Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a global pandemic since its first outbreak in the winter of 2019. An extensive investigation of SARS-CoV-2 is critical for disease control. Various recombinant monoclonal antibodies of human origin that neutralize SARS-CoV-2 infection have been isolated from convalescent patients and will be applied as therapies and prophylaxis. However, the need for dedicated monoclonal antibodies suitable for molecular pathology research is not fully addressed. Here, we produced six mouse anti-SARS-CoV-2 spike monoclonal antibodies that not only exhibit robust performance in immunoassays including western blotting, ELISA, immunofluorescence, and immunoprecipitation, but also demonstrate neutralizing activity against SARS-CoV-2 infection to VeroE6/TMPRSS2 cells. Due to their mouse origin, our monoclonal antibodies are compatible with the experimental immunoassay setups commonly used in basic molecular biology research laboratories, providing a useful tool for future research. Furthermore, in the hope of applying the antibodies of clinical setting, we determined the variable regions of the antibodies and used them to produce recombinant human/mouse chimeric antibodies. The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a threat to global public health and economic development , 4, 5.With the development of humanized mouse antibodies and subsequent generation of fully human antibodies by various techniques, monoclonal antibodies have become widely used in therapy and prophylaxis for cancer, autoimmune diseases, and viral pathogens . Indeed,Isolation of multiple human neutralizing monoclonal antibodies against SARS-CoV-2 has been reported , 28, 29.In addition to the impact of monoclonal antibodies on therapy and prophylaxis, they significantly impact the characterization of SARS-CoV-2. To overcome the long-term battle with the virus, we need a detailed understanding of the replication mechanisms underlying its life cycle, including viral entry, genome replication, budding from the cellular membrane, and interaction with host immune systems. These essential pieces of information are required for drug discovery, vaccine design, and therapy development. Despite the large number of neutralizing antibodies reported to inhibit infection, there is an overwhelming lack of data on a well-characterized antibody available for basic research techniques such as western blotting (WB), immunofluorescence, and immunoprecipitation to study the viral life cycle.Here, we established six monoclonal antibodies against the spike glycoprotein of SARS-CoV-2. The trimeric spike glycoproteins of SARS-CoV-2 play a pivotal role in viral entry into human target cells through the same receptor, angiotensin-converting enzyme 2 (ACE2) as SARS-CoV-1 . We evalet al. . Recombinant RBD proteins tagged with GST or MBP were produced using an E. coli expression system . Both recombinant spike protein RBD and ectodomain (SΔTM) were produced using a mammalian expression system that retained proper protein glycosylation equivalent to that observed during virus replication (C and S1A). Mice were immunized with these recombinant spike proteins to generate antibodies against the SARS-CoV-2 virus, followed by cell fusion to generate a hybridoma-producing antibody. Culture supernatants were prescreened by enzyme-linked immunosorbent assay (ELISA), WB, and immunoprecipitation (IP), and six monoclonal hybridomas were isolated and evaluated.The SARS-CoV-2 spike glycoprotein is a homotrimeric fusion protein composed of two subunits: S1 and S2. During infection, the receptor-binding domain (RBD) on S1 subunit binds to ACE2, resulting in destabilization of the spike protein's metastable conformation. Once destabilized, the spike protein is cleaved into the N-terminal S1 and C-terminal S2 subunits by host proteases such as TMPRSS2 and changes conformation irreversibly from the prefusion to the postfusion state , 33, 34,et al. , in whicet al.   and two from mammalian cells (S1D7 and S3D8) showed remarkable performance. In the ELISA binding assay, all six clones bound glycosylated RBD with high affinity. When tested against spike glycoprotein (SΔTM), two clones (R15 and R52) could not be distinguished from nonimmune IgG (D). We noted that IgG2 subclass members tended to have higher binding affinities. Half maximal effective concentration (EC50) required for these antibodies to bind RBD and SΔTM glycoproteins falls at the low hundreds ng/ml (E). In WB, where target proteins are reduced and denatured, all clones established by E. coli produced-antigens performed well at detecting RBD and SΔTM proteins regardless of glycosylation . Among them, clones R15 and R52 showed higher sensitivity in WB. In addition, R52 was capable of detecting not only artificial spike glycoprotein carrying T4 foldon, but also native spike glycoprotein expressed in 293T cells on WB . However, neither RBD nor SΔTM could be detected by antibody clones established by the mammalian antigen (S1D7 and S3D8) on WB, suggesting a strong preference for intact tertiary structure .To characterize these antibodies in detail, they were first purified from the culture supernatant and examined in terms of ELISA and WB performance. Four monoclonal antibodies derived from the antigen produced by mune IgG D. We notds ng/ml E. In WB,A and S2A). As shown in Figure 2B, our antibodies recognize the spike protein in a glycosylation-independent manner, and the IP efficiencies of R22, R31, S1D7, and S3D8, although mild, outperformed others. Noticeably, although clones S1D7 and S3D8 are not capable of performing WB (F), a strong preference for tertiary structure grants them remarkable performance in IP, where RBD and SΔTM glycoproteins were pulled down in their native conformation. Of note, we found that S1D7 and S3D8 could maintain intact IP efficiency under highly stringent experimental conditions where sodium dodecyl sulfate (SDS) was present (B).An antibody capable of recognizing the intact tertiary structure of spike proteins would contribute to research dissecting the molecular mechanism of SARS-CoV-2 infection, especially cell entry, where these proteins play a significant role. The IP activity of antibodies can be correlated with the activity of capturing the native structure of the target protein and neutralizing the infection. We examined the IP performance of our monoclonal antibodies. Although all clones were capable of immunoprecipitating RBD and SΔTM glycoproteins, clones R22, R31, S1D7, and S3D8 demonstrated superior IP efficiency for SΔTM, whereas R22, S1D7, and S3D8 showed higher IP efficiency for RBD glycoprotein A and S2Arming WB F, a stro present B.Figure A and B), both S1D7 and S3D8 could detect spike proteins expressed homogeneously on the apical side of HeLa cells with a high signal-to-noise ratio (C and S2C). However, their localization pattern is different from that observed for SARS-CoV-1 spike proteins, which are exclusively localized in the Golgi during infection (B). Mouse hepatitis coronavirus spike protein localizes in the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) in a membrane (M) protein-dependent manner . An antibody applicable for IP would also have activity in IF. Cellular localization of spike proteins is essential for elucidating the mechanism of packaging and maturation of virions during release from the cellular membrane. We tested our antibodies' performance in IF using HeLa cells overexpressing spike protein with the transmembrane domain. Consistent with their performance in the above-mentioned assays , A and Bse ratio C and S2Cnfection  . Clones S1D7 and S3D8 clearly inhibited spike–ACE2 binding, as shown by the dimmed spike signal in WB (B). To quantify the inhibition ability, we performed a bead-based neutralization assay by measuring the amount of ACE2 bound to RBD beads after blocking with monoclonal antibodies (C). Antibodies R22 and R31 showed no disruption of ACE2–RBD interaction, whereas S1D7 and S3D8 showed robust hindrance of ACE2–RBD binding with IC50 values of 248.2 ng/ml and 225.6 ng/ml, respectively (D and E). S1D7 and S3D8's abilities to inhibit spike–ACE2 binding was consistent with their superior performance in IP experiments. Four monoclonal antibodies derived from the antigen produced by E. coli  were found to recognize continuous epitope 549-TGVLTESNKKFLPFQQFGRD-568 of spike protein RBD (B–D). In contrast, an epitope of two antibodies from mammalian cells (S1D7 and S3D8) could not be determined (B–D). The fact that they fail to recognize segmented RBD suggests that they recognize an intact tertiary structure of the spike protein.The manner in which antibodies bind and pull down spike glycoproteins in an IP experiment resembles the process of antibody-mediated neutralization, where spike–ACE2 interaction is intercepted by competitive binding between neutralizing antibodies and spike glycoprotein. We then examined whether they were capable of inhibiting spike–ACE2 binding or even neutralizing SARS-CoV-2 infection. First, we performed a spike pull-down assay in which the spike glycoprotein was pulled down by ACE2 in the presence of monoclonal antibodies A and S3Aal in WB B. To quatibodies C. Antiboectively , D and Etein RBD , B–D. Intermined , B–D. ThA). On the other hand, S1D7 and S3D8 were applicable to IF in infected VeroE6/TM2 cells. Spike showed a punctate distribution pattern in the perinuclear region resembling ER and ERGIC . The subcellular localization of spike resembled that of the N protein in Vero cells infected with SARS-CoV-1  cells, which is susceptible to SARS-CoV-2 infection compared with the parental VeroE6 cell line by expressing TMPRSS2 . In WB, Fig. 4nd ERGIC  . In IF, S1D7h could detect spike proteins expressed in HeLa cells . Notably, S1D7h and S3D8h showed robust hindrance of ACE2–RBD binding with IC50 values of 116.3 ng/ml and 137.2 ng/ml, respectively (C).Our mouse antibodies would not be applicable for use in clinical treatment, if not chimeric and humanized, due to their immunogenicity , 43. In Figs. 5ectively C.Figure Emerging SARS-CoV-2 is a global public health threat to society, which is predicted to be long-term for several years . Althoug50 of 405.2 ng/ml and 139 ng/ml) appeared to be lower than those of human antibodies reported previously  tend to underestimate neutralizing activities of our antibodies compared with other research groups. Specifically, we used a high multiplicity of live SARS-CoV-2 virus to infect VeroE6/TM2 cells, which are more prone to virus infection than the commonly adopted VeroE6 cell line. Therefore, it is difficult to compare antibody efficacy among them (in vitro infection, their neutralizing activity in vivo should be examined in animal models that recapitulate SARS-CoV-2 disease. They may be valuable for investigating the mechanism of immune responses to the virus during passive immunization using mouse models for SARS-CoV-2 infection (Our antibodies, S1D7 and S3D8, have been shown to attenuate the interaction of spike proteins with ACE2 and neutralize infection of VeroE6/TM2 cells by SARS-CoV-2. It is worth noting that although their neutralizing activities (ICnfection , 54, 55.Experimental procedures are provided as All data are contained within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Whether or not these observational associations are causal remains uncertain. We aimed to examine the potential causality of these associations using Mendelian randomisation (MR) analysis.Prior research suggested a differential association of 25-hydroxyvitamin D (25(OH)D) metabolites with type 2 diabetes (T2D), with total 25(OH)D and 25(OH)DN = 120,618), 25(OH)D3 , and C3-epi-25(OH)D3  in participants of European descent . We identified genetic variants for MR analysis to investigate the causal association of the 25(OH)D metabolites with T2D . We also estimated the observational association of 25(OH)D metabolites with T2D by performing random effects meta-analysis of results from previous studies and results from the EPIC-InterAct study. We identified 10 genetic loci associated with total 25(OH)D, 7 loci associated with 25(OH)D3 and 3 loci associated with C3-epi-25(OH)D3. Based on the meta-analysis of observational studies, each 1–standard deviation (SD) higher