Patients with chronic kidney disease (CKD) develop anemia largely because of inappropriately low erythropoietin (EPO) production and insufficient iron available to erythroid precursors. In four phase 3, randomized, open‐label, clinical trials in dialysis‐dependent and non–dialysis‐dependent patients with CKD and anemia, the hypoxia‐inducible factor prolyl hydroxylase inhibitor, vadadustat, was noninferior to the erythropoiesis‐stimulating agent, darbepoetin alfa, in increasing and maintaining target hemoglobin concentrations. In these trials, vadadustat increased the concentrations of serum EPO, the numbers of circulating erythrocytes, and the numbers of circulating reticulocytes. Achieved hemoglobin concentrations were similar in patients treated with either vadadustat or darbepoetin alfa, but compared with patients receiving darbepoetin alfa, those receiving vadadustat had erythrocytes with increased mean corpuscular volume and mean corpuscular hemoglobin, while the red cell distribution width was decreased. Increased serum transferrin concentrations, as measured by total iron‐binding capacity, combined with stable serum iron concentrations, resulted in decreased transferrin saturation in patients randomized to vadadustat compared with patients randomized to darbepoetin alfa. The decreases in transferrin saturation were associated with relatively greater declines in serum hepcidin and ferritin in patients receiving vadadustat compared with those receiving darbepoetin alfa. These results for serum transferrin saturation, hepcidin, ferritin, and erythrocyte indices were consistent with improved iron availability in the patients receiving vadadustat. Thus, overall, vadadustat had beneficial effects on three aspects of erythropoiesis in patients with anemia associated with CKD: increased endogenous EPO production, improved iron availability to erythroid cells, and increased reticulocytes in the circulation. The INNO2VATE and PRO2TECT trials both met the prespecified noninferiority criteria (−0.75 g/dL) for the primary efficacy endpoint of mean change in hemoglobin from baseline to Weeks 24 to 36. The INNO2VATE trial also met the prespecified noninferiority criteria (1.25) for the primary safety endpoint of time to first major adverse cardiovascular event ; however, the PRO2TECT trials did not meet the prespecified margin for noninferiority.2TECT trials , was similar in the vadadustat‐ and darbepoetin‐treatment groups.Vadadustat, an investigational oral HIF‐PHD inhibitor that stabilizes intracellular HIFα, increases serum EPO concentrations over baseline in healthy individuals and in patients with CKD.22.12TECT trials included ESA‐untreated patients with NDD‐CKD  or ESA‐treated patients with NDD‐CKD ; the two INNO2VATE trials included ESA‐treated patients with incident DD‐CKD  and prevalent DD‐CKD .2TECT trials in Figure 2VATE trials in Figure We conducted four global, open‐label, sponsor‐blinded, active‐controlled, noninferiority trials in patients with CKD. The two PRO2.22TECT trials, eligible patients were at least 18 years of age and had NDD‐CKD (eGFR ≤60 mL/min/1.73 m2). In the trial involving ESA‐untreated patients, patients were required to have a hemoglobin concentration <10 g/dL and were excluded if they had received any ESA within 8 weeks before randomization. In the trial involving ESA‐treated patients, patients had to be actively receiving maintenance ESA therapy, with at least one dose received within 6 weeks before or during screening and were required to have a hemoglobin concentration of 8–11 g/dL (in the United States) or 9–12 g/dL (in other countries). In both INNO2VATE trials, eligible patients were at least 18 years of age, had CKD and were undergoing dialysis, had a serum ferritin concentration ≥100 ng/mL and a transferrin saturation (TSAT) ≥20%, and had not received an RBC transfusion within the previous 8 weeks. In addition, patients had a hemoglobin concentration between 8 and 11 g/dL  or a hemoglobin concentration between 8 and 11 g/dL (in the United States) or between 9 and 12 g/dL (in other countries) . In all trials, oral or intravenous iron administration was allowed at the discretion of local investigators to maintain serum ferritin ≥100 ng/mL or TSAT ≥20%.In both PRO2TECT and INNO2VATE trials had prespecified primary (Weeks 24–36) and secondary (Weeks 40–52) evaluation periods for efficacy, as measured by change or maintenance of hemoglobin concentrations (target hemoglobin concentrations of 10–11 g/dL in the United States and 10–12 g/dL in other countries). The prespecified noninferiority margins were selected in consultation with regulatory agencies as the lower bound of the 95% confidence interval of −0.75 g/dL for the primary efficacy end point. The primary safety outcome was time to first MACE with a prespecified noninferiority margin of 1.25.The PROIn addition to baseline values, erythrocyte numbers (RBCs) and erythrocyte indices were measured every 2 weeks through Week 12, then every fourth week. Serum ferritin, iron, TSAT, and total iron‐binding capacity (TIBC), were measured every 4 weeks through Week 12, and every 8 weeks through Week 52. Serum bilirubin and lactate dehydrogenase were measured every 4 weeks through Week 28, and every 8 weeks through Week 52. Reticulocyte numbers and serum hepcidin  were measured at Weeks 4, 12, 28, and 52; serum C‐reactive protein  was measured at Weeks 28 and 52. Endogenous serum EPO was measured at Weeks 4, 12, 28, and 52 in patients randomized to receive vadadustat using an ELISA . Endogenous EPO could not be measured in patients randomized to receive darbepoetin because, as a derivative of EPO, darbepoetin is immunologically related to EPO and darbepoetin's erythropoietic activity suppresses endogenous EPO production. However, serum EPO concentrations were also determined in darbepoetin‐treated patients, while recognizing that their values represented combined endogenous EPO and exogenous darbepoetin.2.3t tests. The analyses between groups were based on analyses of covariance with the baseline values of laboratory tests as covariates, and central laboratory baseline hemoglobin group, geographic region, New York Heart Association congestive heart failure class, and treatment group as fixed effects. We considered two‐tailed p < .05 as statistically significant without adjustment for multiple comparisons. All analyses were conducted using SAS statistical software version 9.4 (SAS Institute Inc).We analyzed erythropoietic effects of vadadustat for the safety population . Blood samples for laboratory assays were sent to the trials' central laboratory for analysis. Local investigators were responsible for reviewing laboratory results for clinical significance and adjusting the dose according to the protocol algorithm. Results for RBCs, reticulocytes, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell distribution width , and serum EPO, TIBC, iron, TSAT, hepcidin, and ferritin were compared with respect to mean change from baseline in the primary and secondary evaluation periods. Within treatment group analyses were based on paired 32TECT trials, 1751 ESA‐untreated patients with NDD‐CKD and 1725 ESA‐treated patients with NDD‐CKD  were randomly assigned in a 1:1 ratio to receive vadadustat or darbepoetin. In the INNO2VATE trials, 369 patients in the incident DD‐CKD trial and 3554 patients in the prevalent DD‐CKD trial  were randomly assigned in a 1:1 ratio to receive vadadustat or darbepoetin. Baseline characteristics were generally similar among patients randomized to vadadustat or darbepoetin within all trials  in both the vadadustat‐treatment and darbepoetin‐treatment groups while remaining in the 16–20 mIU/mL range Tables . These iIn both treatment groups, mean serum EPO levels in the ESA‐treated NDD‐CKD trial were in ranges similar to those in the ESA‐untreated trial. In the incident and prevalent DD‐CKD trials, both vadadustat‐treated and darbepoetin‐treated patients maintained their serum EPO levels that were slightly increased at baseline compared with those of patients in the NDD‐CKD trials (20–32 mIU/mL) Tables  and S2.6/μL in both groups at baseline to 3.5 and 3.6 × 106/μL at Week 52 in the vadadustat‐ and darbepoetin‐treatment groups, respectively , although mean MCV and mean MCH were consistently higher in patients randomized to vadadustat compared with those randomized to darbepoetin at each time point in all four trials Table . The inc3.42TECT trials and the trial in prevalent DD‐CKD, serum iron was significantly higher in the vadadustat‐treated group compared with the darbepoetin‐treated groups (Figure In all four trials, serum transferrin concentrations as measured by mean TIBC were significantly increased over baseline at all times in patients randomized to vadadustat relative to those in patients randomized to darbepoetin Figure . Mean bas Figure . Before s Figure .Across all four trials, the generalized increases in TIBC in vadadustat‐treated groups resulted in a pattern of decreased mean TSAT in the early weeks of each trial followed by recovery of mean TSAT only in the incident DD‐CKD trial Figure . In darb3.5Mean serum hepcidin concentrations decreased in both treatment groups in each trial, with more prominent decreases in patients randomized to vadadustat than in those randomized to darbepoetin Figure . Baselin42TECT and INNO2VATE phase 3 clinical trials.2VATE trials but not in the PRO2TECT trials.2TECT trials was similar in the vadadustat‐ and darbepoetin‐treatment groups.As reported previously, vadadustat was noninferior to darbepoetin alfa with respect to hematological efficacy in the PROHIF‐2α regulates EPO gene transcription and subsequent EPO production.Patients randomized to vadadustat achieved and maintained target range hemoglobin concentrations during the primary and secondary evaluation periods in all four trials. The slightly above‐population reference range of serum EPO concentrations in both vadadustat‐treated and darbepoetin‐treated patients indicate that their EPO concentrations were sufficient for achieving the target hemoglobin range but not for achievement of a normal hemoglobin range. The patients' marrow erythroid cells likely had decreased EPO responsiveness compared with healthy individuals. Inflammatory cytokines can directly inhibit proliferation and survival of erythropoietic cells in the marrow,Nearly two‐thirds of total body reticulocytes are normally in the marrow,In the present clinical trials with predetermined target hemoglobin concentrations and nonextensive blood loss, the increased reticulocytes in patients randomized to vadadustat represented 10%–20% of circulating reticulocytes and 0.3% of total circulating erythrocytes, thereby contributing only slightly to the increased mean MCV and MCH. MCVs of non‐reticulocytes in the RBC population of each study at Weeks 28 and 52 Table  show theALAS‐2 geneThese increases in erythrocyte size and hemoglobin content in the patients randomized to vadadustat compared with those randomized to darbepoetin are most consistent with improved iron delivery to erythroblasts, where increased intracellular iron enhances heme synthesis that, in turn, increases hemoglobin accumulation and cell size.Most iron supplied to erythroblasts is recycled from the degraded hemoglobin of senescent erythrocytes that have been phagocytosed by macrophages in a process that is not directly affected by HIF.ERFE from stimulated erythropoiesis may have contributed to decreased serum hepcidin in the first few months after initiating both vadadustat and darbepoetin treatments. At later times in the trials, however, persistent decreases in hepcidin for vadadustat‐treated and darbepoetin‐treated patients, and the relatively more prominent decreases in hepcidin with vadadustat treatment compared with darbepoetin treatment indicate a role for non–ERFE‐mediated effects. In this regard, vadadustat resulted in more pronounced increases in TIBC than darbepoetin at all times in each trial, suggesting that vadadustat induced the transferrin gene, a known transcriptional target of HIF‐1α,2VATE and PRO2TECT trials had elevated baseline serum ferritin and CRP concentrations due to inflammation and oral or intravenous iron supplementation. Furthermore, patients in the trials received oral and/or intravenous iron supplementation to maintain serum ferritin ≥100 ng/mL or TSAT ≥20%, as is common in the care of patients with CKD. Oral or intravenous iron use was similar in the two treatment groups throughout both studies. The serum CRP remaining stable in the 7–11 mg/L range in all four trials suggests that increasing serum ferritin at later times in both NDD‐CKD trials and the incident DD‐CKD trial was likely due to iron administrations rather than changes in inflammation. Because of this ongoing iron supplementation, the effects of vadadustat on oral iron absorption could not be determined. However, HIF‐2α plays key roles in duodenal iron absorption,Like most patients with CKD, participants in the INNOIn conclusion, results from four large phase 3 trials comparing the investigational drug vadadustat with an ESA, darbepoetin, demonstrated that vadadustat can increase endogenous EPO production, improve erythroblast iron availability, and increase circulating reticulocytes in patients with CKD and anemia. Although the underlying physiological mechanisms of these present results were not directly investigated, they are likely due to vadadustat effects on the kidneys, liver, and bone marrow (Figure Contributions: Wolfgang C. Winkelmayer, Glenn M. Chertow, Kai‐Uwe Eckardt, Youssef M. K. Farag, and Patrick S. Parfrey conceived and designed the study. Wenli Luo acquired the data and Mark J. Koury, Wolfgang C. Winkelmayer, Glenn M. Chertow, Kai‐Uwe Eckardt, Todd Minga, and Wenli Luo analyzed the data. Mark J. Koury wrote the first draft and Mark J. Koury, Rajiv Agarwal, Glenn M. Chertow, Kai‐Uwe Eckardt, Steven Fishbane, Tomas Ganz, Volker H. Haase, Mark R. Hanudel, Patrick S. Parfrey, Pablo E. Pergola, Prabir Roy‐Chaudhury, James A. Tumlin, Robert Anders, Youssef M. K. Farag, Wenli Luo, Todd Minga, Christine Solinsky, Dennis L. Vargo, and Wolfgang C. Winkelmayer revised critically for important intellectual content. All authors contributed to the interpretation of the results. All authors provided final approval for the version to be published. The corresponding author attests to having full access to the study data and was responsible for the final decision to submit this manuscript for publication.Steven Fishbane currently serves as a study consultant for Akebia Therapeutics Inc., AstraZeneca, and GlaxoSmithKline. Mark J. Koury, Rajiv Agarwal, Glenn M. Chertow, Kai‐Uwe Eckardt, Tomas Ganz, Volker H. Haase, Mark R. Hanudel, Patrick S. Parfrey, Prabir Roy‐Chaudhury, James A. Tumlin, and Wolfgang C. Winkelmayer currently serve as study consultants for Akebia Therapeutics Inc. Wenli Luo, Robert Anders, Christine Solinsky, and Dennis L. Vargo are employees of Akebia Therapeutics, Inc. Youssef M. K. Farag and Todd Minga were employees of Akebia Therapeutics, Inc., at the time the study was conducted.Appendix S1 Supporting InformationClick here for additional data file.


The median progression‐free survival (PFS) was 18.7 versus 10.2 months, with ixazomib‐Rd versus placebo‐Rd  demonstrating a statistically significant improvement as observed in the primary trial. The median OS improved by 32.6 months with ixazomib‐Rd over placebo‐Rd ; however, the statistically significant benefit seen in PFS was not observed for OS. Improvement with ixazomib‐Rd over placebo‐Rd was observed in overall response  and complete response . Patient‐reported quality of life and use of subsequent therapies were similar across treatment groups. No new safety concerns were identified. Compared with the main cohort, median OS was 10 months longer with ixazomib‐Rd and 21 months shorter with placebo‐Rd in this subgroup, indicating a clinically meaningful survival benefit of ixazomib‐Rd treatment in this patient population with limited access to subsequent novel therapies.Ixazomib‐lenalidomide‐dexamethasone (ixazomib‐Rd) showed clinical efficacy over placebo‐Rd in patients with relapsed/refractory multiple myeloma (MM) in the TOURMALINE‐MM1 trial. Over a median follow‐up of ∼85 months, as patients showed disease progression, they received subsequent novel therapies that confounded the overall survival (OS) benefit. Here, we conducted a post hoc analysis in 148 patients from seven countries defined as emerging markets, with limited access to novel therapies for MM during the trial period, to describe the impact of these therapies on OS. Patients were randomised to ixazomib‐Rd ( In 2020Novel agents have expanded the therapeutic armamentarium for MM, which includes proteasome inhibitors (PIs) such as bortezomib, carfilzomib and ixazomib; immunomodulatory drugs (IMiDs) such as lenalidomide, thalidomide and pomalidomide; monoclonal antibodies such as the anti‐CD38 antibodies daratumumab and isatuximab and the anti‐SLAMF7 CD319) antibody elotuzumab; and chimeric antigen receptor (CAR) T‐cell therapies 9 antibod. Daratump = 0.01) [p = 0.495). The OS benefit in the overall population was confounded by the use of subsequent therapy (≥70% of patients). Specifically, PIs and daratumumab were administered more frequently in the placebo‐Rd group due to earlier disease progression compared with that in the ixazomib‐Rd group. Moreover, greater OS benefit with ixazomib‐Rd over placebo‐Rd was observed in patients who did not receive subsequent therapy compared with those who did [Clinical trials have shown that PIs are effective for treating patients with newly diagnosed, high‐risk MM, and these agents are currently the established regimen for relapsed or refractory (RR) MM in combination with IMiDs and corticosteroids . Ixazomi = 0.01) . However who did .p = 0.035) and median OS  [The China Continuation study, a distinct regional expansion of the TOURMALINE‐MM1 trial comprising 115 patients, showed a statistically significant improvement with ixazomib‐Rd compared with placebo‐Rd in both median PFS  . The med= 0.001) .Therefore, we conducted this post hoc subgroup analysis to assess and describe the OS benefit of ixazomib‐Rd in patients from emerging‐markets where there was limited availability or accessibility of novel agents for MM treatment, specifically daratumumab, elotuzumab, isatuximab and CAR T‐cell therapies, during the TOURMALINE‐MM1 trial .22.1TOURMALINE‐MM1 trial was a randomised, placebo‐controlled, double‐blind, multicentre, phase III trial  . The enrFor the TOURMALINE‐MM1 global trial, patients were randomly assigned 1:1 to receive ixazomib‐Rd or placebo‐Rd using a centralised interactive voice response system (IVRS). The dosage and administration schedule have been described earlier . Randomi2.2OS was defined as the time from the date of randomisation to the date of death or date last known to be alive. PFS was defined as the time from the date of randomisation to the date of first documentation of disease progression or all‐cause death, whichever occurred first. Both OS and PFS were analysed in the intent‐to‐treat (ITT) population and in pre‐specified subgroups defined by age, ISS, cytogenetic risk markers, number of prior therapies, exposure to PIs and IMiDs, refractoriness to last prior therapy and RR status.Other outcomes included disease response, patient‐reported health‐related quality of life (HRQoL) and safety. Disease response was defined and assessed per the International Myeloma Working Group Uniform Response Criteria  for overAdverse events (AEs) were coded using the Medical Dictionary for Regulatory Activities (MedDRA) version 16.0. Drug‐related (any drug in the drug combination) treatment‐emergent AEs (TEAEs) were analysed in the safety population and summarised using the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03. Patients were continuously assessed for new primary malignancies from study treatment initiation until death or study termination. Exploratory outcomes included the classes of subsequent therapies received by both study groups at follow‐up every 12 weeks from disease progression.2.3p values. Response rates were compared using a stratified Cochran‐Mantel‐Haenszel test, and odds ratios (ORs) were estimated using a logistic regression model.The ITT population included all patients from emerging markets who were randomised. The safety population included all patients who received at least one dose of the study drug or placebo. Formal hypothesis testing was not conducted. OS and PFS were evaluated using a closed sequential testing procedure  and asse33.1n = 71) or placebo‐Rd (n = 77). Patient characteristics were well balanced between treatment groups. High‐risk cytogenetic abnormalities occurred in 22% of patients in the ITT population and were similarly distributed in the two treatment groups. Overall, 41%, 43% and 17% of patients had received 1, 2 and 3 prior therapies, respectively; 68% of patients had received bortezomib, and 52% had received IMiDs  from seven countries (emerging markets) who received either ixazomib‐Rd (3.2p = 0.261)  versus the placebo‐Rd group  Figure . The sur) Figure . A signi) Figure . Additiop = 0.008)  Figure . A lower) Figure . All othp = 0.019). Similarly, CR was observed in a significantly higher proportion of patients in the ixazomib‐Rd group (22.5%) versus the placebo‐Rd group . The median TTP was 18.7 and 12.0 months in the ixazomib‐Rd and placebo‐Rd groups, respectively  (Table The ORR was significantly higher in the ixazomib‐Rd group (81.7%) versus the placebo‐Rd group  received subsequent anticancer therapy following study treatment completion. Overall, similar proportions of patients in the ixazomib‐Rd and placebo‐Rd groups were administered corticosteroids (61% vs. 64%), IMiDs (43% vs. 44%), alkylating agents (44% vs. 42%) and PIs (36% vs. 42%) . This is in contrast to the main trial, which showed a similar median OS between the two groups (53.6 vs. 51.6 months) . AlthougThis post hoc analysis demonstrated a greater OS benefit with ixazomib‐Rd over placebo‐Rd among patients with adverse prognostic factors such as ≥2 prior therapies, prior exposure to IMiDs and refractory to prior therapies such as thalidomide. Additionally, a trend towards lower risk of death was observed with ixazomib‐Rd versus placebo‐Rd in patients >75 years of age, with ≥3 prior therapies by EDC and refractory disease. Considering these subgroups represent patients with poor prognosis, this analysis indicated that treatment with ixazomib‐Rd is likely to provide considerable survival benefit in such patients. Further studies should be conducted to confirm these potential predictors for efficacy. In this study, compared with the placebo‐Rd group, the ixazomib‐Rd group had a higher ORR, and more patients treated with ixazomib‐Rd achieved CR. Compared to the main cohort, the emerging‐markets subgroup of ixazomib‐Rd–treated patients had a higher ORR (78.0% vs. 81.7%) and CR (12.0% vs. 22.5%). The DOR with ixazomib‐Rd was not estimable in the subgroup, whereas it was 20.5 months in the main cohort [When compared with the main cohort, the median OS in the emerging‐markets subgroup was approximately 10 months longer in the ixazomib‐Rd group and almost 21 months shorter in the placebo group. Most attributes of the emerging‐market subgroup population were similar to those of the main cohort, with a few key differences. Specifically, this subgroup had a higher proportion of patients who underwent >1 prior therapy (59% vs. 39%) and were refractory to prior IMiDs (42% vs. 23%). In addition, fewer patients had received stem cell transplantation (46% vs. 57%) or had prior exposure to lenalidomide (3% vs. 12%). Although the proportion of patients who received prior PI therapy was similar in the emerging‐markets subgroup and in the main cohort , the proportion of patients refractory to PIs was higher in the emerging‐markets subgroup (9% vs. 2%). A similar proportion of patients in the emerging‐markets subgroup and the main cohort received subsequent therapies ; however, as would be expected, the emerging‐markets subgroup reported lower use of novel therapies versus the main cohort . In the main cohort, 80% of patients were from North America and Europe and had access to novel therapeutic agents for subsequent therapy, which may have confounded the interpretation of OS . Conversp = 0.045) [p = 0.005), which translated to an improved median OS  [p = 0.008) was observed.Triplet regimens containing a PI have demonstrated survival benefits in both clinical and real‐world studies. A combination of carfilzomib, with Rd, was evaluated for clinical efficacy in patients with RRMM who had received 1–3 prior therapies, in the ASPIRE trial . Carfilz= 0.045) . In fact= 0.008) . In thisIn this study, the effect of confounding factors such as subsequent therapy with novel agents was minimised, revealing the potential treatment benefit of ixazomib‐Rd in patients with RRMM, especially among those with a poor prognosis. However, the study had some limitations. The main study was designed to have adequate power to detect benefit in the overall population but was not powered to detect subgroup effects. Owing to the inclusion criteria, the sample size was small and could have led to estimation bias or error in the analysis of survival advantage with ixazomib‐Rd treatment, especially when patients were further divided into smaller subgroups. Nevertheless, the current results provide context on the treatment of patients with RRMM in the emerging markets.5Overall, patients with RRMM from emerging markets showed a clinically meaningful benefit in survival with ixazomib‐Rd compared with placebo‐Rd, with no new toxicity or safety signals. Ixazomib‐Rd, an all‐oral triplet regimen, represents an effective and well‐tolerated treatment option for patients with RRMM, particularly in countries where novel treatment options are limited.Andrew Spencer contributed to data collection. Olga Samoilova contributed to data collection. Wee‐Joo Chng contributed to data analysis and interpretation. Richard Labotka has contributed to study design and data collection. Cong Li contributed to the statistical analysis of data. Kwang‐Wei Wu contributed to study design, concept development and analysis and interpretation of clinical data. Nakul Saxena contributed to data analysis and interpretation. Xu Yan supported with patient enrolment, treatment and evaluation. Jae Hoon Lee supported with patient enrolment. Meral Beksac contributed to data collection and analysis. All authors are accountable for the integrity of this work, provided critical revision of the manuscript and approved the final version for submission.Nakul Saxena is an employee of Takeda Pharmaceuticals International AG, Singapore and holds stocks of Takeda Pharmaceuticals. Kwang‐Wei Wu was an employee of Takeda Pharmaceuticals International AG, Singapore, during the conduct of this study and holds stocks of Takeda Pharmaceuticals. Her present affiliation is Janssen Pharmaceuticals (as of February 2022). Richard Labotka and Cong Li are employees of Takeda Development Center America and hold stocks of Takeda Pharmaceuticals. Meral Beksac has received honoraria from Amgen, Janssen, Sanofi, Oncopeptides, Celgene and Takeda. Andrew Spencer, Olga Samoilova, Wee‐Joo Chng, Xu Yan and Jae Hoon Lee state no conflict of interest.The datasets, including the redacted study protocol, redacted statistical analysis plan and individual participants data supporting the results reported in this article, will be made available within three months from initial request, to researchers who provide a methodologically sound proposal. The data will be provided after de‐identification, in compliance with applicable privacy laws, data protection and requirements for consent and anonymisation.The study was conducted in accordance with the International Conference on Harmonisation Good Clinical Practice guidelines and appropriate regulatory requirements. The study was approved by the local ethics committees or institutional review boards at each centre. All patients provided written informed consent.Supplementary dataClick here for additional data file.


Staphylococcus aureus (S. aureus) is a typical kind of symbiotic bacteria, which can cause human pneumonia, food poisoning, and other health problems. Nowadays, the corresponding prevention and treatment have been a hot issue of general concern in related research areas. However, the mechanism of action against S. aureus is not well understood. In order to tackle such problem, we used broth microdilution to discuss the antibacterial effect of 5-methyl-2-isopropylphenol and determine inhibitory concentration. In addition, membrane potential and lipid peroxidation levels were also measured under experimental conditions. The experimental results suggested that 300 μg/mL thymol might cause cell membrane damage and decrease of NADPH concentration and increase of NADP+ and lipid peroxidation level. In such condition, thymol has the potential to result in membrane rupture and disruption of cellular homeostasis. Furthermore, we also found that NOX2 is involved in maintaining the balance of NADPH/NADP+ in cells. Finally, our work confirms that NOX2 is a potential downstream target for thymol in the cell. Such target can provide specific guidance and recommendations for its application in antifungal activity. Meanwhile, our study also provides a new inspiration for the molecular mechanism of thymol's bacteriostatic action. Staphylococcus aureus (S. aureus) is both a commensal and opportunistic bacterium, causing a variety of infections in humans, such as bacteremia, pneumonia, skin and soft tissue infections, and food poisoning [S. aureus bacteremia or meningitis and osteomyelitis, with an overall mortality of 19% to 30.5% [S. aureus colonization and infection in infants was 24.2 times higher than that in adults, indicating that infants were at high risk for S. aureus colonization [S. aureus is our most urgent task at present.oisoning –4. It waoisoning –7. Data to 30.5% –12. Morenization , 13. At nization –16. Therμmol/L for S. typhimurium (0.15 mg/mL), 1.2 μmol/L for Escherichia (0.18 mg/mL), 2.0 μmol/L for S. aureus (0.31 mg/mL), and 33 μmol/L for E. coli (5.00 mg/mL), respectively [+ and Ca2+ in cells to produce excessive oxygen free radicals, leading to cell death [G. citri-aurantii, the causal agent of citrus sour rot [5-Methyl-2-isopropylphenol (thymol) is a monoterpene phenol, which is mainly isolated from plants belonging to the Lamiaceae family including Thymus, Ocimum, Origanum, and Monardagenera –21. thymectively –32. The ectively , 33–35. ll death –38. Therll death , 39, 40.ll death  suggest ll death ; Serna-Ell death  found thsour rot .S. aureus were explored to elucidate its potential mechanism. Thymol related biofilm dysfunctions were studied by using Scanning Electron Microscopy (SEM) to detect the changes in cell membrane potential, intracellular NADPH/NADP+, ATP concentrations, and membrane integrity. Moreover, the MDA concentration is also discovered as an indicator to track the oxidation level of cell membranes. This study will encourage people to use thymol as a natural antibacterial agent in their daily life.In this study, the antimicrobial effects of thymol against μm filter and were stored at −20°C until analysis. Lipid Peroxidation (MDA) Assay Kit (ab118970) and Cellular Membrane Potential Assay Kit (ab176765) were purchased from Abcam Co., Ltd. All other chemicals used to configure various buffers were maintained in the analytical grade and purchased from the Aladdin Biochemical Technology Co., Ltd .The stock solution of thymol ) was purchased from the Aladdin Biochemical Technology Co., Ltd . Unless otherwise noted, thymol solutions in different concentrations used under all conditions were prepared by mixing medium (Cation-Adjusted Mueller-Hinton II Broth (CA-MHII) or Tryptone Soya Broth (TSB)), PBS, and 2.5% (v/v) ethanol. Propidium iodide (PI) staining solution  was used to measure membrane destruction. These solutions were sterilized using a 0.22 S. aureus ATCC25923 and ATCC43300 were purchased from American Type Culture Collection . S. aureus RN4220 was purchased from Huayueyang biotech co. ltd . To activate the strain, all purchased strains were activated for three generations before use to maintain good physiological status. During the activation process, all strains were inoculated into fresh TSB and replaced medium every 12 hours. After 36 h of incubation at 37°C, the bacteria grow to the logarithmic growth phase or kept stable at the stable phase. The strains used in the experiment were activated strains.S. aureus was determined using broth microdilution method as described elsewhere [S. aureus ATCC25923 and ATCC43300 cells were obtained until OD600 = 0.5 (approximately 108 CFU/mL) by using the method described in μg/mL. The electron carrier solution was diluted 8-fold with sterile water, and the WST solution and the diluted electron carrier solution were prepared in a ratio of 9 : 1. The cell suspension was diluted with sterile water to adjust the cell concentration to about 107 CFU/mL. 10 μl cell suspension and 180 μl thymol solutions at different concentrations were added into each well to maintain a final cell density of 104 CFU/ml. After incubation for 6 h, 10 μL working solution was added to each well and incubated at 37°C for 2 h. For each concentration, microbial growth was determined by microplate reader at 600 nm.The inhibitory concentration of thymol against lsewhere . ActivatS. aureus ATCC25923 was grown to an OD600 value of 0.5 in TSB, and 10 μl cell suspension (∼104 CFU/ml) was added to 96-well plates. 180 μl TSB containing thymol solution was also added to each well to obtain final concentrations of 0, 9.4, 37.5, 75.0, and 150 μg/mL. All plates were placed in a humidified incubator at 37°C for 24/48 hours. Centrifugal 96-well plate and lower ambient temperature are helpful in eliminating steam influence on test results before test [The optical density at 600 nm was measured every 1.0 hour using a full-wavelength scanning multifunction reader  to monitor cell growth. Specifically, ore test .S. aureus cultures at mid-exponential phase were collected by centrifugation and suspended in PBS to the final concentration of ∼105 CFU/mL. PI staining solution was added to the cell suspension, and the final concentration is 1 μg/mL. The mixtures were incubated in darkness at 37°C for 10 min and then divided into the wells of a 96-well plate. Thymol in different concentrations with PBS containing 2.5% ethanol was added to the wells and incubated for 30 min at 37°C. The fluorescence values of cell samples were measured every 60 s for 10 min by using a microreader with the excitation wavelength 536 nm and emission wavelength 617 nm. At the same time, cells with cytoplasmic membrane lesion were also evaluated by using a flow cytometer (Beckman Cyto-FLEX FCM) with a blue argon laser at 488 nm. A control group without thymol treatment was also labeled with PI, and a similar test was performed.PI staining was used to indicate damage to the cytoplasmic membrane, which penetrates only when there is significant damage to membrane. Ex/Em = 530/570 nm.Abcam's Cellular Membrane Potential Assay Kit (Fluorometric-Orange) (ab176764) is a homogeneous assay with fast read time and uses a proprietary membrane potential indicator to detect the membrane potential change that is caused by the opening and closing of the ion channels. These cells were obtained in accordance with the method described in S. aureus ATCC25923, and five titers of thymol solution addition  with PBS containing 2% ethanol, homogeneous samples were obtained by washing, precipitation, preliminary extraction, and other multistep treatment and then treated with either 100 μL of NADP+ extraction buffer for NADP+ determination or 100 μL of NADPH extraction buffer for NADPH determination. Finally, read fluorescence intensity at λex = 530 nm/λem = 585 nm for time “zero” and after a 30-minute incubation at room temperature and plate was protected from light during this incubation.Intracellular NADPH was detected using an NADP+/NADPH assay kits . According to the protocol for collection of S. aureus ATCC25923 as described in Zhou et al. (2019) [S. aureus strains were harvested by centrifugation for 10 min at 500 g, washed twice with phosphate buffered saline (1XPBS), and resuspended in PBS containing 2.0% glutaraldehyde to fix the cells. After 1.5 h fixation at 4°C, the cells were washed with Milli Q water and then dehydrated in a gradient alcohol concentration  for 10 min at each concentration. Finally, the dehydrated samples were sputter coated with gold under vacuum and then observed under a Quanta 200 scanning electron microscope , at an accelerating voltage of 15 KV and a magnification of 8000×. Control samples without thymol treatment were also prepared and examined as mentioned above.Scanning electron microscopy (SEM) analysis was performed to determine the mode of action of thymol on . (2019) . After 66 cells) and washed cells with cold PBS after we mixed 300 μL of MDA Lysis Buffer with 3 μL BHT (100X). Then, homogenized cells were put in 303 μL Lysis Solution (Buffer + BHT) using a Dounce homogenizer (10–50 passes) on ice. When 70–80% of the nuclei do not have the shiny ring, centrifuge at 13,000× g for 10 minutes to remove insoluble material and collect supernatant into 1.5 ml centrifuge tubes. Next, 600 μL of TBA reagent was added into each well containing 200 μL standard and 200 μL sample and incubated at 95°C for 60 minutes. Finally, cool to room temperature in an ice bath for 10 minutes, measuring absorbance immediately on a microplate reader at OD 532 nm for colorimetric assay and RFU at Ex/Em = 532/553 nm for fluorometric assay.We harvested the number of cells needed for each assay ; mL = Original plasma volume used (0.020 mL); B = Correction for using 200 μL of the 800 μL Reaction Mix; C = Sample dilution factor.Concentration of MDA in the test samples is calculated asn = 3), and differences between groups were tested by Student's t-test.All experiments were done in triplicate and repeated at least twice to ensure data credibility. The software R-4.1.0 was used for data analysis. Data were presented as the mean value ± standard deviation , and the concentration of thymol at 300 μg/mL and 200 μg/mL also revealed a noticeable increase. These results suggested that this mechanism of action of thymol might lead to membrane potential depolarization in S. aureus, resulting in membrane damage and bacterial cell death.It was previously reported that thymol is effective against Xanthomonas and has been demonstrated due to increased ROS and activation of apoptosis related proteins . Tian etakazakii . Therefoshown in . Compare+/NADPH and ATP levels in intact cells are in a stable state. However, the destruction of cell homeostasis and integrity may cause changes in intracellular NADP+/NADPH and ATP under stress [S. aureus, but why thymol has such a strong membrane damage ability was not clear. Therefore, it is worthwhile to further study the depolarization and damage effects of thymol. First, we used the NADP+/NADPH detection kit to follow the changes in the concentration of NADPH in S. aureus cells after thymol treatment for 6 hours. As shown in Figures S. aureus was treated with thymol for 6 hours, the concentration of NADPH in the cytoplasm was greatly reduced. But compared with the original level, the concentration of NADP+ was increased by 1.5 times. When the value of thymol concentration is 0, the cells are survived as usual. It has the weakest signal strength. And in the condition of 500 μg/ml, the intracellular homeostasis is destroyed seriously. So, it has the largest volume of expansion and production increase. The difference of axis 0 and 500 is the largest. The ATP bioluminescence method was designed to assessed the effect of thymol on intracellular ATP concentrations. The intracellular ATP concentrations in S. aureus decreased after 300 μg/mL and 500 μg/mL thymol treatment compared with the control group, while no conspicuous variation was found in 50 μg/mL and 100 μg/mL thymol treated cells. In summary, our result demonstrates that 300 μg/mL and 500 μg/mL thymol cause an increase in NADP+ and a decrease in cytoplasmic NADPH and ATP concentration in S. aureus, which indicated the possible leakage of intracellular components induced by the thymol and the disruption of the physiologically NADP+/NADPH balance.The NADPr stress –50. We h+/NADPH ratio is about 0.01, which is mostly in the reduced form to drive reductive biosynthesis reactions [S. aureus cells were cultured in the TSB culture medium containing thymol with 150 pmol of NADPH or ATP to study the effect of thymol on cell viability. Compared with the control group, normal cells incubated with NADPH and thymol showed a significant increase in cell viability, while cell viability did not change in the ATP and thymol groups. More importantly, we found that NADPH did not completely remove the bacteriostatic effect of thymol but effectively alleviated this role (S. aureus within 24 hours after administration of NADPH and measured the contribution of NADPH in the antibacterial effect of thymol. In keeping with the results of previous studies, when thymol and NADPH are cocultured at the same time, the addition of NADPH can ease cell growth stress and improve cell growth state regardless of the concentration of thymol at 100 or 200 μg/mL. In summary, our results showed that thymol administration process will make the cells lose large amounts of NADPH, which is the cause rather than the result of cell membrane polarization and cell death. Although the concentration of ATP has decreased, ATP cannot effectively alleviate the loss of cell viability caused by thymol. This phenomenon indicates that ATP concentration balance may play an important role in other processes such as maintenance of cell membrane potential and energy supply [NADPH is essential in unremitting flux of energy process due to its ability to serve as electron carriers for a large subset of oxidoreductases . For exaeactions . NADPH peactions . S. aurehis role . The heahis role . We alsohis role . There ay supply , 54, 55.μg/mL or 500 μg/mL, the soluble MDA concentration reached 0.4 nmol/mL and 0.5 nmol/mL respectively, and there was a significantly increased difference compared with 0.1 nmol/mL in the control group. It is worth noting that, within the 100 μg/mL concentration range, the observed upward trend is not very prominent (Lipid peroxidation and leakage of cytotoxic enzymes from lysosomes are important intracellular sources of plasma membrane damage in physiological states, and lipid peroxidation is further exacerbated upon reoxygenation . Thymol rominent . For furS. aureus strains were firstly treated with 300 μg/mL of thymol at 37°C and then were collected by centrifugation for 10 min at 4000 g. The collected cells were fixed and dehydrated in the process described in S. aureus after the addition of NADPH and thymol was fewer, and the diameter of the traps also showed a visible decrease compared with that treated with thymol alone (Figures In this part, we subsequently employed the Scanning Electron Microscopy to detect the effect of NADPH on cell membrane trap generation and development. The + is still unknown. In order to more clearly explore the antibacterial effect of thymol, we further analyzed whether several NADPH-related kinases were involved in thymol-induced cell membrane damage. Our unpublished results and other published papers suggest that NOX2-catalyzed oxidation of NADPH to form peroxide free radicals is a possible factor in NADPH depletion. We then used Western blot to detect the expression level of NOX2 when the S. aureus was treated within thymol. After thymol treatment for 6 hours, the expression level of NOX2 shows a positive correlation with the concentration of thymol [2 expression increased nearly fourfold after treatment with 300 μg/mL thymol compared with 100 μg/mL sample. Next, we used si-RNA silencing to knock down the expression of NOX2 in S. aureus to observe the effect of NOX2 on cytoplasmic NADPH and NADP + concentration. We were pleasantly surprised to find that when NOX2 was knocked down, and S. aureus was treated with thymol, the concentration of soluble NAPH in the cytoplasm was significantly increased compared to the control (+. These results directly indicate that thymol can change or destroy the morphology of the cell membrane by changing the concentration of NADPH/NADP+ in the cytoplasm, thereby mediating the death of S. aureus. And Although it has been previously stated that thymol induces cell membrane damage because of NADPH depletion, how NADPH in cells transfers to NADPf thymol , 58. As  control . And we S. aureus can be effectively inhibited by thymol, which is firstly confirmed in this paper. Furthermore, the inhibitory concentration of thymol is also measured by Broth Micro-Dilution method. Thymol might lead to membrane potential depolarization in S. aureus, resulting in membrane damage and cell death. In order to further understand the corresponding antibacterial mechanism, the concentration of soluble NADP+/NADPH and ATP in the cytoplasm is detected. Thereafter, an increase in NADP+ and a decrease in cytoplasmic NADPH and ATP are caused by thymol. Such phenomenon indicated the possible leakage of intracellular components and the disruption of the physiologically NADP+/NADPH balance. Then, the biochemical experiments and SEM results suggested that the cell membrane lipid oxidation levels are significantly increased by using thymol treatment. Besides, a certain amount of NADPH supplementation can not completely alleviate the peroxidation reaction and reduce the number and size of cell membrane traps. The restricted catalytic enzyme NOX2 can regulate the balance of NADPH/NADP+. So, NOX2 can be a potential downstream target of thymol in the cell. Finally, a specific guidance is provided in food preservation and storage about the use of thymol.The growth of


Germ cell tumor with malignant transformation is extremely rare. We present a case of testicular primitive neuroectodermal tumor with multiple metastases that was effectively managed by surgery, irradiation, and second‐line chemotherapy.A 22‐year‐old man was diagnosed as having teratoma including primitive neuroectodermal tumor with lymph node and multiple bone metastases. Five months afterwards the first‐line therapy, his skull metastasis recurred. Vincristine, doxorubicin, and cyclophosphamide therapy followed by vincristine, actinomycin D, and cyclophosphamide therapy was given as second‐line chemotherapy. Computed tomography revealed no disease progression 3 months after the treatments.Metastatic primitive neuroectodermal tumor may be successfully managed by multidisciplinary cancer treatment. A left high inguinal orchiectomy, first‐line chemotherapy, and RPLND were given. Five months later, the skull metastasis had increased in size. Radiation of the metastatic lesions and VDC followed by VAC therapy was effective in managing the metastatic lesions.Testicular GCT is the most common solid tumor diagnosed in young men.2, and cisplatin 20 mg/m2) therapy were given in combination with denosumab 120 mg every 4 weeks. After chemotherapy, tumor markers returned to normal range and the metastasis in the para‐aortic lymph node had decreased in size. Subsequent RPLND led to a pathological diagnosis of viable teratoma without PNET. One month afterwards, PET‐CT revealed strong accumulation in the sternum and pelvic bone. Since no additional metastases or enlargements in bone metastatic lesions were evident, he was followed closely under continued denosumab administration. Five months after RPLND, his serum lactate dehydrogenase and neuron‐specific enolase had increased to 260 U/L and 19.9 ng/mL, respectively. CT revealed soft tissue mass formation with skull destruction  therapy. Afterwards, 12 courses of VAC  therapy were added due to the limited dose of doxorubicin. CT revealed no disease progression 3 months after all 17 courses of VDC and VAC therapy and no other metastatic lesions. The patient is currently under close follow‐up with continued denosumab administration.A 22‐year‐old man with a half‐year history of swelling of the left scrotum presented with anterior chest pain. CT revealed a large 14 × 9 cm mass in the left scrotum and metastatic lesions in the para‐aortic lymph node, skull, pelvic bones, sternum, and vertebrae Fig. . The tumion Fig. . As the ion Fig. . PET‐CT ion Fig. . ExternaPNET is an immature malignant tumor derived from the neuroectoderm, and classified into two groups: cPNET originating from the central nervous system and pPNET deriving from other tissues. PNET from testicular GCT is predominantly classified as cPNET,PNET can only be diagnosed by histopathological examination. Tumors exhibit small round cells with round‐to‐oval nuclei and may show rosette‐like arrangement.et al. described that 8 of 10 PNET patients achieved an objective response to chemotherapy of VAC alternating with ifosfamide plus etoposide.et al. found that VAC therapy for CD99‐positive metastatic PNET malignantly transformed from testicular teratoma provided asymptomatic relief for 18 months.The treatment of PNET remains uncertain. Although cisplatin‐based chemotherapy regimens have good outcomes in non‐transformed GCT, they were found to be ineffective for PNET.The presented case had a primary testicular tumor and metastatic lesions in the para‐aortic lymph node and multiple bones. Pathological examination of the primary lesion and a skull biopsy revealed teratoma including PNET. In immunohistopathological analysis, the neuroendocrine markers CD56, synaptophysin, and chromogranin A were all positive. As CD99 was weakly positive in the former examination and difficult to judge in the latter, the tumor could not be conclusively determined as pPNET. The first‐line chemotherapy was ineffective for PNET, as evidenced by the metastatic lesions. However, VDC followed by VAC therapy was able to control the metastatic lesions following the surgery, radiation, and first‐line chemotherapy. VDC following VAC therapy may therefore represent a therapeutic option for metastatic pPNET. However, careful follow‐up for metastatic lesions using CT and PET/CT remains essential. Further investigation is needed to establish successful testicular PNET treatment regimens.Takuya Takasawa: Writing – original draft. Tomonori Minagawa: Writing – review & editing. Takahisa Domen: Resources. Toshirou Fukushima: Resources. Yu Fujii: Resources. Mai Iwaya: Resources. Tomonobu Koizumi: Resources. Teruyuki Ogawa: Resources. Osamu Ishizuka: Resources.The authors declare no conflict of interest.Not Applicable.Written informed consent was obtained from patient for publication of this case report and accompanying images.Not Applicable.


We report a case of abdominal compartment syndrome due to hydroperitoneum after endoscopic combined intrarenal surgery.A 56‐year‐old woman with a left staghorn calculus underwent endoscopic combined intrarenal surgery as a two‐staged procedure and developed a distended abdomen, cyanosis of both legs, and hypotension immediately after the second operation. A computed tomography scan showed hydroperitoneum. We performed urgent laparotomy and evacuated approximately 2 L of nearly transparent fluid. No peritoneal injury was detected. Postoperatively, she required intensive care for shocked liver and acute kidney injury.Hydroperitoneum after endoscopic combined intrarenal surgery is a rare complication and may lead to abdominal compartment syndrome or a condition where intra‐abdominal pressure exceeds 20 mmHg, causing impaired organ perfusion. Delayed drainage can be fatal. Hydroperitoneum is a rare complication of endoscopic combined intrarenal surgery. Immediate drainage is desirable.ACM‐IGPair‐capsule‐based measurement of intra‐gastric pressureACSabdominal compartment syndromeCTcomputed tomographyECIRSendoscopic combined intrarenal surgeryPNLpercutaneous nephrolithotripsyECIRS or PNL is widely performed to treat staghorn calculus. We report a case of hydroperitoneum, or leakage of irrigation fluid into the peritoneal cavity, after ECIRS. In this case, hydroperitoneum resulted in ACS, or a condition in which intra‐abdominal pressure exceeds 20 mmHg resulting in impaired organ perfusion.A 56‐year‐old woman was referred to our hospital because of asymptomatic gross hematuria. An abdominal radiograph revealed a left staghorn calculus measuring 40 × 30 mm Fig. . It invoThe patient was placed in the modified Valdivia position. A 0.035‐mm guidewire was placed as a safety guidewire in the ureteral lumen . We inserted a 12/14‐Fr 35‐cm ureteral access sheath .We subsequently punctured the middle posterior calyx under ultrasound guidance. A 0.035‐mm guidewire  was inserted through the puncture needle, received by the flexible ureteroscope, and exited through the urethra. We dilated this percutaneous tract to 24 Fr using a balloon (Boston Scientific NephroMax®).The antegrade‐side surgeon used a 22‐Fr nephroscope and Swiss LithoClast® . The retrograde‐side surgeon used a 9.5‐Fr flexible ureteroscope (Boston Scientific LithoVue®) and a Holmium laser system (Lumenis Pulse 120H® and SlimLine SIS® 200 μm [Boston Scientific]). Irrigation fluid was located approximately 50 cm above the patient.Although lever operation was not forceful, the flexible ureteroscope suddenly broke when operated at a downward angle, making a retrograde approach to the inferior calyx challenging. Thus, the first‐stage operation was terminated, and several stones remained in the inferior renal calyx. A 6‐Fr, 24‐cm ureteral stent (Boston Scientific Polaris Loop®) and a 24‐Fr nephrostomy tube were inserted. The operation time was 107 min, with an uneventful postoperative course.We conducted the second‐stage surgery under general anesthesia on September 10, 2021. We achieved stone‐free status with an operation time of 114 min.The patient was placed in the modified Valdivia position. The antegrade‐side surgeon placed a 24‐Fr sheath as the percutaneous tract . The retrograde‐side surgeon inserted a 12/14‐Fr, 35‐cm ureteral access sheath (BARD Proxis®). Irrigation fluid was located approximately 50 cm above the patient. It was challenging for the antegrade‐side surgeon to approach the residual stone in the inferior renal calyx due to the renal column. To avoid injury to the kidney, the nephroscope was moved as little as possible. Therefore, we fragmented the residual stone using the ureteroscope and the fragments were removed via the percutaneous tract .Intraoperative bleeding was minimal, and renal pelvic or ureteral injury was not detected. Hemodynamic parameters remained stable, and airway pressure did not increase intraoperatively. However, when the drapes were removed, the patient's abdomen was markedly distended, and cyanosis in both legs was observed. Heart rate increased to 100 beats/min, and systolic blood pressure dropped to 70 mmHg. Diagnosis was difficult using only bed‐side ultrasonography; hence, a CT scan was performed Fig. . ImagingWe performed an urgent laparotomy. Approximately 2 L of nearly transparent fluid was drained, but no peritoneal injury was detected.2O or 36.8 mmHg. Thus, the case was diagnosed as ACS.It was difficult to inflate the bladder when placing a ureteral stent at the end of the second surgery. Therefore, intra‐vesical pressure, which reflects intra‐abdominal pressure, was inferred as more than 50 cmHPostoperatively, the patient required intensive care for shocked liver and acute kidney injury. She was extubated after 9 days. The nephrostomy tube and the ureteral stent were removed on the 26th and 39th postoperative days, respectively. She was discharged on the 43rd day after the second‐stage operation.We encountered a case of ACS due to hydroperitoneum after ECIRS. ACS is characterized by intra‐abdominal pressures exceeding 20 mmHg and impaired organ perfusion.et al. reported that risk factors for hydroperitoneum include an increased amount of irrigation fluid, operative time of more than 60–90 min, renal pelvis mucosal tear, and peritoneal scarring caused by previous ECIRS or PNL.Two mechanisms may result in hydroperitoneum. First, mucosal tearing may cause intravascular absorption of irrigation fluid. Second, a technical error in percutaneous tract placement may cause iatrogenic peritoneal injury. Khalil In this case, a minor peritoneal injury could have been overlooked. However, fluid overload should not be ruled out. It was reported that irrigation fluid is absorbed in all candidates who undergo PNL.In terms of preventing ACS due to hydroperitoneum, it is crucial to consider which position is superior between the prone and modified Valdivia positions. Renal calyx puncture is easier in the prone position,According to previous reports, increased airway pressure, distended abdomen, or tachycardia, was an early sign of hydroperitoneum.Intravesical or intra‐gastric pressure measurement is recommended to diagnose ACS.During the second surgery, 22 L of irrigation fluid was infused, and the output was 10 L. However, irrigation fluid leaked outside the operative field, making these measurements inaccurate. Proper draping of the operative field may prevent such occurrences.Percutaneous drainage was conducted for treatment in all previously reported cases.Hydroperitoneum is a rare complication of ECIRS. It might lead to ACS or a condition in which intra‐abdominal pressure exceeds 20 mmHg resulting in impaired organ perfusion. Delayed drainage could be fatal.Shuhei Okada: Conceptualization; data curation; investigation; project administration; writing – original draft. Takuro Saito: Conceptualization; supervision. Yasushi Ichimura: Conceptualization; supervision. Masahiro Iinuma: Conceptualization; supervision; writing – review and editing.The authors declare no conflict of interest.Not applicable.Informed consent for publication was obtained from the patient.Not applicable.


Fibroblast‐activating protein (FAP) is expressed in cancer‐associated fibroblasts (CAFs) in many human carcinomas and in some types of carcinoma cells. Here, we examined the proportion of FAP protein expression in non‐small cell lung carcinoma (NSCLC) and investigated the correlation of FAP expression with clinicopathological background.In total, 344 NSCLC tissues were examined. Tissue microarrays were constructed, and FAP expression was analyzed using immunohistochemistry. The status of FAP expression in tumor cells and CAFs was correlated with clinicopathological background, molecular features, and patient outcomes.p < 0.001) and recurrence‐free survival . Based on combinations of FAP expression in tumor cells and CAFs, patients with LowT/LowCAFs had better OS and RFS than did those in the other subgroups. By contrast, patients with HighT/HighCAFs had poor OS and RFS compared with those in the other subgroups.A total of 280 patients (81.4%) had low FAP expression, and 64 patients (18.6%) had high FAP expression in tumor cells. In CAFs, 230 patients (66.9%) had low FAP expression, and 114 patients (33.1%) had high FAP expression. In multivariate analyses, high FAP expression in tumor cells was an independent predictive factor of both overall survival  and recurrence‐free survival .A total of 280 patients (81.4%) had low FAP expression, and 64 patients (18.6%) had high FAP expression in tumor cells. In CAFs, 230 patients (66.9%) had low FAP expression, and 114 patients (33.1%) had high FAP expression. In multivariate analyses, high FAP expression in tumor cells was an independent predictive factor of both overall survival (OS; HR = 2.57, 95% CI: 1.49–4.42,  Lung cancer (LC) is the leading cause of cancer‐related death in developed countries.Fibroblast‐activating protein , a cell surface glycoprotein belonging to the serine protease family, is a 170‐kDa dimer that is catalytically active and has dipeptidase and gelatinase activities.Accordingly, in this study, we examined FAP protein expression in NSCLC and investigated correlations with clinicopathological factors, including patient outcomes.A retrospective review of a prospectively maintained surgical database was performed to identify patients who underwent primary LC resection with curative intent from 2015 to 2017. The histopathological diagnosis was made according to the eighth edition of the TNM Classification of the Union for International Cancer Control and the 2015 World Health Organization classification.In total, 344 formalin‐fixed, paraffin‐embedded samples from consecutive resected NSCLC collected from 2015 to 2017 were used for the preparation of TMAs. We searched for the representative tumor area (the area of predominant subtypes as for adenocarcinoma) and the area of proliferation of spindle‐shaped cells in stroma. Then, we set up the area which fills with two criterions as previously stated and arrayed a cylindrical 3‐mm tissue core from the corresponding paraffin blocks into a recipient block using a tissue arrayer . Hematoxylin and eosin staining was used to evaluate both tumor cells and CAFs in each TMA specimen.FAP protein expression was examined by immunohistochemistry. TMA blocks were sliced into 4‐μm‐thick sections, deparaffinized, and stained for FAP  using a DAKO Autostainer Universal Staining System (Dako). Two pathologists (N.Y. and N.U.) evaluated the slides. FAP protein expression was scored for both extent of immunopositivity and intensity, as previously described, with modifications.We performed immunohistochemical staining using antibodies against ALK  and p53  in the TMAs. The immunohistochemical results were evaluated as follows: strong granular cytoplasmic staining of ALK in tumor cells (any