In this study, we explore further the applicabilityof NAT/AT anodes for oxidizing pharmaceutical compounds using carbamazepine(CBZ) and fluconazole (FCZ) as model probe compounds. Details of theoxidation reaction kinetics and subsequent reaction products are investigatedin the absence and presence of chloride (Cl–) andsulfate (SO42–). In all cases, fasteror comparable degradation kinetics of CBZ and FCZ are achieved usingthe double-layered NAT/AT anode coupled with a stainless steel (SS)cathode in direct comparison to an identical setup using a boron-dopeddiamond anode. Production of O3 on NAT/AT enhances theelimination of both parent compounds and their transformation products(TPs). Very fast CBZ degradation is observed during NAT/AT-SS electrolysisin both NaClO4 and NaCl electrolytes. However, more reactionproducts are identified in the presence of Cl– thanClO4– (23 TPs vs 6). Rapid removal ofFCZ is observed in NaClO4, while the degradation rate isretarded in NaCl depending on the [Cl–]. In SO42–-containing electrolytes, altered reactionpathways and transformation product distributions are observed dueto sulfate radical generation. SO4·– oxidation produces fewer hydroxylated products and promotes theoxidation of aldehydes to carboxylic acids. Similar trend in treatmentperformance is observed in mixtures of CBZ and FCZ with other pharmaceuticalcompounds in latrine wastewater and secondary WWTP effluent.Electrochemical oxidationprovides a versatile technique for treatingwastewater streams onsite. We previously reported that a two-layerheterojunction Ni–Sb–SnO Thus,residual pharmaceutical products have been detected in virtually allenvironmental waters including groundwater, surface water, and wastewatertreatment plant (WWTP) influents and effluents.4 This level of discharge of untreatedor partially treated pharmaceutical products is problematic for aquaticsystems and drinking water supplies. While traditional WWTPs are inadequatefor degrading many commonly used pharmaceutical compounds,7 advanced oxidation processes (AOPs) involving reactive species suchas ozone (O3), hydroxyl radicals (•OH),and free reactive chlorine provide an attractive alternative for compounddegradation.10 Meanwhile, transformation products of pharmaceuticalsalso are an area of increasing research interest. The identificationof specific transformation products allows us to further understandtheir ultimate environmental fates, including the formation of transformationproducts that are more toxic than their parent compounds.13Pharmaceuticals represent an importantgroup of emerging environmentalcontaminants due to excretion of ingested medicines in urine and fecesand through the intentional disposal of unused or expired medicines.15 The composition of the electro-activeanode materials is a key factor in determining energy consumptionand electrolytic treatment efficiency. Ideal nonactive electrodessuitable for wastewater treatment that promote complete oxidationof organic pollutants include boron-doped diamond (BDD) and antimony-dopedtin oxide (AT: Sb–SnO2) anodes.17 While large-scale application of BDD electrodesis hindered by high manufacturing costs, typical AT electrodes offera less expensive option due to the absence of platinum-group metalcomponents and lower production costs. Moreover, when nickel is co-dopedwith AT (NAT: Ni–Sb–SnO2), the NAT anodesproduce ozone,19 which provides an additionalanodic oxidation pathway.Electrochemical oxidation (EO) is applied indecentralized andsmall-scale wastewater treatment systems when proceeded by a biologicalpretreatment step.2),20 mixed metal-oxide (Ti/IrxTayO2/[Bi2O3]z[TiO2]z1–),15 and mostly BDD electrodes22 in pure electrolytes, latrinewastewater, and biologically treated hospital wastewater. However,degradation with Sb–SnO2-based anodes has not beenwell studied, and transformation product analysis is limited. In ourprevious study, a heterojunction Ni–Sb–SnO2 anode (NAT/AT) was prepared that simultaneously produces ozone (O3) and hydroxyl radical (•OH) at the anode.23 NAT/AT electrolysis was shown to be effectivefor treating a mixture of pharmaceutical compounds in toilet wastewater.An anodic O3 activation mechanism was proposed by Zhanget al. to explain the accelerated degradation kinetics of some aromaticcompounds that are not highly reactive with O3 .8 Since aromaticrings and/or N and S atoms with nonbonded electrons are ubiquitousin pharmaceutical compounds, NAT/AT electrolysis should prove to beeffective for treating pharmaceuticals in waste effluents.Electrochemical oxidation has beeninvestigated for various pharmaceuticalcompounds using dimensionally stable anodes .26 The added impact of electrochemical O3 production onthe observed degradation kinetics and pathways in the NAT/AT systemis compared to degradation using BDD electrodes as a reference anodematerial. The influence of pH and common ions present in wastewaters(Cl– and SO42–)28 is investigated. Kinetic models to predict overall reaction kineticsand removal efficiencies are presented. Furthermore, pharmaceuticaldegradation at the NAT/AT anode is evaluated in actual toilet wastewaterand secondary effluent where the occurrence of these compounds isfrequently reported.Theprimary goal of this study is to investigate both the kineticsand the possible mechanistic pathways for product formation via NAT/AT-SSelectrolysis in undivided cells as a promising AOP for pharmaceuticalcompound treatment in contaminated water. Degradation kinetics andtransformation products (TPs) formation are studied in detail forcarbamazepine (CBZ) and fluconazole (FCZ). CBZ and FCZ are persistentlyfound in natural water bodies around the world.All pharmaceutical compounds,acridine, carbamazepine 10,11-epoxide, and 9-acridinecarboxylic acidstandards were purchased from Sigma-Aldrich. Actual latrine wastewaterwas collected from a public electrochemical toilet prototype on thecampus of Caltech . Secondary effluent was obtainedfrom Sanitation Districts of Los Angeles County  andstored in the dark at 4 °C for less than 1 month prior to use.Stock solutions of individual pharmaceuticals were prepared at20 μM in relevant electrolytes and stored under room temperaturein the dark. Wastewater samples were filtered with 0.45 μm glassfiber membranes and amended with pharmaceuticals (2 μM) beforetreatment.23 BDD electrodeswere purchased from NeoCoat (Switzerland). All electrochemical testswere performed in an undivided electrolysis cell. A stainless steelcathode (6 cm2) was coupled in parallel to an NAT/AT orBDD anode (6 cm2) with a 5 mm separation. An Ag/AgCl/Sat.NaCl reference electrode  was placed at the same spacingclose to the anode. Electrolysis experiments were conducted in 25mL solutions at a fixed current density of 10 mA/cm2 (specificsurface area = 24 m2/m3).Two typesof electrodes wereused in this study. NAT/AT anodes were prepared by a dip-coating methodas previously described.4), hydrochloric acid (HCl), or sodiumhydroxide (NaOH).Linear sweepvoltammetry (LSV) was conducted using a Biologic VSP-300 potentiostatin relevant solutions using a scan rate of 0.05 V/s. For some experiments,solutions were buffered with 5 mM phosphate and pH was adjusted withperchloric acid reagent . Dissolved O3 producedby NAT/AT was quantified using the indigo method.29 Current efficiencies (η) for chlorine and O3 evolution were calculated using the equationn is the number ofelectrons required for 1 mole formation of Cl2 from Cl– (n = 2) or O3 from O2– (n = 6), V is theelectrolyte volume (25 mL), F is Faraday constant, and I is the current (A). Commonwastewater ions  were quantifiedby ion chromatography.Free chlorine concentrations were measuredby DPD (Carbamazepine (CBZ), fluconazole (FCZ),and other pharmaceutical compounds were quantified using high-performanceliquid chromatography coupled with a UV detector (HPLC-UV) and equippedwith an XDB-C18 column . CBZ and FCZ were monitored at 285 and 205 nm, respectively.Eluent flowed at 0.4 mL/min and consisted of acetonitrile (ACN) andwater with 0.1% formic acid. A gradient was used to resolve peaks:0 min, 5% ACN; 2 min, 5% ACN; 6 min, 95% ACN; 8 min, 95% ACN; 9 min,5% ACN; 12 min, 5% ACN.4 (50 mM), Na2SO4 (5 mM) + NaClO4 (50 mM), NaCl (50 mM),or Na2SO4 (5 mM) + NaCl (50 mM). Products wereanalyzed using an ultrahigh-performance liquid chromatography system(Waters Acquity UPLC) coupled to a time-of-flight mass spectrometer(Waters Xevo GS-2 TOF) equipped with an Acquity BEH C18 column . Eluent consisting of ACNand water with 0.1% formic acid flowed at 0.5 mL/min. The appliedACN/H2O gradient profile was as follows: 0 min, 5% ACN;0.2 min, 5% ACN; 3.2 min, 95% ACN; 3.5 min, 95% ACN; 3.6 min, 5% ACN;5 min, 5% ACN. Products were monitored under positive electrosprayionization (ESI+) in resolution mode (measurement accuracy betterthan 1 ppm RMS) with 0.2 kV capillary voltage. Other mass spectrometerconditions include: cone voltage 50 V, source offset 80 V, sourcetemperature 120 °C, desolvation temperature 400 °C, conegas 40 L/h, desolvation gas 800 L/h, 0.3 s scan time in continuummode, collision energy 1.0 eV, and second acquisition channel collisionenergy scanned from 0 to 30 eV. A leucine lock-mass (m/z = 556.2771) was used to correct for accuratemass values.For transformation product identification,electrolysis of CBZand FCZ was conducted at an elevated compound concentration (20 μM)and in solutions containing: NaClOParent compounds and transformation products (TPs)were identifiedwith the Masslynx software (Waters) and quantified with Quanlynx.TP chemical formulas were obtained using accurate mass determinationsand known parent compound elemental compositions. Tentative structureswere proposed based on fragmentation patterns, isotopic patterns (whenchloride present), authentic standards (when available), and comparisonto the literature.30 A total of 117 elementary reactions were considered withrate constants obtained from the literature. The model was evaluatedin all electrolytes to examine the influence of salts on degradationkinetics (more details in Text S1 and Table S1).Kinetic modelingand predictionof pharmaceutical degradation kinetics were achieved using the chemicalkinetics computational program, Kintecus 6.80.Degradationwas investigated indetail for carbamazepine  and fluconazole .4 electrolytes, which appears to be due to directoxidation by O3. Our kinetic model also predicted completeremoval in 30 s . When Cl– is present in the system, O3 productionis inhibited due to competition for active sites between Cl– and OH–,23 althoughrapid removal is achieved with 100% removal