level of 25(OH)D was associated with a 20% lower risk of T2D , but a genetically predicted 1-SD increase in 25(OH)D was not significantly associated with T2D ; this result was consistent across sensitivity analyses. In EPIC-InterAct, 25(OH)D3 (per 1-SD) was associated with a lower risk of T2D , while C3-epi-25(OH)D3 (above versus below lower limit of quantification) was positively associated with T2D , but neither 25(OH)D3  nor C3-epi-25(OH)D3  was causally associated with T2D risk in the MR analysis. Main limitations include the lack of a non-linear MR analysis and of the generalisability of the current findings from European populations to other populations of different ethnicities.We performed a meta-analysis of genome-wide association studies for total 25(OH)D D with T2D risk. The findings based on MR analysis in a large sample of European ancestry do not support a causal association of total 25(OH)D or 25(OH)D metabolites with T2D and argue against the use of vitamin D supplementation for the prevention of T2D. Using both observational and genetic analyses, Ju-Sheng Zheng and colleagues investigate the relationship between vitamin D metabolites and type 2 diabetes among European individuals. There is ongoing uncertainty on whether the body’s vitamin D status indicated by blood 25-hydroxyvitamin D (25(OH)D) is relevant to the prevention of type 2 diabetes. There are conflicting findings from observational studies and a limited number of randomised controlled trials.3 and C3-epi-25(OH)D3, an isomer of 25(OH)D3.Prior research did not distinguish between different metabolites of total 25(OH)D, including 25(OH)DIt is not clear whether the associations of 25(OH)D metabolites with type 2 diabetes are causal, with conflicting findings from prior Mendelian randomisation studies of total 25(OH)D and no previous Mendelian randomisation studies appraising 25(OH)D metabolites.The current research compared observational estimates of the association between 25(OH)D metabolites and incident type 2 diabetes with Mendelian randomisation estimates based on genetic instruments.3 and 3 loci for C3-epi-25(OH)D3.Using multiple data sources, we performed genome-wide association studies among 120,618 individuals for total 25(OH)D, and among 40,562 individuals for the other vitamin D metabolites. Among participants of European descent, 10 genetic loci were identified for total 25(OH)D, 7 loci for 25(OH)D3, but for C3-epi-25(OH)D3, a positive association with type 2 diabetes was found.In meta-analysis of observational studies, we found that each 1–standard deviation higher level of total 25(OH)D was associated with 20% lower risk of type 2 diabetes. The result was similar for 25(OH)DWith up to 80,983 type 2 diabetes cases and 842,909 controls, we assessed the association of genetically predicted differences in total 25(OH)D and its metabolites with type 2 diabetes. Neither genetically predicted higher total 25(OH)D level nor genetically predicted higher levels of 25(OH)D metabolites were significantly associated with type 2 diabetes.There were conflicting findings for a link with type 2 diabetes for the observational analysis of biochemically measured 25(OH)D metabolites versus the genetically predicted levels of these metabolites.The null findings based on Mendelian randomisation analysis indicate that blood levels of 25(OH)D or its metabolites are not likely to be causal factors for the development of type 2 diabetes.The current findings together with other evidence from randomised controlled trials do not support the use of vitamin D supplementation for the prevention of type 2 diabetes. Similarly, we estimated the association of genetically predicted higher C3-epi-25(OH)D3 level (above versus below the LLQ) with T2D risk. For each of the 25(OH)D variables, we used an IVW method, MR-Egger method, and weighted median method to pool the estimates from multiple SNPs ) ) . The autanalysis  cannot banalysis .3 at SHQ1, which encodes H/ACA ribonucleoprotein assembly factor and has functions in the processing of ribosomal RNAs, modification of spliceosomal small nuclear RNAs, and stabilisation of telomerase [SDR9C7 for C3-epi-25(OH)D3. SDR9C7 encodes short chain dehydrogenase, whose relationship to the epimerisation of 25(OH)D3 to C3-epi-25(OH)D3 or other metabolites is unknown. Since the biological role of the epimerase is yet to be discovered [Described for the first time, to the best of our knowledge, we found a genome-wide significant signal for 25(OH)Dlomerase . In addiscovered , the pre3 is usually the major component of total 25(OH)D [3, on its own, with T2D risk has not previously been evaluated in MR analysis. Prior research mainly focused on total 25(OH)D as a biomarker of vitamin D status, which included a combination of 25(OH)D2 and 25(OH)D3, and, less frequently, C3-epi-25(OH)D3, the epimeric form of 25(OH)D3, depending on the assay methods. Blood 25(OH)D3 is derived both from diet and from biosynthesis in the skin upon exposure to sunlight, and 25(OH)D2 is mainly derived from diet, while C3-epi-25(OH)D3 is a metabolite of 25(OH)D3 via a C3 epimerisation process [3 from 25(OH)D3 and thus include it within the definition of 25(OH)D3 [3 had an inverse association with T2D risk, while C3-epi-25(OH)D3 was positively associated. Therefore, these current findings are an important extension to previous research, by including MR analysis for T2D for total 25(OH)D as well as for 25(OH)D3 and C3-epi-25(OH)D3 separately, as the results for total 25(OH)D may be potentially confounded by other vitamin D metabolites.Although 25(OH)D process . Traditi25(OH)D3 . Our recN = 120,618), and used the most comprehensive genetic instrument for the MR estimate for 25(OH)D and T2D risk (based on 10 SNPs versus the 4 or 2 used previously [3 and C3-epi-25(OH)D3—and used results from these in the MR analysis.There are several strengths of this study. To the best of our knowledge, this is the largest GWAS meta-analysis to date of total 25(OH)D N = 20,618, aeviously ,12). We 3) in MR analysis can potentially lead to violation of the exclusion restriction assumption: The genetic variant can influence the outcome via the continuous risk factor even if the binary exposure does not change [A limitation of this study is the lack of generalisability of our results from European populations to other populations of different ethnicities. Another limitation is that we combined studies with different study designs to maximise sample size and power for both MR analysis and observational analysis, even though definitions of endpoints  and exposure measurements D measurement) were not standardised across these studies. In addition, we tested a linear causal association using the MR approach, but we cannot rule out a potential weak causal association among people with vitamin D deficiency or insufficiency. Furthermore, although we did not find convincing evidence for an association of these variants with a variety of metabolic markers and lifestyle and demographic factors, we cannot rule out the possibility that horizontal pleiotropy exists for the genetic variants used in the MR analysis. Finally, use of a binary exposure (C3-epi-25(OH)Dt change .The public health implication of the present study, together with prior evidence of null findings from RCTs of vitamin D supplementation, is that focusing on population-wide supplementation to raise blood vitamin D levels is not likely to be an effective strategy for the prevention of T2D in European populations. However, our current limitations also indicate there are unresolved issues with important implications for further research. Next steps to address these issues include, but are not limited to, investigating the reasons for the discrepancy between the observational and MR findings, which likely reflect residual confounding as discussed above; including populations from different ethnic groups with varying distributions of 25(OH)D levels; and further increasing sample size and hence statistical power for conducting MR analysis of non-linear associations. The last of these would help to address whether there may be effects specifically in those who have hypovitaminosis D or are vitamin D deficient.In summary, the present findings using MR do not support a causal association of 25(OH)D and related metabolites with T2D, despite the strong association seen in observational studies. The totality of the available evidence from our study and RCTs to date does not justify the use of vitamin D supplementation for the prevention of T2D.S1 STROBE Checklist(DOCX)Click here for additional data file.S1 FigFor each of the genetic loci, we used LocusZoom software to draw the regional association plot.(TIF)Click here for additional data file.S2 FigGWAS, genome-wide association study; QQ, quantile–quantile.(TIF)Click here for additional data file.S3 FigEffect estimate  of each forest plot represents the change (in standard deviation unit) in total 25-hydroxyvitamin D per allele of the corresponding genetic variant across participating cohorts. GWAS, genome-wide association study.(TIF)Click here for additional data file.S4 Fighttp://www.phenoscanner.medschl.cam.ac.uk/). We extracted the results with the largest sample size if results from multiple data sources were available in the PhenoScanner database. The corresponding databases in PhenoScanner were UK Biobank for body mass index and diastolic and systolic blood pressure, GIANT for waist-to-hip ratio (PMID: 25673412), GLGC for the 4 lipid traits (PMID: 24097068), MAGIC for the 6 glycaemic traits (PMID: 20081857), SSGAC for years of educational attainment (PMID: 27225129), and TAG for ever smoker (PMID: 20418890). p < 0.003 was considered statistically significant after correction for multiple testing within each genetic variant, and none of the results were significant. HbA1c, glycated haemoglobin; HOMA-B, homeostatic model assessment of beta cell function; HOMA-IR, homeostatic model assessment of insulin resistance.Effect estimate  of each forest plot represents the change in each trait per allele of the corresponding genetic variant. The summary statistics shown in the present figure were extracted from the PhenoScanner database ((TIF)Click here for additional data file.S5 Fig3 variable (above versus below the lower limit of quantification). None of the results show significant heterogeneity (p = 0.661 from Q-test) or directional horizontal pleiotropy (p = 0.153 from test of Egger intercept). For multivariable MR analysis, the result of total 25(OH)D or 25(OH)D3 was adjusted for the genetic variants of C3-epi-25(OH)D3, while the multivariable MR result of C3-epi-25(OH)D3 was adjusted for total 25(OH)D, as the definition of total 25(OH)D includes only 25(OH)D3 and 25(OH)D2, not C3-epi-25(OH)D3. For C3-epi-25(OH)D3, MR-PRESSO result was not available due to limited number of genetic variants.The Mendelian randomisation estimate is per 1-SD increase in vitamin D metabolite, except for the binary C3-epi-25(OH)D(TIF)Click here for additional data file.S6 FigFor each of the genetic loci, we used LocusZoom software to draw the regional association plot.(TIF)Click here for additional data file.S7 FigGWAS, genome-wide association study; QQ, quantile–quantile.(TIF)Click here for additional data file.S8 Fig3 per allele of the corresponding genetic variant across participating cohorts. GWAS, genome-wide association study.Effect estimate  of each forest plot represents the change (in standard deviation unit) in total 25-hydroxyvitamin D(TIF)Click here for additional data file.S9 FigFor each of the genetic loci, we used LocusZoom software to draw the regional association plot.(TIF)Click here for additional data file.S10 FigGWAS, genome-wide association study; QQ, quantile–quantile.(TIF)Click here for additional data file.S11 Fig3 per allele of the corresponding genetic variant across participating cohorts. GWAS, genome-wide association study.Effect estimate  of each forest plot represents the change (log odds) in C3-epi-25-hydroxyvitamin D(TIF)Click here for additional data file.S12 Fighttp://www.phenoscanner.medschl.cam.ac.uk/). We extracted the results with the largest sample size if results from multiple data sources were available in the PhenoScanner database. The corresponding databases in PhenoScanner were UK Biobank for body mass index and diastolic and systolic blood pressure, GIANT for waist-to-hip ratio (PMID: 25673412), GLGC for the 4 lipid traits (PMID: 24097068), MAGIC for the 6 glycaemic traits (PMID: 20081857), SSGAC for years of educational attainment (PMID: 27225129), and TAG for ever smoker (PMID: 20418890). p < 0.003 was considered statistically significant after correction for multiple testing within each genetic variant, and none of the results were significant. HbA1c, glycated haemoglobin; HOMA-B, homeostatic model assessment of beta cell function; HOMA-IR, homeostatic model assessment of insulin resistance.The summary statistics shown in the present figure were extracted from the PhenoScanner database ((TIF)Click here for additional data file.S13 Fighttp://www.phenoscanner.medschl.cam.ac.uk/). We extracted the results with the largest sample size if results from multiple data sources were available in the PhenoScanner database. The corresponding databases in PhenoScanner were UK Biobank for body mass index and diastolic and systolic blood pressure, GIANT for waist-to-hip ratio (PMID: 25673412), GLGC for the 4 lipid traits (PMID: 24097068), MAGIC for the 6 glycaemic traits (PMID: 20081857), SSGAC for years of educational attainment (PMID: 27225129), and TAG for ever smoker (PMID: 20418890). p < 0.003 was considered statistically significant after correction for multiple testing within each genetic variant, and none of the results were significant. HbA1c, glycated haemoglobin; HOMA-B, homeostatic model assessment of beta cell function; HOMA-IR, homeostatic model assessment of insulin resistance.Effect estimate  of each forest plot represents the change in each trait per allele of the corresponding genetic variant. The summary statistics shown in the present figure were extracted from the PhenoScanner database ((TIF)Click here for additional data file.S14 Fig3 variable: above versus below the lower limit of quantification) and T2D risk. For 25(OH)D3, 4 prior known genes are GC (rs4588), CYP2R1 (rs116970203), NADSYN1/DHCR7 (rs28364617), and CYP24A1 (rs17216707). Two genes in the 25(OH)D synthesis pathway are CYP2R1 (rs116970203) and NADSYN1/DHCR7 (rs28364617). Two genes related to 25(OH)D metabolism are GC (rs4588) and CYP24A1 (rs17216707). Three genes, SHQ1, AMDHD1, and SULT2A1, were recently identified in the present GWAS meta-analysis or by another recent study. For C3-epi-25(OH)D3, we did sensitivity analysis stratified by the SDR9C7 variant and the other variants D), as SDR9C7 is a unique locus associated with C3-epi-25(OH)D3. The SDR9C7 SNP was rs11172066. 25(OH)D, 25-hydroxyvitamin D; SNP, single nucleotide polymorphism.Mendelian randomisation (MR) estimate represents the association between a genetically predicted 1–standard deviation increase in 25-hydroxyvitamin D metabolites (except for the binary C3-epi-25(OH)D(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.


ARID1A) encodes a key component of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex that participates in gene expression. ARID1A alterations are quite common among cancer patients, although their role remains debated. The aim of this article was to study ARID1A-mutated cancer patients.AT-rich interaction domain 1A . Cancer patients with at least one molecular alteration in ARID1A gene were identified as ARID1A+.Molecular and clinical data of cancer patients evaluated at Phase 1 Unit of Fondazione Policlinico Universitario A. Gemelli IRCCS were collected. Molecular analyses were performed using FoundationOne+ population was higher compared to ARID1A– population (6 vs. 4), as well as tumor mutational burden (TMB) [20 mutations/megabase (mut/Mb) vs. 1.26 mut/Mb]. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphatase and tensin homolog (PTEN), catenin beta 1 (CTNNB1), and lysine methyltransferase 2D (MLL2) mutations were enriched in ARID1A+ population. In this cohort, ARID1A did not display any relation with response to platinum chemotherapy. Cancers with double alterations in ARID1A (ARID1A2+) were all gynecological cancers .Among the 270 patients undergoing molecular analysis, we found 25 (9%) with at least one pathogenic alteration in ARID1A. The vast majority of these patients were female (84%). The median age at diagnosis was 59; most of the cancers  were gynecological , diagnosed at an early stage. Frameshift alterations in ARID1A were the most common  alterations. The median number of mutations in ARID1A+ cancers, in particular the subgroup of gynecologic cancers. The high frequency of concurrent mutations in the phosphoinositide 3-kinase (PI3K) pathway among endometrioid endometrial cancers may support the proposal of a new treatment strategy based on the combination of ataxia telangiectasia and Rad3-related (ATR) inhibitor and PIK3CA inhibitor.This analysis provides clinical and molecular details about the phenotypes of ARID1A ARID1A) gene, also known as Brahma-related associated factor 250a (BAF250a), is located on chromosome 1p36.11. It encodes a key component of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex . ECOG PS, at the time of our evaluation, was good (0 or 1) in 92% of the study population although they were pre-treated with a median of 2 lines of chemotherapy.+ cancer have a high TMB (≥ 20 mut/Mb), with 12 (48%) showing stability of the microsatellite system. PIK3CA, TP53, PTEN, CTNNB1, and MLL2 were the most concurrent mutations  , the molecular analysis was performed on the archival sample referring to the diagnosis time (average time from sample collection 11 months). More than half of the patients with ARID1Actively) .Endometrioid cancer of endometrium was the prevalent tumor, representing 24% of the cases, followed by clear cell ovarian cancer . Among the pathogenic molecular alterations in ARID1A, frameshift alterations were the most common  . Interes+ population was significantly higher compared to ARID1A– population , as well as TMB . Also, the median number of VUS was higher in ARID1A+ cancers . In PIK3CA, PTEN, CTNNB1, and MLL2) in ARID1A+ population. On the contrary, TP53 was prevalent in ARID1A– population (63% vs. 48%), although not statistically significant.The median number of mutations in ARID1A+ tumors vs. ARID1A– tumors, the presence of alterations did not relate with a different time to platinum treatment failure . OS for ARID1A+ patients was 66 months.Comparing the response to platinum for gynecologic patients with ARID1A+ (ARID1A2+). They were all women with gynecological cancer, mainly of endometrial origin. Five (83%) had endometrioid endometrial cancer and the other one was diagnosed with clear cell ovarian cancer. ECOG PS was 0 or 1. TMB was 24.5 mut/Mb, the higher found among the ARID1A+ patients, with the majority  displaying microsatellite instability.Interestingly, the authors found six patients with 2 different alterations in ARID1AThe response to immunotherapy of the endometrial cancer patients was variable: two patients with high TMB (48/20 mut/Mb) and MSI-H progressed within 6 months; other two patients with MSI-H and TMB intermediate/high (14/29 mut/Mb) reached 14 months and 27 months of PFS, with one of them being still on treatment. The last patient with TMB low/MSS progressed after two cycles of immunotherapy. To note that one patient with MSI-H/high TMB endometrioid endometrial carcinoma progressed on immunotherapy very fast but showed a partial response to PI3K inhibitor lasting for 7 months.+ cancers, without any drug approval so far. These medicines include immune checkpoint blockades, PI3K/AKT/mTOR inhibitors, PARP inhibitors, ATR inhibitors, enhancer of zeste homolog 2 (EZH2) inhibitors, and pan-histone deacetylase (HDAC) inhibitors. In ARID1A is being configured as a potential new target for cancer treatments. A variety of compounds are being tested in ARID1AARID1A deficiency was defined as the absence of tumor expression of its gene product (BAF250a) by IHC staining. Of note, tumors harboring pathogenic mutations in ARID1A do not necessarily have a loss of expression of BAF250a, which was reported in 67% of the study population [In particular, a significant interest in this gene is rising in the gynecological field, due to the high percentage of ARID1A alterations among these patients. For instance, ATARI trial is a multicenter, international, phase II study (NCT0405269) testing the ATR inhibitor ceralasertib as a single agent and in combination with olaparib in ARID1A gynecological cancers. pulation . The intpulation .+ tumors may guide the design of future clinical trials targeting this gene. Therefore, we retrospectively analyzed our cancer population undergoing molecular evaluation with FoundationOne® CDx assay.A deeper insight into the clinical and molecular landscape of ARID1A+ cancers, a percentage comparable to that reported in the literature (6%) [+ population, especially in the ARID1A2+ group, whose median TMB reached 24.5 mut/Mb. PIK3CA, TP53, and PTEN were the most common concurrent mutations in ARID1A+ population, although TP53 was slightly increased in ARID1A– cancers (63% vs. 48%).In the population of patients being offered an NGS test for their advanced tumor, we found 9% of ARID1Aure 6%) . The sli% . The sure (6%) , endometPIK3CA or PTEN, frequently co-occurring, may cooperate with ARID1A loss to drive cancer and downstream activation of the PI3K/AKT pathway [Previous evidence suggested that ARID1A inactivation alone is insufficient to drive ovarian tumorigenesis . Defects pathway . Moreove pathway .TP53, Kirsten rat sarcoma viral oncogene homologue (KRAS), and adenomatous polyposis coli (APC) occurred most frequently in ARID1A+ GI cancers [In a previous experience with GI cancers , TP53, K cancers . This obIn our experience, ARID1A alterations were found both in solid tissue (primary diagnosis and relapsed disease) and blood. A recent report among 71,301 patients suggests that liquid biopsies identified ARID1A alterations at a frequency similar to that found in primary tumor material . If muta2+, among the patients who underwent liquid biopsy.Double alterations in ARID1A were observed only in solid tissues. Probably, it may be linked to the absence of gynecological cancers, the ones identified as ARID1A– tumors. Moreover, we observed a poor outcome among ARID1A+ tumors compared to the ARID1A WT cohort, consistently the largest report so far [With the limitation of the small sample size, our study did not show any differences in terms of duration of response to platinum-based chemotherapy among gynecological cancer displaying ARID1A alterations as compared with ARID1At so far .It is uncertain if monoallelic inactivations share the same phenotype (i.e. loss of ARID1A expression) as biallelic inactivations . This an+ cancers, no predictive role for platinum-based chemotherapy). In addition, we provide clinical and molecular details in both ARID1A+ and ARID1A2+ cohorts, highlighting Q372fs*19 as the most frequent alteration in solid tissue.In conclusion, this analysis confirms and strengthens existing literature , we propose to combine ATR inhibitor or PARP inhibitor with PIK3CA inhibitors in a new trial to test the synergic action of these drugs. Genomic analysis and protein expression of cancer tissue, as well as liquid biopsy, need to be performed and correlated with response to therapy.ARID1A among the key genes mutated in clear cell ovarian cancer may offer new treatment opportunities in this orphan disease, poorly responsive to chemotherapy. Moreover, among ARID1A2+ endometrioid endometrial carcinomas with a high value of TMB, a combination or new immunotherapies may be proposed in future studies.The identification of


This article has been retracted: In Figures 3A, 8D, and 7D, multiple wound healing assay images of gastric cancer cell lines overlap with the images from another published paper studying the effect of different miRNA in liver cancer [r cancer , which wr cancer ; image or cancer ; Figure r cancer  HepG2/miIn Figures 3B, 7E, 8E multiple transwell assay images are the duplication of transwell assay images from unrelated papers of different authors, for the example - Figures 3C/3D and 7C/7D from  and Figu95880-95895. https://doi.org/10.18632/oncotarget.21431Original article: Oncotarget. 2017; 8:95880–95895.


