On reoxidation, U was associated with an iron (oxyhydr)oxidephase either as an adsorbed uranyl (approximately 65%) or an incorporatedU (35%) species. These findings support the overarching concept ofiron (oxyhydr)oxides acting as a barrier to U migration in the environment,even under fluctuating redox conditions.Over 60 years of nuclear activity have resulted in aglobal legacyof contaminated land and radioactive waste. Uranium (U) is a significantcomponent of this legacy and is present in radioactive wastes andat many contaminated sites. U-incorporated iron (oxyhydr)oxides mayprovide a long-term barrier to U migration in the environment. However,reductive dissolution of iron (oxyhydr)oxides can occur on reactionwith aqueous sulfide (sulfidation), a common environmental species,due to the microbial reduction of sulfate. In this work, U(VI)–goethitewas initially reacted with aqueous sulfide, followed by a reoxidationreaction, to further understand the long-term fate of U species underfluctuating environmental conditions. Over the first day of sulfidation,a transient release of aqueous U was observed, likely due to intermediateuranyl(VI)–persulfide species. Despite this, overall U wasretained in the solid phase, with the formation of nanocrystallineU(IV)O 2.Reaction of U(VI)−goethite withaqueous sulfide resultsin transient remobilization of trace levels of aqueous uranium withlong-term solid phase retention of U(IV)O Sulfidation of iron (oxyhydr)oxides is a complex multi-stepprocess,28 which typically results in reductivedissolution at the mineral surface, forming elemental sulfur (S80) and releasing Fe(II) into solution.45 The resultant aqueous Fe(II) may then react with aqueous HS– to form secondary iron sulfide phases, such as mackinawite(FeS).45 Aqueous HS– may also reactwith S80 to form surface-associated polysulfide(Sn2–) species.46 Consequently, these complex processes may influencethe behavior and fate of radionuclides, including U.50The formation of U-incorporated iron (oxyhydr)oxide phasesis thoughtto occur via substitution of U(VI/V) for an Fe(III) within the mineralstructure, potentially immobilizing U in the long-term.24 Transient aqueous U release was also observed during abiotic sulfidationreactions, including the reaction of an abiotic sulfide solution withU(V)-incorporated magnetite,51 U(VI)-adsorbedhematite, and U(VI)-adsorbed lepidocrocite.53 These abiotic studies suggested that U release may be a result ofpoor U(VI) affinity for a sulfidized surface.53 Recently, furtherinsight was provided by the sulfidation of U(VI)-adsorbed ferrihydrite,where aqueous U(VI) speciation was attributed to the formation ofan intermediate uranyl(VI)–persulfide species, which had onlya weak adsorption affinity for FeS.54 However,in all abiotic sulfidation systems, overall reduction to U(IV) wasobserved, with U being retained mainly as either nanoparticulate uraninite(U(IV)O2)54 or non-crystalline U(IV).52A field study at Rifle (USA) observed a release of aqueousU followingthe onset of microbial sulfate reduction.2 to oxygen, oxidation to U(VI)typically occurs.42 However, nanocrystalline mackinawite(FeS) has previously been shown to protect U(IV)O2 fromreoxidation;55 synthetic nanocrystallineFeS acted as an oxygen scavenger, transforming to nanogoethite andlepidocrocite, with no U(IV)O2 dissolution observed priorto complete FeS depletion.55 U(IV)O2 oxidative dissolution, under carbonate rich conditions , then led to the remobilization of aqueous U(VI)–carbonatocomplexes and subsequent U(VI) adsorption (25%) onto goethite/lepidocrocite.Consequently, following sulfidation, the long-term fate of U(IV)O2 will be dependent on the oxygen scavenging capability ofthe formed FeS phase and the ambient physicochemical conditions, withcarbonate concentration being a significant control.55 Simultaneous reoxidation of FeS and U(IV)O2 mayresult in partial U incorporation into the forming iron (oxyhydr)oxidephase. Alternatively, FeS may scavenge oxygen, as already observedwith nanocrystalline mackinawite,55 therebydelaying U(IV)O2 reoxidation and potentially preventingU incorporation into newly formed Fe(III)-bearing (oxyhydr)oxides.This may lead to delayed U(IV) oxidation, presumptively to U(VI),which would then likely be retained as a more labile adsorbed phasewith potentially higher mobility in the environment.Onexposure of U(IV)O51 ferrihydrite,54 hematite, and lepidocrocite53 for iron (oxyhydr)oxides to be considered as long-term sequestersof U in sub-surface environmental systems, further understanding ofU behavior during sulfidation and associated redox cycling  is needed. In particular, the mechanism for the transientU release during iron (oxyhydr)oxide sulfidation is still unclear,as is the long-term fate of the reduced U(IV)O2 phase formedduring sulfidation. Here, highly controlled sulfidation and air reoxidationexperiments were performed on U(VI)-incorporated goethite using achemostat system.54 In contaminated land and wastedisposal scenarios, dissolved sulfide may originate from microbialreduction of sulfate or from groundwater sources.26 The rate of sulfide ingress in sub-surface systems is consequentlycontrolled by groundwater flow or in situ biotic formation. Therefore,to make this system more environmentally relevant, sulfide was slowlyadded to the experiment over 4 h at a constant rate,54 with an aqueous sulfide concentration similar to that measured inanoxic sediments.56 Throughout, reactionswere monitored at selected timepoints using geochemical analyses ,X-ray absorption spectroscopy (XAS), and transmission electron microscopy(TEM).Given theobserved transient release of U during the sulfidationof U-associated magnetite,23 The resultant slurry was thenwashed several times with DIW and washed with 4 mM HCl to remove adsorbedU(VI).59 After several more washes with DIW, thesolid was left to dry overnight (40 °C), and the mineral phasewas confirmed by powder X-ray diffraction (XRD).U(VI)-incorporated goethite (approximately0.2 wt % U) was formed via a hydrothermal synthesis method, as previouslydescribed.54 Samples were periodicallycollected under anoxic conditions, with all sample manipulations conductedwithin an anaerobic Coy cabinet under a mixed nitrogen/hydrogen (95%:5%)atmosphere. A U(VI)–goethite slurry was prepared  and transferred to the Applikon Bioreactor under a flow of N2 and then left to equilibrate overnight. A sodium sulfidesolution (0.4 M) was prepared in the Coy cabinet from sodium sulfidenonahydrate (Na2S·9H2O), with the concentrationconfirmed through the methylene blue assay using the radiello RAD171standard.57 The resulting sodium sulfidesolution was then added to the vessel at a constant rate (0.1 mL/min)over 4 h to reach a final total S(−II)/Fe(III) molar ratioof 2:1. The experiment was kept anoxic under a constant flow of N2, and the pH was maintained at pH 7 via the automated additionof 1 M HCl. The reaction was controlled in the chemostat vessel for72 h and then transferred to a Schott bottle in the Coy cabinet forlong-term anaerobic storage. During sulfidation, the experiment wassampled periodically, and the slurry was filtered to <1.5 nm using3 kDa Nanosep Centrifugation Ultrafilters (PES). The filtrate wasthen preserved for analysis by either acidification for cation analysis(U and Fe) or by reaction with a zinc acetate solution (82 mM) forsulfide analysis. Aqueous Fe and U were monitored by inductively coupledplasma mass spectrometry , with aqueoussulfide analyzed using the methylene blue assay and the radiello RAD171standard.57Experiments were performed underanoxic conditions in an Applikon Bioreactor (nitrogen atmosphere),which monitored and/or controlled the pH, Eh, temperature, and reagentadditions as required.Section S3).For solid-phase analysis,samples were studied by TEM, conventional XAS, and XAS in a high energyresolution detection (HERFD) mode. For TEM, sample slurry was droppedonto TEM grids (holey C film on Au 300 mesh) and dried inside theanaerobic cabinet prior to analysis on either FEI Tecnai TF20 or FEITitan3 Themis 300 (LEMAS). For XAS analysis, solid samples were obtainedby filtration  and then storedand transported at −80 °C under anoxic conditions to theDiamond Light Source (UK) for analysis on either the I20-scanningor B18 beamline; for HERFD-XANES, select solid filtrate samples weretransported frozen and under anoxic conditions to the European SynchrotronRadiation Facility (ESRF) in Grenoble ,pH, Eh, temperature, and reagent additions. After sampling, samplemanipulations were conducted within an anaerobic Coy cabinet. Briefly,after 5 months of aging under anoxic conditions, the sulfidized U(VI)–goethiteslurry was diluted in de-oxygenated water , transferredto the Applikon Bioreactor, and left to equilibrate overnight undera flow of nitrogen. The pH was maintained at pH 7 via addition of0.05 M HCl or 0.05 M NaOH, and the reaction was initiated by the introductionof laboratory air, with the chemostat set to maintain a DO level of5% within the solution. Samples were collected periodically over 4days, with aqueous (U and Fe) and solid phase samples collected as above for analysis.Figure S7) to confirm that goethitewas the only crystalline phase present. Previous analysis has confirmedthat U(VI) was incorporated within the goethite structure by substitutioninto an Fe(III) site (∼0.2 wt % U), forming a distorted octahedralcoordination.23The U(VI)-incorporated goethitewas initially characterized byXRD . However, after aqueous sulfidehad been removed (24 h), aqueous Fe steadily increased (150 μMby 72 h). These trends are characteristic of the reported sulfidationmechanism, with the steady decrease in aqueous sulfide from 4 h likelydue to sulfide oxidation on reaction with, and concomitant reductivedissolution of, the U(VI)–goethite.60 However, given the excess aqueous sulfidein this system (HS–/Fe(III) molar ratio of 2:1),it is likely that immediate precipitation of FeS occurred followingthe release of Fe(II) into solution. This would explain the low levelsof aqueous Fe initially detected, followed by an increase in aqueousFe after all aqueous sulfide was removed from the system. As withprevious studies of U associated with iron (oxyhydr)oxides,54 the reductive dissolution ofU(VI)–goethite also displayed a transient release of U duringsulfidation  at 6 h, followed by a gradual depletion over a further26 h. As aqueous samples were collected using 3 kDa ultrafilters , released U is assumed to be an aqueousU(VI) speciation .62 Interestingly, past work ascribed the transient U release duringU(VI)–ferrihydrite sulfidation to the formation of a uranyl(VI)–persulfidespecies.54Sulfidation ofthe U(VI)–goethite slurry  was initiatedby a controlled 4 h addition of aqueous sulfide (22.5 mM) to reacha final total S(−II)/Fe(III) molar ratio of 2:1. Over the 4h of addition, the concentration of aqueous sulfide increased steadilyto a peak of 12.1 mM at 4 h 1, followFigure S8).63 In addition, XRD patterns collected at 4.5 h and 3 days revealed a significant depletionin goethite over time, along with an ingress of poorly ordered FeS.Elemental sulfur (S80), an important intermediateduring sulfide oxidation in sub-surface systems,64 was also identified after 3 days . This confirms that rapid reductive dissolution of U(VI)–goethite,followed by secondary FeS formation, had occurred by 1 day. Furthermore,a selected area electron diffraction pattern collected after 7 monthsof aging  was also consistentwith nanocrystalline FeS.63 However, thepresence of persistent rod-shaped iron (oxyhydr)oxide phases can alsobe seen in the samples at 7 months , which are likely refractory U–goethite crystals. This isconsistent with previous similar work that observed residual goethiteparticles after 6 months despite an initial excess of sulfide.65 Overall, TEM images confirm that although rapidsulfidation and reductive dissolution of U(VI)-incorporated goethiteoccurred, a residual U–goethite component was still presentafter 7 months.The mineral transformations that occurred duringthe sulfidationof U(VI)–goethite were monitored by TEM, with images collectedat selected timepoints. After 1 day of sulfidation, sheet-like particleswere observed that matched well with nanocrystalline mackinawite (FeS)morphology .66 As expected,there was a continuous decrease in U(VI) and increase in U(IV) overtime, from predominantly U(VI) after 1 h to predominantly U(IV) by9 months (Supporting Information). Thisseems to correlate with results from a recent study investigatingthe reduction of U(VI) by magnetite.67 Onreaction with magnetite, U(VI) was initially reduced to a mixed U(IV)/U(V)oxide phase, with the formation of U(IV)O2 nanoparticlesdominant by 4 weeks.67 In addition, thereduction of surface-associated U(VI) by FeS has previously been suggestedto result in mixed valence U oxide phases, such as U3O8 or U4O9, as well as U(IV)O2.68 Therefore,we suggest that although U(IV)O2 formation may be dominantin the long-term (due to reducing agents such as Fe(II) or HS–), during the early stages of U(VI)–goethitesulfidation, surface-associated U(VI) on FeS may be reduced to mixedvalence U bearing oxide particles .To monitor U speciation during U(VI)–goethitesulfidation,U MIII-edge EXAFSto determine changes in the local coordination environment (Supporting Information), with the fit matching closely to previous EXAFS studies on U(VI)incorporated into goethite (23 Given that there wasno aqueous U detected at 1 h (1 (1.82(1)–1.88(1) Å),23 possibly reflecting the formation of U(V), asindicated by the ITFA analysis of the U MIV-edge HERFD-XANESdata (–) may have partially reduced incorporated U(VI) toU(V) in goethite, as previously observed during the reaction of Fe(II)with U(VI)–goethite.23 By 4 h, thebest fit had 3 U–O distances and a S backscatterer (2.67 Å), with U–Fe shells (0.5Fe backscatterers) at 3.21 and 3.44 Å. The diminished occupancyof the U–Fe shells indicates a decrease in a long range ordersurrounding the U, likely from significant reductive dissolution ofU(VI)–goethite by 4 h  indicated the presence of a uranyl persulfide species.54 Formation of the proposed uranyl persulfidespecies is likely favored by the uranyl adsorbing to persulfide anions(S22–), which form at the goethite surfaceduring sulfidation.46 In addition, densityfunctional theory (DFT) calculations in past work suggested a weakadsorption affinity for uranyl(VI)–persulfide and the mackinawitesurface.54 Therefore, the formation ofa uranyl persulfide complex distributed between the solid and solutionmay explain the transient release of aqueous U during the first severalhours of reaction. Interestingly, this transient uranyl persulfidespecies has only been observed during the sulfidation of U(VI) systems(here as U(VI)–goethite and during U(VI)–ferrihydritesulfidation54) but was not observed duringU(V)–magnetite sulfidation.51 Inaddition, the remobilization of U to solution was relatively short-livedin the circumneutral U(VI) systems –magnetite system, U remained in solution for over a week.51 A possibleexplanation for these observations could be related to the overallcharge of the uranyl persulfide species. For U(VI), a uranyl ion (UO22+) is bound to a disulfide species (S22–) and coordinated by water molecules, resultingin a neutral complex that is relatively weakly bound to the FeS surface.54 This is thought to result in a short-lived transientaqueous U species, which is partially adsorbed onto the FeS surface,thereby enabling the uranyl(VI) persulfide species to be observedby EXAFS of the solid phase. However, a U(V) uranyl ion (UO2+) binding to a disulfide (S22–) species would result in a negatively charged complex, as previouslymodeled using DFT calculations.54 As thepoint of zero charge for magnetite is 6.55,69 with nanomagnetite being the only identified phase (by TEM) after8 h of reaction,51 a negatively chargedU(V)–persulfide species would be repelled from the surface,not be associated with the solid EXAFS sample, and may persist inthe solution phase for an extended period of time, as observed duringU(V)–magnetite sulfidation.51 Overall,this suggests that although U(VI) and U(V) may both form an aqueousuranyl persulfide complex, the neutral U(VI) species is expected tobe partially adsorbed to the solid phase at a circumneutral pH, andthere is consequently only a transient release of U(VI) before reductionto U(IV) occurs.U speciation was further probed by U Lironment 3. After goethite 1.23 Gived at 1 h 1, this sNESdata 2. AsU ae by 4 h 3. The prs <32 h, 1,54 yet 2 formation, with the low U–Ucoordination number, indicating a poorly crystalline UO2 phase .62 However, the 3O backscatterers at 2.24 Å may suggest the presence of a U4O9 (U(V)2U(IV)2O9) or U3O7 phase .72 This further supports the hypothesis that a mixed-valenceU oxide species is present, in addition to poorly crystalline UO2, providing a possible explanation for the U(IV) and U(V)components observed in the MIV-edge HERFD-XANES spectra–and U(VI)–goethite includes O backscatterers at approximately2.2 and 2.4 Å (cf. 2.24 and 2.38 Å after 1 day of sulfidation),and given the highly reducing conditions in the sulfidic system, bothU(V)– and U(VI)–goethite incorporated species may contributeto the persistent U(V) and U(VI) components of the MIV-edgeHERFD-XANES spectra (–) and/or the formation of mixed-valence U oxides  formed upon reduction of U(VI) by FeS.After 1 day of sulfidation, the best fit model contains3 O backscatterersat 2.24 Å, 3 O backscatterers at 2.38 Å, 0.5 Fe backscatterersat 3.29 and 3.47 Å, and 1 U backscatterer at 3.71 Å, suggestinga complex mixture of uranium coordination environments and oxidationstates 2. First, spectra2. In add9 months 2, and go spectra 2. Conseq2 phase.73 After 3 months,the best fit model contained 4 O backscatterers at 2.31 Å, 2O backscatterers at 2.46 Å, 5 U backscatterers at 3.86 Å,and 8 distal O backscatterers at 4.42 Å. This suggests that at3 months, U is still present as a complex mixture of nanocrystallineUO2, mixed valence U oxide and/or U–goethite. Specifically,nanocrystalline UO2 contains a U(IV) coordinated by 8 Oions at 2.37 Å, which is an approximate mid-point between thefitted O backscatterers at 2.31 and 2.46 Å. In addition, the5 U backscatterers (3.86 Å) and 8 distal O backscatterers (4.42Å) correlate well with nanocrystalline UO2 (U(IV)O2 = 12 U at 3.87 Å, 24 O at 4.53 Å),62 which supports the progressive U(IV) formation observedin both LIII-edge XANES  and MIV-edge HERFD-XANES  and to monitor the redox potential during reoxidation.Given fluctuating redox conditionsin the environment, to better understand the long-term fate of U-incorporatediron (oxyhydr)oxide phases, a controlled reoxidation experiment wasperformed . A chemostat system was utilized to maintainthe pH (via acid/base additions) and DO content , and there was a gradual increase in Ehof 14 mV over the 4 h . However, Eh then rapidlyincreased, with an Eh of +2.7 mV by 24 h. Moreover, from 11.5 h onwards,a repeated intermittent flow of nitrogen was needed to maintain a5% DO level with minimal laboratory air , suggesting that the redox buffering capacity of the experimentwas limited from 11.5 h onward.First, despite a constant flow of laboratory air, DO levels initiallydecreased over the first 5 h to a minimum of 2.5%, followed by a gradualincrease up to the set-point of 5% DO 7.5 h; 4B. DurinFigure S14). This was corroborated in TEM images, which showedclusters of goethite rods and lepidocrocite laths .39 Therefore, the observed geochemical behavior  was likely due to the redox buffering effect of FeS, whichhas been shown to oxidize by the following equation55After 4 days of oxygen ingress (at 5% DO), XRDrevealed that amixture of lepidocrocite (γ-FeOOH), goethite (α-FeOOH),and elemental sulfur had formed in the system O2 reoxidation.55 In the current study, the oxidation of FeS wasindicated by the release of aqueous Fe, which reached a maximum concentrationof 184 μM after 3 h  will have thenbeen immediately adsorbed and/or incorporated onto the newly formediron (oxyhydr)oxide (FeOOH) phase.74In the past work on synthetic nanocrystallineFeS mixed with uraninite,FeS was shown to preferentially oxidize before U(IV)Ofter 3 h 4A. This fter 3 h 4A. This 75 or a sequential acid extraction (for adsorbedU(VI)/U(IV)O2).23 Here, following 4 days of reoxidation, 65% U was found to be bicarbonate-extractable,consistent with 62% for a 0.1 M HCl extraction . Therefore, after 4 days of reoxidation, approximately35% U appears to be resistant to bicarbonate or acid leaching andis potentially incorporated within an iron (oxyhydr)oxide , with 65% U likely adsorbed.To assessthe association of U with the iron (oxyhydr)oxide species, surface-bound U is often removedusing techniques such as a bicarbonate extraction (for adsorbed U(VI)/non-crystallineU(IV))III-edge XANES were collected at selected timepointsduring reoxidation . The collected spectra reveal a small but progressive increasein the XANES edge position over the first 11 h of reaction (∼0.6eV), indicating partial U oxidation . By 4 days, there was a significant increase in the XANES edgeposition (∼2.7 eV), suggesting that oxidation to U(VI) wascomplete. Overall, this confirms that simultaneous reoxidation ofU(IV)O2 and FeS occurred. During FeS oxidation (approximately11 h), a fraction of U was oxidized to U(VI); this U(VI) may havethen been incorporated into the growing iron (oxyhydr)oxide phases(up to ∼35% U incorporation from the bicarbonate and acid leaching).The majority of U reoxidation then occurred on depletion of FeS andthe associated redox buffering to form U(VI)-adsorbed iron (oxyhydr)oxidesas the predominant species. In addition, XANES were collected on asample after acid extraction of surface-bound  U;the edge position of the U LIII-edge XANES decreased inenergy, suggesting that a more reduced U species was present in theacid-leached sample, where adsorbed U(VI) had been preferentiallyremoved . This suggests thatalthough the adsorbed fraction (65% U) consists of a U(VI) species,the incorporated fraction (35% U) has a lower overall oxidation state,potentially with a U(V) component. Interestingly, during the sulfidationof U(VI)-incorporated goethite, there was evidence for U(V)-incorporatedgoethite formation -incorporated goethite componentafter reoxidation further supports the hypothesis that U(V)-incorporatedgoethite was formed during U(VI)–goethite sulfidation.To further probethe speciation of the U-associated iron (oxyhydr)oxidespecies, U Lxidation S19A–C. TIII-edge EXAFS. Firstly, EXAFS best fits for 3–5and 7.5–11 h are very similar, with 2 O backscatterers at 2.19–2.21Å, 4 O backscatterers at 2.37 Å and 3 U backscatterers at3.82–3.85 Å . Therefore, although there was a slight increasein the energy of the XANES edge position, which indicates U oxidation, there was still significantUO2, and possibly mixed valence U oxides , retained after 11 h of reoxidation. By 4 days ofair reoxidation, the EXAFS best fit contained 2 O at 1.81 Å,2 O at 2.29 Å, 2 O at 2.44 Å and 0.5 Fe at 3.44 Å,confirming that U(VI) was predominantly in a uranyl coordination (Table S3). Therefore, the dominant U speciationwas likely an adsorbed uranyl goethite/lepidocrocite phase, whichis consistent with the chemical extraction results. Given that U(VI)–goethitehas undergone a substantial dissolution and recrystallization process, incorporated U(VI) was expected to be transferredinto a more labile phase . Therefore, finding a significant fractionof U (35%) still retained within the iron (oxyhydr)oxide followingredox cycling, despite the substantial reductive dissolution of U(VI)–goethite,is a significant result. This suggests that under ambient environmentalconditions, even when subject to dissolution and transformation reactions, iron (oxyhydr)oxides willhinder U mobility /(V)). The newly formedlabile fraction ) has the potential to beremobilized  or maybecome re-incorporated during further recrystallization of iron (oxyhydr)oxidesduring subsequent redox cycling.39The U behavior and speciation during reoxidation were further exploredusing U Lx2+. Interestingly, TEM images indicatedthat a modest fraction of residual U-incorporated goethite was alsoretained after several months. During controlled air reoxidation,mackinawite then transformed to a mixed goethite/lepidocrocite phase,which contained adsorbed U(VI) (approximately 65%) and U(V/VI)-incorporated(approximately 35%) iron (oxyhydr)oxide species. In contaminated landand waste disposal scenarios, a complex combination of biotic andabiotic interactions will occur, where sulfate-reducing bacteria willproduce aqueous sulfide in-situ and microorganisms can also directlyreduce both U(VI) and iron (oxyhydr)oxides.2 Inaddition, sulfide is present in some groundwaters56 and may be transported in the sub-surface. This study focuseson further understanding the abiotic interactions that may occur betweenaqueous sulfide  and U-associated iron (oxyhydr)oxides. Whilereaction with aqueous sulfide may initially release a modest fractionof aqueous U , the aqueous U species is short-lived andrapidly reduced to solid-phase UOx2+.In addition, despite excess aqueous sulfide, a fraction of U(V/VI)-incorporatedgoethite was resistant to reductive dissolution, and partial reincorporationof U with goethite/lepidocrocite may have occurred during reoxidation.Consequently, although U is released from the goethite structure duringsulfidation, under the environmental conditions , a U-incorporated goethitecomponent may persist in the sub-surface in the long term.Using a combination of XASand geochemical analysis, the behaviorand speciation of U during the sulfidation and subsequent reoxidationof U(VI)-incorporated goethite have been explored. Initially, remobilizationof a transient aqueous U species was observed during sulfidation,with EXAFS analysis indicating the formation of a U(VI)–persulfidespecies, which was partially adsorbed to the solid phase at 4 h. However,in the long term, U was largely retained in the solid phase as nano-crystallineU(IV)O


Clade 2.3.4.4 H5Nx influenza viruses have further diversified into several subclades. Sub-clade 2.3.4.4b H5N1 viruses have been widely circulating in wild birds and detected in Europe, Africa, Asia, and North America since October 2020. In this study, we report the first detection of highly pathogenic avian influenza H5N1 clade 2.3.4.4b viruses in wild birds and domestic ducks from live bird markets in Egypt. Phylogenetic analysis revealed that the Egyptian H5N1 virus retained the genomic composition of Eurasian strains. Mutations in the viral proteins associated with zoonotic potential and pathogenicity were detected in Egyptian isolates. Egypt is considered a hot spot for the evolution of the influenza virus, so active surveillance of avian influenza viruses in Egypt is warranted. Infectious diseases and pandemics in humans are often caused by pathogens transmitted from non-human animal reservoirs. Influenza A viruses (IAVs) spread among a variety of different hosts and cross species barriers to create new viral strains. Waterfowl serves as the primary reservoir and can perpetuate many avian influenza virus (AIV) subtypes via asymptomatic shedding, which plays an important role in the reassortment and transmission of influenza subtypes to domestic poultry . In 1996The phylogenetic clade 2.3.4.4 of H5Nx viruses has caused extensive outbreaks across the globe and has further evolved into eight subclades (2.3.4.4a–2.3.4.4h) , and sevHighly pathogenic AIV (H5N1) was initially introduced into Egypt in 2005 and became endemic in poultry in 2008. Since then, many outbreaks have been reported in domestic poultry farms. Multiple clades of H5N1 Gs/Gd lineage viruses  were identified . The 2.3To monitor the influenza viruses with pandemic potential at the human–animal interface, in this study, we identify the genetic and antigenic characteristics of HPAI H5N1 viruses that were introduced into Egypt through active surveillance of AIVs in live bird markets (LBMs) and migratory wild birds.Active surveillance of avian influenza viruses has been conducted in Egypt through collaborative efforts of the Center of Scientific Excellence for Influenza viruses, National Research Centre, Egypt, and Center of Excellence for Influenza Research and Surveillance at St. Jude  since 2009. In April 2021, we sampled poultry and wild birds sold in LBMs in Egypt. We collected cloacal and oropharyngeal swab samples, which were kept chilled in virus transport medium until they reached the laboratory.Samples were thoroughly vortexed prior to viral nucleic acid extraction from 200 μL of viral transport media using either the automated MagNA Pure 96 platform, KingFisher Flex instrument  or the QIAamp Viral RNA Mini kit , according to the manufacturer’s instructions. Nucleic acid extracts were screened by real-time RT-PCR (rRT-PCR) for the presence of the AIVs  , and samViral RNA extracted from allantoic fluid was subjected to reverse transcription to synthesize the first cDNA strand using a SuperScript IV first-strand synthesis kit  and the Uni12 influenza primer. Then, Phusion high-fidelity DNA polymerase  and Uni12/13 primers were used for multiplex PCR of all eight gene segments, and PCR products were purified. