multiple activators . This caWith the aim of identifying and characterizing new compounds with lead-like properties that could modulate YAP-TEAD activity, in the present study, we followed a repurposing strategy, by selecting and screening a small library of 27 compounds. This library contains preclinical compounds contributed by our academic laboratories in different medicinal chemistry programs. Thus, most of the collection is comprised of lead-like molecules, which offer excellent opportunities for repurposing. A few commercially available compounds, including known modulators of the Hippo pathway, were acquired as an internal reference. Since the biological crosstalk with the YAP/TAZ–TEAD pathway is mediated by PPI +. UPLC-MS purity (UV at 254 nm) 98%.A solution of The physicochemical properties were calculated using the molecular descriptor calculator tool included in the QikProp module of the Canvas 3.2 platform ,28. The k-means clustering method as a function of six molecular descriptors. Consequently, PC score plot was generated [A PCA multivariate statistical method was followed to reduce to a 2D-visual representation the chemical space covered by the compound collection, as a function of the six molecular descriptors using the PCA module of the Canvas 3.2 platform . The twoenerated ,31. Basehttps://rstudio.com/, accessed on 20 September 2021), to visually represent, as a dendrogram, the chemical similarities between molecules. With this aim, it was necessary to use the hclust algorithm, in order to translate the Canvas clustering matrix (csv file) to tree file format [http://itol.embl.de/, accessed on 20 November 2021) for displaying the clustering treemap [A similarity-based clustering analysis was performed to identify putative compound groupings sharing the same chemical core structure using the hierarchical clustering tool in the Canvas 3.2 platform . A single format . Sixteene format . The che treemap ,37.2. All studies were performed in Mycoplasma-negative cells. All cell lines were obtained from ATCC and the European collection of cell cultures, ECACC via Sigma [HaCat cells were maintained in DMEM medium  supplemented with 2 mM L-Glutamine and 10% FBS . A2780 and A2780/CP cell lines were maintained in RPMI medium, containing 10% FBS. HT29, HCT116, and HCA46  were maintained in DMEM medium. Cells were incubated at 37 °C in a humidified atmosphere containing 5% COia Sigma . Vertepoia Sigma . 5 cells per well. After 24 h, cells were exposed to increasing concentrations of the compounds for 18 and 48 h. 3--2,5-diphenyltetrazolium bromide reagent (MTT) (Sigma Aldrich) was added to the cells at a final concentration of 0.5 mg/mL, and then the cells were incubated for 4 h at 37 °C in a humidified incubator with 5% CO2, until purple precipitates were visible. The medium was removed, and the cells were washed once with PBS. To dissolve the formazan precipitates, DMSO was added to each well and the absorbance was read at OD = 535 nm. The drug concentration resulting in 50% inhibition of cell growth (EC50) was calculated. A growth inhibition assay was performed to assess cell viability. Cells were seeded in 24-well plates at a density of 5 × 105 cells/well). The day after, the cells were transiently co-transfected with pGL4 Firefly luciferase ‘experimental’ vector (125 ng) (Promega), in which was inserted a YAP/TEAD response element  at the level of the upstream promoter of luciferase gene; pGL4 Renilla luciferase ‘control’ vector (10 ng) (Promega) and YAP expressing plasmid ‘activator’ plasmid (42 ng), using Lipofectamine 2000 Reagent (Invitrogen). After twenty-four hours from the transfection, the cells were treated with the compounds for 18 h. The concentrations used for the treatment were the IC50 for each compound derived from the cell viability assay at 48 h. Verteporfin was used as a reference compound. The dual luciferase reporter assay was performed according to the manufacturer’s instructions (Promega).Twenty-four hours before the transfection, HaCat cells were seeded into 24-well plate  (Sigma-Aldrich Corporation). The resulting suspension was centrifuged for 30 min, 14,000 rpm at 4 °C. The supernatant was collected, and the protein concentration was determined using Bradford Reagent (Sigma Aldrich). Cell lysates were incubated with 4× SDS sample buffer for 5 min at 100 °C and then were run on SDS-PAGE gels and transferred to PVDF membranes. Western blot analysis was performed using anti-YAP , anti phospho-YAP Ser127 , and anti-Actin . Protein bands were detected by enhanced chemiluminescence . Band density was calculated by using Image J software.Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and then lysed in RIPA Lysis Buffer , 150 mM NaCl, 1% NP-40, 1 mM EGTA, 1 mM Na5 cells/well) and the day after were treated with the compounds for 24 h. Cells were fixed for 10 min in 4% paraformaldehyde (PFA). The fixed cells were permeabilized with 0.3% Triton X-100 in PBS for 2 min on ice. After blocking in 3% BSA for 30 min, slides were incubated with the first antibody diluted in 1% BSA for 1.5 h. After washing with PBS, the slides were incubated with Alexa Fluor 568-conjugated secondary antibodies (1:1000 dilution) for 1.5 h. Nuclei were stained with DAPI for 3 min. Then, the slides were washed and mounted with antifade reagent .HT29 cells were seeded in multi 24-well plates , according to the manufacturer’s instructions. Reverse transcription was performed with 0.5 μg of total RNA using SuperScript first-strand, synthesis for RT-PCR (Invitrogen). The cDNA strand generated was used for PCR amplification in a total volume of 20 μL. Real-time PCR was performed with 100 ng of cDNA using the Power SYBR green PCR master mix (Applied Biosystems). Samples were amplified in a CFX96 Touch™ Real-Time PCR Detection System. Each sample was run independently in triplicate. The amplified were analyzed using Biorad CFX software. For each sample, three replicates were acquired, and Student’s t test was performed to evaluate statistical significance. The amount of target genes expressed was normalized using r18s.t-test. A p-value of <0.05 was considered significant. Analyses were performed using GraphPad Prism v7 .All data were obtained using the mean of three experiments. Statistical analysis was performed by using an unpaired www.string-db.org, accessed on 25 January 2022) and PANTHER . This work was preliminary to the compound selection and is reported in detail in With the goal of identifying YAP-TEAD inhibitors in a timely and cost-effective manner, and tailoring them for further development, we assembled a small chemical library of 27 entries, selected from a collection of about 2000 compounds within the databases of our academic laboratories. We included a few commercial compounds, such as pristimerin, digoxin, deslanoside, and verteporfin with known connection with the Hippo pathway. The chemical structures of the included compounds are reported in Our library includes registered drugs (~7%) and approximately 93% preclinical compounds coming from in-house molecular design expertise and synthesis efforts in various drug discovery projects . Particuhttps://ippidb.pasteur.fr, accessed on 25 January 2022) [All compounds had never been studied in the context of the Hippo pathway. The compounds selected for the library carry structural features such as those reported in the iPPI-DB database containing protein–protein interactions modulators (ry 2022) .2, with between 1 and 28 rotatable bonds, with a mean of 8.4. A chemoinformatic in silico analysis was performed to characterize the physical and chemical properties of the 27-compound collection using the QikProp module of the Canvas 3.2 platform. Verteporfin and dasatinib were added to the library as reference compounds with a known biological profile in YAP–TEAD activity modulation. For each molecule, the calculated descriptors Molecular Weight (MW), AlogP, number of H-bond acceptors (HBA) and H-bond donors (HBD), Topological Polar Surface Area (TPSA), and number of Rotatable Bonds (RBs) were analyzed . The MW Principal component analysis (PCA) was performed for evaluating the chemical space coverage of our molecule collection as a function of the six physicochemical properties previously defined. In PCA analysis, each principal component (PC) is a linear combination of all original variables involved, and PCs are defined in such a way that the variance of the scores is maximal. Based on the screen-plot in ULA26; cluster 2 (persian blue): UCM871, ST11cis, ST11trans, UPR1260, IA5, ACG35, JCM24; cluster 3 (navy blue): EU11, pristimerin, UPR1320, URB694, URB913; cluster 4 (violet-purple): memoquin, ADC3, RS34, PCM129, UPR1355, BAT33, UPR1268, dasatinib, DA15, SVT118, verteporfin; cluster 5 (magenta-cherry-red): digoxin, CDM38, deslanoside. Based on the map-distance, our library spans different regions of the chemical space in the 2D reference frame. In this graph, single dots located at opposing region of the score plot correspond to chemical compounds being strongly dissimilar and occupying different and far away regions of the chemical space. Based on the distribution of PCA scores, it was possible to identify five main compound clusters, occupying similar regions of the chemical space. The five identified clusters are the following: cluster 1 (blue): antipao, http://denovoprofiling.xielab.net, accessed on 20 November 2021), which provides structural identification and chemical space exploration relative to compounds in the public database, i.e., PubChem, and the collected results were very similar (data not shown). The compound collection was then analyzed in details in the following sections [In addition, aiming to identify putative core structures shared by the molecules populating the entire collection, a similarity-based hierarchical-clustering analysis was performed. Sixteen compound clusters were first identified based on functional type and hybridization grade of atoms and bonds, respectively. Among these, four clusters were singlet clusters, while the molecules populating the remaining 12 clusters were further visually inspected, and compound groupings sharing the same chemical core-structure were grouped together: i—3-vinylpyridine; ii—1,3-dimethoxybenzene; iii—2,5-diamino-cyclohexa-2,5-diene-1,4-dione; iv—N-(2-((3-methoxy-phenyl)amino)ethyl)acetamide; v—phenol; vi—chlorobenzene; vii—6,7-dimethoxyquinazoline-2,4-diamine; viii—acridin-9-amine; ix—1H-pyrrol-1-amine; x—1H-indole; xi—5α,14α-androstane; xii—methylcyclohexa-2,4-dienecarboxylate . We alsosections ,64,65,66SVT118 [IA5 [EU11 [CDM38, BAT33, UPR1268) [ACG35) [JCM24) [RS34) [ADC3); carbazole (ULA26) [PCM129, UPR1355, digoxin, deslanoside) [URB694, URB913) [UCM765, UCM871) [DA15) is unpublished, and we report the synthetic procedure below. As depicted in 2 was obtained through the reductive amination of 2-(benzyloxy)benzaldehyde with ethylamine. Then, the nucleophilic substitution reaction between 2 and N-(6-bromohexyl)phthalimide afforded compound 3. Removal of the phthalimide protecting group was performed, with hydrazine leading to the deprotected primary amine 4, which was then reacted with 2,5-dimethoxy-1,4-benzoquinone, providing 1 (DA15) in a good yield (85%). 1′ identity was confirmed by analytical (HPLC), full scan MS (ESI+), and spectroscopic data  ,50,51; sUPR1268) ; thiazol [ACG35) ; diketop [JCM24) ; tetrahy) [RS34) ; dibenzo (ULA26) ; steroidanoside) ,60,61,62 URB913) ,59; anil UCM871) ,64 , inhibited cell growth at concentrations lower than 8 µM. To identify efficient cell growth inhibitors, we screened the chemical library of 27 compounds  with theTT assay . To seleTT assay , we eval at 48 h . Three oDA15, IA5, or quinolines, such as UPR1268, were pinpointed as inhibiting PPI, by abolishing pivotal π-π or π-cation interactions, deemed to be the hot spots for protein–protein interface formation [DA15), has an additional chance of recognizing multiple anionic sites of the protein–protein interface by assuming different protonated conformations.The most active compounds were progressed to further studies . Among tormation , or by bormation . QuinoneUPR1268 is effective against malignancy transformation and progression, through pathways inferred from the literature [Chemical entities belonging to the steroid class, such as the triterpenoid pristimerin  has demoterature  in gefitterature ) can proterature , and decterature . As prevterature , the binterature ; howeverIA5, DA15, memoquin, UPR1268, pristimerin, digoxin, and deslanoside for 18 h, at their IC50 value calculated at 48 h. VP, known to disrupt YAP–TEAD complex at 18 h in uveal melanoma cells, was used as a reference compound in all experiments [We next tested the compounds’ ability to specifically modulate YAP–TEAD activity. We performed a specific gene-reporter assay based on Luciferase signal variation detection . By cloneriments ,71,72. W50 values at nanomolar concentrations. IA5, DA15, memoquin, and UPR1268 inhibited cell growth in all cancer cell lines, showing an IC50 value between 1 and 14 µM. Much evidence highlights that YAP-TEAD activity is essential for cancer initiation and the growth of most solid tumors ,7. To as50 values. The compounds IA5, UPR1268, digoxin, and deslanoside reduced the total YAP and pYAP-Ser127 protein levels, DA15 increased the total YAP protein levels but not its phosphorylated form (pYAP-Ser127), and memoquin enhanced both the total YAP protein and pYAP-Ser127  and connective tissue growth factor (CTGF), were evaluated using quantitative PCR (qPCR), in cells treated for 48 h . We foun results B and the results B–D, suggn of YAP C and incn of YAP A. It is 50 by MTT assay. Among these compounds, we selected seven out of 27, as able to inhibit the cell growth at low concentration. Next, we tested their capability to reduce the YAP–TEAD complex activity in cells, using an luciferase assay. We found that the tested compounds reduced the YAP-TEAD activity with respect to the control cells. It is well known that YAP plays an important role in regulating cell tumor proliferation [IA5 reduced the total YAP protein levels and increased the pYAP-Ser127/YAP ratio in HT29 cells, with respect to controls, indicating that IA5 might inhibit the nuclear YAP translocation, followed by a reduction of YAP-TEAD activity. This might be an important feature for a potential compound capable of targeting the YAP–TEAD complex, inhibiting the cancer proliferation. Moreover, we found that digoxin and deslanoside reduced the total protein YAP, but also the pYAP-Ser127/YAP ratio, suggesting that they might operate similarly to digitoxin, inducing the YAP nuclear retention and enhancing YAP activity [Since the YAP–TEAD complex is an interesting target in cancer, modulating the interaction of YAP/TAZ with transcriptional factor, TEAD, may be an effective strategy for cancer therapy . Currentferation . Consequactivity .IA5, digoxin and deslanoside for 24 h, displayed a drastic reduction of YAP protein when cells were treated with IA5 with respect to control cells, while the two digitoxin derivative molecules (digoxin and deslanoside) increased the YAP protein translocation into the nucleus. Notably, YAP interacts with TEAD in the nucleus and induces expression of genes involved in the regulation of cell growth, proliferation, and survival, including connective tissue growth factor CTGF and cysteine-rich angiogenic inducer 61, CYR61 [IA5 reduced the mRNA level of CYR61 and CTGF1, with respect to control cells. The reduction was comparable with the reference molecule, VP. This result may suggest IA5 as a potential inhibitor of TEAD activity. Quinone moieties are widely distributed in nature and are the key pharmacophore referred to the 2-(4-hydroxyanilino)-naphthoquinone scaffold -naphthoquinone scaffold of IA5 may establish hydrogen bonds, aromatic contacts, and a favorable interaction between the central electron-poor quinone ring and the electron-rich peptidic carbonyls aromatic residues; all recognized as common key motifs in iPPI [IA5 has been reported for its capability to affect the cell cycle [IA5 compound will be explored in future experiments, to guide further medicinal chemistry work, leading to more selective YAP-TEAD activity modulators.The putative interference behind the YAP-TEAD activity inhibition by  in iPPI . While Ill cycle  and for ll cycle , to the


In addition to advanced non-small cell lung cancer, nanoparticle albumin-bound paclitaxel (nab-PTX) may also harbor potential benefit for patients with relapsed small cell lung cancer (SCLC), since weekly paclitaxel (PTX) shows modest activity for relapsed SCLC. We evaluated the efficacy and safety of both weekly nab-PTX and PTX for relapsed SCLC.We retrospectively reviewed 52 consecutive relapsed SCLC patients who were treated with weekly nab-PTX or PTX at our hospital.The response rate, median progression-free survival and overall survival with nab-PTX and PTX were 5.6 vs 8.8%, 3.2 vs 1.7 months, and 5.4 vs 4.5 months, respectively. No statistically significant differences were observed. There was no statistical difference between the 2 groups for ≥Grade 3 adverse events.Weekly nab-PTX and PTX showed similar activity for relapsed SCLC. The toxicity profile of nab-PTX was equally tolerable to that of PTX. Small cell lung cancer (SCLC) accounts for about 14% of all lung cancer.nd-line options for sensitive relapse patients, defined as those with relapse at an interval of 90 to 180 days after the completion of initial chemotherapy.–4 In contrast, treatment options are limited in patients with refractory relapse, defined as no response to initial chemotherapy or relapse within 90 to 180 days after the completion of initial chemotherapy. Based on the data from phase II trials, paclitaxel (PTX), irinotecan, and nivolumab ± ipilimumab (if immuno-checkpoint inhibitor is not used in 1st-line treatment) are recommended for 2nd-line options in the National Comprehensive Cancer Network Clinical Practice Guidelines. Although the efficacy of these agents is modest, toxicity induced by 2nd-line chemotherapy is a clinical issue associated with treatment termination because relapsed SCLC patients often show poor tolerability due to deterioration of performance status. Additionally, some patients have both SCLC and interstitial lung disease (ILD), but irinotecan and immune-checkpoint inhibitors are difficult to use for patients with ILD due to lung toxicity. Therefore, less toxic treatments are sought.However, almost all extensive-stage patients and 75% of limited-stage patients experience a disease relapse after initial therapy. In the treatment of relapsed SCLC, topotecan and cisplatin plus etoposide with irinotecan are the standard 2 Weekly nab-PTX therapy also shows modest activity, with a 31.7% response rate in patients with pre-treated advanced NSCLC in 1 study. In addition to advanced NSCLC, nab-PTX could harbor potential benefit for patients with relapsed SCLC since weekly PTX shows modest activity for relapsed SCLC.,8Nanoparticle albumin-bound paclitaxel (nab-PTX), a nanoparticle conjugate of PTX to albumin, is designed to improve PTX solubility. This results in reduced toxicity from PTX, such as infusion reaction. In advanced non-small cell lung cancer (NSCLC), carboplatin plus weekly nab-PTX has been shown to be equal to carboplatin plus PTX combined with carboplatin, with fewer neurological adverse events (AEs).–14 Based on such evidence, we conducted the current retrospective study to evaluate the efficacy and safety of both weekly nab-PTX and weekly PTX regimens for relapsed SCLC.Several reports of weekly nab-PTX and weekly PTX treatment for relapsed SCLC have described its efficacy and safety, but they are limited cases and has not been fully discussed.22.1This retrospective cohort study was carried out under the principles of the Declaration of Helsinki and approved by the Tosei General Hospital Institutional Review Board, Seto, Japan (IRB No. 789). We retrospectively reviewed consecutive relapsed SCLC patients who were treated with weekly nab-PTX or weekly PTX at Tosei General Hospital between January 2008 and March 2019. The exclusion criteria of this study were inadequate organ function and ECOG-PS of 4. The aim of this study was to evaluate the efficacy and safety profile of both weekly nab-PTX and weekly PTX. Objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were set as efficacy outcomes. All AEs during weekly nab-PTX or weekly PTX therapy were recorded according to Common Terminology Criteria for Adverse Events (version 4.0). The data cut-off date for this analysis was June 30, 2019.Patients were selected according to the following criteria: histologically diagnosed as SCLC, Eastern Cooperative Oncology Group Performance Status (ECOG-PS) of 0 to 3, at least 1 measurable lesion defined by Response Evaluation Criteria in Solid Tumors version 1.1.2.22, days 1, 8, 15/q4 weeks and that of weekly PTX was 80 mg/m2, days 1, 8, 15, 22, 29, 36/q8 weeks, until radiographically confirmed disease progression, unacceptable toxicity, withdrawal, or death. Nab-PTX became available for use in Japan on February 21, 2013, and has been used sequentially at our hospital thereafter.Patients were treated with nab-PTX alone or PTX alone. The treatment regimen of weekly nab-PTX was 80 mg/m2.3We analyzed the subjects’ clinical characteristics, treatment courses, the clinical efficacy of weekly nab-PTX or weekly PTX, and AEs. ORR, DCR, PFS, and OS were set as efficacy outcomes. ORR was evaluated using Response Evaluation Criteria in Solid Tumors version 1.1. OS was defined as the time from the first administration of the chemotherapy until the final visit or death. PFS was defined as the time from the first administration of the chemotherapy to the date of confirmation of disease progression or death.2.4P values of <.05 were considered statistically significant. All statistical analysis was performed using the EZR .Survival curves were prepared using the Kaplan-Meier method and the differences in PFS and OS between weekly nab-PTX and weekly PTX were compared using the log-rank test. Mann-Whitney test and Fisher exact test were used to analyze the difference in the 2 groups. Differences with 2-sided 33.1P = .456). The proportion of patients who had undergone previous thoracic radiation therapy, and those with sensitive relapse in the nab-PTX group were also statistically significantly lower than those in the PTX group . The PTX group patients had more previous chemotherapy regimens than the nab-PTX patients (P = .031).A total of 52 patients with relapsed SCLC were reviewed; 18 and 34 patients received weekly nab-PTX and weekly PTX, respectively. The patients’ characteristics are shown in Table Respiratory comorbidities included 10 (55%) patients with ILD and 4 (22%) with chronic obstructive pulmonary disease in the nab-PTX group, and 16 (47%) patients with ILD and 7 (21%) with chronic obstructive pulmonary disease in the PTX group.3.2P = .999), and the DCR was 56% and 38% (P = .257) (Table P = .353)   with weekly nab-PTX and 1 (range = 1–13 cycles) with weekly PTX. The ORR with nab-PTX and PTX was 5.6% and 8.8%, respectively (–8 cycles3.3P = .758). No acute exacerbation of ILD or treatment-related mortality was observed in either group.The AE profile is summarized in Table 4We examined the efficacy and safety of weekly nab-PTX and weekly PTX for relapsed SCLC. The efficacy of weekly nab-PTX in our study seems to be similar to that of weekly PTX. Of note, in terms of neurotoxicity, weekly nab-PTX showed a favorable profile compared to weekly PTX, consistent with the reported data on nab-PTX in NSCLC.,8 As a result, weekly PTX is considered to be one of the treatment options. Nab-PTX is expected to have the same treatment potential for relapsed SCLC. Naito et al reported on nab-PTX monotherapy (n = 9) for relapsed SCLC in a retrospective study, and found that ORR, DCR, median PFS, and median OS were 11%, 44%, 2.0 months, and 4.0 months, respectively. Another study that included PTX (n = 27) and nab-PTX (n = 4) regimens reported the ORR, DCR, median PFS, and median OS to be 5%, 58%, 2.2 months, and 4.4 months. Recently, a prospective phase II study of nab-PTX in relapsed SCLC showed 8% ORR, 30% DCR, 1.8 months median PFS, and 3.6 months median OS. In our study, survival with both nab-PTX and PTX was comparable to these previous results.The disadvantage of weekly PTX therapy is seen especially in terms of neurotoxicity. Weekly PTX was reported in 2 prospective phase II studies, which showed ORR of 23% to 29%, and median OS of 3.3 to 5.8 months.,14 In a study of weekly PTX, pneumonitis occurred in 1 of 21 (4.7%) patients, but ILD was excluded from the study cohort. In this study, grade 3 pneumonitis was observed in 6% of patients with both nab-PTX and PTX. Acute exacerbation of ILD was not observed, while 10 of 18 patients in the nab-PTX group and 16 of 34 patients in the PTX group had underlying ILD. These results were consistent with those of previous studies on weekly nab-PTX and weekly PTX for relapsed SCLC.The AEs in the present study in each group were acceptable in terms of severity. According to 2 previous studies, both of which included ILD, pneumonitis caused by nab-PTX was 0% to 5%. and 5 of 9 patients  with nab-PTX monotherapy. With weekly PTX regimens, the AE of neuropathy was reported in 12 of 21 patients  and 18 of 24 patients . In the present study, 5 of 18 patients developed neuropathy with the nab-PTX regimen, which were only grades 1–2. Consistent with previous reports, the rate of neurotoxicity profile might be lower with the weekly nab-PTX than with weekly PTX. Collectively, nab-PTX regimens are well tolerated as PTX regimens.In previous studies, neuropathy was seen in 3 of 9 patients 2, was lower than in clinical trials of NSCLC (100 mg/m2). In this study, the dose of nab-PTX was set to be the same as that of PTX. However, it is unclear whether this dose was more appropriate than 100 mg/m2. Thus, in the future it will be necessary to examine whether weekly nab-PTX and weekly PTX are appropriate at a dose of 80 mg/m2. Second, this study was a small-sized, single-center, retrospective study. We think that more cases and more data are needed to examine the efficacy of weekly nab-PTX for relapsed SCLC.This study has some limitations. First, the dose of nab-PTX, which was 80 mg/mIn conclusion, the weekly nab-PTX regimen and weekly PTX regimen showed similar activity for relapsed SCLC. In terms of toxicity, the weekly nab-PTX might be less toxic than weekly PTX. Although this is a retrospective single-center study, nab-PTX might be a treatment option alongside PTX for relapsed SCLC.The authors wish to thank all the study participants and their families.Conceptualization: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Masahiro Morise, Yasuhiko Yamano, Toshiki Yokoyama, Kensuke Kataoka, Yasuhiro Kondoh.Data curation: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Masahiro Morise, Yasuhiko Yamano, Toshiki Yokoyama, Kensuke Kataoka, Yasuhiro Kondoh.Formal analysis: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Masahiro Morise, Yasuhiko Yamano, Toshiki Yokoyama, Kensuke Kataoka, Yasuhiro Kondoh.Funding acquisition: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Masahiro Morise, Yasuhiko Yamano, Toshiki Yokoyama, Kensuke Kataoka, Yasuhiro Kondoh.Investigation: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Yasuhiro Kondoh.Methodology: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Masahiro Morise, Yasuhiro Kondoh.Project administration: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Yasuhiro Kondoh.Resources: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Yasuhiro Kondoh.Software: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Yasuhiro Kondoh.Supervision: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Masahiro Morise, Yasuhiko Yamano, Toshiki Yokoyama, Kensuke Kataoka, Yasuhiro Kondoh.Validation: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Masahiro Morise, Yasuhiko Yamano, Toshiki Yokoyama, Kensuke Kataoka, Yasuhiro Kondoh.Visualization: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Masahiro Morise, Yasuhiko Yamano, Toshiki Yokoyama, Kensuke Kataoka, Yasuhiro Kondoh.Writing – original draft: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Masahiro Morise, Yasuhiro Kondoh.Writing – review & editing: Hajime Oi, Toshiaki Matsuda, Tomoki Kimura, Masahiro Morise, Yasuhiko Yamano, Toshiki Yokoyama, Kensuke Kataoka, Yasuhiro Kondoh.


PPARG). Notably, the expression levels of PPARγ target genes were frequently deregulated in PTC compared to benign tissues and were closely associated with disease-specific survival (DSS) outcomes in a TCGA-PTC cohort. Machine learning-powered personalized scoring index comprising 10 PPARγ targets, termed as PPARGi, achieved a near-perfect accuracy in distinguishing cancers from benign tissues, and further identified a small subpopulation of patients at high-risk across different profiling platforms.Through targeted next-generation sequencing of thyroid cancer-related genes in monozygotic twins with papillary thyroid cancer (PTC), we identified common variants of the gene encoding peroxisome proliferator activated receptor gamma  consisting exclusively of thyroid cancers and normal tissues.In most cases, papillary thyroid cancer (PTC) is highly curable and associated with an excellent prognosis. Yet, there are several clinicopathological features that lead to a poor prognosis, underscoring the need for a better genomic strategy to refine prognostication and patient management. We hypothesized that PPARγ targets could be potential markers for better diagnosis and prognosis due to the variants found in  The worldwide incidence of thyroid cancer has been rising rapidly in the past three decades, with the largest contribution being in papillary thyroid cancer (PTC) in all countries analyzed in a global population-based assessment study . While iBRAF (V600E) and RAS (NRAS involving codons 12 and 61) mutations, RET gene fusions, and PAX8-PPARG gene fusions ..PPARG geARID1A, ALK, MSH2, FN1, KMT2A, TUBA3C, BRAF, STRN, KMT2D, and NF1 (MSH2. They were discordant for missense variants in TUBA3C (p. Tyr262Cys) and BRAF . For TP2, concordant variants were found in BRAF  and STRN , while missense variants in KMT2D (p. Gln827His) were present in only one of the twins. TP3 harbored missense variants in FN1 (p. Asn172Asp) and NF1 (p. Glu836Ala), which was present only in one of the twins who had recurrent cancer. To explore the frequency of nonsynonymous mutations in these identified genes in thyroid cancers, we leveraged cBioPortal-derived MSKCC and TCGA data (see n = 500) and poorly differentiated and anaplastic thyroid cancers . Except for BRAF mutation occurring in 60% and 37% of PTC and ATC, respectively, nonsynonymous variants of ARID1A, ALK, MSH2, FN1, KMT2A, TUBA3C, STRN, KMT2D, and NF1 were found in about 0.2–1.4% and 2.6–6% of patients in TCGA-THCA and MSKCC-ATC, respectively  and was significantly higher than that of normal tissues  and PPARGilow  tumors stratified by the optimal cut-off index determined by Cutoff Finder . Through differential expression (DE) analysis between the two stratified groups, we found a total of 1775 DE genes with fold change (log2-base) > 1 and adjusted p-value < 0.05, as shown in low tumors , which were found to highly express DIO1, TPO, IYD, DIO2, TG, DUOXA2, FOXE1, PAX8, SLC5A5, and DUOX2 .PPARGind DUOX2 H. Additiw tumors I. These w tumors J. In addn = 52), poorly-differentiated thyroid cancer , papillary thyroid cancer , and normal tissues (n = 78). As depicted in UMAP representation , as previously carried out for other major cancer types ,31,32,33entation A, the neentation B.p-value ≤  0.0001), of which PTC showed the highest mean of PPARGi among thyroid cancers  would be comparable to TCGA stratification. DE analysis identified a total of 549 DE genes , which were found to highly express DIO1, TPO, IYD, PAX8, DIO2, FOXE1, DUOXA2, TG, and SLC5A5 , highlighting their robust association with PPARGi-comprising genes  derived from PTC tissues from 14 patients. Seurat (v4.0.1) identified a total of 15 cell clusters in PTC comprising 4045 QC-passed cells, including pericytes, plasma cells, group 1 innate lymphoid cells (ILC), T cells, B cells, endothelial cells, fibroblasts, epithelial cells, and macrophages A. Each cf PPARGi E. GO anaf PPARGi F, consisn = 564) and MMD-THCA  and to a lesser extent in MMD-THCA , as shown in To evaluate the diagnostic performance of expression profiles of PPARGi-comprising genes, we applied different machine learning (ML) algorithms to TCGA-THCA and MMD-THCA data annotated with the origin of tissue see . As PPARWe next aimed to demonstrate the clinical utility of PPARGi genes across different profiling platforms by developing the ML model from RNA-seq-acquired TCGA-PTC data and applying it to the microarray-acquired MMD-PTC data. Notably, a k-nearest neighbors (kNN) model developed from TDM-transformed TCGA-PTC data achieved the best AUC value of 0.942 in classifying TT from NT tissues in MMD-PTC, while the rest of the tested ML models achieved the AUC ranging from 0.890 (SVM) to 0.925 , showing the clinical applicability of PPARGi genes regardless of profiling platform C,D.high and PPARGilow groups, which were found to have significantly different prognosis in terms of DSS , of which four transcripts lack exon 5 and 6, which encode LBD  via a flexible hinge region, which interacts physically with the DNA in PPARγ . It is acode LBD . Skippindiseases .In cancer, the tumorigenic role of PPARγ remains highly controversial. Studies have observed the inhibitory effects of PPARγ-RXRα signaling pathway on tumor growth, angiogenesis, differentiation, and that of production of inflammatory cytokines and tumor invasiveness, suggesting the anti-tumorigenic role of PPARγ in several cancer types including colon, lung, pancreas, prostate, and breast cancer ,45. By cPPARG gene with varying degree of impaired ability in inducing transactivation of target genes [low tumors. In line with the findings supporting the role of PPARγ-RXRα pathway in modulating immune cell-infiltration, genomic alterations of PPARγ-RXRα complex  induced evasion of immunosurveillance and partial resistance to immunotherapies in muscle-invasive bladder cancer [The cancer-related loss-of-function PPARγ mutations have been found predominantly throughout the LBD of the et genes . Here, wr cancer .low PTC tumors might be attributed in part to the suppressed repression of the production of these pro-inflammatory genes in which the expression levels of many of the genes are potential predictors for metastasis or shorted survival time in thyroid carcinoma [Through analysis of microarray- and RNA-seq-generated, patient-derived, tissue-level gene expression and scRNA-seq data, we assessed the expression profiles of diagnostic and prognostic PPARγ targets comprehensively in a cell type-specific manner and identified specific cell populations contributing the most to the computation of PPARGi in PTC. The last decade has seen the emergence of PPARγ as a key regulator of inflammatory and immune responses particularly in monocytes and macrophages, with anti-inflammatory effects in several disease models and clinical studies ,49,50. Tarcinoma ,55.By curating multiple independent GEO datasets comprising a total of 216 patient-derived PTC and PTC-free tissues, we generated a merged PTC-specific MMD annotated with clinical features across different subtypes of thyroid carcinomas, including anaplastic thyroid cancer, poorly-differentiated thyroid cancer, and papillary thyroid cancer. These unified data processed using a uniform R pipeline in this paper source would allow parallel cross-platform analyses with TCGA, as previously shown in other major types of cancer ,31,32,33PPARG gene would induce loss-of-function effects on PPARγ or impaired PPARγ-RXRα signaling pathway. Further, our sample size was small and might only represent a small subset of PTC cohort in Korea, although the expression levels and prognostic performance of PPARGi-comprising genes were extensively validated using public transcriptomic databases. We thus aim to assess the predictive power of PPARGi in a larger validation patient cohort or in a prospectively conducted study. Altogether, the functional PPARGi personalized scoring system may represent a powerful and effective genomic tool to improve patient management in PTC.It remains to be investigated whether the identified intronic variants found in monozygotic twins in the LBD of the


Decreased functional capacity and HRQoL were found in 36%  and 52% , respectively. On average, 8 out of 10 of the patients had returned to work or reported no work impairment. Conclusion: Post-COVID-19 patients may experience persistent respiratory symptoms, fatigue, decreased functional capacity and decreased quality of life up to 6 months after infection. Further studies are needed to establish the extent to which post-COVID-19 effects continue beyond 6 months, how they interact with each other, and to clarify their causes and their effective management.Background: The long-term impact of COVID-19 is still unknown. This study aimed to explore post COVID-19 effects on patients chest computed tomography (CT), lung function, respiratory symptoms, fatigue, functional capacity, health-related quality of life (HRQoL), and the ability to return to work beyond 3 months post infection. Methods: A systematic search was performed on PubMed, Web of Science, and Ovid MEDLINE on 22 May 2021, to identify studies that reported persistent effects of COVID-19 beyond 3 months follow-up. Data on the proportion of patients who had the outcome were collected and analyzed using a one-group meta-analysis. Results: Data were extracted from 24 articles that presented information on a total of 5323 adults, post-infection, between 3 to 6 months after symptom onset or hospital discharge. The pooled prevalence of CT abnormalities was 59% (95% CI 44–73, I The novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has spread rapidly worldwide reaching over 191 million confirmed cases, including 4.1 million deaths as of 21 July 2021 .Evidence from literature on viral pneumonia indicates that many recovered patients have significant lung changes and are affected by acute respiratory distress syndrome (ARDS), which can negatively impact their lung, physical function and quality of life for months or years ,5. PreviIn recent months, the scientific literature has used the term “Long COVID” to describe illness in people who have reported lasting effects of the infection or have had the usual symptoms for far longer than would be expected . AbnormaThis systematic review of the literature was conducted using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . It was A systematic review was performed in the electronic bibliographic databases of PubMed, Web of Science and Ovid MEDLINE on 22 May 2021. The key words and the search strategy used were: (“COVID-19” OR “COVID19” OR “SARS-2” OR “Severe acute respiratory syndrome 2”) AND (“follow-up” OR “follow-up studies” OR “lingering symptoms” OR “persistent symptoms” OR “long-term symptoms”). The search was limited to full text articles published in English. In addition, the reference lists of the selected studies were checked to retrieve relevant publications that were not found with the computerized search.A total of 24 publications met the inclusion criteria of: (1) reporting the effect of COVID-19 on one or more of the following outcomes in adult patients’ lung imaging, lung function, respiratory symptoms, fatigue, functional capacity, activity limitations, overall health, quality of life, or return to work; (2) the mean or median study follow-up was longer than 3 months; (3) the publication was not a meta-analysis or systematic review. The articles selected were appraised by two review authors (RT and YZ) using the tool recommended by the National Heart, Lung, and Blood Institute (NHLBI) for the quality assessment of observational cohort and cross-sectional studies . The systematic search retrieved 5054 references and 13 references were identified from other sources such as automatic email alerts on COVID-related databases, reference lists of identified studies, links shared on social media, suggested by a researcher, etc. After removing duplicates, two researchers (DS and KN) screened 2390 titles and abstracts and read in full the text of 74 articles. Both researchers independently reviewed the articles and selected 24 publications that met the inclusion criteria . PrimaryInformation from the 24 articles is synthesized in 2 index which describes the percentage of variation across the studies in the pooled analysis that is due to inconsistency rather than by chance. The I2 statistic was used to present between-study heterogeneity, where I2 ≤ 30%, between 30% and 50%, between 50% and 75%, and ≥75% were considered to indicate low, moderate, substantial, and considerable heterogeneity, respectively [Review Manager  software was used to pool the individually included studies. The analysis is presented as prevalence based on the presence of the outcome using one-group meta-analysis, and random-effects methods. To investigate heterogeneity between studies, the authors used the Iectively .This review identified a total of 24 articles that explore persistent effects of COVID-19 beyond 3 months of follow-up on patients lung imaging, lung function, respiratory symptoms, fatigue, functional capacity, HRQoL, and/or the ability to return to work ,48,49,50The NHLBI’ Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies was used to evaluate the quality of the 24 studies included in the review. Nineteen studies were rated as “fair” and five rated as “poor” .2 = 96%) (2 = 97%), followed by interstitial thickening or interlobular septal thickening in 33% , parenchymal band or fibrous stripe in 31% , bronchovascular bundle distortion or bronchiectasis in 26% , thickening or adjacent pleura in 11% , and consolidations in 6% . One study reports crazy pavement in 5% of participants a mean of 93 days after discharge from hospital. Residual lung lesions at follow-up were associated with disease severity during the infectious period in some studies [Thirteen articles report the results of chest CT scans in post COVID-19 patients at a mean of 3.8 (SD 1.3) months follow-up ,41,43,452 = 96%) . The mos studies ,41,45 (S2 = 94%) of the patients (2 = 89%) of the patients assessed, followed by restrictive pattern in 12% , and obstructive pattern in 8% . DLCO was lower in hospitalized vs. non-hospitalized patients, however, abnormal values were observed in both groups [Thirteen articles explored the effect of COVID-19 on lung function at follow-up ,43,45,46patients . Impaireh groups ,45. No ih groups ,41.2 = 98%) (2 = 9%), chest pain or tightness by 16% , cough by 13%, , sputum by 12% , and sore throat by 4% . One study reports a higher prevalence of fatigue in patients with severe COVID-19 [Fifteen studies explored persistent post COVID-19 fatigue ,47,49,502 = 98%) , dyspneaCOVID-19 , and anoCOVID-19 . HoweverCOVID-19 ,41,42,442 = 97%) of the patients at a mean of 4.2 (SD 1.3) months follow-up using data from five studies which presented a proportion of decreased functionality assessed with the work productivity and impairment questionnaire (WPAI) [Decreased functional capacity was identified in an average of 36%  ,48, the e (WPAI) ,48, and e (WPAI) ,45 (Suppe (WPAI) . Lower pe (WPAI) ,34,47,49e (WPAI) . 2 = 94%) of the participants at a mean of 4.5 (SD 1.7) months follow-up  months follow-up ,40,44,49Results of this literature review and meta-analysis indicate that post-COVID-19 patients may experience persistent chest CT abnormalities, decreased lung function, persistent fatigue, and respiratory symptoms, decreased functional capacity, and decreased quality of life up to 6 months after symptom onset or hospital discharge. On average, 8 out of 10 patients had returned to work or reported no work impairment at around 3 months of follow-up.Prevalence of chest CT abnormalities and decreased pulmonary function post-COVID-19 aligned with evidence from previous studies, which report abnormal outcomes in patients with viral pneumonias up to 64 months after illness onset ,52. PersHigh prevalence of cough, dyspnea, and fatigue were reported in post COVID-19 patients. Cough and dyspnea can be indicators of impaired lung function  and may Decreased functional capacity was found in one third of post COVID-19 patients at follow-up. CT abnormalities are not associated with functional performance , howeverPatients with severe COVID-19 disease had higher prevalence of chest CT abnormalities ,34,41,45This study helps fill a significant knowledge gap about the lingering effects of COVID-19 on patient outcomes between 3 to 6 months after infection, which is very important considering the large number of COVID-19 patients who will potentially need help managing the impact of the disease beyond the infectious period. Outcomes of this study may help to understand the prevalence of persistent post-COVID-19 effects and their potential causes, which may guide the development of rehabilitation programs tailored to the needs of COVID-19 patients. From a public health perspective, the results of this study may help inform future health care planning and resources allocation. Some factors should be considered when interpreting these findings. First, there is no data available on outcomes studied before the patients contracted COVID-19, therefore, it is unclear whether the abnormalities may have existed before the diagnosis of COVID-19. Second, it is possible that the presence of pre-existent comorbidities may have affected the results of the studies. However, Wong et al.  report tThe results indicate that post-COVID-19 patients may experience persistent respiratory symptoms, fatigue, decreased functional capacity, and decreased quality of life up to 6 months after infection and, on average, around 8 out of 10 of the patients had returned to work at around 3 months follow-up. Outcomes suggest that, in addition to disease severity and lung injury, other potential causes of persistent symptoms, such as post-viral autonomic dysfunction and fatigue syndrome should be assessed and considered when planning post-COVID-19 rehabilitation interventions. This study may guide the development of rehabilitation programs tailored to the needs of COVID-19 patients and inform future health care planning and resource allocation. Further studies are needed to establish the extent to which post-COVID-19 effects continue beyond 6 months, how they interact with each other, and to clarify their causes and their effective management.


Accumulating evidence has indicated that augmented SOCE is related to the generation and development of cancer, including tumor formation, proliferation, angiogenesis, metastasis, and antitumor immunity. Therefore, the development of compounds targeting SOCE has been proposed as a potential and effective strategy for use in cancer therapy. In this review, we summarize the current research on SOCE inhibitors and blockers, discuss their effects and possible mechanisms of action in cancer therapy, and induce a new perspective on the treatment of cancer.Store-operated Ca Cells respond to this depletion of ER intraluminal Ca2+ by opening Ca2+ channels to allow cellular influx, which is also the provenance of “SOCE.” Researchers have been exploring the mechanisms and functions of SOCE since its discovery and definition, and it has been found that stromal interaction molecules (STIMs) and ORAI family proteins are the two major participants in SOCE  is typically activated by ligands of cell surface receptors such as G proteins that activate phospholipase C (PLC) to cleave phosphatidylinositol 4, 5-bisphosphate (PIP2) and produce inositol 1, 4, 5-trisphosphate (IP3). IP3 binds to IP3 receptors (IP3R) on the endoplasmic reticulum (ER) membrane, leading to the release of Ca in SOCE .2+ entry, thereby maintaining basal cytoplasmic Ca2+ levels by preventing uncontrolled activation of ORAI proteins (STIM proteins (STIM1 and STIM2) are located on the ER membrane and are essential for SOCE . STIM1 cproteins .2+ entry during SOCE. Three homologs of ORAI have been identified in humans, namely, ORAI1, ORAI2, and ORAI3 , among which, ORAI1 is the most potent and has been extensively studied. ORAI1 contains four transmembrane helices (TM1-TM4), an intracellular location domain  and an extracellular location domain (including an epitope tag introduced into the TM3-TM4 loop). The N-terminus and C-terminus of ORAI1 intracellular sites are essential for the interaction with STIM1 and the opening of the ORAI1 channel (ORAI proteins are plasma membrane (PM) channels that can be gated by STIMs for Ca channel . The hom channel , and the channel , leading channel .2+ decrease in the ER, it initiates conformational changes and oligomerization, overcoming the intracellular autoinhibition mediated by CC1 to expose SOAR/CAD and the PB C-tail. Activated STIM1 multimerizes and migrates toward the PM, where it interacts with the intracellular regions of ORAI1, resulting in ORAI1 activation and Ca2+ influx  contain similar structures, consisting of six transmembrane-helical domains (TM1-TM6) with a loop between TM5 and TM6 and cytoplasmic N- and C-termini . TRP chaactivity . Howeveractivity . Furtherchannels . Therefo+ and CD8+ T cells. For example, the inhibition of SOCE by genetic deletion of ORAI1, STIM1, or STIM2 in murine CD4+ T cells impaired Th17 cell function, causing a decrease in the production of IL-17, which is believed to be proinflammatory factor important for tumor progression , the Ca2+ influx that is independent of depolarization and caused by receptor occupation, leading to a significant increase in [Ca2+]i biologically, with no impact on internal Ca2+ release in platelets, neutrophils or endothelial cells (2+ entry (CCE) in astrocytes  , it activated SOCE pathway, while at high concentrations (≥10 µM), it blocked SOCE pathway completely  . Subsequmpletely . The mecmpletely . Moreovempletely . 2-APB ampletely . It was mpletely , howevermpletely . Among tmpletely .in vivo by inhibiting VEGF production and endothelial tube formation through the blockage of SOCE , nasopharyngeal carcinoma (NPC), prostate cancer, ovarian cancer, breast cancer, glioblastoma, lung cancer, melanoma, and cervical cancer . For ins of SOCE . In anotutophagy .These effects suggest that 2-APB is attractive as a potentially potent therapy for primary cancer and metastatic cancer.2+ channels, TRPC channels, or Ca2+ pumps when exerting maximal inhibitory effect on Ca2+ entry , both DPB-162AE and DPB-163AE could facilitate Orai3 currents, and at high concentrations (>300 nM), they transiently activated ORAI3 currents and then deactivated them. DPB compounds have been proven to activate ORAI3 in a STIM1-dependent manner, but they could not change the pore diameter of ORAI3, which is different from the mechanisms of 2-APB. It is speculated that because they are larger than 2-APB, DPB compounds are unable to enter the pore of ORAI3 . In addihibition .Although there have been no studies on DPB compounds with respect to cancer treatment to date, considering the specific inhibition of SOCE, DPB compounds are expected to be developed as potential anticancer drugs.2+-activated cation channel that decreases Ca2+ influx by depolarizing lymphocytes, is the main mechanism for the suppression of cytokine release. Furthermore, it has been reported that the mechanism of inhibiting TRP channels, such as TRPC3 and TRPC5, involved in reducing their open probability rather than changing their pore properties without affecting the other Ca2+ signals in T cells including Ca2+ pumps, mitochondrial Ca2+ signaling and ER Ca2+ release  signal transduction cascade . BTP2 al release .BTP2 has exhibited inhibitory effects on several types of allergic inflammation, including autoimmune and antigen induced diseases through the suppression of cytokine release  and T cell proliferation . AlthougIn summary, the mechanism and effect of BTP2 on cancer are multi-aspect, and it is necessary to carry out further research on them for clarification.2+-dependent signaling pathways, and structure-function relationship studies showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3 -MAPK signaling cascade , and cytokines  in human lung mast cells (HLMCs), and inhibit the expression of T-bet and the production of IL-2, IL17, and IFN-γ in lamina propria mononuclear cells (LPMCs) and biopsy specimens obtained from inflammatory bowel disease (IBD) patients  mouse model . These mAbs potently inhibited the SOCE, NFAT translocation and cytokine secretion from Jurkat T cells and in human whole blood . Anotherse model , suggest2+ influx and related signaling processes, such as receptor-associated tyrosine phosphorylation, arachidonic acid generation, and nucleotide biosynthesis, CAI displayed effective anticancer effects, including antiproliferative, antimigratory, antiangiogenic and antimetastatic properties, in a broad range of human tumors, including melanoma, glioblastoma, head and neck squamous cell carcinoma (HNSCC), hepatoma, small cell lung cancer (SCLC), chronic myelogenous leukemia (CML), and ovarian, breast, colon and prostate cancer (+ T cells or stimulating the IDO1-Kyn metabolic circuitry (CAI (carboxyamidotriazole), also called L-651582, is one of the SOCE inhibitors that has been evaluated in clinical trials. It was initially developed as a coccidiostat and was confirmed to inhibit calcium influx, including SOCE, RMCE and VDCE . Throughe cancer . Moreoveircuitry .As a potential anticancer drug, CAI has been tested in phase I/II/III clinical trials, some of which showing that CAI could stabilize and improve the condition of patients with pancreaticobiliary carcinomas, melanoma, non-small cell lung cancer, epithelial ovarian cancer, prostate cancer, glioblastoma and other anaplastic gliomas while exhibiting a limited toxicity profile . However2+ oscillations in a dose-dependent manner, leading to the inhibition of NF-κB/p65 translocation to nuclei, thus impeding the proliferation and in ESCC xenograft tumor growth sulfonyl-1,4-diazepane) was initially described as an inhibitor of myosin light chain kinases (MLCKs) that binds at or near the ATP-binding site at the active center of kinases with or without Calmodulin . Shortly+ stores , and it + stores . Further+ stores , causing+ stores . In anototherapy .2+ influx in Jurkat cells in a dose-dependent manner (NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid), frequently used as a blocker of chloride channels, could also reduce CCE in endothelial cells . It was t manner . In ovart manner .2+ influx by binding to the C-terminus of ORAI1 directly to perturb the interaction between STIM1 and ORAI1 by reducing the affinity of ORAI1 for STIM1. Through SOCE inhibition, AnCoA4 blocked T cell activation and inhibited the T cell-mediated immune response in vitro and in vivo, indicting the possibility that it can be used in therapeutic areas, including immunomodulation, inflammation and cancer -8,8-dimethylpyranochromen-4-one) was identified by screening small-molecule microarray (SMM) using minimal functional domains of STIM1 and ORAI1, and it was found to inhibit Cad cancer .+ T cells, resulting in a decrease in TCR-triggered gene expression, cell proliferation and cytokine production in T cells -3,6-dihydro-2H-pyridin-5-yl]pyrazin-2-yl]benzamide) could specifically block SOCE by inhibiting the IP3-dependent CRAC current in native RBL-2H3 cells, CHO cells with STIM1/ORAI1 overexpression and human primary CD4 T cells .2+ influx and NFAT nuclear entry through an automated high-content screening platform and it was found to exhibit potent anti-tumor activity against glioblastoma. It could bind to HSP90 directly and antagonize its calcineurin-chaperoning effect to reduce NFAT nuclear translocation and inhibit other key proto-oncogenic pathways, including hypoxic and glycolytic pathways and the PI3K/AKT/mTOR axis, thus leading to cell cycle arrest in G2/M phase of glioblastoma and promoting apoptosis and inhibition of tumor cell proliferation and migration (YZ129 (4-(isoquinolin-6-ylamino)-naphthalene-1,2-dione) was identified because of its inhibition of thapsigargin-triggered Caigration .MRS-1844 (DHPs-32) and MRS-1845 (DHPs-35) are compounds in the 1,4-dihydropyridine (DHP) family, and they were confirmed to inhibit store-operated calcium channels and reduce voltage-dependent L-type calcium channels. In addition, their potency in SOCE inhibition was much smaller than that of common SOCE inhibitors (the reported order is Synta66 > 2-APB > GSK-7975A > SKF96365 > MRS1845) .2+ influx through SOCE indicates the importance of learning more about their pharmacological properties and mechanisms, and the potential to extend their clinical indications.In addition to the abovementioned compounds targeting SOCE, other compounds that have been approved for clinical use in corresponding diseases were confirmed to inhibit SOCE. The ability of these compounds to inhibit Ca2+ influx in a variety of cell types without affecting the whole-cell monovalent cation current mediated by TRPM7 channels. Trans-stilbene, a close structural analog of DES that lacks hydroxyl and ethyl groups, had no effect on CRAC current, further suggesting the specificity of DES for SOCE -4-(4-hydroxyphenyl)-hex-3-en-3-yl]-phenol) is a potent synthetic estrogen used for estrogen therapy in prostate and breast cancer. It was reported to inhibit SOCE and Cafor SOCE .2+ uptake and promotes the Ca2+-dependent inactivation of SOCE, thus inhibiting the proliferation of HT29 cells  are commonly used to relieve pain, fever and inflammation. Epidemiological studies worldwide have demonstrated that NSAIDs also have cancer-protective effects, as these drugs are associated with a reduced risk of various types of cancer. In colon cancer, it has been reported that NSAIDs could exert antiproliferative effects through SOCE inhibition, and salicylate, the main aspirin metabolite, is considered a mild mitochondrial uncoupler that prevents mitochondrial Ca29 cells . Another29 cells .2+ channel blocker that was initially developed as a cardiovascular drug, was recently found to inhibit SOCE by blocking ORAI channels in a dose-dependent and reversible manner to significantly inhibit cell proliferation, induce cell apoptosis and arrest cell cycle in S and G2/M phases in HEK-293T-REx cells -2-[2-[3-(1H-benzimidazol-2-yl)propyl-methylamino]ethyl]-6-fluoro-1-propan-2-yl-3,4-dihydro-1H-naphthalen-2-yl]2-methoxyacetate), a T-type CaEx cells .2+ signaling is involved in almost all cellular activities in organisms. As a major route of Ca2+ entry in mammalian cells for replenishing the depleted intracellular Ca2+ store, SOCE regulates a diverse array of biological processes. Accumulating evidence has shown that STIM/ORAI-mediated SOCE is excessive in cancer tissues, and it is becoming clear that augmented SOCE promotes the malignant behavior of cancer cells, including tumor growth, angiogenesis, and metastasis. Therefore, SOCE could be a potential therapeutic target for the treatment of cancer.Ca2+ entry, cells could adopt other ways to obtain sufficient Ca2+ even after complete SOCE inhibition. Under this condition, drug combinations could be considered. Furthermore, due to the universal roles of Ca2+ signaling in cells, the cytotoxicity of SOCE inhibitors on normal cells and some anticancer immune cell inhibitors should also be considered in clinical anticancer applications. Developing SOCE inhibitors that could specifically target tumor tissues in certain circumstances is a hopeful therapeutic orientation toward cancer in the future.As we summarized above, multiple compounds targeting SOCE have been developed and their efficiency in the inhibition of proliferation and migration of cancer cells has been evaluated. However, rare SOCE inhibitors have been approved for clinical use in cancer treatment due to their poor selectivity, which urgently needs to addressed. In addition, as SOCE is not the only channel for Ca


The liver plays a pivotal role in nutrient/energy metabolism and storage, anabolic hormone regulation, ammonia detoxification, and cytokine production. Impaired liver function can cause malnutrition, hyperammonemia, and chronic inflammation, leading to an imbalance between muscle protein synthesis and proteolysis. Patients with chronic liver disease (CLD) have a high prevalence of sarcopenia, characterized by progressive loss of muscle mass and function, affecting health-related quality of life and prognosis. Recent reports have revealed that osteosarcopenia, defined as the concomitant occurrence of sarcopenia and osteoporosis, is also highly prevalent in patients with CLD. Since the differentiation and growth of muscles and bones are closely interrelated through mechanical and biochemical communication, sarcopenia and osteoporosis often progress concurrently and affect each other. Osteosarcopenia further exacerbates unfavorable health outcomes, such as vertebral fracture and frailty. Therefore, a comprehensive assessment of sarcopenia, osteoporosis, and osteosarcopenia, and an understanding of the pathogenic mechanisms involving the liver, bones, and muscles, are important for prevention and treatment. This review summarizes the molecular mechanisms of sarcopenia and osteosarcopenia elucidated to data in hopes of promoting advances in treating these musculoskeletal disorders in patients with CLD. Sarcopenia is a syndrome characterized by decreased muscle mass and function  ,2,3,4,5.Several studies revealed a close relationship between sarcopenia and osteoporosis in community-dwelling older adults. This finding has fueled the concept of osteosarcopenia, defined as the concomitant occurrence of sarcopenia and osteoporosis ,12,13,14The assessment criteria for sarcopenia have changed over time and vary among different working groups. In our study of 142 patients with LC, we compared the diagnostic performance of sarcopenia using the JSH, AWGS , and EWGSOP2 criteria . AccordiIn general, the prevalence of sarcopenia increases with the progression of CLD stage . HoweverIn general, age-related loss of skeletal muscle mass occurs at a rate of 1% per year up to 70 years of age and increases to 1.5% after that. However, among patients with LC, those categorized as Child–Pugh class A, B, and C have a rate of 1.3%, 3.5%, and 6.1%, respectively . In largSkeletal muscle is the most major reservoir for protein. Under normal physiological conditions, skeletal muscle mass and protein turnover are maintained through a controlled balance between protein anabolism (synthesis) and catabolism (breakdown or proteolysis). Decreased protein synthesis and increased proteolysis cause muscle loss. Although sarcopenia is a common complication in patients with LC, it is caused by the interrelationship of multiple factors, such as attenuated protein anabolism due to impaired hepatic function, increased gluconeogenesis due to depleted glycogen reserves, excessive muscle protein breakdown, disease-related malnutrition, hyperammonemia due to impaired detoxification ability, reduced levels of anabolic hormones , and increased levels of inflammatory and catabolic cytokines ,40,41,42IGF-1  is produced by hepatocytes and myocytes. Its production is stimulated by growth hormones. IGF-1 has various paracrine, autocrine, and endocrine functions. The multimodal functions of IGF-I include the positive regulation of skeletal muscle growth ,38,39,41Serum IGF-1 levels, which decrease with age and progression of liver disease stage, are especially low in patients with LC ,43. Low mTOR is a pleiotropic protein kinase that forms two distinct multicomponent complexes (mTORC1 and mTORC2). These mTOR complexes sense growth factors , nutrients , and cell stressors  and regulate cell growth (increase in cell mass/size), proliferation, metabolism, protein synthesis, autophagy, and survival ,37,40,45In skeletal muscle cells, mTORC1-mediated signaling promotes protein synthesis by activating the ribosomal protein S6 kinase 1 (S6K1) and inactivating the eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1) . S6K1, pMeanwhile, mTORC2 demonstrates non-overlapping functions with mTORC1. Moreover, mTORC2 does not regulate protein synthesis and does not respond to the cellular nutrient/energy status. However, mTORC2 plays a key role in cell proliferation and survival via the activation of AKT.The rate of protein synthesis is directly dependent on amino acid uptake. Amino acid sufficiency stimulates mTORC1 more directly compared to the aforementioned TSC-mediated pathway ,37,45. ABCAAs  are essential amino acids that the body cannot produce and must be obtained from dietary protein. BCAAs are involved in the formation and maintenance of the skeletal muscle . As suchInsufficient cellular energy attenuates mTORC1 signaling ,40. ElevPatients with CLD  have disease-related malnutrition, characterized by a combination of phenotypical  and etiological criteria . They are predisposed to decreased glycogen storage, synthesis, and breakdown, promoting increased gluconeogenesis using amino acids derived from myoprotein breakdown and decreased skeletal muscle mass ,41. SuchMyostatin, a transforming growth factor beta (TGF-β) superfamily cytokine, is one of several myokines produced and released by skeletal muscle cells, and serves as a key inhibitor of skeletal muscle growth  ,42. IGF-LPL), and downregulates lipogenesis (SREBP1c) and glycolysis (PDK4). Meanwhile, the SMAD2/3 complex-suppressed AKT/mTOR signaling pathway promotes the inhibition of skeletal muscle growth. The activation of SMAD pathway signaling inhibits the proliferation of satellite cells (tissue stem cells) and differentiation of myoblasts into myotubes by suppressing MYOD1, MYF5, and MYOG in a SMAD3-dependent manner [Myostatin signaling phosphorylates the SMAD2/3 complex via the activin receptor type IIB (ActRIIB), ubiquitously expressed on skeletal muscle cells . The actt manner .In patients with LC, excessive muscle autophagy is mediated by hyperammonemia and oxidative stress or promoted via the upregulated UPS due to increased levels of proinflammatory cytokines  and interleukin-6 (IL-6)), which can cause skeletal muscle loss and sarcopenia ,53,56,57Ammonia is normally detoxified via conversion into urea through the urea cycle in the liver. It can also be alternatively detoxified using glutamine synthetase in the skeletal muscle. In patients with LC, the skeletal muscle compensates for the decreased hepatic ability to detoxify ammonia. Therefore, hyperammonemia occurs when ammonia production exceeds the skeletal muscle’s and liver’s detoxification capacity. Hyperammonemia increases the expression of myostatin by activating nuclear factor kappa B (NF-κB) ,61. In iPhysical activity and exercise are critical determinants of muscle anabolism, particularly in patients with LC . RegularTestosterone is an anabolic–androgenic steroid hormone synthesized from cholesterol, mainly in the gonad and adrenal gland ,68. CircLC disturbs the hypothalamic–pituitary–gonadal axis, causing hypogonadism (low testosterone), although the mechanism has yet to be fully understood and is probably multifactorial ,73,74. PNutrition- and inflammation-related disorders are caused by prolonged acute and chronic diseases and lack of nutrient intake and/or absorption, promoting compromised body composition and function  ,41,48,75Dysgeusia and hyposmia are frequently noted in advanced liver diseases and may diminish nutrient intake, promoting nutrient deficiency. Zinc insufficiency or deficiency has been associated with altered senses of taste and smell as well as dysorexia . MalabsoIn 2009, Binkley and Buehring advocated the concept of sarco-osteoporosis, which was then defined as the concomitant occurrence of sarcopenia and osteoporosis and has now evolved into the term “osteosarcopenia” . PatientAs shown in Given that muscles and bones are closely related during their development and growth, it is conceivable that sarcopenia, osteoporosis, and osteosarcopenia often progress in conjunction with each other ,16,17,18Increasing the mechanical load on the skeletal muscles leads to protein synthesis and muscle hypertrophy, while