in less than 1 min inNaCl electrolytes. Despite lower aqueous-phase O3 concentrations,a similar CBZ removal efficiency is achieved due to the formationof the chlorine radical anion Cl2·– .11 Prediction using the kinetic modelalso gave similar results taking into account the kinetics of thechlorine radical anion .Fast degradation of CBZ was observed using theNAT/AT anode 1a. For e3 production.7 However, due to the low concentration of SO42– and thus SO4·– present, CBZ degradationwas dominated by O3, chlorine radical species, and •OH.Degradation of CBZ with BDD was slower in both electrolytes comparedto that with NAT/AT due to the absence of Ooduction1b. React4 vs NaCl electrolytes . This result suggeststhat, similar to ibuprofen,23 FCZ degradationis promoted by anodic O3 activation on the NAT/AT anode.In NaCl solutions, on the other hand, FCZ removal was retarded comparedto that in NaClO4 given that only 60–70% removalwas observed after 1 h of electrolysis. This removal rate did notdeviate significantly from our kinetic model predictions , indicating that anodic O3 activation had little impact in this case.Very different degradation rateswere obtainedusing the NAT/AT anode in NaClOtrolytes 1c. The r4 and 80–90% removal in 1 h in NaCl electrolytes (•OH production levels at BDD coupledwith an added contribution from direct electron transfer (vide infra). For FCZ, degradation with both electrodes wasenhanced in the presence of SO42– inNaCl electrolytes. We propose that such enhancement in removal kineticsresults from oxidation by sulfate radicals (SO4·–), which have a comparable or slightly higher redox potential (E0 = 2.5–3.1 VNHE)31 than that of •OH (E0 = 2.7 VNHE).32 Formation of SO4·– occursvia the following two pathways33• (E0 = 2.4 VNHE)34 formsfrom Cl– in solution.Even though [HSO4–] (pKa = 1.92)35 is negligible inthe bulk solution at pH ∼9, sulfate radical production via •OH, SO4·– is a more selective oxidant that reactsprimarily via electron transfer.36 While •OH also reacts readily and rapidly viaaddition and H-abstraction pathways, SO4·– generally has lower rate constants for those reactions. For manypharmaceutical compounds, however, reaction rates with SO4·– are comparable to or occasionally fasterthan those with ·OH.7 Inaddition to FCZ, the presence of SO42– in wastewater matrices has been reported to accelerate the removalof other pharmaceutical compounds including ciprofloxacin, sulfamethoxazole,22 and ketoprofen21 usingBDD anodes. With FCZ, the effect of SO42– was prominent in NaCl electrolytes. This result suggests that SO4·– played a more important role incases where O3 and/or ·OH-mediated oxidationwas less important. In NaClO4 electrolytes, in contrast,FCZ removal appeared to be unaffected in the presence of SO42–.Electrolysis usingthe BDD anode resulted in ∼80% FCZ removal in 20 min in NaClOtrolytes 1d. Highe·OH is slightly lower 26 suggests that additional oxidation pathways are operative.Given that direct electron transfer (DET) is known to happen at thesurface of BDD (but not NAT/AT), it was suspected that DET made asubstantial contribution to the removal of FCZ. DET from FCZ to BDDwas confirmed using linear sweep voltammetry (LSV) in 50 mM NaClO4 (control) and in 50 mM NaClO4 containing 20 μMCBZ or FCZ . While no obviousfeature is observed for CBZ compared to the NaClO4 control,a notable peak was recorded at ∼3.0 VNHE in theFCZ voltammogram, which implies that DET is taking place.The higher removal percentage of FCZthan CBZ at BDD over 20 mineven though the reaction rate constant between FCZ and – concentrations. When [Cl–] is decreased from 50 to 5 mM, removal of FCZ after1 h increased from 60 to >80%  as well as experiments atNAT/AT with variable Cl to >80% 2a, which4, pH had negligible influence on FCZ removal for NAT/AT andBDD . Similar degradation kineticsat all pH values indicate a consistent ·OH production on theanode surface coupled with O3 production on NAT/AT. InNaCl, different kinetic profiles for BDD compared to NAT/AT were observed.For example, degradation on BDD anode was not influenced by the variationin electrolyte pH in that similar removal levels were observed aspH was lowered from 9 to 2 . However, the trend did not continueto pH 2, where the slowest removal was observed . With NAT/AT, on theother hand, decreased chlorine evolution (CER) and lower current efficiencieswere observed as the pH was decreased . Meanwhile, the aqueous O3 concentrations followedan opposite trend in that during 15 min of electrolysis, the steadystate [O3] increased from ∼0.5 mg/L at pH 9 to ∼2mg/L at pH 2 . These resultssuggest that, at NAT/AT in the presence of Cl–,lower pH values favor the production of O3 over chlorineevolution, which can explain the FCZ degradation trend in the pH rangefrom 9 to 5. At pH 2, despite higher O3 production, theslowest FCZ removal can be attributed to the different reactivitiesbetween deprotonated and protonated species with O3.38 In many cases, the reaction rate of the deprotonatedspecies of a compound can be several orders of magnitude higher thanthat of its protonated species. FCZ has pKa values of 2.6, 2.9, and 11.0, where 2.6 and 2.9 correspond to thetwo nitrogens in the triazole rings.39 AtpH 2, a protonated nitrogen could have significantly lower or no reactivitywith O3, resulting in retarded removal. Though kineticdata is not available for FCZ, the deprotonated form of imidazole,with a similar structure to triazole, reacts with O3 4orders of magnitude faster compared to its protonated form.38To explain the differencesin FCZ degradation vs pH, chlorine evolutionwas measured for each anode, and Om/z ratios, majorfragment ions, mass error to theoretical m/z ratios, calculated chemical formulas, and proposed structuresand their confidence levels. Reaction pathways for TP formation areproposed in videinfra). The pathways presented, as will be shown later, maynot be comprehensive, and formation of many TPs is possible via multiplepathways.Liquid chromatography–mass spectrometry(LC-MS) analysisshowed that there were 23 distinct transformation products (TPs) observedduring CBZ oxidation, 10 of which have not been previously reported.Properties of the TPs as documented in 4 electrolytes, 5 TPs were detected using NAT/AT and 5  using BDD  showed an MS response 1 order of magnitude higher than allof the other TPs in NaClO4-based electrolytes . While various structures have been proposed for the same m/z ratios in the literature, based onits fragmentation patterns, TP267 was identified as the carboxylicacid of TP251 (BaQM). The oxidation of BQM (TP251) to BaQM (TP267)has been reported before during ozonation.43 Other pathways for BaQM formation were also seen during biological40 and ferrate44 oxidationof CBZ, where BQM was not detected. Similar to the oxidation of BQMto BaQM, an aldehyde-to-carboxylic-acid pathway has been reportedin a variety of treatment options including ozonation,45 UV/chlorine,42 UV/TiO2 photocatalysis,47 ferrate,44 and biodegradation with white-rot fungus Pleurotus ostreatus.46 In our case,the abundance of TP267 increased significantly when SO42– was added in the electrolyte solution , which implies that SO4·– is also capable of oxidizing aldehydesto carboxylic acids.49 Following TP251 and TP267, TP147 was formed via bond cleavage. Due to the fastreaction between CBZ and O3, oxidation by ·OH played a minor role in oxidation with NAT/AT. The hydroxylatedproduct, TP253a , was detectedwith the lowest response among all 5 TPs, with its maximum peak area3 orders of magnitude less than that of TP251.In NaClOtrolytes 4a. This m/z = 253.0972 (TP253b) was detected, which was not seen duringNAT/AT electrolysis. This product is carbamazepine 10,11-epoxide (CBZ-EP)based on direct comparison to commercial standard. The peak area ofCBZ-EP stayed relatively constant during 20 min of electrolysis . In the case of BDD-induced ·OH-mediated oxidation, in comparison to the direct formationof TP251 (BQM) via ozonation, we propose that CBZ is first oxidizedto its hydroxylated product (TP253a), which is in turn oxidized toBQM , which was confirmed using a reference standardto be acridine, a known mutagenic and carcinogenic compound.11 TP180 is relatively stable. It forms in CBZstock solutions and, in cases where the stock was kept for longertimes, was actually detected without electrolysis. TP180 is also transformedslowly by ·OH. Comparing to the relatively fast removalby O3 with NAT/AT, it was only slowly removed using BDD.Finally, electrolysis in NaClOS9a). For BDD, the TP concentrations leveled off after 20min, after which their concentrations started to decrease due to furtheroxidation (S9b). This result indicates thatlonger treatment times are required for further oxidative removalof the TPs where ·OH is the sole oxidant.Removal of the TPswas slower in comparison to parent compounddegradation. The 5 TPs detected using NAT/AT were formed and basicallycompletely removed within 5 min of electrolysis  had a response of ∼0.74 and was identified as thechlorination byproduct of TP251. TP253b (CBZ-EP), the epoxide productmediated by ·OH oxidation, was also detected at substantiallevels in NaCl compared to that in NaClO4. Another setof interesting TPs included TP208 , TP224 , TP180, and TP196 . TP208 was determined to be 9-acridine-carboxaldehyde.Possible precursors for it include TP253a and TP253b (videinfra). The pathway TP253b → TP208 has been reportedduring ozonation,43 ClO2 oxidation,51 and biotransformation51 by ring contraction and loss of the carbamoyl group. Oxidation ofthe aldehyde group on TP208 gives the corresponding carboxylic acidproduct TP224 (9-acridinecarboxylic acid), which can undergo decarboxylationto give TP180, acridine. TP208 and TP224 represent another aldehyde–carboxylicacid pair like TP251 and TP267. Hydroxylation and oxidation of TP180then leads to the formation of TP196, acridone, which also had a relativeresponse factor of >0.05. The oxidation of acridine to acridonehasbeen confirmed in electrochemical oxidation39 and represents a known biological detoxification process.46 The sequence of pathways from TP208 to TP196has been observed in biotransformation in WWTP biological processes40 as well as with the white-rot fungus P. ostreatus.46 Direct formation ofTP180 from TP208 by cleavage of the aldehyde group is also possible.51 In addition to the above 4 TPs, TP226  could be another hydroxylated precursor of TP208, and TP253c  is proposed to form via an intramolecularcyclization at the carbamoyl group. Overall, since O3 productionis inhibited in the presence of Cl–, other