Streptococcus pneumoniae (S. pneumoniae) is understudied. We show that S100A9 was significantly increased in BAL fluids of patients with bacterial but not viral pneumonia and correlated with procalcitonin and sequential organ failure assessment scores. Mice deficient in S100A9 exhibited drastically elevated Zn2+ levels in lungs, which led to bacterial outgrowth and significantly reduced survival. In addition, reduced survival of S100A9 KO mice was characterized by excessive release of neutrophil elastase, which resulted in degradation of opsonophagocytically important collectins surfactant proteins A and D. All of these features were attenuated in S. pneumoniae-challenged chimeric WT→S100A9 KO mice. Similarly, therapy of S. pneumoniae-infected S100A9 KO mice with a mutant S100A8/A9 protein showing increased half-life significantly decreased lung bacterial loads and lung injury. Collectively, S100A9 controls central antibacterial immune mechanisms of the lung with essential relevance to survival of pneumococcal pneumonia. Moreover, S100A9 appears to be a promising biomarker to distinguish patients with bacterial from those with viral pneumonia.S100A8/A9 has important immunomodulatory roles in antibacterial defense, but its relevance in focal pneumonia caused by Trial registration: Clinical Trials register (DRKS00000620). in vivo and in vitro by binding important metal ions like manganese and zinc. The observed drop in bacterial loads was further accompanied by reduced release of neutrophil elastase in lungs of mice, which in turn protected the lungs of mice from infection-driven lung damage. Collectively, S100A8/A9 proteins are important antibacterial effector proteins of the lung and thus indispensable for survival of pneumococcal pneumonia.Acute bacterial infections of the lower respiratory tract remain a major cause of morbidity and mortality worldwide. They are usually associated with massive recruitment of neutrophilic granulocytes to the lungs, which release specific proteins called S100A8 and S100A9 in addition to many other inflammatory mediators. S100A8 and S100A9 belong to the family of calcium-binding proteins and preferentially form S100A8/A9 heterodimers. When released by neutrophils upon infection, S100A8/A9 proteins are capable of binding several metal ions. In this report, we show that S100A9 is significantly elevated in the lungs of patients with bacterial but not viral pneumonia, suggesting that this alarmin might be a promising new biomarker in bacterial pneumonia. In addition, we show in a preclinical mouse model that S100A8/A9 reduces bacterial growth  Streptococcus pneumoniae (S. pneumoniae) is the most prevalent pathogen causing community-acquired pneumonia (CAP) in humans [S. pneumoniae still represents a life-threatening clinical condition especially in young children (< 5 years) and elderly or immunocompromised individuals [n humans –4. Despiildren <  years anividuals . Particuividuals –13).2+ binding proteins comprising approximately 25 members [2+ ions [S100A8 and S100A9 are part of the EF-hand superfamily of Ca members –16. The [2+ ions . Both S1[2+ ions ,15,18. S[2+ ions –23. S100[2+ ions –31. In t[2+ ions –35.S. pneumoniae is still understudied. Here we characterized the role of functional S100A8/A9 deficiency in a model of pneumococcal pneumonia in mice. We found that the heterodimer regulates essential antibacterial immune mechanisms by controlling levels of divalent cations in the lung and thereby inhibiting bacterial outgrowth. Second, neutrophil elastase (NE) release by recruited neutrophils was limited in the presence but not the absence of S100A9, with major impact on NE-dependent degradation of surfactant proteins SP-A and SP-D and overall survival. Finally, S100A9 appears to be a promising biomarker to distinguish bacterial from viral pneumonia patients.The role of S100A8 and S100A9 in bacterial infections caused by S100A8/A9 heterodimer, also known as calprotectin (CP), is a protein complex serving as a biomarker for different inflammatory diseases like inflammatory bowel disease (i.e. Crohn’s disease and ulcerative colitis), and rheumatoid arthritis –39. AcutMoreover, S100A9 protein levels were positively correlated with C reactive protein (CRP) in BALF of patients with bacterial but not viral pneumonia  . In addiS. pneumoniae. At baseline, we found low levels of S100A9 protein in plasma and BALF of WT mice, which increased significantly in response to pneumococcal challenge with peak values observed at days 1–2 post-infection  confirmed the striking susceptibility of S100A9 KO mice to pneumococcal challenge , and pneumococcal challenge did not alter Zn2+ levels in lungs of WT→WT mice , while > 80% KO→KO mice succumbed to pneumococcal pneumonia  , pneumococcal growth was significantly inhibited in vitro  deficient mice triggered substantial degradation of alveolar collectins surfactant protein A (SP-A) and D (SP-D) in a neutrophil elastase dependent manner, with fatal consequences for the infected host [S. pneumoniae-infected WT as compared to S100A9 KO mice. As shown in S. pneumoniae infection, NE was only weakly detectable in BALF of WT mice, but strongly upregulated in BALF of S100A9 KO mice  were incubated with exogenous NE, followed by analysis of SP-D by western blotting. As shown in S. pneumoniae-infected WT and S100A9 KO mice. While SP-D protein was degraded in BAL fluids of S. pneumoniae-infected WT mice in a dose dependent manner  of WT or S100A9 KO bone marrow into WT or KO mice. As shown in S. pneumoniae, and drastically increase once S100A9 deficient neutrophils are recruited upon S. pneumoniae infection. At the same time, low levels of BALF NE were accompanied by easily detectable levels of SP-A and SP-D in WT→WT and WT→KO mice, whereas BAL fluids of KO→KO and KO→WT mice with increased NE levels completely lacked collectins SP-A and–D  is an important cholesterol-binding virulence factor of pneumococcus, that also serves as a pathogen-associated molecular pattern (PAMP) binding to and activating professional phagocytes via TLR4 . Pneumolh groups . At the  KO mice . As expen groups . At the  KO mice . The datS. pneumoniae: 1) Increased levels of S100A9 protein were found in BALF of patients with bacterial, but not viral ARDS, which were correlated with CRP, PCT and SOFA scores, suggesting S100A9 as novel biomarker for bacterial pneumonia. 2) Increased levels of S100A9 correlated with low levels of Zn2+ and bacterial loads in lungs of S. pneumoniae-infected mice, and S100A8/A9 heterodimer inhibited Zn2+ and Mn2+ dependent pneumococcal growth in vitro. 3) Lack of S100A9 (and thus heterodimeric S100A8/A9) led to increased NE dependent degradation of surfactant proteins SP-A and -D, which was accompanied by poor outcome in KO mice, and corrected in chimeric S100A9 KO mice harboring the hematopoietic system of WT mice. Together, S100A8/A9 limits pneumococcal outgrowth in focal pneumonia by chelation of divalent cations, which in turn limits pneumolysin-driven release of neutrophil-derived NE and subsequent NE-dependent degradation of surfactant proteins, thus favoring survival of pneumococcal pneumonia  has been assigned several immunomodulatory activities and also serves as biomarker in inflammatory diseases including, but not limited to, ulcerative colitis, Crohn’s disease and rheumatoid arthritis –39,48,49in vivo . Bacteriin vivo . Therefoin vivo .Therefore, we wondered whether not CP, but rather S100A9 protein itself would be an appropriate biomarker for discrimination between bacterial and viral ARDS. Others have reported increased S100A9 levels in the serum of patients with CAP, which positively correlated with inflammatory cytokines and blood routine parameters . MoreoveS. aureus and K. pneumoniae pneumonia, and increased bacterial loads and inflammatory responses were observed in S100A9 KO mice in a model of invasive pneumococcal disease (IPD) [S. pneumoniae [The role of S100A8/A9 in experimental models of bacterial lung infection is controversial. Detrimental effects of S100A8/A9 deficiency were observed in se (IPD) –63. In ceumoniae . Howevereumoniae . For a seumoniae  may morePrevious studies found that neutrophil recruitment was diminished in the absence of S100A8/A9 in preclinical models of invasive pneumococcal disease ,63 and t2+ and Mn2+ [in vitro, and that deficiency of S100A9 (and thus of heterodimeric S100A8/A9) was accompanied by increased levels of divalent metal ions in pneumococcal pneumonia, leading to bacterial outgrowth. In contrast, previous studies suggested that S100A8/A9 may contribute to worsening of IPD by complexing and thereby reducing zinc-mediated toxicity against the pathogen, hence fostering bacterial outgrowth [Staphylococcus aureus outgrowth in locally confined tissue abscesses, due to complexing of essential Mn2+ and Zn2+ ions [S. pneumoniae in highly susceptible S100A9 KO mice by lowering Zn2+ levels in lungs of mice accompanied by attenuated bacterial outgrowth. Again, the contradictory results regarding the role of S100A8/A9 in lowering divalent metal ions may be related to the different pathogenicity profiles of the pneumococcal strains and thereof developing infection phenotypes employed.It is known that S100A8/A9 is able to prevent bacterial and fungal growth by binding essential divalent metal ions like Znand Mn2+ –22,62. Hutgrowth . Opposedn2+ ions ,21. In tWe recently showed in a mouse model of AAT deficiency how effectively uncontrolled proteolytic activity impaired lung antibacterial immunity in pneumococcal pneumonia, specifically due to NE-mediated degradation of alveolar collectins SP-A and -D . The abiAll patients and healthy volunteers enrolled in this study, or their closest relatives provided written informed consent. Collection of biosamples was registered at the German Clinical Trials register (DRKS00000620) and the biosampling was approved by the institutional review board of Hannover Medical School (#2516–2014 and #8146_BO_K_2018).Handling of animals was done according to institutional guidelines of the Central Animal Facility of Hannover Medical School. All animal experiments were approved by the Lower Saxony State Office for Consumer Protection and Food Safety following the European Council Directive 2010/63/EU and the German Animal Welfare Act.WT (C57BL/6) mice were purchased from Janvier Lab . S100A9 KO mice on a C57BL/6 background were generated as described elsewhere . Mice usin vitro, MgCl2, ZnCl2 and CaCl2 were purchased from Carl Roth  and MnSO4 was purchased from Sigma-Aldrich . For Western blot analysis, polyclonal rabbit anti-mouse SP-A antibody  was purchased from antibodies online . Monoclonal rabbit anti-mouse SP-D antibody (clone ERP 21774–143) was purchased from Abcam  and monoclonal rat anti-mouse Neutrophil elastase antibody was obtained from R&D Systems . Recombinant S100A8/A9 heterodimer was generously provided by Thomas Vogl, University of Muenster, Germany, or was purchased from BioLegend . For immunofluorescence staining, Ly6G AF647 (clone 1A8) and CD11b BV421 (clone M1/70) were purchased from BioLegend .For pneumococcal growth experiments Within this study, patients with bacterial and viral ARDS as well as healthy volunteers serving as controls were analyzed. ARDS patients with malignancies and immunosuppression were excluded from the study.ARDS patients: Diagnosis of ARDS was made in accordance with the Berlin 2012 definition [finition . All patThe study group of patients with viral ARDS consisted of 11 patients including 8 males and 3 females. The average age was 54 years, with a range from 20–87 years. Six of the included patients were smokers and 5 were non-smokers. All patients underwent BAL for diagnostic reasons.Seven healthy volunteers were included in this study, all of them were males. The average age was 52 years with a range from 43–59 years. All participants underwent respiratory workup including thorough physical examination, pulmonary function testing and capillary blood gas analysis. All patients were asymptomatic with normal pulmonary function testing and did not receive any medication.BAL was performed in the middle lobe or lingula. BAL samples were rested on ice until timely further laboratory work-up. Macro-impurities in the BAL were removed by filtration of the fluid through sterile gauze. BAL samples were centrifuged at 500 g for 10 minutes at 4°C and subsequently the BAL fluid was aliquoted and stored at -80°C.S. pneumoniae was carried out as described [S. pneumoniae serotype 19F were inoculated in Todd Hewitt broth  supplemented with 20% fetal bovine serum  [S. pneumoniae was done by plating tenfold serial dilutions onto sheep blood agar plates  followed by incubation at 37°C and 5% CO2 for 18 h. Afterwards colony forming units (CFU) were counted. Additionally, CFU were also determined in mouse inoculation aliquots prior to infection of mice to accurately determine the applied infection dose.Culture and quantification of escribed . Brieflyts, USA) ,65,74. PGeneration of mutant murine S100 proteins was done analogous to the generation of mutant human S100 proteins, as recently described in detail . S100A8 S. pneumoniae (~6 x 106 CFU/mouse) as published elsewhere [Mice were orotracheally infected with lsewhere ,75. In bS. pneumoniae-infected mice were euthanized and bronchoalveolar lavage (BAL) as well as lung homogenates were prepared as reported earlier [At indicated time points,  earlier ,76. BrieS. pneumoniae as described above and S100A9 protein levels were determined in plasma and BAL fluid of mice on days 1, 2, 3 and 4 post-infection. The effect of S100A9 deletion on outcome of pneumococcal pneumonia was evaluated in S100A9 KO versus WT mice, which served as disease controls. Mice were infected orotracheally with S. pneumoniae and bacterial loads as well as lung histopathology were determined at indicated time points. Moreover, disease progression was monitored for an observation period of 6 days.Four treatment groups were established: WT mice were infected with S. pneumoniae and bacterial loads were determined at indicated time points. The course of pneumococcal pneumonia was analyzed over a time period of 10 days. In additional experiments, Zn2+ levels were determined in lung homogenates of chimeric mice, as indicated.Four different groups of bone marrow chimeric mice were generated to monitor the effect of hematopoietic deletion or reconstitution of S100A9 on lung CFU and outcome in bone marrow transplanted WT➔WT, KO➔KO, WT➔KO, and KO➔WT mice. Chimeras were infected orotracheally with S. pneumoniae-infected S100A9 KO mice was analyzed. To this end, S100A9 KO mice were orotracheally infected with S. pneumoniae and treated twice with 50 μg mutant S100A8/A9 protein i.p. or vehicle. Zn2+ levels in lungs, lung bacterial clearance and neutrophil recruitment were analyzed at 8 hours after pneumococcal challenge.In selected experiments, the effect of mutant S100A8/A9 protein application on lung bacterial loads in 7 bone marrow cells/mouse via lateral tail veins. Recipient chimeric mice were then kept under pathogen-free conditions with free access to sterilized food and water and were monitored twice daily for a time period of 6 weeks. Engraftment efficiencies of more than 97% were accepted for subsequent infection studies [Generation of bone marrow chimeric mice was done as described earlier ,78. Brie studies .5) were suspended in 50 μl RPMI/ 10% FCS and seeded into 96-well plates. Following a 30 minute resting time at 37°C/5% CO2, 2 mM of luminol was added to each well. Cells were incubated again for 1 hour at 37°C/5% CO2 and cells were infected with S. pneumoniae at a multiplicity of infection (MOI) of 5 to trigger respiratory burst induction followed by monitoring emitted luminescence signals expressed as relative light units (RLU) over time using a Flx800 fluorescence/luminescence reader .Analysis of respiratory burst induction in bone-marrow derived neutrophils (BM-PMN) was carried out as described previously ,79,80. B5 cells per well) were seeded into 96 well plates and infected with S. pneumoniae at MOI 26. Following a 30 min incubation in RPMI/10% FCS/1% Glutamine at 37° C and 5% CO2, non-phagocytosed bacteria were repeatedly washed off the wells, and residual extracellular bacteria were killed by short incubation of cells in RPMI/10% FCS/1% Glutamine supplemented with 20 μg/ml gentamicin . After 10 minutes of incubation at 37° C and 5% CO2, cells were washed again and lysed at indicated time points post-infection. Determination of CFU was performed by plating ten-fold serial dilutions of cell lysates onto sheep blood agar plates, followed by incubation for 18 h at 37° C and 5% CO2.Phagocytosis of professional phagocytes from WT and S100A9 KO mice was examined as described recently ,81. ShorS. pneumoniae-infected WT and S100A9 KO mice were used to determine the content of SP-A, SP-D and NE in BAL supernatants of mice of the respective treatment groups.Western Blot analysis was performed as described recently ,73,76. ADetermination of NE activity was done essentially according to recently published protocols .2, 1 μM ZnCl2, 2.5% Triton X-100). Next, washing buffer was changed to incubation buffer  and gels were shaken for 10 min at 37°C. Afterwards, the incubation buffer was changed and gels were shaken again for 18 h at 37°C. Following incubation, casein-containing gels were stained for 30 min with Coomassie Blue G250 . Subsequently, gels were de-stained until white bands appeared on blue background. White bands within gels therefore represent the location of casein degradation by active BAL fluid proteases.Protein-content of BALF from respective treatment groups was determined using Pierce BCA protein assay kit . Equal protein amounts were supplemented with Laemmli sample buffer, loaded onto casein-containing SDS gels and were run for 1.5 h at 150 V under non-reducing conditions. To get rid of SDS, casein gels were washed twice for 30 min at RT in washing buffer  on growth of S. pneumoniae was analyzed in THB-Chelex medium supplemented with magnesium (200 μM) only, or combinations of magnesium (200 μM) and manganese (50 μM) or magnesium and zinc (50 μM).To examine the roles of defined divalent cations on pneumococcal growth ablished ,82–84. TLung homogenate supernatants were generated as outlined above. Zinc concentrations were analyzed using the LT-SYS Zinc assay  implemented on a Cobas 8000 c502 clinical chemistry analyzer .S. pneumoniae-infected WT and S100A9 KO mice were examined for the occurrence of interstitial and/or alveolar infiltrating neutrophils and mononuclear cells, hemorrhage as well as interstitial/alveolar edema formation. Percent inflamed lung tissue represents the mean value of lung inflammation recorded for each lung lobe per mouse lung examined.Lung histopathology was evaluated as described previously ,76. BrieS. pneumoniae-infected WT and S100A9 KO mice was done following a previously published protocol [Immunofluorescence analysis of frozen lung tissue sections of untreated and protocol . BrieflyS. pneumoniae-infected WT and S100A9 KO mice was done by flow cytometric analysis as described previously [S. pneumoniae-infected WT and S100A9 KO mice were subjected to BAL, followed by removal of individual lung lobes and digestion of lung tissue with collagenase A (5 mg/ml) and DNAse I (1 mg/ml) for 45 min at 37° C on days 0, 1 and 2 post-infection. For immunophenotypic analysis of macrophages in lung tissue, cell suspensions of CD11cpos cells were enriched using a magnetic cell separation kit . Leukocyte subsets purified from BAL fluids and lung tissue were seeded into 96-well plates (5 x 105 cells/well) and treated with Fc receptor blocking agent Kiovig . Afterwards, cells were stained with fluorochrome-conjugated mAbs specific for F4/80 , Ly6G , CD45 , CD11c  and CD11b  for 20 min at 4°C followed by two washing steps. Subsequently, cells were subjected to FACS analysis of specific cell surface Ag expression using a BD LSR Fortessa flow cytometer .Identification and quantification of macrophages and neutrophils recovered from BAL fluids and lung tissue of eviously ,81,86. BS. pneumoniae-infected WT and S100A9 KO mice using commercially available ELISA Kits . Pro- and anti-inflammatory cytokines CXCL1, CXCL2, G-CSF, TNF-α, IL-1β, IL-10 were measured in plasma and BALF of untreated and S. pneumoniae-infected WT and S100A9 KO mice using Luminex bead arrays .Human S100A9 and calprotectin levels were measured in plasma and BALF of ARDS patients using commercially available ELISA kits  according to the manufacturer’s instructions. Mouse S100A9, neutrophil elastase and Surfactant protein D levels were measured in plasma or BALF of untreated and U test or Kruskal-Wallis test. Survival data were analyzed using log-rank test. P values lower than 0.05 were assumed significantly different.Data analysis was performed with GraphPad Prism software and data are shown as mean ± SD. Differences between treatment groups were calculated by Mann-Whitney S1 Fig(A) Positive correlation between BAL fluid S100A9 and C-reactive protein (CRP) in BAL fluids of patients with bacterial pneumonia (n = 17 patients). (B) Endogenous S100A9 protein in BAL fluids of patients with viral pneumonia was negatively correlated with C-reactive protein (n = 11 patients).