the opposite causes muscle atrophy . The maiGLYAT) gene, which is essential for the regulation of glucose and energy metabolism, were associated with bone size and muscle mass [METTL21C as a pleiotropic gene for bones and muscles [METTL21C is highly expressed in muscles and plays an important role in myoblastic differentiation, calcium homeostasis, and survival of osteocytes against apoptosis through the modulation of NF-κB signaling [MEF-2C) and α-actinin 3 (ACTN3) are candidate genes with a pleotropic effect on bones and muscles [MEF-2C results in increased bone mass [ACTN3 is highly expressed in fast glycolytic muscle fibers and contributes to the differentiation of muscle fibers toward the fast-twitch type [ACTN3 is expressed in osteoblasts, and the deletion of ACTN3 reduces bone mass [During embryogenesis, muscles and bones originate from a common mesenchymal precursor, and their development is controlled by common genes and growth factors . Therefocle mass . A subse muscles . METTL21ignaling ,93. In a muscles . MEF-2C  muscles . MEF-2C one mass . ACTN3 itch type . Additioone mass .The production of reactive oxygen species (ROS) and proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α, is increased in chronic disease conditions, including CLD ,100. ChrSkeletal muscle cells secrete various endocrine molecules, such as IGF-1, myostatin, irisin, beta-aminoisobutyric acid (BAIBA), fibroblast growth factor 2 (FGF2), IL-6, IL-7, IL-15, and osteoglycin, which influence bone metabolism ,17,18. I−/−) mice showed reduced muscle mass, and treatment with exogenous osteocalcin increased muscle mass in older mice, whereas deletion of osteocalcin resulted in increased bone mass [−/− mice demonstrated that osteocalcin was not involved in the regulation of bone quantity and muscle mass [2+-induced muscle force production [As bones secrete various substances that affect other organs, they may also be considered as endocrine organs. Bones secrete various osteokines such as osteocalcin, sclerostin, Wnt, TGF-β, fibroblast growth factor 23 (FGF23), and prostaglandin E2 (PGE2), which affect the metabolism of not only bones but also muscles ,16,17,18one mass ,118. In cle mass . Therefocle mass ,121. Sclcle mass . In paticle mass . A Koreacle mass . Howevercle mass . Therefocle mass , inducescle mass . In a mooduction .2C12 skeletal muscle cells reported that vitamin D promoted the differentiation of myogenic cells by increasing the expression and nuclear translocation of the vitamin D receptor (VDR) and modulating promyogenic and antimyogenic factors [The liver plays a crucial role in vitamin D metabolism. When the liver’s function is impaired, bone and muscle homeostases are dysregulated through vitamin D metabolism ,130. Vit factors . VDR kno factors . Reporte factors . In one  factors . Our stu factors . These fOptimal physical exercise has positive effects on skeletal muscle and bone mass. In one study of community-dwelling older adults with osteopenia or increased fall risk, a 12-month supervised, multicomponent exercise program consisting of progressive resistance, weight-bearing impact, and balance training thrice a week improved the bone mineral density (BMD), muscle strength, and physical function of the patients . In anotAs described above, BCAAs activate the mTOR pathway for the proliferation of satellite cells, thereby promoting muscle protein synthesis and growth. A systematic review revealed that administration of leucine or leucine-enriched proteins mainly increases lean muscle mass, thereby improving sarcopenia in older adults . In patiOsteoporosis is caused by impaired bone homeostasis due to an imbalance between bone remodeling and resorption. Pharmacological treatment for osteoporosis includes anti-resorptive , anabolic , anti-sclerostin , and hormonal agents  . The rec


However, the association of the PBRM1 immunohistochemical expression with histomorphological features of ccRCC and the endothelial expression of tumor vasculature, which is an important role of the tumor microenvironment related to treatment response, is little known. Recently, our research team has established a vascularity-based architectural classification of ccRCC correlated with angiogenesis and immune gene expression signatures, which could provide prognostic information and function as a surrogate for treatment selection. In the present study, we found the PBRM1 expression was correlated with the architectural patterns. Furthermore, we demonstrated that endothelial expression tended to be lost in cases with low PBRM1 expression. This correlation implied the orchestrated expression of PBRM1, raising the possibility that the cancer cells and their microenvironment interact in ccRCC.The PBRM1 protein, whose gene is the most frequently mutated one in clear cell renal cell carcinoma (ccRCC) following p < 0.001). PBRM1 loss in both cancer and endothelial cells was associated with a lower recurrence-free survival rate (p < 0.001). Our PBRM1 expression profile indicated that PBRM1 expression in both cancer and endothelial cells may be regulated in an orchestrated manner.Loss of the polybromo-1 (PBRM1) protein has been expected as a possible biomarker for clear cell renal cell carcinoma (ccRCC). There is little knowledge about how PBRM1 immunohistochemical expression correlates with the histomorphological features of ccRCC and the endothelial expression of tumor vasculature. The present study evaluates the association of architectural patterns with the PBRM1 expression of cancer cells using a cohort of 425 patients with nonmetastatic ccRCC. Furthermore, we separately assessed the PBRM1 expression of the endothelial cells and evaluated the correlation between the expression of cancer cells and endothelial cells. PBRM1 loss in cancer cells was observed in 148 (34.8%) patients. In the correlation analysis between architectural patterns and PBRM1 expression, macrocyst/microcystic, tubular/acinar, and compact/small nested were positively correlated with PBRM1 expression, whereas alveolar/large nested, thick trabecular/insular, papillary/pseudopapillary, solid sheets, and sarcomatoid/rhabdoid were negatively correlated with PBRM1 expression. PBRM1 expression in vascular endothelial cells correlated with the expression of cancer cells (correlation coefficient = 0.834,  In addifunction , novel sfunction ,5. Howevfunction .PBRM 1 gene encoding the protein polybromo-1, which is a subunit of the SWI/SNF chromatin remodeling complex, is a second major ccRCC cancer gene, following the VHL gene [PBRM1 is considered not only a key driver gene of ccRCC but also a key regulator of tumor cell-autonomous immune response in ccRCC, the influence of PBRM1 loss on the response to immune checkpoint inhibitors (ICIs) has been investigated [Recent genomic advances using exome sequencing revealed that the VHL gene ,8. SeverVHL gene ,10. SubsVHL gene . Furtherstigated ,12,13.Recently, we first demonstrated that histological phenotypes, such as clear or eosinophilic types, were significantly correlated with survival outcomes and response to TKIs and ICIs in patients with ccRCC, which could be applied as a predictive marker for treatment selection . AdditioTo evaluate the association of the PBRM1 expression with histomorphological features, we semiquantitatively re-evaluated the expression by using the PBRM1-stained slides used in our previous study . In addiThis study was performed under the institutional review board’s approval at Kansai Medical University Hospital (No. 2018109 and No. 2020222). As in our previous report , data foAll histomorphological features were evaluated by C.O., blinded to clinical outcomes, using whole-tissue sections of H&E-stained slides. Histological phenotype, based on cytoplasmic features, such as clear, mixed, or eosinophilic, and vascularity-based architectural classification, based on nine architectural patterns, such as compact/small nested, macrocyst/microcystic, tubular/acinar, alveolar/large nested, thick trabecular/insular, papillary/pseudopapillary, solid sheets, and sarcomatoid and rhabdoid, were determined at the highest-grade area as previously described .As previously described ,23,24, Tp < 0.05 was considered statistically significant. A Chi-squared test for categorical variables was used to evaluate the statistical significance among two or more groups. The t-statistic in linear regression analysis and one-way ANOVA analysis were used to evaluate the statistical significance among the architectural patterns. Interobserver agreement was statistically assessed using kappa statistics. Correlations between the two variables were evaluated using Spearman’s rank correlation test. Recurrence-free survival  was assessed using the Kaplan–Meier method with the log-rank test.Statistical analyses were performed using EZR version 1.54  . A two-sThe median age of the patients was 65 years . The male to female ratio was 2.8:1 . The rate of TNM stage III or IV, WHO/ISUP grade 3 or 4, and the presence of necrosis was 24.0% (102/425), 32.3% (137/425), and 15.3% (65/425), respectively. Of the 425 patients, 57 (13.4%) experienced a recurrence of ccRCC during a median follow-up of 62.6 months .Cases with PBRM1 loss and PBRM1 retention were observed in 148 (34.8%) and 277 (65.2%) patients, respectively. The interobserver variability showed good agreement between the two pathologists (kappa = 0.84). The PBRM1 expression of clinicopathological factors is shown in p < 0.001; p < 0.001; Loss of PBRM1 expression was significantly associated with worsened pathological prognostic factors, such as TNM stage, WHO/ISUP grade, and the presence of necrosis  of compact/small nested, 1/36 (2.8%) in macrocyst/microcystic, 7/63 (11.1%) in tubular/acinar, 20/47 (42.6%) in alveolar/large nested, 37/55 (67.3%) in thick trabecular/insular, 10/20 (50%) in papillary/pseudopapillary, 8/9 (88.9%) in solid sheet, and 15/18 (83.3%) in sarcomatoid/rhabdoid patterns . Represep < 0.001), tubular/acinar , and compact/small nested  were positively correlated with the PBRM1 expression although compact/small nested was not statistically significant. On the other hand, thick trabecular/insular , sarcomatoid/rhabdoid , solid sheets , alveolar/large nested , and papillary/pseudopapillary  were negatively correlated with the PBRM1 expression. To evaluate the correlation between architectural patterns and PBRM1 expression (H-score), multiple linear regression analysis was performed . Macrocyp < 0.001, p < 0.001, and p < 0.05, respectively) (Of 403 cases where two cores were assessed for PBRM1 expression (22 out of 425 cases were missing one core), 77 (19.1%) showed heterogeneity of PBRM1 expression (H-score ≤ 20 vs. >20) between cores. Therefore, we examined whether PBRM1 expression was correlated with the architectural patterns of the corresponding area by assessing a