Y APAP. Since oxidative stress, mitochondrial damage and 64849-39-4 site disrupted calcium homeostasis play an important role in APAP-mediated hepatotoxicity, it is not exceptional that we identified SOD1 and CaM as proteins with biomarker potential. The involvement of superoxide dismutases in APAP-induced liver injury has previously been demonstrated by the increased toxicity of APAP in mice with reduced activity of SOD2 [29,30]. The exact role of SOD1 in APAP-mediated hepatotoxicity remains controversial as both protective and damaging effects have been reported, but SOD1 nitration and reduction in SOD1 activity appear to be involved [31]. A role for CaM in APAP-induced liver injury has not beenclearly described; however, CaM does play a key role in maintaining intracellular calcium buy SPDP balance. Binding of NAPQI to mitochondrial proteins can cause mitochondrial permeability transition, after which mitochondrial Ca2+ is released into the cytosol [32]. The cytosolic Ca2+ concentration is tightly regulated and any excess Ca2+ will be effluxed via the plasma membrane Ca2+ ATPase transporter (PMCA), using CaM as ultimate cofactor [33]. However, the peroxynitrite formed during APAPinduced oxidative stress can oxidize specific methionine positions of CaM, after which CaM is no longer able to activate PMCA, which results in reduced excretion of cytosolic Ca2+ [34]. Previous studies showed decreased activity of PMCA during APAP-induced liver injury [35]. With sustained high cytosolic Ca2+ concentrations, Ca2+ will be translocated to the nucleus by CaM, where it will cause DNA fragmentation and ultimately lead to cell death [36]. CaM is thus involved in the initiating events of APAPinduced liver injury and may, therefore, be a potential early biomarker. Based on these data, the next step would be to investigate the dynamics of the suggested biomarkers on hepatic regulation and excretion in urine with DILI, by including multiple time points of measurement after drug administration. This will also allow better comparison of the urinary proteins as non-invasive biomarkers with the conventional plasma ALT measurements, including the predictive value for DILI. Continuous urine sample collection of patients with APAP-induced liver injury and DILI caused by other drugs is needed to assess further the suitability of the biomarkers suggested for acute DILI in general. In summary, using a translational approach we identified CA3, SOD1 and CaM as novel urinary biomarkers in relation to APAPinduced liver injury in both mouse and human urine samples. These results allow further clinical validation to assess their applicability as non-invasive biomarkers for acute DILI.AcknowledgmentsWe’d like to thank the people of the Nijmegen Proteomics Facility for their support with the proteomics analysis and M. Stitzinger and M. van Tuyl for coordinating the plasma liver enzyme analysis performed at NOTOX B.V.Author ContributionsConceived and designed the experiments: RPLvS CMML RM FGMR. Performed the experiments: RPLvS 
CMML. Analyzed the data: RPLvS CMML RAW. Contributed reagents/materials/analysis tools: ECvdK EMK RAW KDA DJT MHS. Wrote the paper: RPLvS ECvdK EMK RAW KDA RM FGMR. Coordination of human sample collection: KDA DJT MHS.
The ability of certain highly soluble proteins to enhance the solubility of their fusion partners is often exploited for the production of recombinant proteins [1]. Escherichia coli maltosebinding protein (MBP) falls into this category and has been used exte.Y APAP. Since oxidative stress, mitochondrial damage and disrupted calcium homeostasis play an important role in APAP-mediated hepatotoxicity, it is not exceptional that we identified SOD1 and CaM as proteins with biomarker potential. The involvement of superoxide dismutases in APAP-induced liver injury has previously been demonstrated by the increased toxicity of APAP in mice with reduced activity of SOD2 [29,30]. The exact role of SOD1 in APAP-mediated hepatotoxicity remains controversial as both protective and damaging effects have been reported, but SOD1 nitration and reduction in SOD1 activity appear to be involved [31]. A role for CaM in APAP-induced liver injury has not beenclearly described; however, CaM does play a key role in maintaining intracellular calcium balance. Binding of NAPQI to mitochondrial proteins can cause mitochondrial permeability transition, after which mitochondrial Ca2+ is released into the cytosol [32]. The cytosolic Ca2+ concentration is tightly regulated and any excess Ca2+ will be effluxed via the plasma membrane Ca2+ ATPase transporter (PMCA), using CaM as ultimate cofactor [33]. However, the peroxynitrite formed during APAPinduced oxidative stress can oxidize specific 
methionine positions of CaM, after which CaM is no longer able to activate PMCA, which results in reduced excretion of cytosolic Ca2+ [34]. Previous studies showed decreased activity of PMCA during APAP-induced liver injury [35]. With sustained high cytosolic Ca2+ concentrations, Ca2+ will be translocated to the nucleus by CaM, where it will cause DNA fragmentation and ultimately lead to cell death [36]. CaM is thus involved in the initiating events of APAPinduced liver injury and may, therefore, be a potential early biomarker. Based on these data, the next step would be to investigate the dynamics of the suggested biomarkers on hepatic regulation and excretion in urine with DILI, by including multiple time points of measurement after drug administration. This will also allow better comparison of the urinary proteins as non-invasive biomarkers with the conventional plasma ALT measurements, including the predictive value for DILI. Continuous urine sample collection of patients with APAP-induced liver injury and DILI caused by other drugs is needed to assess further the suitability of the biomarkers suggested for acute DILI in general. In summary, using a translational approach we identified CA3, SOD1 and CaM as novel urinary biomarkers in relation to APAPinduced liver injury in both mouse and human urine samples. These results allow further clinical validation to assess their applicability as non-invasive biomarkers for acute DILI.AcknowledgmentsWe’d like to thank the people of the Nijmegen Proteomics Facility for their support with the proteomics analysis and M. Stitzinger and M. van Tuyl for coordinating the plasma liver enzyme analysis performed at NOTOX B.V.Author ContributionsConceived and designed the experiments: RPLvS CMML RM FGMR. Performed the experiments: RPLvS CMML. Analyzed the data: RPLvS CMML RAW. Contributed reagents/materials/analysis tools: ECvdK EMK RAW KDA DJT MHS. Wrote the paper: RPLvS ECvdK EMK RAW KDA RM FGMR. Coordination of human sample collection: KDA DJT MHS.
The ability of certain highly soluble proteins to enhance the solubility of their fusion partners is often exploited for the production of recombinant proteins [1]. Escherichia coli maltosebinding protein (MBP) falls into this category and has been used exte.