oxidationpathways played more important roles in the formation of the otherTPs.In NaCl electrolytes, moreTPs were detected compared to those found during electrolysis in NaClO 0.5 min 5a. In thm/z =301.0380, C15H10N2O3Cl),TP181 , TP289 ,49 and TP230 were identified as the chlorinated byproducts of TP267, TP147, TP253b,and TP196, respectively. TP271  was identified as 10,11-dihydro-10,11-cis-dihydroxy-carbamazepine(cis-diOH-CBZ). TP205 and TP211  were two other products, besidesTP253c, that may have formed after carbamoyl group intramolecularcyclization. This cyclization pathway has also been proposed in thetreatment of CBZ by UV/chlorine and UV/H2O2.52 Finally, TP230 ,TP244 , and TP278 are proposed to be the singly and multiply hydroxylated and chlorinatedproduct of TP196, acridone.Many additional TPs were observed using NAT/AT although with lowerrelative responses. In addition to TP285, which is the chlorinatedbyproduct of TP251, TP301  along with its corresponding chlorinated byproduct, TP301 . Another carboxylic acid product,TP224, was also detected in higher abundance when SO42– was added to the base electrolyte despite TP208 havinga lower response . Meanwhile,lower response factors for several other TPs were observed. The moreprominent ones include TP226, TP271, and TP253a . These collective observations indicate that,in the presence of SO42–, oxidation wasprimarily promoted by ·OH and SO4·– simultaneously along with subsequent chlorination. However, ·OH played a smaller role in oxidation comparing to thecases in which SO42– was absent. Sinceelectron transfer is the preferred oxidation pathway by SO4·– as opposed to addition or H-abstraction,the hydroxylated products produced by ·OH oxidationwere formed in lower yields.Formation patterns of several TPswere different when SO4 electrolytes,the maximum MS peak area was also 1 order of magnitude smaller thanthat for NAT/AT. TP147 and TP267, as well as their chlorinated byproductsTP181 and TP301, were not detected during BDD electrolysis at measurablelevels in both solutions. Furthermore, TP251 appeared to be a lessimportant TP, which suggests that other oxidation pathways predominatedover route 2 in In the case of BDD electrolysis,the product with the highest responsewas TP208 with an intensity that peaked at 0.5 min 5b. TP208S11a). For BDD, 100%CBZ removal occurred around 5 min, whereas most of the TPs peakedbetween 2 and 5 min and were then partially removed after 20 min ofelectrolysis (S11b). These results indicate theeffectiveness of O3 in eliminating the intermediate TPsin addition to the initial oxidation step of CBZ. Responses of allTPs in MS at the two electrodes in all four electrolyte combinationsare summarized in Table S3.CBZ was completely degraded within 1 minof electrolysis usingNAT/AT. Most of the 22 TPs detected during NAT/AT electrolysis peakedbetween 0.5 and 1 min and then were either removed completely or toa high degree after 5 min . In all electrolytesand in secondary effluent (discussed below), these 3 TPs did not constitutea major part (<15%) of the transformed CBZ in terms of mass balance.Mass balance is not closed here due to the variety of TPs detectedand a lack of reference standards. However, identification over completequantification of the TPs and of their removal trends can be moreimportant for practical engineering purposes, since TP distributionscan vary a lot depending on the wastewater composition, yet TP identificationcan help facilitate treatment efficiency evaluation and toxicity assessmentin general.The moreimportant TPs of CBZ were confirmed and quantified where commercialstandards are available: TP180 (acridine), TP253b ,and TP224 (9-acridinecarboxylic acid). TP180 and TP224 are also knownto be toxic. More of all of the three TPs was detected in NaCl thanin NaClOFigures S13 and S14). In general, the formation of many TPs is possible viamultiple pathways.Electrolysis using the TP253b and TP224 as parentcompounds was also conducted to further elucidate and confirm thetransformation pathways in 56 ranging from 0 detected in constructedwetlands56 to 6 under UV/chlorine.12 In NAT/AT and BDD systems, only 1 TP, TP224, was detected in the MS for FCZdegradation in NaClO4 electrolytes. It formed from cleavageof the parent molecule have been reportedfor FCZ in the literature compared to those for CBZ,molecule 6a. This  ∼20min 6b. Thea3 and ·OH.26 CBZand FCZ fall into group Ia with the fastest kinetic rates and groupIV with the slowest kinetic rates, respectively. To obtain a quantitativeranking of pharmaceutical degradation with NAT/AT, five other pharmaceuticalsin the top 100 list1 were selected fromthe different categories. They included atenolol ,gabapentin , trimethoprim , sulfamethoxazole, and ibuprofen . The physical–chemicalproperties of the seven target compounds including their pKa values and rate constants with O3 and ·OH are summarized in Table S4. Individual degradation kinetics of ATL, GBP, and IBP atNAT/AT and their kinetic modeling predictions are shown in Figure S15.Theoxidative degradation of CBZ and FCZ and formation of their transformationproducts were also carried out in actual latrine wastewater and insecondary effluent along with a mixture of common pharmaceutical compounds.Previously, Lee et al. characterized micropollutant elimination duringozonation into five categories based on their reaction rate constantswith O1 The chemical compositionsof the wastewaters are given in Table S5. In latrine wastewater, due to the presence of high background levelsof COD (440 mg/L) and NH4+ (31 mM), and theresulting competitive consumption of oxidants, degradation was retardedcompared to that in pure electrolytes. In latrine wastewater, >80%removal of the spiked pharmaceutical compounds was achieved in 75min electrolysis except for FCZ, GBP, and IBP . In domesticsecondary wastewater treatment effluent, the pharmaceuticals weredegraded faster in comparison to latrine wastewater due to low initialCOD (80 mg/L) and NH4+ (0.3 mM) concentrations.Complete removal of all pharmaceuticals except for FCZ was achievedin 5 min of electrolysis, while the time required for FCZ removalwas 15 min . Comparedto NAT/AT, BDD, while achieving similar COD reduction , demonstrated better performancefor pharmaceutical degradation in latrine wastewater and worse performancein secondary effluent . This result indicates the advantage of NAT/AT application in systemswith lower chloride concentration. Potential improvement could alsobe achieved by including pretreatment units to lower COD and removeNH4+ and other interfering components from wastewatersbefore electrochemical treatment.The 7 pharmaceuticals werespiked into the wastewaters at 2 μM each, which is higher thanthe typical concentrations detected in wastewater sources , to investigate treatment with elevatedconcentrations. and IBP 7a, indic and IBP S16. In d3) as well as their transformation products.The NAT/AT-SS system, requiring lower cell voltage than BDD (Table S5) under the same applied current density,could achieve similar or better removal with lower energy consumption.It thus potentially represents a more economical and efficient methodfor water treatment practices that is capable of large-scale implementation.Moreover, the secondary effluent used herein had similar chemicalcompositions to a biologically treated hospital wastewater previouslyinvestigated,22 suggesting that electrolyticoxidation with NAT/AT could also provide a suitable treatment alternativefor the control of pharmaceuticals in hospital wastewaters.Insummary, the NAT/AT anodes demonstrated promising performancein degrading a range of pharmaceutical compounds (with very differentreactivities with O


Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor patient prognosis. Remarkably, PDAC is one of the most aggressive and deadly tumor types and is notorious for its resistance to all types of treatment. PDAC resistance is frequently associated with a wide metabolic rewiring and in particular of the glycolytic branch named Hexosamine Biosynthetic Pathway (HBP).Transcriptional and bioinformatics analysis were performed to obtain information about the effect of the HBP inhibition in two cell models of PDAC. Cell count, western blot, HPLC and metabolomics analyses were used to determine the impact of the combined treatment between an HBP’s Phosphoglucomutase 3 (PGM3) enzyme inhibitor, named FR054, and erastin (ERA), a recognized ferroptosis inducer, on PDAC cell growth and survival.Here we show that the combined treatment applied to different PDAC cell lines induces a significant decrease in cell proliferation and a concurrent enhancement of cell death. Furthermore, we show that this combined treatment induces Unfolded Protein Response (UPR), NFE2 Like BZIP Transcription Factor 2 (NRF2) activation, a change in cellular redox state, a greater sensitivity to oxidative stress, a major dependence on glutamine metabolism, and finally ferroptosis cell death.Our study discloses that HBP inhibition enhances, via UPR activation, the ERA effect and therefore might be a novel anticancer mechanism to be exploited as PDAC therapy. Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor patient prognosis . ConventN-acetyl-D-glucosamine (UDP-GlcNAc), the main substrate for O- and N-protein glycosylation. Both post-translational modifications (PTMs) play critical roles in protein folding, stability, activity, macromolecular interactions, function, and nuclear translocation. Therefore, enhancement of HBP flux, responsible for the aberrant protein glycosylation often observed in different types of tumors including PDAC . Indeed, HBP flux reduction, decreasing UDP-GlcNAc availability, causes a reduction in protein N-glycosylation, an accumulation of misfolded proteins, and finally irremediable cell damage, which drives CHOP-dependent apoptotic signaling , 12. WorTherefore, in this study, we aimed to further detail the mechanism of FR054-induced cell death, in order to verify whether its pro-apoptotic ability could be enhanced by co-targeting a specific protein or cellular pathway. For this purpose, we performed an RNA-seq analysis in two different PDAC cell lines, namely, MiaPaCa-2 and BxPC3, treated with FR054 to provide unbiased mechanistic insights. Results demonstrate that FR054 treatment induces expression of genes related to UPR, to the NRF2 pathway, and to glutathione biosynthesis and ferroptosis. Consequently, we show that inhibition of glutathione biosynthesis, by using the specific inhibitor for the Solute Carrier Family 7 Member 11/xCT (SLC7A11), ERA, in combination with FR054, significantly enhanced the FR054 effect causing a noteworthy increase in cancer cell proliferation arrest and death. Remarkably, the latter effect was tightly associated with a higher expression of the pro-apoptotic protein DNA Damage Inducible Transcript 3/CHOP (CHOP), reduced activity of SLC7A11 transporter, an alteration of intracellular glutathione (GSH) and glutathione disulfide (GSSG) levels, and a strengthening of lipid peroxidation, indicating that SLC7A11 inhibition is synthetic lethal with FR054. Notably, the metabolic analysis supported the role of FR054 in favoring glutamine metabolism over glycolysis. Therefore, our work reveals that simultaneous inhibition of HBP with FR054 and GSH metabolism could have therapeutic benefits in the treatment of pancreatic cancer.N-Acetyl-L-cysteine (NAC), DL-Buthionine-sulfoximine (BSO), and BPTES were purchased from Sigma-Aldrich , while erastin (ERA) was purchased from Selleckchem . FR054 was synthesized either by our laboratories or by WuXi AppTec Co., Ltd.  , 11.2. The medium was replaced every 2–3 days and cells were split or seeded for experiments when they reached the sub-confluence. MiaPaCa-2, PANC1, and BxPC-3 were originally obtained from the American Type Culture Collection (ATCC).Human pancreatic ductal adenocarcinoma cell lines MiaPaCa-2 and PANC1 were routinely cultured in high glucose Dulbecco’s medium Eagle’s medium (DMEM), while the BxPC3 cell line was cultured in RPMI . Both media were supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin , and 10% fetal bovine serum . The cells were grown and maintained according to standard cell culture protocols and kept at 37°C with 5% COWhere not differently specified, for experiments, cells were seeded in the complete growth medium, and after 24 h, cells were washed twice with phosphate buffer saline 1X  and incubated in a complete medium with or without FR054. Twenty-four hours later, cells were also treated with or without 10 µM ERA.4 (MiaPaCa-2 and PANC1) and 8 × 104 (BxPC3) viable cells per well in 12-well dishes, respectively. Inhibition of proliferation and cell death were assessed at different time points (48 and 72 h), counting harvested cells with Burker chamber after staining with trypan blue 0.4% . In particular, cells were incubated in a complete medium with or without 350 µM FR054 for 24 h and then, after further 24 h, were also treated with or without 10 µM BPTES or 10 µM erastin. The MiaPaCa-2 cell line was also tested with two different concentrations of FR054, namely, 350 µM or 500 µM, to define an optimal concentration for all the other experiments in all PDAC cell lines. Cell death was also measured at 72 h of treatments as previously described, using a different treatment scheme: 24 h after seeding, cells were incubated in a complete medium with or without 350 µM FR054, and at 48 h post-seeding, cells were also treated with or without 10 µM ERA and 5 mM NAC or 5 mM BSO.Trypan blue vital assay was performed by seeded 4 × 105 cells) and BxPC3 (1.2 × 106 cells) were seeded onto 100-mm dishes in the complete growth medium. After 48 and 72 h, cells were harvested and disrupted in a buffer containing 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% (v/v) Triton X-100, 0.2% (v/v) sodium dodecyl sulfate (SDS), 0.5% (v/v) sodium deoxycholate, 1 mM MgCl2, 1 mM EDTA, protease inhibitor cocktail, and phosphatase inhibitors ; 20 µg of total proteins was resolved by SDS-PAGE and transferred to the nitrocellulose membrane, which was incubated overnight with specific primary antibodies: cleaved caspase-3 , GRP78 , phospho-eIF2α Ser 51  from Cell Signaling Technology Inc. , eIF2α  from Santa Cruz Biotechnology Inc. , ATF4 , CHOP  from Cell Signaling Technology Inc. , NRF2  from Santa Cruz Biotechnology Inc. , SLC7A11  from Abcam , HO-1  from Cell Signaling Technology Inc. , and Keap1  and Vinculin  from Santa Cruz Biotechnology Inc. . Levels of protein expression on Western blots were quantified by densitometry analysis using ImageJ software.MiaPaCa-2 (6 × 104 (MiaPaCa-2 and PANC1) and 8 × 104 (BxPC3) per well in 12-well dishes. The media used for subsequent HPLC analyses were collected after 48 h and 72 h of treatment. Glutamic acid and cystine concentrations were determined by chromatographic analysis using a Jasco HPLC system  under the control of the ChromNAV 2.0 software. The analyses were carried out using the method described by Henderson et al. with some modifications. The derivatized amino acids were separated using a Waters  XTerra RP18 Column  equipped with a precolumn Security Guard . The flow rate was set at 1 ml/min with the column oven at 40°C. Twenty microliters of the sample, appropriately diluted, was injected into the system and the chromatogram was monitored at 338 nm. The pre-column derivatizing reagent o-Phtalaldehyde (OPA) was prepared by dissolving 25 mg of OPA and 25 mg of 3-Mercaptopropionic acid in 5 ml of 0.2 M borate buffer, pH 10.2, and stored at 4°C until any sign of degradation. The samples were prepared by adding 5.5 μl of the derivatization reagent to 5.5 μl of the samples and bringing the volume up to 200 μl with water. HPLC separation employed mobile phases A  and B (30% acetonitrile:60% methanol:10% H2O). The column was equilibrated with 94.5%/5.5% (v/v) mobile phase A/B for 10 min before each sample analysis. The elution gradient consisted of 94.5%/5.5% (A/B) for 0.85 min; 2.15-min linear gradient to 13% phase B; 23-min linear gradient to 54% phase B; 94.5%/5.5% (A/B) for 4 min. Amino acid peaks were identified by comparing their retention times with that of the standard mixture. At the end of every set of samples, the column was washed with 20% acetonitrile–80% water for 30 min to remove any trace of salts, and then with 80% acetonitrile–20% water for 20 min and stored in solution.Cells were plated at a density of 4 × 105, 6 × 105, and 1.2 × 106 per well, and they were treated with 350 µM FR054 after 24 h. At 48 h post-seeding, the cells were treated with 10 µM ERA. The images were collected with Olympus CX40 phase contrast microscope using a 20X objective using X_entry Alexasoft Imaging Software.MiaPaCa-2, PANC1, and BxPC3 cells were plated onto 100-mm dishes in the complete growth medium at a density of 6 × 105, 1 × 105, and 2 × 105 per well, respectively. Intracellular GSH and GSSG levels were determined after 48 h of FR054 and erastin in single or combined treatment by VICTOR X3 multimode plate reader  using a Total GSH/GSSG colorimetric kit  according to the manufacturer’s instructions. The sample absorbance was measured at 412 nm. Relative GSH and GSSG content was normalized to the cell number.To measure reduced glutathione (GSH) and oxidized glutathione (GSSG) content, MiaPaCa-2, PANC1, and BxPC3 cells were plated in six-well dishes at a density of 1 × 103 cells) and BxPC3 (1 × 104 cells) were seeded onto a black 96-well plate in the complete growth medium. Then, cells were incubated in the complete medium with or without 350 µM FR054. After 24 h, cells were also treated with or without 10 µM ERA. All cell lines were also treated with 5 mM NAC or 5 mM BSO as controls for the presence of oxidative stress. Lipid peroxidation level was measured after 48 h of treatment. Cells were washed twice with PBS 1X and incubated in PBS 1X with 10 µM BODIPY C11 dye for 15 min at 37°C. Then, cells were washed again with PBS 1X, and the dye fluorescence was measured. Oxidation of BODIPY C11 resulted in a shift of the fluorescence emission peak from 590 nm to 510 nm proportional to lipid ROS generation and was analyzed using FLOUstar® Omega .To evaluate lipid peroxidation, the BODIPY 581/591 C11 dye  staining was carried out. One day before treatment, MiaPaCa-2, PANC1 (5 × 105 (MiaPaCa-2 and PANC1) or 1.2 × 106 (BxPC3) onto 100-mm dishes in the complete growth medium. The MDA levels in the cells were measured after 48 h of FR054 and ERA in single or combined treatment using MDA Colorimetric assay kit  according to the manufacturer’s instructions. The sample absorbance was measured at 532 nm by VICTOR X3 multimode plate reader  and the relative MDA concentration was normalized to the protein content.To evaluate the formation of the lipid peroxidation product, malondialdehyde (MDA), the cells were seeded at a density of 6 × 104 cells/well for MiaPaCa-2 and PANC1 cell lines or 1.1 × 105 cells/well for the BxPC3 cell line. After 24 h of seeding, the media were replaced with DMEM for mass spectrometry  for MiaPaCa-2 and PANC1 cell lines, and with MS-Grade SILAC RPMI 1640  medium, without Phenol Red, L-Arginine, and L-Lysine, which were manually added at a concentration of 200 mg/ml and 40 mg/ml, respectively, for the BxPC3 cell line. In both media, 10% sterile-filtered dialyzed FBS was added . For stable isotope labeling, in media, U13C Glucose and unlabeled Glutamine or U13C Glutamine and unlabeled Glucose  were also added. After 48 h of FR054 and ERA in single or combined treatment, the medium of each condition was collected, and wells were washed with 0.9% NaCl solution to perform metabolite extraction following standard laboratory procedure as described in Ref  starting from 5 × 106 cells/sample. RNA-seq experiments, data extraction, and analysis were performed in outsourcing . In particular, total RNA was quantified using a spectrophotometer and RNA integrity was evaluated using the Agilent 4200 TapeStation. RNA-stranded libraries were generated starting from 500 ng of total RNA, and mRNA was selected with poly-T oligo attached magnetic beads using an Illumina TruSeq® Stranded mRNA Library Prep kit . Libraries were quantified and quality checked on an Agilent 4200 TapeStation system (High Sensitivity D1000), and then sequenced on an Illumina NovaSeq 6000 platform with paired-end reads 150 bp long, with a depth of 30 million clusters/sample. Fastq reads were quality checked using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for overall and per-base read quality and presence of adaptor sequences. Paired reads were subsequently aligned to the human reference genome (GRCh38/hg38) using the splice-aware aligner STAR v 2.5.0c and indexed with Samtools. Per-gene read counts were generated using the STAR quantMode geneCounts option. Raw per-gene read counts were then processed using the Bioconductor package DESeq2 package v. 1.30 ] < 0.05 and log2FC <−2 or >2 were considered as differentially expressed. Sorted, indexed bam files were finally used for manual quality check of the alignment profiles. The RNA-seq data have been deposited at the NCB-GEO database, accession number GSE223303.Two biological replicates were used for gene expression analysis. In particular, MiaPaCa-2 and BxPC3 cell lines were seeded at a density of 5 × 10 v. 1.30  in orderp < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) was determined using one-way ANOVA with Tukey’s multiple comparisons test. For GC/MS analysis performed with stable isotope labeling, statistical significance  was determined with Student’s t-test. Instead, for all other experiments, statistical significance  was determined using two-way repeated measures ANOVA with Tukey’s multiple comparisons test.All statistical analyses were performed using GraphPad Prism v 8.0.2  and data are presented as mean ± SD and as mean ± SEM from three or more independent experiments. For the inhibition of proliferation evaluated after treatment with different concentrations of FR054 and/or ERA/BPTES and for GSH, GSSG, and MDA determination, statistical significance , Glutamate-Cysteine Ligase Catalytic Subunit (GCLC), Glutamate-Cysteine Ligase Modifier Subunit (GCLM), Spermidine/Spermine N1-acetyltransferase 1 (SAT1), Aldo-Keto Reductase Family 1 Member C1 (AKR1C1), Oxidative Stress Induced Growth Inhibitor 1 (OSGIN1), Glutathione-Disulfide Reductase (GSR), SLC7A11, Thioredoxin Reductase 1 (TXNRD1) Ferritin Light Chain (FTL), Activating Transcription Factor 3 (ATF3), Aldehyde dehydrogenase 3A1 (ALDH3A1), and Alpha/beta-hydrolase domain containing 4 (ABHD4), were the top enriched genes in treated cell lines compared with untreated cells. It should also be noted that the other top enriched genes were related to the anti-oxidant response regulated by NRF2 since MAF BZIP Transcription Factor G (MafG) heterodimerizes with NRF2 and NmrA Like Redox Sensor 1 (NMRAL1P1), which encodes for an NADPH sensor protein, is tightly associated with the NRF2 pathway. In addition, as expected for the well-known capacity of FR054 to induce ER stress and UPR, some transcription factors (TFs), tightly linked to this response, including DNA Damage Inducible Transcript 3/CHOP (DDIT3), Early Growth Response 1 (EGR1) and Activating Transcription Factor 3 (ATF3), were more upregulated in MiaPaCa-2-treated cells. To computationally confirm the specific enrichment of some TFs as responsible for the observed changes in gene expression upon FR054 treatment, upregulated DEGs for both cell lines were used as a query to interrogate the ChEA3-ChIP-X Enrichment analysis libraries using the web-based software Enrichr (2FC ≥ 2 and the ReMap library (2FC ≥1.5 (upregulated genes used: 137) and the Top rank analysis that gives as readout the integrated results from all the ChEA3-ChIP-X libraries. As shown in p-values smaller than 0.05) related to NRF2-dependent activation mechanism as well as genes related to ferroptosis. In particular, we identified six different cellular processes linked to NRF2 activity and/or ferroptosis, namely gluthatione metabolism, iron metabolism, ROS metabolism, lipid metabolism, metabolism, and autophagy as well as TFs associated with NRF2. As shown in the dendrograms presented in cystine/glutamate antiporters SLC7A11 and SLC3A2 and Glutathione-Disulfide Reductase (GSR)]; glutamine, cysteine, and glycine metabolism ; NADPH production ; and genes involved in anti-ferroptosis mechanisms such as iron transport and storage (FTH1 and FTL), Fe2+ sequestration (NUPR1 and LCN2), membrane repair mechanisms , and autophagic genes associated with both NRF2 activation and ferroptosis . Conversely, genes associated with lipid metabolism, generally repressed by NRF2, such as ACACA, SCD, LPCAT3, FADS2, and ELOVL6 were downregulated, further confirming an FR054-dependent activation of the NRF2 axis. The latter effect was also confirmed by the concomitant upregulation of different TFs related to the NRF2 pathway including MAFA, MAFG, MAFF, CEBPB, ATF4, and NRF2 as well. Of note, FR054 also induced a few important ferroptosis genes including CHAC1, a γ-glutamyl cyclotransferase toward glutathione, a well-known ferroptosis marker, and HMOX1, the major intracellular source of iron Fe2+ , we used mRNAs showing twofold changes with  Enrichr . In part library . ConversNRF2 is a master regulator of antioxidative responses and plays critical roles in maintaining redox balancing; thus, NRF2 activators attenuate oxidative stress. Indeed, NRF2 is one of the key regulators of GSH metabolism. GSH synthesis enzyme glutamate-cysteine ligase, glutathione synthetase, and reductase are target genes of NRF2 . GlutamiIn MiaPaCa-2 cells, as result of vital cell count, ERA treatment reduced cell proliferation by around 30%, BPTES by approximately 43%, and FR054 by approximately 52% compared to untreated cells  and analyzing both proliferation and cell death by vital trypan blue staining. As shown in Previously published data indicated that prolonged treatment with FR054 causes cell death by inducing ER stress and activation of UPR , 12. On Kelch-like ECH-associated protein 1 (KEAP1), a well-known negative regulator of NRF2 protein stability  mRNA was significantly upregulated only in this cell line upon FR054 treatment. Altogether, these findings suggest that FR054 alone increases in both cell lines SLC7A11 glutamate/cystine exchange and intracellular GSSG level. Conversely, when combined with ERA, MiaPaCa-2 cells might promote cell death by reducing glutamate/cystine exchange and GSH synthesis, while in BxPC3, its proapoptotic effect, despite being linked to a significant reduction of glutamate/cystine exchange, does not appear to be related to GSH level reduction.To better understand the relation between the observed level of SLC7A11 protein and its function as an exchanger of the anionic form of cystine and glutamate, we measured the amount of these two amino acids into cultured media in both cell lines. While ERA, as expected, inhibited glutamate release in both cell lines and at both time points analyzed, FR054 alone, as predicted by the transcriptional data, albeit not statistically significant, caused a time-dependent increase in the amount of released glutamate . SurprisN-acetyl-L-cysteine , a cysteine prodrug, able to replenish intracellular GSH levels, or 24 h of cotreatment with BSO (5 mM) to block de novo synthesis of GSH. NAC cotreatment, as measured by trypan blue vital assay, led to decreased cell death in both cell lines and in all the conditions analyzed as compared to control cells  at 48 h as a hallmark of cystine depletion as well as ferroptosis by using the fluorescent probe C11-BODIPY. As expected from previous results, treatment with ERA or FR054 alone increased fluorescence, especially in ERA-sensitive BxPC3 and slightly in FR054-treated MiaPaCa-2 cells. Conversely, combined treatment induced a significant increase in fluorescence in both cell lines  or in combination with ERA , suggesting a reduced activity of both pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) , M5 α-ketoglutarate , and M4 citrate  upon all treatments and especially in the combined treatment  7C. The ed cells .To further reinforce the notion that ERA and FR054 treatment could cooperate in inducing ferroptosis in PDAC cell models, we treated human pancreatic cancer PANC1 cells, characterized by K-Ras mutation and a great sensitivity to cysteine depletion, with ERA and FR054 alone or in combination along a time course of 72 h. We observed a significant effect of all treatments on proliferation , M5 α-ketoglutarate , and M4 citrate  upon all treatments and especially in combined treatment was observed  and M5 citrate  when cells were treated with ERA and FR054 alone or in combination, suggesting a reduced activity of both PDH and PC , which acts as a negative feedback regulator of the ATF4-dependent transcription during the UPR , suggesting a possible TRIB3-dependent mechanism of ATF4’s function. Furthermore, it has been recently shown that Glycogen Synthase Kinase 3 Beta (GS3K) can target ATF4 for degradation. Such mechanism is controlled by the state of activation of AKT Serine/Threonine Kinase (AKT). Indeed, attenuation of the AKT pathway, leading to GSK3 activity, causes ATF4 degradation  a significant stronger activation of UPR and of the anti-oxidative response but a lower level of ATF4 as compared to ERA. However, at a later time point (72 h), only the UPR remained significantly active as the anti-oxidative response as well as ATF4 significantly declined. On the other hand, the regulation of the same proteins in BxPC3 cells indicated their different behavior following the different treatments. Indeed, in ERA- and FR054-treated cells, the UPR, ATF4, and anti-oxidative responses were slowly activated and in a minor measure as compared to MiaPaCa-2 cells. Nonetheless, also in this cell line, the combined treatment induced a persistent UPR and a significant decrease of the anti-oxidative response as well as of ATF4, suggesting a minor cysteine depletion, as we observed, and slower intracellular oxidative stress. From these data, we can argue that the lower sensibility to ERA of MiaPaCa-2 cells as compared to BxPC3 cells, as previously described , must be the UPR , or its  the UPR , 58. In radation . Notablyradation , thereforadation , 61. Furradation  that ultradation , 64. In radation , 66. Intradation , 68. Theradation . Indeed,radation . Of interadation . In thisradation , 12, tharadation S7, an iIn cancer cells, inhibition of system SLC7A11-mediated cystine uptake by ERA may be sufficient to initiate ferroptosis by interfering with GSH and GSSG intracellular levels. Our results in the three PDAC cell lines indicated that ERA was able to induce a small but significant decrease in GSH level only in PANC1 and BxPC3 cells 8F and G2+), generated by HMOX1 activity could cause further lipid peroxidation in the combined treatment. Indeed, Western blot analysis indicated that both MiaPaCa-2 and BxPC3 cells were also induced at the protein level  and 6-Phosphogluconate Dehydrogenase (PGD) or Phosphoserine Aminotransferase 1 (PSAT1) and Phosphoglycerate Dehydrogenase (PHGDH), necessary for the recycling of the oxidized GSH through GSR activity in order to maintain the cellular redox homeostasis  can be found below: GEO database, accession number GSE223303.Conception and design: AW and FC. Development of methodology: BZ, MG, TL, AP, LB, AW, and FC. Performing research: BZ, MB, TL, VB, and AP. Analysis and interpretation of data: BZ, MG, TL, AP, LB, AW, and FC. Writing the draft of the manuscript: FC. Review of the manuscript: BZ, VB, AW, and FC. Revision of the manuscript: all authors. All authors contributed to the article and approved the submitted version.