total of 828 cores. It was revealed that PBRM1 expression was correlated with the architectural patterns among all of the evaluated cores. Notably, this association between PBRM1 expression and the architectural patterns assessed in the highest-grade area, namely, macrocyst/microcystic, tubular/acinar, and compact/small nest, had significantly higher PBRM1 expressions (H-score) compared to the other patterns (ctively) . p < 0.001; A positive correlation between PBRM1 expression in cancer cells and endothelial cells was confirmed  compared to the other patterns , which sPBRM1 mutation status of ccRCC has also been investigated [p = 0.035) [The role of PBRM1 protein expression for clinical decisions is not only being a biomarker of prognostic prediction but also providing information on molecular mechanisms and potential therapeutic targets. In the present study, we showed the prognostic predictive ability of PBRM1 loss in nonmetastatic ccRCC, while Cai et al. also showed that PBRM1 could improve the predictive accuracy for survival outcomes of metastatic RCC patients treated with tyrosine kinase inhibitors (TKIs) . Recentlstigated ,35,36,37stigated ,35, datastigated ,36,37. Astigated . We have= 0.035) .PBRM1 mutations were associated with increased angiogenesis, decreased immune infiltrates, and poor response to ICIs [PBRM1 mutation does not directly determine the loss of the corresponding protein or function [PBRM1 mutation and PBRM1 IHC expression have been reported, a comprehensive investigation, including PBRM1 mutation, PBRM1 expression, and histomorphological features, should be conducted. Recently, Lin et al. evaluated the influence of PBRM1 loss for treatment response, focusing on the “immunogenic” tumor microenvironment [Contrary to our findings, however, some studies showed that  to ICIs ,37. Whilfunction . Becauseironment . Howeverironment ,39. NeveTo the best of our knowledge, we are the first to have demonstrated that the PBRM1 IHC expression of endothelial cells is correlated with the expression of cancer cells, which suggests that the vascular endothelial cells may also be genetically or immunohistochemically abnormal . AlthougPBRM1 mutation status. Despite these limitations, we comprehensively showed the association of the PBRM1 expression profile with clinicopathological factors, including detailed histomorphological features.Our current work has some limitations. The PBRM1 expression was evaluated using only TMA, including the highest-grade area. Even considering intratumoral heterogeneity, however, we showed that the PBRM1 expression was correlated with the architectural patterns. Next, we semiquantitatively assessed PBRM1 IHC expression in cancer cells using an H-score. Furthermore, we could not validate the association of architectural patterns with We demonstrated that PBRM1 expression of cancer cells correlated with histomorphological features of ccRCC and correlated with the expression of vascular endothelial cells. Our PBRM1 expression profile indicated that PBRM1 expression in both cancer and endothelial cells may be regulated in an orchestrated manner.


Objectives: Uncovering the genetic basis of COVID-19 may shed insight into its pathogenesis and help to improve treatment measures. We aimed to investigate the host genetic variants associated with COVID-19.Methods: The summary result of a COVID-19 GWAS  was obtained from the COVID-19 Host Genetic Initiative GWAS meta-analyses. We tested colocalization of the GWAS signals of COVID-19 with expression and methylation quantitative traits loci  using the summary data-based Mendelian randomization (SMR) analysis. Four eQTL and two mQTL datasets were utilized in the SMR analysis, including CAGE blood eQTL data , GTEx v7 blood (n = 338) and lung (n = 278) eQTL data, Geuvadis lymphoblastoid cells eQTL data, LBC-BSGS blood mQTL data , and Hannon blood mQTL summary data . We conducted a transcriptome-wide association study (TWAS) on COVID-19 with precomputed prediction models of GTEx v8 eQTL in lung and blood using S-PrediXcan.Results: Our SMR analyses identified seven protein-coding genes  associated with COVID-19, including two novel risk genes, CCR5 and tau-encoding MAPT. The TWAS revealed four genes for COVID-19 , including two novel risk genes, CCR5 and PIGN.Conclusion: Our study highlighted the functional relevance of some known genome-wide risk genes of COVID-19 and revealed novel genes contributing to differential outcomes of COVID-19 disease. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and resultant COVID-19 have created a public health crisis worldwide. The majority of infected persons are either affected mildly or stay asymptomatic. It was reported that ~10–20% of people with COVID-19 infection need hospitalization .OAS1, OAS2, and OAS3), chr19p13.2 (TYK2), chr19p13.3 (DPP9), and chr21q22.1 (IFNAR2)  is one of the global efforts to promote human genetic variance research of COVID-19 by platform building, analytical activities, and data sharing . Genome-(IFNAR2) . These tTo get a clear understanding of these GWAS outputs and gain more insight into the SARS-CoV-2 pathophysiology, we performed summary data-based Mendelian randomization (SMR) and transcriptome-wide association analyses. We prioritized the genes co-localized with the COVID-19 GWAS hits and mapped additional involved genes. The resultant list of genes suggests potential therapeutic targets for symptomatic COVID-19.The summary result of a COVID-19 GWAS  was obtained from the COVID-19 Host Genetic Initiative (HGI) GWAS meta-analyses round 5  . All theP ≤ 5.0 × 10−8) and of independence (r2 < 0.6). For each group of IndSigSNPs, lead SNPs were identified when they were in LD with each other at r2 < 0.1 within a 500 Kb window. The merging of lead SNPs into genomic risk loci was performed when they were located at a distance of <500 kb from each other. Clumping was carried out according to the European 1,000 Genomes Project phase 3 reference panel, with the entire MHC locus being merged into one region (chr6:25-35Mb).FUMA was used to map SNPs to genes and identify genomic regions independent of linkage disequilibrium (LD) . All genn = 2,765) (n = 338) and lung (n = 278) eQTL data (n = 373)    which is embedded in the SMR analysis workflow.Colocalization of GWAS signal with expression and methylation quantitative traits loci  was performed in a framework of the SMR v1.03 . In this= 2,765) , GTEx v7QTL data , Geuvadin = 373) , LBC-BSG= 1,980) , and Han= 1,175) . Bonferrhttp://predictdb.org/. Bonferroni procedure was employed to adjust P-values for multiple testing.Putatively causal genes were prioritized by a TWAS procedure, which was conducted for the lung and the whole blood cells. The gene-level association results were calculated from GWAS summary statistics using S-PrediXcan , 15. We LIMD1, SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, XCR1, CCR3, FLT1P1, CCR1, UQCRC2P1, CCR2, LRRC2, VSTM2A, ABO, OAS1, OAS3, OAS2, DPP9, and IFNAR2. Two of these genes, FLT1P1 and UQCRC2P1, are non-coding genes.A total of six genomic loci were identified in the COVID-19 dataset, respectively , Table 1TYK2, IFNAR2, OAS1, OAS3, XCR1, CCR5, and MAPT) and four non-coding genes  , OAS1 (eQTL of Geuvadis lymphoblastoid cell and mQTL of Hannon blood), and XCR1 (mQTL of Hannon blood and mQTL of mQTL of Hannon blood). Two protein-coding genes, CCR5 and MAPT, are novel susceptibility genes for COVID-19, which were implicated by mQTL of Hannon blood and mQTL of LBC-BSGS blood, respectively  . Severalectively .CXCR6 and CCR5) and two genes associated with the blood eQTL dataset (CCR9 and PIGN)  . Among tCCR5, MAPT, and PIGN).Together, our SMR analysis and TWAS of COVID-19 identified a total of 14 genes associated with COVID-19, comprising seven genes implicated by the input or previous GWASs and seven novel genes  encodes a subunit for interferons alpha and beta binding receptors. Notably, IFNAR2 is capable of producing soluble receptors, which binds and regulates endogenous production of type I IFNs , while another gene VSTM2A maps to newly identified loci in the 7q31.1.Out of 18 protein-coding genes associated with COVID-19, twelve have been reported previously , 6, inclCCR5 and MAPT. Our TWAS analysis identified four genes significantly associated with predicted expression levels in the lung or the blood. Two of these genes, CCR9 and CXCR6, were also found within the set of COVID-19 associated genes, while CCR5 and PIGN genes were novel. Together, our SMR and TWAS analyses identified three novel protein-coding genes for COVID-19, namely, CCR5, MAPT, and PIGN.Our SMR analysis identified seven protein-coding genes significantly associated with predicted expression levels in the lung or the blood. Among these genes, two are novel, including CCR5 is located in 3p21.31 and encodes chemokine receptors expressed in macrophages and T cells. In macrophages, CCR5 protein serves as a gateway for many viruses including HIV , with no homozygotes found among the patients, compared to 1% of the controls . Independent identification of CCR5 as overexpressed in hospitalized COVID-19 patients supports the validity of our TWAS analysis.ding HIV . In a recontrols . In addiMAPT gene encodes the microtubule-associated protein tau. Genetic variation within MAPT was reported to be associated with multiple neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease , an anchor that tether proteins to the extracellular face of eukaryotic plasma membranes (Mutations in gene embranes . Notablyembranes , 37. In embranes . The metembranes . It seemThe strengths of this study include the use of the largest COVID-19 dataset available. Furthermore, we have diminished the potential population heterogeneity by limiting our analysis to individuals of European ancestry. Among the noticeable study limitations are that TWAS associations are considered to be noisy and that we have limited ourselves to testing only the genetic factors associated with COVID-19 risk, rather than taking into account the social and the environmental variables as well. As the findings from our study may be relevant to the European population only, uncovered associations certainly require further validation and detailed investigation.In summary, our study highlighted some known and revealed some novel genes contributing to differential outcomes of COVID-19 disease.https://www.covid19hg.org/results/.Publicly available datasets were analyzed in this study. This data can be found here: FZ designed the study and performed the statistical analyses. FZ and AB contributed to data interpretation and wrote the manuscript. HC contributed to the data preparation. All the authors approved the final manuscript for submission and publication and agreed to be accountable for all aspects of the work.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.