percentage) was defined as ALK positivity; staining of p53 in greater than or equal to 10% of tumor cells was defined as p53 positivity. For PD‐L1, TMAs were stained for PD‐L1 using PD‐L1 IHC 22C3 pharmDx assays  and an Autostainer Link 48 using an automated staining protocol. If the membrane of the tumor cells was stained, the cells were considered positive for PD‐L1 protein expression. The tumor proportion score (TPS) of PD‐L1 in tumor cells was defined as the percentage of PD‐L1‐positive tumor cells in the TMA tumor sections. If the TPS was greater than or equal to 1%, the section was defined as having positive expression.EGFR mutations were detected using real‐time polymerase chain reaction .2 tests or Fisher's exact tests, as appropriate. Recurrence‐free survival (RFS) and overall survival (OS) were analyzed using the Kaplan–Meier method, and differences in variables were calculated using log‐rank tests. RFS was defined as the time from surgery to recurrence, death, or the last follow‐up. OS was defined as the time from surgery to death or the last follow‐up. The last follow‐up observation was censored if the patient was alive or lost to follow‐up. A multivariate survival analysis was performed using the Cox proportional hazards model. All statistical analyses were performed using EZR , a modified version of R commander  designed to add functions frequently used in biostatistics.p‐values less than 0.05 were considered statistically significant.Statistical comparisons were performed using χT/LowCAFs), high expression in tumor cell/low expression in CAFs (HighT/LowCAFs), low expression in tumor cell/high expression in CAFs (LowT/HighCAFs), and high expression in tumor cell/high expression in CAFs (HighT/HighCAFs) were as follows: 198 (57.6%), 32 (9.3%), 82 (23.8%), and 32 (9.3%), respectively.Scoring of FAP expression in tumor cells and CAFs is summarized in Table EGFR mutation analysis was performed in 243 patients, and 114 (46.9%) of these patients had EGFR mutations. p53 and PD‐L1 immunohistochemical expression was positive in 73 (32.8%), and 39 (16.6%) patients, respectively. Detailed EGFR mutation, ALK immunohistochemistry and adjuvant therapy are shown in supplementary Table Patient, clinicopathological, and molecular characteristics are summarized in Table p = 0.013) and positive PD‐L1 expression (p < 0.001).The associations of clinicopathological and molecular characteristics with FAP expression in tumor cells are shown in Table p < 0.001), smokers (p < 0.001), and patients with more advanced stage disease (p < 0.001), SCC (p < 0.001), lymphocytic invasion (p = 0.002), vascular invasion (p < 0.001), pleural invasion (p = 0.004), positive p53 expression (p < 0.001), and positive PD‐L1 expression (p < 0.001).The associations of clinicopathological and molecular characteristics with FAP expression in CAFs are also shown in Table The mean follow‐up period was 44.3 months ; 66 of the 344 patients died during the follow‐up period. Of these, 43 died as a result of cancer recurrence, and the remaining 23 died of other causes. Of the 278 patients who were alive at the time of analysis, 44 had recurrent disease, and 234 had no evidence of disease.p < 0.001)  Table .p < 0.001)Table .T/LowCAFs had a better OS and RFS than those in the other subgroups. In contrast, patients with HighT/HighCAFs had a poor OS and RFS compared with those in the other subgroups. In addition, patients with HighT/LowCAFs and patients with LowT/HighCAFs had almost the same OS and RFS, and their survival curves were located at  between survival curve of patients with LowT/LowCAFs and survival curve of patients with HighT/HighCAFs.OS and RFS curves according to combinations of FAP expression in tumor cells and CAFs are shown in Figure Previous cancer research has mainly focused on cancer cells. However, in the last 20 years, the stromal cells surrounding cancer cells have also been shown to be important factors for cancer development. Cancer cells interact in complex ways with surrounding stromal cells, making up the TME.Interestingly, FAP expression was found not only in CAFs but also in LC cells for the first time. In other cancers, FAP expression has been found in cancer cells of the stomach,T/HighCAFs was associated with significantly poorer OS rates than LowT/HighCAFs and LowT/LowCAFs, and both HighT/LowCAFs and LowT/HighCAFs were associated with significantly poorer OS rates than LowT/LowCAFs. Thus, the combination of FAP expression in tumor cells and CAFs should be assessed.FAP expression in CAFs has been described in some types of cancers. To date, three studies have reported FAP expression in CAFs from patients with NSCLC.Recently, immunotherapy using immune checkpoint antibodies targeting PD‐1 (programmed cell death‐1/PD‐L1 [programmed cell death‐1 ligand]) has been shown to improve outcomes in patients with various malignant tumors.This study had some limitations. First, we used TMAs rather than large tissue sections. Because TMAs may not always be representative of the entire tumor, heterogeneity of FAP expression is a major issue. Second, this was a retrospective study performed at a single institution; thus, the possibility of bias cannot be excluded.In conclusion, our results showed that FAP expression in tumor cells and the combination of FAP expression in tumor cells and CAFs was strongly associated with patient survival. Thus, we suggest that FAP expression in these cells may be a useful predictive biomarker for clinical outcomes in patients with NSCLC.The authors declare that they have no conflicts of interest.Table S1 Relationships of FAP expression in tumor cells and CAFs with clinicopathological and molecular characteristics.Click here for additional data file.


Decreasing the dietary intake of methionine exerts robust anti‐adiposity effects in rodents but modest effects in humans. Since cysteine can be synthesized from methionine, animal diets are formulated by decreasing methionine and eliminating cysteine. Such diets exert both methionine restriction (MR) and cysteine restriction (CR), that is, sulfur amino acid restriction (SAAR). Contrarily, SAAR diets formulated for human consumption included cysteine, and thus might have exerted only MR. Epidemiological studies positively correlate body adiposity with plasma cysteine but not methionine, suggesting that CR, but not MR, is responsible for the anti‐adiposity effects of SAAR. Whether this is true, and, if so, the underlying mechanisms are unknown. Using methionine‐ and cysteine‐titrated diets, we demonstrate that the anti‐adiposity effects of SAAR are due to CR. Data indicate that CR increases serinogenesis (serine biosynthesis from non‐glucose substrates) by diverting substrates from glyceroneogenesis, which is essential for fatty acid reesterification and triglyceride synthesis. Molecular data suggest that CR depletes hepatic glutathione and induces Nrf2 and its downstream targets Phgdh (the serine biosynthetic enzyme) and Pepck‐M. In mice, the magnitude of SAAR‐induced changes in molecular markers depended on dietary fat concentration (60% fat >10% fat), sex , and age‐at‐onset (young > adult). Our findings are translationally relevant as we found negative and positive correlations of plasma serine and cysteine, respectively, with triglycerides and metabolic syndrome criteria in a cross‐sectional epidemiological study. Controlled feeding of low‐SAA, high‐polyunsaturated fatty acid diets increased plasma serine in humans. Serinogenesis might be a target for treating hypertriglyceridemia. Phgdh. (3) Increase in the transcription and translation of Phgdh and Pck‐2 results in higher hepatic serinogenesis . (4) Higher serinogenesis competes with glyceroneogenesis as both pathways use the same set of substrates, which results in lower fatty acid re‐esterification. Note: broken arrows represent the presence of other biochemical intermediates not shown in the figure. Up and down arrows adjacent to the metabolic intermediates represent an increase and decrease, respectively. 3PG, 3‐phosphoglycerate; OAA, oxaloacetic acid; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; FA, fatty acids.The proposed mechanism of CR‐specific effects of SAAR on adipose metabolism. (1) Due to the lack of Cys in SAAR diets, it imposes both MR and CR. CR specifically results in decreased biosynthesis of the tripeptide glutathione. (2) Lower hepatic glutathione increases the abundance of the transcription factor Nrf2, which translocates to the nucleus and induces the transcription of  Although the metabolic requirement of Cys is essential, its dietary requirement is dispensable, as healthy adult humans and non‐human animals can convert Met to Cys through the transsulfuration pathway  supplemented with 0.5% w/w Cys, SAAR‐induced decreases in body weight, adipose depot weights, plasma triglycerides, leptin, total cholesterol, and LDL cholesterol were reversed , Igf1 , and leptin , but increased food consumption , Fgf21 , and adiponectin . Growth rates , food intake , Igf1 , Fgf21 , and leptin  exhibited a strong dose‐response to MR, but adiponectin did not  to CR than to MR. Plasma adiponectin, which did not respond to MR, exhibited a strong dose response to CR . Overall, our data confirm that among the changes induced by the SAAR diet, changes in growth rates, food intake, plasma Igf1, and leptin were MR‐dependent , lysine , histidine , serine , and threonine , and decreased the levels of phenylalanine  and glycine , histidine , Ser , threonine , and tryptophan  showed a dose response to CR but not to MR . Due to the technical difficulty of accurately quantifying Cys, we did not quantity it. Since Ser alters lipid metabolism markers, we focused our additional analyses on it  but not on MR. To confirm if hepatic Ser increased due to its de novo biosynthesis, we quantified the mRNA levels of the rate‐limiting enzyme for Ser biosynthesis, phosphoglycerate dehydrogenase (Phgdh). Hepatic Phgdh expression was approximately 6‐ to 9‐fold higher in SAAR than in CD . A robust dose response was found in Phgdh levels with CR but not MR . Similar to our findings, previous studies based on nuclear run‐off assays show that dietary protein regulates Phgdh mRNA post‐transcriptionally. Higher Cys, but not Met, concentrations destabilized Phgdh mRNA . These data demonstrate that the SAAR‐induced increase in de novo hepatic Ser biosynthesis is specifically due to CR.To understand the role of Ser in lipid metabolism, we wanted to identify its source. Although the kidney and liver are the two major sources of plasma Ser, we quantified only hepatic Ser, as kidneys were not collected , which indirectly suggests that glycolysis is not contributing to Ser biosynthesis. Another likely reason for the non‐glycolytic origin of Ser is that hepatic glycolysis is minimal during fasting states; plasma and tissues were obtained after an overnight fast. Since 3PG can also be derived from gluconeogenesis, we probed for the mRNA expression of two enzymes that regulate this pathway, phosphoenolpyruvate carboxykinase‐C  and glucose‐6‐phosphatase‐C . SAAR did not alter either of these enzymes  is required. Under fed conditions, the canonical pathway for Ser biosynthesis is the “phosphorylated pathway,” in which the substrate 3PG is derived from glucose through glycolysis  increased Ser biosynthesis, we quantified hepatic Pck2 mRNA  but also that it exhibited dose response to CR (CRβ < 0.05) but not to MR. In addition, we also confirmed that the CR‐induced increase in the Pck2 transcript is reflected in protein levels  and that the changes in Pepck‐M are CR‐dependent . These data indicate that CR increases the utilization of mitochondrial oxaloacetate to replenish cytosolic phosphoenolpyruvate, the substrate, and product of Pepck‐M, respectively. Based on these data, we infer that the pathway involved in CR‐induced Ser biosynthesis is serinogenesis (Ser biosynthesis from non‐glucose substrates) and not the typical phosphorylated pathway.Based on a recent report that the mitochondrial form of Pepck ] < 0.01). Rats on SAAR had lower hepatic G3P concentrations than those on CD . Earlier, it was reported that administering ethionine, the ethyl analog of Met and an inhibitor of Met‐dependent functions, including transsulfuration, decreased G3P concentration and thus lipogenesis in both liver and adipose tissue . We previously demonstrated that SAAR decreases hepatic non‐protein‐bound total GSH (tGSH = rGSH [GSH in reduced form] + GSSG [GSH in oxidized form]) and found a CR‐specific decrease in hepatic GSH of rats on Cys‐titrated diets Figure . Low GSHPhgdh and Pck2  with a 1% increase in plasma tCys and decreased by 0.30%  with a 1% increase in plasma Ser .Association of plasma amino acids  with triglycerides and the occurrence of MetS criteria were investigated from a previously conducted epidemiological study  with an increase in each MetS criterion . Plasma Ser decreased with an increase in the number of MetS criteria in both models ; however, the association of tCys/Ser with MetS criteria was more robust than the association with either amino acid alone. In the unadjusted model, the ratio of tCys/Ser increased by 5.31% with an increase in each MetS criteria . Overall, findings from the epidemiological study are consistent with mechanisms suggested by our animal data, that is, tCys deficiency results in higher Ser biosynthesis and lower triglycerides  or high (SAAhigh) SAA  while in SAAhigh, the percent of saturated fatty acids was increased (SAAhigh+SFA). Both studies were conducted for 1 week, and changes in plasma Ser over time  were compared between diet groups. Dietary composition and sample sizes are provided in Table To test if low SAA diets alter plasma Ser levels, we used data from two previously conducted short‐term controlled feeding studies  and SAAhigh (from 103.7 to 100.7 mmol/L) groups in Study 1. The time‐related changes between the two diet groups were similar . In Study 2, there was an increase in estimated marginal mean plasma Ser concentrations from 106 to 118 mmol/L in the SAAlow+PUFA group and a decrease from 101.2 to 96.1 in the SAAhigh+SFA group; these time‐related changes between the two diet groups were significantly different . The associations between SAA, Ser, triglycerides, and MetS criteria in human studies are consistent with mechanistic findings from rodent studies.Compared to plasma Ser on day 0, the estimated marginal mean Ser concentration on day 7 slightly decreased in both SAA3Improvement of lipid metabolism by SAAR is well documented in rodent models, but mechanisms are unclear. To this end, we conducted dietary SAA depletion‐repletion bioassays. Data show that MR and CR exert discrete and non‐nutritive biological effects. MR‐dependent SAAR‐induced changes include decreases in growth rate, blood glucose, plasma Igf1, and leptin, and increases in food consumption and Fgf21 Figures . CR‐depeOur current understanding of the roles Cys plays in energy metabolism is relatively less than that of its functions associated with thiol‐disulfide exchanges. In addition to affecting energy metabolism by contributing to its carbon moiety, Cys acts as a signaling molecule. Cys contributed up to 10% of intracellular pyruvate in pancreatic cancer cells and up to 20% upon knocking out pyruvate kinase  at 4°C and transferred to 80°C the next day. The second rat cohort, obtained after analyzing data from the first cohort, was treated the same way as the first cohort except that the diets used were CD, SAAR, CR1, CR2, CR3, CR4, and CR5.All procedures were conducted according to the Institutional Animal Care and Use Committee guidelines of the Orentreich Foundation. Animals were singly housed and maintained on a 12:12 light–dark cycle at 20 ± 2°C and 50 ± 10% relative humidity. Food and acidified water were provided ad libitum. Food intake and body weight were recorded weekly. To find the discrete effects of MR (experiment 1) and CR (experiment 2), two cohorts of 8‐week‐old male F344 rats  were fed a Met‐titrated or Cys‐titrated diet for 12 weeks. In experiment 1, rats were randomly assigned to experimental diets . Following an overnight fast, the rats were anesthetized with isoflurane, and blood was collected from the retro‐orbital plexus. Plasma obtained from blood  was stored at −80°C. Rats were sacrificed by COThe effects of dietary fat content, sex, and age‐at‐onset on SAAR‐induced changes were determined by feeding three cohorts of C57BL/6J mice . The first  and second  cohorts of 8‐week‐old mice were fed CD and SAAR diets containing 10% Kcal and 60% Kcal fat for 16 weeks. The third cohort of male and female mice was aged in‐house until 18 months old (experiment 5) and fed CD and SAAR diets with 10% Kcal fat for 12 weeks. At the study's conclusion, blood and livers were obtained from 4‐hr fasted mice and were processed in the same manner as for rats. For all rodent experiments, the sample size was eight per group.All diets fed to rats were isocaloric with the same total amino acid content Table ; Covance4.1.2The population used for the current study was a subset of the parent study, which consisted of 278 coronary artery disease patients and 591 controls. Parent study objectives and subject recruitment details are described elsewhere . Unlike the manufacturer's instructions, we could not collect and quantify all the filtrate from the spin column. Hence, concentrations were expressed as the pmol/uL filtrate rather than the pmol/mg liver.4.4Non‐protein‐bound tGSH in the liver was determined, as described earlier, by an enzymatic recycling method using Ellman's reagent  and western blots (tissue proteins). Growth factors (Igf1 and Fgf21) and adipokines (leptin and adiponectin) were determined by quantitative sandwich ELISAs following the manufacturer's recommendation  and expressed as mg/dl.Fasting (16 h in rats and 4 h in mice) blood glucose in rats and mice was determined from decapitated and retro‐orbital blood, respectively, using FreeStyle Lite4.8Plasma triglycerides were quantified in microplates using Infinity Triglycerides Liquid Stable Reagent  and a Triglycerides Standard from Pointe Scientific (T7531‐STD). Triglycerides Reagent (200 μl) was added to wells containing 10 μl plasma and incubated at 37°C for 5 min. Absorbance (500 nm) was measured and extrapolated to a standard curve. Triglyceride concentrations were expressed as mg/dl.4.9t test. For quantitative data, CD and SAAR groups from the two cohorts were combined to increase the statistical power and for easier graphical representation; however, semi‐quantitative data such as mRNA and protein expression from the two cohorts were tested individually and represented in two different panels. Data were analyzed in R (v 4.1.0) and GraphPad Prism. A p‐value ≤0.05 was considered statistically significant in all analyses.Mean differences between CD and SAAR in the two rat cohorts were assessed using the two‐tailed Student's 4.9.1p‐Values were adjusted for multiple comparison errors using false discovery rate (FDR).Linear mixed‐effect regression was used to test if there were any significant differences in the mean for each of the MR and the CR groups with respect to SAAR. Separate regressions were run for MR and CR diets. A random intercept was added to models to consider the heterogeneous effects contributed by each sample. 4.9.2t test (without the assumption of homogeneity).Two sets of estimated mean differences were extracted from the linear mixed models, namely MR groups versus SAAR and CR groups versus SAAR. Assuming a linear trend, two simple linear regressions were fitted for each of the two sets. We tested for any significant difference between the two estimated slopes using the Welch two‐sample 4.9.3t tests were conducted , equivalence tests in the form of two‐one‐sided  Lakens, . Followi4.9.4p‐values were <0.05. p‐Values for interaction between the two independent variables were represented as μint, and pairwise comparisons were represented by μ.Data from mouse models were analyzed by two‐way ANOVA followed by Sidak's multiple comparison test. mRNA expression was considered significantly different from SAAR only if the fold‐change was >2 and 4.9.5Data from the epidemiological study were log‐transformed due to non‐normal distribution. The association of Met, tCys, and Ser with triglycerides was investigated with and without adjusting for age, sex, and BMI (log[triglycerides] ~ log[amino acid] + age + sex + BMI). We also investigated whether plasma amino acids changed with the number of MetS criteria with and without adjusting for age and sex (log[amino acid] ~ MetS criteria + age + sex). BMI was not considered for the second regression analysis as MetS criteria include obesity. Estimates were expressed as a percentage increase in triglycerides per percentage increase in amino acids. Estimates from the second model were expressed as a percentage change in amino acids with an increase in one MetS criterion. All data were expressed as geometric means and geometric standard deviations. In controlled‐feeding studies, differences in plasma Ser between groups were analyzed using linear mixed model regression with Ser as the outcome and group, visit, and their interaction term (group × visit) as predictors. Models were adjusted for baseline differences.Conceptualization, original manuscript draft, data visualization, supervision, project administration: SNN; methodology: CT, HR, KJV, and TO; formal analysis: SNN, VM, JPR, CT, TO, MP, and JS; investigation: SNN, DALM, DC, VLM, WM, GPA, BO, NEB, HR, KJV, AKS, MP, JS, and VK; writing (reviewing and editing): all authors.No authors have any conflict of interest.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Appendix S1Click here for additional data file.