Insecticide-treated nets (ITNs) using pyrethroids have been the main vector control tools deployed in malaria endemic countries and are responsible for the dramatic reduction in African malaria cases in the early 2000s. The World Health Organization (WHO) cone test was designed to assess the rapid toxicity effects of pyrethroid exposure on mosquito vectors but has yielded no insights beyond 60-min knockdown and 24-h mortality. As dual-active-ingredient (AI) ITNs become more widespread, bioassays that can provide realistic assessment of single- and dual-treated ITNs (i.e. nets with more than one active ingredient) are urgently needed.Anopheles gambiae sensu lato (s.l.) were exposed to four ITNs  and untreated nets in these modified cone tests. Life history data  for individual mosquitoes were recorded.We present an augmentation of the cone test that enables accurate quantification of vector behavioural responses  to ITNs using video recording and bespoke software that uses background segmentation methods to detect spatial changes in the movement of mosquitoes within the cone. Four strains of All mosquitoes responded to the presence of ITNs, spending from 1.48 to 3.67 times more time in the upper region of the cone, depending on the ITN type. Of all ITNs, PermaNet 2.0 provoked the smallest change in behavioural response. Activity in the cone influenced observed post-exposure longevity, and in resistant strains exposed to Interceptor G2, the higher the activity, the greater the risk of dying, as long as the proportion of activity at the net surface was less than 50%. All ITNs inhibited blood-feeding, and smaller blood meals were taken when mosquitoes fed.The additional mosquito behaviour data obtained by using this modification to the WHO cone test provides unique insight into the innate responses of different mosquito strains on untreated nets and the entomological mode of action of ITNs, important evidence when evaluating ITN characteristics.The online version contains supplementary material available at 10.1186/s13071-023-06029-z. Anopheles gambiae sensu lato (s.l.) and Anopheles funestus in at-risk communities across Africa , Olyset Net , and Interceptor G2  and N’gousso (NG) (Anopheles coluzzii) strains and pyrethroid-resistant Banfora (BF) and VK7 strains (both An. coluzzii). Both insecticide-resistant strains are maintained under 6-monthly selection pressure with deltamethrin and have been previously described , followed by OS [48.57% (17/35)] and IG2 [61.86% (73/118)]. At 24 h PE, approximately one third of BF mosquitoes  that were unfed at 1 h after IG2 exposure, and all mosquitoes that were unfed at 1 h after exposure to P2 and OS tests, blood-fed .Over 92% of mosquitoes successfully blood-fed after tests with UT nets, with most mosquitoes feeding at 1 h . After ITN tests, none of the mosquitoes exposed to P3, or the susceptible strains exposed to P2 and OS, survived long enough to feed. Of the few KS and NG mosquitoes that lived long enough to take a blood meal after IG2 tests, approximately half fed at 1 h PE (PE) . In the resistant strains, compared to UT nets, BF mosquitoes were at least 90% less likely to feed at 1 h PE when exposed to ITNs ; OS: 87.50% (42/48); IG2 62.07% (18/29)].χ2 = 5.47; df = 1; P = 0.0193; VK7 χ2 = 13.94; df = 3; P = 0.0030), where, compared to UT, at least 4.0 µg less blood was ingested . Activity in the LHC during tests was significantly associated with smaller blood meal weights in VK7 mosquitoes, and blood meals became 9.3 times smaller as activity increased  regardless of treatment.The mean total weight of blood meals taken after tests with UT netting was 12.47 µg SD = 7.64). BMW per strain on UT netting was as follows: KS 12.26 µg (SD = 7.89), NG 11.74 µg (SD = 6.13), BF 10.48 µg (SD = 6.42) and VK7 13.10 µg (SD = 8.93). After ITN exposure, blood meal weights decreased . The sizes of individual mosquitoes had a significant effect on blood meal weights in the NG, BF and VK7 strains  and as size increased, so did the size of the blood meal .In VK7 mosquitoes that could not feed at 1 h PE but which recovered to feed at 24 h PE, blood meal weights were 1.45 times smaller blood meals compared to those that fed at 1 h PE  and as the proportion of activity in the LHC increased, the likelihood of dying increased for UT and P2 . In VK7 mosquitoes, observed longevity also was influenced by the activity in the cone during tests . The amount of blood ingested had a slight impact on longevity: BF and VK7 mosquitoes were 7% and 2% less likely to die as the BMW increased, respectively . The effect of treatment on longevity varied with wing length for all strains, for example, smaller mosquitoes lived longer PE to UT netting  and were more likely to die if they were exposed to IG2 for all the strains except BF  of individual mosquitoes may only be inferred . The stricter measure of resting used in this report (consecutive frames where none of the five mosquitoes moved) demonstrated large differences in resting between susceptible and resistant strains  and the steeply inward tapering of the cone impede the absolute determination of vertical mosquito flight, and thus changes in cone region occupancy are best evaluated as relative occupancy proportions. A more bespoke equipment setup such as that used in the baited box study  would reBy using a simple augmentation of the WHO cone bioassay combined with automated video analysis we were able to demonstrate a wide range of complex behavioural responses of mosquitoes in response to insecticide-treated nets during the bioassay. Such behavioural responses  have already been proven invaluable in the translation of this information to bednet designs , 50, witAdditional file 1: Figure S1. Flowchart of image analysis pipeline during a ViCTA experiment (top) and output files exported at the end of an experiment (bottom).Additional file 2: Figure S2. Treatment effect on longevity as the wing length measurement increases by An. gambiae strain based on the fitted mixed-effects Cox regression model. a Effect by treatment, b contrast (insecticide-treated net [ITN] vs untreated netting) effect, KS Kisumu, NG N’Gousso, BF Banfora, UT untreated net, IG2 Interceptor® G2 net, OS Olyset net, P2 PermaNet 2.0 net, and the solid black horizontal line is the reference  with values above 1 showing more likelihood of dying when exposed to an ITN compared to UT and vice versa. Figure S3. Treatment effect on longevity as the activity at the lower half of the cone increases based on the fitted mixed-effects Cox regression model (VK7 strain only). a Effect by treatment, b contrast (insecticide-treated net [ITN] vs untreated netting) effect, UT untreated net, IG2 Interceptor® G2 net, OS Olyset net, P2 PermaNet 2.0 net, and the solid black horizontal line is the reference  with values above one showing more likelihood of dying when exposed to an ITN compared to UT and vice versa. Table S1. Total movement activity, insecticide treated net versus untreated netting comparisons within An. gambiae strain from a linear regression model. Multiple pairwise comparisons 95% confidence intervals and P-values corrected using the Dunnett adjustment. UT untreated net, IG2 Interceptor® G2 net, OS Olyset net, P2 PermaNet 2.0 net, P3 PermaNet 3.0 net, KS Kisumu, NG N’Gousso and BF Banfora. Table S2. Treatment comparisons for blood-feeding success post-exposure based on generalised linear mixed models (GLMMs) with a binomial distribution fitted for each An. gambiae strain separately within the time the blood meal was offered (1 or 24 h). Only the predictor variables in the final model for each strain are provided. KS Kisumu, NG N’Gousso, BF Banfora, UT untreated net, IG2 Interceptor® G2 net, P2 PermaNet 2.0 net, OS Olyset net, Ref reference group, OR odds ratio, CI confidence interval, *significant at 5% significance level. Random effects include testing day and replicate. Table S3. Blood meal size results based on linear mixed effects models fitted for each An. gambiae strain separately. Only the predictor variables in the final model for each strain are provided. KS Kisumu, NG N’Gousso, BF Banfora, UT untreated net, IG2 Interceptor® G2 net, P2 PermaNet 2.0 net, OS Olyset net, Ref reference group, CI confidence interval, SD standard deviation, cone activity = predicted proportion of activity at the lower half of the cone during exposure (behaviour data), DE during exposure, *significant at 5% significance level. Random effects include testing day and replicate. Table S4. Mortality at 24-h results based on generalised linear mixed models (GLMMs) with a binomial distribution fitted for each An. gambiae strain separately within the time the blood meal was offered (1 or 24 h). Only the predictor variables in the final model for each strain are provided. KS Kisumu, NG N’Gousso, BF Banfora, UT untreated net, IG2 Interceptor® G2 net, P2 PermaNet 2.0 net, OS Olyset net, Ref reference group, OR odds ratio, CI confidence interval, *significant at 5% significance level. Random effects include testing day and replicate. Table S5. Longevity results based on the final model mixed-effects Cox regression models fitted for each An. gambiae strain separately. Only the predictor variables in the final model for each strain are provided. KS Kisumu, NG N’Gousso, BF Banfora, UT untreated net, IG2 Interceptor® G2 net, P2 PermaNet 2.0 net, OS Olyset net, Ref reference group, CI confidence interval, SD standard deviation, min minimum, max maximum, HR hazard ratio, cone activity = predicted proportion of activity at the lower half of the cone during exposure (behaviour data), DE during exposure, *significant at 5% significance level and +overall descriptive results. Random effects include testing day and replicate.