(TIF)Click here for additional data file.S2 FigS. pneumoniae. Macrophages and neutrophils in BALF and lung tissue were analyzed by FACS on days 0, 1 and 2 after pneumococcal challenge.  Flow cytometric gating strategy for identification of macrophages and neutrophils in BAL fluids of WT and S100A9 KO mice. (A) Macrophages were identified according to their forward scatter area (FSC-A) versus side scatter area (SSC-A)  and FCS-A/FCS-H characteristics (P2) followed by hierarchical subgating according to their FSC-A versus F4/80 cell surface expression (P3). Alveolar macrophages were characterized as CD11cpos/CD11bneg (C) while exudate macrophages were identified as CD11cpos/CD11bpos (D). (B) Neutrophils were gated according to their FSC-A/SSC-A profile (P1) followed by hierarchical subgating according to their FSC-A versus Ly6G cell surface expression (P2) and were then identified as Ly6Gpos/CD11bpos cells (E).  Flow cytometric gating strategy for identification of macrophages and neutrophils in lungs of WT and S100A9 KO mice. (F) Macrophages were identified according to their FSC-A/SSC-A (P1) and FCS-A/FCS-H characteristics (P2) followed by hierarchical subgating according to their FSC-A versus F4/80 cell surface expression (P3). Lung macrophages were characterized as CD11cpos/CD11bneg (H) while exudate macrophages were identified as CD11cpos/CD11bpos (I) cells. (G) Neutrophils were gated according to their FSC-A versus SSC-A profile (P1) followed by hierarchical subgating according to their CD45 versus Ly6G cell surface expression (P2) and were then identified as Ly6Gpos/CD11bpos cells (J). (K) Bacterial loads in BAL fluid of WT and S100A9 KO mice on day 1 and day 2 after pneumococcal challenge. Values are shown as mean ± SD (n = 3–5 mice per time point and treatment group). *p ≤ 0.05, **p ≤ 0.01 compared to WT mice (Mann-Whitney U test).WT and S100A9 KO mice were either left untreated or were infected orotracheally with (TIF)Click here for additional data file.S3 FigS. pneumoniae-infected WT and S100A9 KO mice on days 1 and 2 post-infection (n = 5–8 mice per time point and treatment group). Data are shown as mean ± SD and are representative of two independently performed experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared to WT mice (Mann-Whitney U test).(A-C) Proinflammatory TNF-α (A) and IL-1beta (B) and anti-inflammatory IL-10 (C) cytokine levels in BAL fluids of untreated and (TIF)Click here for additional data file.S4 FigS. pneumoniae at a multiplicity of infection (MOI) of 25.  Phagocytosis capacity of resident AM (A) and bone marrow-derived neutrophils (BM-PMN) (B) at 30 or 60 minutes after infection of cells with S. pneumoniae. (C) Burst induction in purified BM-PMN of WT and S100A9 KO mice by S. pneumoniae (MOI 5). Data are representative of two independently performed experiments.Cells were purified as described in Materials and Methods followed by infection with (TIF)Click here for additional data file.


Second, in multiple breast cancer cell lines with high A3B, expression levels were observed to oscillate throughout the cell cycle and again associate with Cyclin B1. Third, induction of A3B expression is potently repressed throughout G0/early G1, likely by RB/E2F pathway effector proteins. Fourth, in cells with low A3B, induction of A3B through the PKC/ncNF-κB pathway occurs predominantly in actively proliferating cells and is largely absent in cells arrested in G0. Altogether, these results support a model in which dysregulated A3B overexpression in breast cancer is the cumulative result of proliferation-associated relief from repression with concomitant pathway activation during the G2/M phase of the cell cycle.APOBEC3B (A3B) is aberrantly overexpressed in a subset of breast cancers, where it associates with advanced disease, poor prognosis, and treatment resistance, yet the causes of A3B dysregulation in breast cancer remain unclear. Here, A3B mRNA and protein expression levels were quantified in different cell lines and breast tumors and related to cell cycle markers using RT-qPCR and multiplex immunofluorescence imaging. The inducibility of A3B expression during the cell cycle was additionally addressed after cell cycle synchronization with multiple methods. First, we found that A3B protein levels within cell lines and tumors are heterogeneous and associate strongly with the proliferation marker Cyclin B1 characteristic of the G PIK3CA, KMT2C, ARID1A, NF1, and CDH1 have been attributed to APOBEC enzymatic activity and are enriched in metastatic breast cancer, supporting a model in which APOBEC proteins are promoting tumor evolution [Normal and cancerous mammary epithelial cells are subject to a multitude of endogenous and exogenous sources of mutagenesis, scarring their genome with a barrage of genetic alterations ,2,3. Welvolution ,11. The volution ,14 and ivolution .A3B, which is expressed at a low level in normal mammary tissue, is overexpressed in a subset of cancerous mammary tissues [A3B expression in breast cancer is associated with aggressive clinicopathological characteristics [Humans express up to seven different APOBEC3 (A3) enzymes, namely A3A, A3B, A3C, A3D, A3F, A3G, and A3H. To date, the most likely family members to contribute to APOBEC mutagenesis are A3A and A3B, with abundant evidence supporting a role for A3B in breast cancer ,16,17,18 tissues ,15,20, a tissues . In addieristics ,16,17. TA3B transcription is facilitated through two distinct repressive E2F complexes, at least one of which associates with the cell cycle [0 and G1 phases), whereas pathway activation during the proliferative stage (G2 and M phases) contributes to the overexpression of A3B found in a subset of breast cancer cell lines and tumors. Together, these results provide a molecular explanation for the association of A3B expression and cancer cell proliferation and provide further insights into the transcriptional (dys)regulation of this cancer genomic DNA deaminase.Earlier work by our group and others has pointed out that both A3B mRNA and protein levels associate with proliferation markers in multiple tumor types ,26,27,28ll cycle ,25,29. H2. MCF10A cells and the derived HDR and control clones were previously described and cultured using an identical medium recipe [Mycoplasma using the MycoAlert Mycoplasma Detection Kit . All cell counting took place using automated cell counters . PMA was always used at 25 ng/mL for 6 h. Knockdown of A3B was performed using shRNA technology, as described in [All cell lines used in this study were cultured at 37 °C under 5% COm recipe . MCF7 cehttps://www.coreon.org/wp-content/uploads/2020/04/coreon-code-of-conduct-english.pdf, accessed on 14 April 2023) and the new European General Data Protection Regulation (GDPR). This protocol states that the use of coded left-over material for scientific purposes and, therefore, for the greater good, does not require informed consent according to Dutch law. This waiver for informed consent was acknowledged by the medical ethics committee of the Erasmus Medical Centre Rotterdam, the Netherlands, in MEC 02.953.Cell lines used in conventional and multiplex IHC assays were part of an in-house cell line microarray (CMA), which contains 54 formalin-fixed paraffin-embedded (FFPE) breast cancer cell lines. Primary breast cancer specimens were obtained under the protocol to study biological markers associated with disease outcome and in accordance with the Code of Conduct of the Federation of Medical Scientific Societies in the Netherlands  was performed. Subsequently, multiplex staining was performed for A3B, Cyclin B1, Cyclin E2, and pan-Cytokeratin. In short, non-specific epitopes were blocked in blocking solution  for 10 min at RT, and rabbit anti-A3B  was addn = the total cells per cell line;k = the number of double positives ;p = the chance of a double-positive cell based on the occurrence of the individual Cyclin per cell line, as calculated using the following Formula (2):Spectral library mapping was performed using single-stained sample slides of BT474 cells using the Vectra 3.0 system. This library was used for spectral overlap compensation and to determine the set exposure time per filter channel of the Vectra microscope. Grey-scale images of samples were taken using inform advanced image analysis software package (PerkinElmer) and exported to a TME-Analyzer version 2.4 beta , which was utilized to segment cells based on cytokeratin  staining and appearance . A Python code was written (available upon request) to process data provided by the TME-Analyzer and determine co-localization at a cellular level. To determine the likelihood of the found co-localization occurring naturally, binomial distribution was performed using the following Formula (1):p-value of 0.05 was corrected with a Bonferroni correction. The null hypothesis was rejected when p-values were lower than 0.05.The null hypothesis of the binomial test stated that the amount of double-positive cells  was caused by chance, and the alternative hypothesis stated that co-localization was caused by a yet-to-be-determined underlying biological mechanism. The chosen 0 by a combined method involving contact inhibition and growth factor withdrawal. In short, cells were plated in T75 or T175 flasks (according to experiment size), and growth medium was refreshed after 2 days. Once cells had reached full confluence, growth medium was once again replaced and contact inhibition was initiated for 24 h. Cells were then harvested, washed in HBSS twice, counted, and split at a 1:5 ratio into separate T25 or T75 flasks (according to experiment size) into mitogen-free medium (MCF10A medium without horse serum and EGF). After 24 h of growth inhibition, mitogen-free medium of all flasks except t = 0 was replaced with normal MCF10A growth medium. For each time point, cells were harvested from individual flasks. Double-thymidine block experiments were performed by plating cells in individual T25 flasks and adding thymidine at a concentration of 2.0 mM after 24 h (48 h for HCC1143). After 18 h of incubation, all flasks were washed with pre-warmed, CO2-equilibrated HBSS and allowed to progress in pre-warmed, CO2-equilibrated culture medium. After 8 h, thymidine was added again at a concentration of 2.0 mM and was removed by washing twice with pre-warmed, CO2-equilibrated HBSS after 18 h. Thymidine was not removed for the t = 0 time point, which was harvested directly. Of note, due to excessive cytotoxicity, MCF10A cells could not be reproducibly synchronized using double-thymidine. The double-thymidine block was also unsuitable for the synchronization of BT474 cells, which is likely due to their low proliferation rate [g for 10 min, or 800× g for 15 min for final sample collection.MCF10A cells were synchronized in Gion rate . Serum wg, 10 min), and ethanol was removed. Cells were washed twice in PBS before staining for 45 min with staining solution (PBS supplemented with 0.1% (w/v) Triton X-100, 20 µg/mL propidium iodide, and 200 µg/mL RNase A) at 1–2 × 106 cells per m: at 37 °C. Cells were then analyzed immediately using the PE channel of a FACS Canto analyzer . To observe mitosis, live cells were stained with 0.3 µg/mL Hoechst in PBS for 5 min at room temperature, enclosed, and analyzed using conventional fluorescence microscopy. Fluorescent immunocytochemistry was performed using rabbit anti-53BP1, 1:1000 .Single-cell suspensions were fixed in ice-cold 70% ethanol while vortexing and incubated on ice for at least 30 min. Cells were pelleted  using a validated antibody against A3B ,27,28. Wh levels ,31. Two A-MB-330 ,31,32,33A-MB-330 ,34. IntrA3B with markers of proliferation in breast cancer  with Cyclin B1, but not Cyclin E2 , we opted to further diversify the cell line panel. Two additional breast cancer cell lines, MCF7 and HCC1143, as well as the previously featured HCC1954 cell line, were therefore included in cell cycle synchronization experiments. These cell lines represent the range of A3B expression found in clinical samples, with MCF7 expressing low levels, HCC1954 expressing moderate levels, and HCC1143 expressing high levels of A3B (CCNE2 (Cyclin E2) and CCNB1 (Cyclin B1) was included to monitor cell cycle progression. Next, multiple cell synchronization methods were used to investigate the degree to which s of A3B . Each ces of A3B , and perA3B mRNA levels rise in all three cell lines, in a manner that associates positively with CCNB1 expression and inversely with CCNE2 expression , indicative of the G2/M phase of the cell cycle. Additionally, and as defined previously [A3B from being expressed, even during active cell cycling and proliferation (discussed further below).To obtain a view of e arrest ,40. Compter 16 h D. Comparn values . These veviously , a repreA3B were associated previously with cell proliferation [A3B transcription is repressed by the RB/E2F pathway [A3B expression. To investigate whether the RB/E2F pathway controls A3B expression, MCF10A and BT474 cells were subjected to prolonged treatment with the CDK4/6 inhibitor palbociclib. Inhibition of the RB/E2F pathway through inhibiting CDK4/6 for 24 h has been shown to induce growth arrest in early G1 . It is ts ssDNA ,52, whicA3B expression, we first set out to select cell lines with differential responses to induction. For this purpose, the chemical compound phorbol myristic acid (PMA) was used to induce PKC/ncNF-κB-dependent expression of A3B in MCF10A and HCC1143. MCF10A cells show high A3B inducibility, whereas HCC1143 breast cancer cells, which have much higher baseline levels of endogenous A3B, show appreciably lower A3B induction potential . We havE2F site . To inveished in ) were syin CCNE2 E. IntrigA3B expression levels oscillate throughout the cell cycle in a manner that associates positively with Cyclin B1 (G2/M phase) and negatively with Cyclin E (G0/G1 phase). Surprisingly, expression and induction of A3B during growth arrest are tightly repressed by the RB/E2F pathway in all cell lines tested. Conversely, A3B induction is a prominent feature during cell cycle progression of A3B-overexpressing breast cancer cells, and only possible during proliferation in cells with low constitutive expression. Thus, our study offers new insights into the mechanism of A3B regulation during the cell cycle. Building on our previous work [This study constitutes the first detailed insights into the cell cycle dynamics of A3B expression in breast cancer. Starting with the observation that A3B expression is heterogeneous in cultured breast cancer cells, we show that A3B expression associates strongly with Cyclin B1 and not Cyclin E2 on a single-cell level in cell lines and clinical breast cancer specimens. Furthermore, breast cancer cell line synchronization studies indicate that ous work ,25, we pous work ,22,23,54A3B. Since its first association, the PKC/ncNF-κB pathway has continued to take a central role in A3B regulation [A3B promoter [A3B can also be stimulated directly using PMA. Conversely, transcriptional repression is facilitated by the RB/E2F pathway and is enforced by the binding of E2F-based repressive complexes to an E2F site within the A3B promoter [A3B during viral infections [Over the recent years, various publications have reported on the transcriptional regulation of gulation ,47,55. Tpromoter ,47,49. Apromoter ,25. Disrfections . The datA3B expression. We now show that a decrease in A3B expression coincides with arrest in G0/early G1, strongly implicating proliferative status with the expression dynamics of A3B. In follow-up research, it would be informative to include a transcription factor screen of the A3B promoter during palbociclib treatment and during promoter activation in response to PMA, which would further elucidate the transcriptional dynamics of A3B. Secondly, repression of A3B during G0 appears to be largely intact in all cell lines included in this study, including cells with high expression of A3B. We previously proposed that overexpression of A3B, in part, may be attributable to the failure of some breast cancer cells to repress A3B through the RB/E2F pathway. Although this is still supported by multiple lines of evidence, the current study offers more detailed insights and indicates that cancer cells with high A3B expression reside disproportionally in cell cycle phases where endogenous and exogenous stimuli are free to activate the promoter. Thus, we propose that overexpression of A3B is the culmination of untimed proliferation and activation of (inflammatory) pathways, including the PKC/ncNF-κB pathway . Such tumors are not only actively dividing but are also therefore likely to be undergoing active DNA deamination, damage, and mutagenesis caused by A3B . This finding is corroborated by other work that shows that A3B is preferentially expressed during G2/M in myeloid cells [2-arresting drugs. Inducing G2 arrest in A3B-positive tumors to be treated with IR may also provide clinical benefit since tumor cells are most sensitive to DNA damage in G2, during which A3B expression may be induced further to possibly toxic levels. Future cell-based assays should determine the dosage, timing, and feasibility of such an approach. Second, this work shows the applicability of multiplex IHC with an A3B antibody and two cell cycle proteins. In essence, any other clinically relevant marker may be co-stained using this approach, which may then inform the tumor microenvironment of A3B-expressing tumors. For example, the infiltration of CD4+/CD8+ T-cells into A3B-expressing tumor mass could be directly observed in clinical biopsies. This is exceptionally relevant since recent evidence indicates that APOBEC-dominated tumors might be receptive to immune-based anticancer therapies [The clinical relevance of this work is at least two-fold. First, it explains the heterogeneity observed in tumor A3B protein expression by IHC, with strong positivity associating with active cell cycling and specifically with the Gid cells . Since Aid cells ,57, it mherapies ,59,60.In conclusion, the data presented here consolidate the proposed association between cellular proliferation and A3B expression and offer further insights into the cell cycle dynamics of the regulation of this cancer genomic DNA deaminase. In breast cancers, A3B overexpression appears to be the cumulative result of proliferation-associated relief from repression with concomitant pathway activation .


A total of 11 PEX-GSH and7 PEX-NH2OMe adducts were identified in HLM/MLM using anLC–MS-based metabolomics approach. Additionally, 4 PEX-GSHadducts were detected in the PHH. CYP3A4 and CYP3A5 were identifiedas the primary enzymes responsible for the formation of these adductsusing recombinant human P450s and CYP3A chemical inhibitor ketoconazole.Overall, our studies suggested that PEX metabolism can produce reactivemetabolites mediated by CYP3A, and the association of the reactivemetabolites with PEX hepatotoxicity needs to be further studied.Pexidartinib , a selective and potent inhibitor ofthe macrophage colony-stimulating factor-1 receptor, has been approvedfor the treatment of tenosynovial giant cell tumor. However, frequentand severe adverse effects have been reported in the clinic, resultingin a boxed warning on PEX for its risk of liver injury. The mechanismsunderlying PEX-related hepatotoxicity, particularly metabolism-relatedtoxicity, remain unknown. In the current study, the metabolic activationof PEX was investigated in human/mouse liver microsomes (HLM/MLM)and primary human hepatocytes (PHH) using glutathione (GSH) and methoxyamine(NH PEX is availableto patients only through a restricted program under a Risk Evaluationand Mitigation Strategy,4 and it is essentialto monitor liver function prior to the initiation of PEX treatment.12Pexidartinib ,a novel small molecule tyrosine kinaseinhibitor, has highly selective and potent inhibitory activity againstmacrophage colony-stimulating factor-1 receptor (CSF1R).14 It is well appreciated that reactivemetabolites play a critical role in idiosyncratic adverse drug reactions.15 According to FDA documents, PEX is rapidly metabolized, primarilyby the enzymes CYP3A and UGT1A4, which are involved in phase I andphase II metabolism, respectively.12 PEXwas recovered in the feces as the unchanged form (44%) and in theurine (27%) as metabolites (≈10% as N-glucuronide).5 Our previous studies focused on the carbon–carbon bond cleavagein PEX metabolism, provided a comprehensive profile of the phase Imetabolism of PEX in human liver microsomes (HLM), and identifiedits stable metabolites.16 However, it isstill unclear whether PEX metabolism is capable of generating reactivemetabolites and what specific roles they may play in causing its severehepatotoxicity.To date, the mechanism(s) of PEX-related toxicity remainsmostlyunidentified. Drug metabolism is closely connected to both drug efficacyand adverse effects.2OMe) as trapping reagents in HLM and mouse liver microsomes(MLM).20 The drug metabolizing enzymes contributing to the generation ofPEX reactive metabolites were determined using recombinant human P450sand a specific chemical inhibitor in HLM/MLM and primary human hepatocytes(PHH). Our studies revealed 11 PEX-GSH and 7 PEX-NH2OMeadducts in HLM/MLM. Four PEX-GSH adducts were observed in PHH treatedwith PEX. These findings from this study may facilitate the understandingof PEX hepatotoxicity from the perspective of drug metabolism.In the present study, we employed LC–MS-basedmetabolomicapproaches to investigate the metabolic bioactivation of PEX in invitro systems. We used reduced glutathione (GSH) and methoxyaminemethyl]-N-[[6-(trifluoromethyl)pyridin-3-yl]methyl]pyridin-2-amine),was purchased from Cayman Chemical . Ketoconazole (KCZ),NH2OMe hydrochloride, GSH, formic acid, and β-nicotinamideadenine dinucleotide 2′-phosphate reduced tetrasodium salthydrate (NADPH) were purchased from Sigma-Aldrich .HLM , MLM , recombinant human P450s (EasyCYP Bactosomes), and PHHwere obtained from XenoTech . All solvents for liquidchromatography and mass spectrometry were of LC–MS grade .PEX,  was added to initiate the reactions. The incubationcontinued for 40 min at 37 °C with gentle shaking. Incubationswithout NADPH or GSH/NH2OMe were used as controls. Co-incubationsof PEX (30 μM) and KCZ in HLM and MLM with GSH or NH2OMe were performed to determinethe role of CYP3A in the formation of GSH or NH2OMe adductsrelated to PEX. Reactions were quenched by adding 100 μL ofice-cold acetonitrile and vortexing for 30 s, and the mixtures werethen centrifuged at 15,000 rcf for 15 min. The supernatant was transferredto an autosampler vial, and 3 μL was injected into an ultrahigh-performanceliquid chromatography (UHPLC) Q Exactive MS system for analysis. Incubationswere performed in duplicate for cDNA-expressed P450 enzymes and intriplicate for LM experiments. The PEX concentration (30 μM)used in liver microsomes is clinically relevant according to the Cmax in human subjects.Incubations were conducted in 1× phosphate-buffered saline containing 30 μM PEX, 0.1 mg LM (HLM or MLM),or 1 pmol of each cDNA-expressed human P450 enzymes  and 2.5 mM GSH or2.5 mM NHCmax of PEX is around 4 μg/mL (∼9.6μM) in human subjects at 400 mg per day.21 Typically, the concentration of drugs in the liver is severaltimes greater than that in the bloodstream.The average plasma 2.35 cells/well.The cells were cultured in complete HepatoZYME medium, containingGlutaMAX, Insulin-Transferrin-Selenium, and penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2 for 24 hbefore PEX treatment. The PHH were treated with 20 μM PEX withor without 10 μM KCZ for 6 h. The medium was then transferredinto Eppendorf tubes and centrifuged at 100 rcf for 5 min to removethe suspending cells. The cells were washed with 1× DPBS  3 times, harvested in 500 μL of methanol–water(v/v 1/1), and lysed with a probe ultrasonicator . Twenty microliters of culture medium was added to 60 μLof ice-cold acetonitrile containing 0.1 μM agomelatine as theinternal standard (IS), or 50 μL of cell lysate was added with100 μL of IS solution. After vortexing and centrifugation at15,000 rcf for 15 min, the supernatants were transferred to samplevials and 3 μL was injected into a UHPLC-Q Exactive MS systemfor analysis.PHH  were thawed and platedin 12-well plates  according to the protocolof the vendor with the density of 6.95 × 102.42OMe adducts in the samples were resolved, analyzed,and relatively quantified by a UHPLC-Q Exactive MS system . The column for analyte separationwas an Acquity 100 mm × 2.1 mm BEH C-18 column , whose temperature was maintained at 40 °C.The flow rate was 0.3 mL/min with gradient ranging from 2 to 95% inthe water-acetonitrile mobile phase system both containing 0.1% formicacid in a 15 min run. Q Exactive MS was operated in positive modewith electrospray ionization. Ultrapure nitrogen was applied as thesheath (45 arbitrary unit), auxiliary (10 arbitrary unit), sweep (1.0arbitrary unit), and the collision gas. The capillary gas temperaturewas set at 350 °C, and the capillary voltage was set at 4.3 kV.MS data were acquired from 80 to 1200 Da in the profile mode. Theion at m/z 371.1012 was used asa reference for positive mode during acquisition. The MS/MS of GSHand NH2OMe adducts associated with PEX was performed withthe normalized collision energy set at 10–35 arbitrary unitswith an isolation width of 2 m/z.PEX and its GSHor NH2.5m/z 80 to 1200 wereacquired in profile format by Xcalibur software . These data were processed by Compound Discoverer 3.1software  to generate a multivariatedata matrix. Data matrices were exported into SIMCA14  for multivariate data analysis. Orthogonal projection to latentstructures-discriminant analysis (OPLS-DA) was performed on Pareto-scaleddata.20 For chemometric analysis, matrixdata were processed from m/z 80to 1000.Chromatograms and massspectra from 33.1Figure S1) was the most prevalent adduct, constituting 83% of the total PEX-GSHadducts. In contrast, M2 was the adduct with the largest peak in thePHH medium representing 51% of the total, while M9 accounted for 6.3%. Protonated molecules with exact masses at m/z 723.1722 were observed for M1 and M2,eluting at 8.86 and 8.98 min, respectively . Thus, it is likely that GSH in M1 and M2 is not linked to the(6-(trifluoromethyl)pyridin-3-yl)methyl moiety in PEX. The fragmentions were interpreted in the inlaid structural diagrams  and oxidized PEX (M3–M7)in the presence of NADPH and GSH in HLM and MLM. Their formation isNADPH- and GSH-dependent, as they were not detected in control groups   2G. The MH + GSH) 2I. The M308.0915 2J, which16 M8 and M9 were generated by conjugating GSHwith cleaved parts (S1). M10 was formed by GSHreacting with the N-dealkylated PEX metabolite . M8 eluted at 6.27 min with a protonated m/z value of 559.1581 related to the carbon–carbon cleavage in PEX phase I metabolism,which was investigated in detail in our previous study.ed parts 2L and S1559.1581 2K. The Mdiagram 2L.M9 ha3.32OMe. The principal component analysis resulted in three distinctclusters, as shown in Figure S5A. The S-plotgenerated from OPLS-DA  with larger peak areas associated with PEX in LMwere determined in LM based on the metabolomic analysis  in LM.16 As proposed in Figure S7, the formation of amine-NH2OMe adducts M14 and M15 followedsimilar pathways to the formation of M12 and M13. Oxime M17 has theprotonated m/z value of 205.0583and was eluted at 12.41 min, which was identified as the NH2OMe adduct of 4-(trifluoromethyl)-benzaldehyde . The MS/MS analysisof M17 produced fragment ions at 147.0282 and 174.0390, which wereelucidated in the inlaid structural diagram . M16 and M18 were formed by capturing the aldehydes producedfrom products of carbon–carbon cleavages of PEX with NH2OMe . Their formationpathways have been extensively studied, and their structures wereconfirmed with standard compounds in our previous work.16M12 is the dominant adduct in both HLM andMLM, followed by M13 3B. M12 h9.50 min 4A. The M diagram 4B. M13, 463.1261 4C. The M diagram4D. The puced M13 5. M14 ha4.74 min 4E.The M diagram 4F. M15 e3.52OMe adductsusing a panel of human recombinant P450 enzymes and the specific chemicalinhibitor in HLM. Using human recombinant P450 enzymes, CYP3A4 andCYP3A5 were identified as the primary enzymes responsible for theformation of adducts . Additionally,the CYP2A6 isoenzyme contributed to the formation of M4 (31%) .In PHH lysate and medium, KCZ at a concentration of 10 μM inhibitedthe formation of PEX-GSH adducts , with inhibitionranging from 65 to 100% (We investigated the role of P450senzymes in the formation of PEX-GSH and PEX-NHM4 (31%) 2. SeveraM4 (31%) 6A,D. In  to 100% 6B,C.422 Reactive drug metabolites are well appreciated as significant contributorsto drug adverse effects due to their potential for covalent modificationof important biological macromolecules such as proteins and DNA.17 Comprehensive understanding of the metabolic profile and characterizingreactive metabolites of drug benefit drug safety evaluation and improvement.In our previous work, we identified stable phase I metabolites ofPEX, indicating extensive CYP3A-mediated metabolism of PEX in vitro.16 In this study, we employed an LC–MS-basedmetabolomic approach to screen for trapped reactive metabolites relatedto PEX. A total of 11 PEX-GSH and 7 PEX-NH2OMe adductswere identified in HLM or MLM incubations. The formation of GSH adductsassociated with PEX suggests the presence of active metabolites duringin vitro metabolism, indicating the possibility of a similar reactionoccurring between PEX and free sulfur groups in proteins, which couldimpair the functions of important macromolecules in vivo. In PHH lysateand medium, M1 and M2 were identified, showing a similar pattern tothose in HLM/MLM, while the major adduct M6 observed in HLM/MLM wasnot detected in PHH  was likely producedfrom M1 or M2 by the loss of the glutamic acid motif. The exact mechanismsof formation for M1, M2, and M11 remain unclear. Adducts M8–M10are three GSH adducts formed by conjugating GSH with cleaved partsof PEX . Previous studiessuggest that PEX can form phenol metabolites through carbon–carboncleavage.16 We suspected that the phenolmetabolite could react with GSH, followed by the loss of H2O to form M8; however, incubation of the phenol metabolite with GSHin HLM did not yield M8, indicating that M8 formation from PEX occurredvia alternative unknown pathways. We have determined the formationof M9 in the study of CYP3A-mediated carbon–carbon cleavageof PEX.16 In brief, the formed cation conjugateswith GSH to form M9 . Following further oxidization and hydrogenation, the resultingmetabolite can be modified by GSH to form M10 , theMS/MS spectra of high quality were not obtained due to their low abundance.The tentative structures were speculated based on their exact masses.The GSH adductsidentified in both HLM and MLM were M1, M2, M6,and M9 1C.Speci2OMe to produce oxime M12. M13 can be formed fromM12 by eliminating H2O. An alternative pathway involvesthe aldehyde losing H2O first and subsequently reactingwith NH2OMe to produce M13. As shown in Figure S7, the formation of M14 and M15 followed a similarprocess when the amine served as a substrate. Among the oximes M16–M18,the formation of M16 has been detailed in our carbon–carboncleavage paper.16 The aldehyde M16 itselfhas also been detected in both HLM and MLM . M17 is the product of the condensation of 4-(trifluoromethyl)benzaldehydewith NH2OMe. The formation of benzaldehyde in PEX metabolismwas further confirmed by the detection of its corresponding acid andalcohol. The structures of these compounds were also validated usingtheir standard compounds.The formation of theoximes (M12–M18) in HLM and MLM indicatedthe generation of reactive aldehydes during PEX metabolism. The formationof M12 and M13 is proposed in 16 .A total of 11 GSH adducts and 7 NH2OMe adducts associatedwith PEX were identified (In summary, we conducted a comprehensivestudy on the reactivemetabolites of PEX in HLM, MLM, and PHH using an LC–MS-basedmetabolomic approach and trapping agents (GSH and NHentified 7. Among