Thermal injury often leads to prolonged and excessive inflammation, which hinders the recovery of patients. There is a notable absence of suitable animal-free models for investigating the inflammatory processes following burn injuries, thereby impeding the development of more effective therapies to improve burn wound healing in patients.In this study, we established a human full skin equivalent (FSE) burn wound model and incorporated human peripheral blood-derived monocytes and T cells.+ and elevated production of IL-8 (CXCL8), in comparison to uninjured FSEs. Among the T cells that actively migrated into the FSEs, the majority were CD4+ and CD25+. These T cells demonstrated augmented expression of markers associated with regulatory T cell, Th1, or Th17 activity, which coincided with significant heightened cytokine production, including IFN-γ, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IP-10 (CXCL10), and TGF-β1. Burn injury did not impact the studied effector T cell subsets or cytokine levels.Upon infiltration into the FSEs, the monocytes differentiated into macrophages within a span of 7 days. Burn-injured FSEs exhibited macrophages with increased expression of HLA-DRCollectively, this study represents a significant advancement in the development of an immunocompetent human skin model, specifically tailored for investigating burn-induced innate or adaptive immune reactions at the site of burn injury. GainiImmediately after burn injury, pro-inflammatory neutrophils and macrophages accumulate at the wound site –7. TheseIn vitro human skin models are promising alternative experimental tools for studying various aspects of skin injury based on the behavior of keratinocytes and fibroblasts . With a 19% burn, the effect of the injury is clearly present, while there is enough material and cells that is not destroyed so that cells can enter. We hypothesized that burn injury alters the inflammatory state of immune cells in these FSEs, which can be detected by changes in cell phenotype and cytokine expression. Cells from the innate and the adaptive immune system, namely monocytes and T cells, were isolated from human buffy coats and cultured in the FSE. We examined alterations in marker expression on immune cells and the secretion of cytokines in the culture medium.In this study, we aimed to develop a human full skin equivalent (FSE) based on the collagen-elastin matrix MatriDerm® , 29, as ® . MatriDe® –34. Our 22.1https://www.coreon.org/) and approved by the institutional privacy officers. Subjects were actively informed of this procedure and were able to easily withdraw at any point. Split-thickness samples of 0.3 mm were harvested using a dermatome .Skin samples were obtained from adult patients who underwent abdominoplasty at the Red Cross Hospital in Beverwijk, Medical Clinic in Velsen or Spaarne Gasthuis in Haarlem. Samples from 17 different donors were used . Consent for the use of these anonymized, post-operative residual tissue samples was received through an informed opt-out protocol, in accordance with the national guidelines  and cultured at 37°C with 5% CO2. For keratinocyte isolation, the epidermis was transferred into 0.05% trypsin (Gibco) and incubated for 20 min at 37°C. The cell suspension was poured through a 70 µm cell strainer and centrifuged for 10 min at 110 × g. Next, the cell pellet was washed in culture medium and centrifuged for 10 min at 160 × g. The cell pellet was then resuspended in CnT-07 medium  and keratinocytes were transferred onto a 1 µg/cm2 collagen type IV -coated culturing flasks (Starstedt) at 37°C with 5% CO2.See 2.3®  with a thickness of 3 mm was cut into circular pieces of 1.13 cm2. At day one, 2 × 105 fibroblasts were seeded onto the matrix and the matrix was submerged in culture medium containing 65 µg/mL ascorbic acid for 4 days at 37°C with 5% CO2  and 0.5 ng/ml EGF  for 4 days at 37°C with 5% CO2. Next, the FSE was transferred to a transwell (Starstedt) and cultured air-exposed in deep well plates  with FSE II medium containing 4 ng/ml KGF and 1 ng/ml EGF. From day 11 onward the FSE was cultured in FSE III medium containing 4 ng/ml KGF and 1 ng/ml EGF and from day 15 onward in FSE III medium that was refreshed twice weekly. At day 22, the FSE was ready to use for immune cell culture. Cell numbers and culture conditions are based on preceding experiments (Our FSE development protocol was based on previous experiments . MatriDeeriments .2.4Our burn injury procedure was based on previous experiments . A coppe2.5g for 15 min (without brakes), the PBMCs were collected in FSE I medium. Cells were resuspended in 50% fetal bovine serum (Gibco) + 40% FSE I medium + 10% dimethyl sulfoxide. After 24 h storage in Mr. Frosty (ThermoFisher scientific) with isopropanol at -80°C, cells were stored in liquid nitrogen until use.PBMCs were isolated from buffy coats obtained from healthy donors  by density gradient centrifugation using Lymphoprep . The buffy coat was diluted in 0.5% Bovine serum albumin in PBS and layered over the density gradient medium. After centrifugation at 1000 × 2.65 cells were added to the dermal side of the FSE. For burn-injured FSEs, the cells were added directly after burn injury was inflicted. Inverted FSE with monocytes was incubated at 37°C for 2 h and subsequently placed back into the transwell  at a bead/cell ratio of 2.5:1 at 2-8°C for 20 min on a tube roller. Monocytes were isolated from the PBMCs using a magnet . Monocytes were resuspended in FSE I medium and 2.5 × 102.75 cells were placed between the transwell membrane and the dermal side of the FSE  at a bead/cell ratio of 5:1 at 37°C for 4 h. After the activation, cells were resuspended in FSE I medium and 2.5 × 10 the FSE . Of thes2.8The FSE dissociation procedure was based on a protocol from He et al. . Macroph2.9Single cell suspensions were stained using the macrophage or T cell panel  for 2 h at 4°C. The FSEs were then put in 20% sucrose (Sigma) in PBS solution overnight at 4°C. FSEs were embedded in Tissue Tek OCT  and sections of 10 µm were cut using a cryotome . Dried sections were washed in PBS and incubated with LDH solution (2 mM Gly-Gly (Sigma); 0.75% NaCl (Sigma); 5% polypep (Sigma); 1.75 mg/ml β-nicotinamide adenine dinucleotide (Sigma); 3 mg/ml nitroblue tetrazolium (Sigma) in demineralized water of pH 8) for 3 h at 37°C. Sections were washed in tap water at 50°C and in PBS and then stained with Eosin Y for 4 min. Sections were then put in PBS for 1 sec, acetone for 30 sec, acetone/xylene (1:1) for 1 min and xylene for 1 min, before embedding with Eukit Mounting Medium.2.12Microscopic visualization was performed with a Zeiss Axioskop40FL microscope . Images were acquired using a Nikon Eclipse TS2 camera and the NIS-Elements software version 4.4 .2.13Length of re-epithelization in the FSEs was measured in microscopic images of H&E-stained sections by two assessors using NIS-Elements software. The mean of the two wound sides was used for analysis.2.14Cytokines, chemokines and growth factors were analyzed in samples of medium. Neat samples were measured using the Human Essential Immune Response LegendPlex Multi-analyte Flow Assay kit , according to the manufacturer’s instructions and were acquired on the flow cytometer. This 13-plex immunoassay included: IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-12p70, IL-17A, IP-10 (CXCL10), MCP-1 (CCL2), TNF-α and TGF-β1. Concentrations were determined using FlowLogic software. When cytokine levels were lower than the standard range, the lowest level of quantification was used. When cytokine levels were higher than the standard range, the levels were estimated based on the fluorescent signal in the assay.2.15We used the Shapiro-Wilk test in R  to determine distribution of data and found that the majority of data were not normally distributed. Therefore, differences in cell number/percentages and cytokines levels between different modeling conditions were explored using Mann-Whitney U test in R . Data was visualized using R  and significant  differences were indicated by asterisks.33.1FSEs were generated by seeding human keratinocytes and fibroblasts into a collagen-elastin containing matrix from MatriDerm see . After 33.25) were administered directly to the dermal side of the FSEs (see Unstimulated monocytes were introduced to full-established (burn-injured) FSEs to simulate an innate immune response. To prevent the cells from adhering to the transwell membrane, monocytes , CD14 (monocyte marker), CD11b (activation marker), HLA-DR (M1 differentiation marker) and CD163 (M2 differentiation marker). In uninjured FSEs, an average of 8.0 ×103.3At day 7 (when FSEs were terminated), the levels of 13 inflammatory cytokines in the culture media were analyzed Figure 43.45 T cells were placed between the transwell membrane and the dermal side of the FSEs, and they were cultured for a duration of 3 days  FSEs. Approximately 2.5 × 10days see . Pre-act3) of T cells had migrated into the FSEs , CD4 (effector T cell marker), CD25/CD127 (activation marker and regulatory T cell marker), CXCR3 (Th1 differentiation marker) and CCR4/CCR6 (Th17 differentiation marker). Only a small portion , MIP-1β (CCL4), and RANTES (CCL5) . Exploriin vivo situation. Furthermore, unlike microscopy-based studies, our research employed flow cytometry for quantification and analysis of macrophages and T cells. Although our flow cytometry panel did not include markers specific to keratinocytes and fibroblasts, investigating their expression of markers such as elafin, CK10, CK17, CD10, Ki67, FAP, or α-SMA, could provide more detailed insights into the effect of monocytes or T cells on burn wound healing processes. Although the effect of burn injury on the studied monocyte and T cell markers appeared limited in our current set-up, further investigations involving other time points, longer culture periods, distinct immune cell activation methods or different burn techniques (i.e. burn temperature or duration) are warranted. In addition, our model can be used to study the effect of burn injury on specific immune cell subsets, or a combination thereof. This can be achieved by differentiating monocytes into M1 or M2 macrophages or skewing T cells towards Tregs, Th1, Th2, or Th17 cells before introducing them into the FSEs. Including neutrophils in the model, despite the challenges involved, would also contribute to a better understanding of the burn-induced immune response and their role in wound healing  and institutional guidelines of the local hospitals in Beverwijk. Buffy coats from healthy donors were purchased at the Dutch Blood bank , in accordance with the national and institutional guidelines. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements because in this study, tissue samples were obtained from planned elective plastic surgeries  that were part of routine patient treatment. Therefore, these tissues are residual materials and no evaluation by the medical ethical committee was required for the collection of these samples. As described in the Materials and Methods section, we used an opt-out protocol to receive tissue samples. Subjects were informed of this procedure and were able to withdraw at any point. This procedure is in accordance with the national guidelines (https://www.coreon.org/) and institutional guidelines of the local hospitals in Beverwijk. Buffy coats from healthy donors were purchased at the Dutch Blood bank , in accordance with the national and institutional guidelines.Ethical approval was not required for the studies involving humans because in this study, tissue samples were obtained from planned elective plastic surgeries  that were part of routine patient treatment. Therefore, these tissues are residual materials and no evaluation by the medical ethical committee was required for the collection of these samples. As described in the Materials and Methods section, we used an opt-out protocol to receive tissue samples. Subjects were informed of this procedure and were able to withdraw at any point. This procedure is in accordance with the national guidelines (PM: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Software, Supervision, Visualization, Writing – original draft. MV: Conceptualization, Formal Analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – review & editing. AE: Formal Analysis, Investigation, Methodology, Validation, Writing – review & editing. LR: Formal Analysis, Investigation, Software, Visualization, Writing – review & editing. LM: Formal Analysis, Investigation, Visualization, Writing – review & editing. EM: Conceptualization, Formal Analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review & editing. IJ: Conceptualization, Formal Analysis, Funding acquisition, Methodology, Supervision, Writing – review & editing. HK: Conceptualization, Formal Analysis, Funding acquisition, Methodology, Supervision, Writing – review & editing. BB: Conceptualization, Formal Analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review & editing.


Multivariate regression analysis indicated L5 tilt angle as a preoperative risk factor for CIB (p = 0.007) and indicated L5 tilt angle and age as a preoperative independent risk factors for iatrogenic CIB (p = 0.01 and p = 0.008). Patients with a preoperative trunk shift towards the convexity of the main curve (type C) are more prone to postoperative CIB and leveling the L4 and L5 vertebrae is the key to achieve coronal alignment preventing the “takeoff phenomenon”.The aim of the present study is to elucidate preoperative risk factors for inadequate correction of coronal imbalance and/or creation of new postoperative coronal imbalance (iatrogenic CIB) in patients who undergo surgery for Adult Spinal Deformity (ASD). A retrospective review of adults who underwent posterior spinal fusion (>5 levels) for ASD was performed. Patients were divided into groups according to the Nanjing classification: type A (CSVL < 3 cm), type B (CSVL > 3 cm and C7 plumb line shifted to major curve concavity), and type C (CSVL > 3 cm and C7 plumb line shifted to major curve convexity). They were also divided according to postoperative coronal balance in balanced (CB) vs. imbalanced (CIB) and according to iatrogenic coronal imbalance (iCIB). Preoperative, postoperative, and last follow-up radiographical parameters and intraoperative data were recorded. A multivariate analysis was performed to identify independent risk factors for CIB. A total of 127 patients were included . They all underwent long (average levels fused 13.3 ± 2.7) all-posterior fusion. Type C patients were more at risk of developing postoperative CIB ( Maintaining or restoring global alignment is a fundamental goal of Adult Spinal Deformity (ASD) surgery ,2,3. Ide®, Version 2.0.6, New York, NY, USA) by two experienced surgeons. Postoperative complications  and revisions were recorded up to the last follow-up. Patients were stratified in two groups  based on absolute postoperative CVA. Then, the iatrogenic CIB (iCIB) subgroup of patients was identified: iatrogenic CIB was defined as a postoperative CVA > 30 mm in patients with a preoperative normal alignment (CVA < 30 mm). Iatrogenic CIB patients were compared with those whose CVA was normal both preoperatively and postoperatively. Moreover, according to the Nanjing classification [After Institutional Review Board approval, a retrospective cohort study was conducted including consecutive patients who underwent posterior fusion for Adult Spinal Deformity (ASD) in our institution between 1 January 2012 and 1 March 2020. Inclusion criteria were age at surgery > 18 years; a diagnosis of Adult Spinal Deformity (ASD) Aebi types 1 or 2, with main curve Cobb angle > 30°; posterior fusion of at least 5 levels with all-screws constructs extended distally to L5 or below; available preoperative, postoperative and last follow-up X-rays; and minimum 2-year follow-up. Patients with congenital or neuromuscular scoliosis, prior spinal operations, no coronal deformity (main curve Cobb angle < 10°), ankylosing spondylitis, Parkinson disease or other neurological disorders, and spinal neoplasms were excluded. Basic demographic and surgical data were recorded. The following radiographical parameters were assessed preoperatively, postoperatively (just before discharge), and at the last follow-up on long-cassette standing X-rays of the entire spine: Cobb angle of the main curve and of the lumbosacral fractional curve (LSF), main curve apex, coronal vertical axis , sagittal vertical axis (SVA), lumbar lordosis (LL), thoracic Kyphosis (TK), and spinopelvic parameters . Coronal imbalance (CIB) was defined as a CVA > 30 mm. All the measures were taken with Surgimap Software .Descriptive statistics were used to summarize patients’ demographic characteristics and radiographic data. Categorical data were presented as frequencies and percentages, while continuous data were presented as mean ± standard deviation (SD). The present study was divided into steps. First, all pre- and postoperative radiological parameters were analyzed with a paired t-test to compare the differences. Then, comparisons between the groups  were carried out by independent t-tests or Fisher exact test. Chi-squared tests were performed to analyze the relationship between pre- and postoperative coronal imbalance. Bonferroni post-hoc correction of significance levels was used when there were multiple comparisons. To calculate the measure of effect for continuous variables among three groups , Analysis of Variance (ANOVA) was used, with ηOne hundred twenty-seven patients  were included. Baseline demographics and surgical data are reported in Surgery consisted of a long (average levels fused 13.3 ± 2.7) all-posterior thoracolumbar fusion with high density pedicle screw constructs that terminated above T9 in 113/127 patients (88.9%) and in S1 or pelvis in 69/127 (54.3%) patients. All patients were operated on with the same technique; the fusion area was preoperatively defined according to the Lenke criteria; in the presence of disc degeneration at the level distal to the planned Lower Instrumented Vertebra (LIV), the fusion area was extended distally; when the LSF curve showed low flexibility (<20° Cobb at supine radiographs), the fusion area was extended to the pelvis. The mean number of posterior column osteotomies per patient was 6.02 ± 1.9; three column osteotomies were performed on five patients (3.9%). Posterior lumbar interbody fusion was performed on 84 patients (66.2%). Coronal, sagittal, and spinopelvic parameters improved after surgery , and thePreoperatively, most patients  had Type A coronal alignment, while 30 (23.6%) were shifted to the concavity of the main curve (type B) and 12 (9.4%) were shifted to the convexity (type C), p = 0.04, η2 0.3). Patients with type B coronal imbalance had a significantly more severe main curve  and higher SVA  and CVA , while type A patients had higher LL , L4-S1 LL , and SS  and lower PT . As for L4-S1 Lumbosacral Fractional (LSF) curve and L4 and L5 tilt, there was no preoperative difference between the groups; however, a strong difference was recorded in postoperative L4 tilt , with type C patients showing the highest value (15.7 ± 5.2).Type C patients were more at risk of having a higher postoperative CVA  had good postoperative coronal balance (CB), while 43 (33.9%) had coronal malalignment (CIB) , Table 4p > 0.05). The two groups were significantly different in preoperative L5 tilt . Multivariate regression analysis indicated L5 tilt angle as a preoperative risk factor for CIB (p = 0.007). Both groups showed significant improvements in all coronal and sagittal parameters after surgery, without significant differences. However, postoperative L4 and L5 tilts were significantly lower in the balanced group . There was no difference in complications and revision rate between the two groups (p = 0.9 and p = 0.4).Preoperatively, the average CVA and main curve Cobb angle were 25.2 ± 22.6 mm and 47.3 ± 17.4° in the CB group, and 30.7 ± 30.5 mm and 49.4 ± 15.9° in the CIB group , 28 (33%) had iatrogenic CIB .p = 0.04) and a greater postoperative L4 tilt . Multivariate regression analysis indicated L5 tilt angle and age as a preoperative independent risk factor for iatrogenic CIB . Both groups showed improvements in all coronal and sagittal parameters after surgery, without significant differences. There was no difference in complications and revision rate between the two groups (p = 0.9 and p = 0.8).The two groups were not different in preoperative CVA, SVA, major curve, and lumbosacral fractional curve Cobb; however, patients with iCIB showed a greater preoperative L5 tilt  and the highest L4 tilt angle , while the major curve Cobb angle was similar between the groups: this might indicate that, in type C patients, correction of the major curve has been excessive and not tailored to match correction of the LSF curve, failing to restore a good balance [p = 0.007) and iatrogenic imbalance . These results are in line with the findings of other authors [p = 0.004). This finding, which is in line with Zuckerman et al. [The aim of the present study was to elucidate preoperative risk factors for inadequate correction of coronal imbalance and/or creation of new postoperative coronal imbalance (iatrogenic CIB) in patients who undergo surgery for Adult Spinal Deformity (ASD). Our main findings are twofold: first, patients with a preoperative trunk shift towards the convexity of the main curve (type C) are at higher risk of postoperative CIB; second, preoperative L5 tilt is an independent risk factor for both coronal imbalance and iatrogenic coronal imbalance. In the present study, preoperatively balanced (type A) and imbalanced (type B and C) patients did not differ significantly on the coronal plane . However, postoperatively, type C patients showed the highest postoperative CVA  are more prone to postoperative CIB and that leveling the L4 and L5 vertebrae is the key to achieve coronal alignment, preventing the “takeoff phenomenon” : this is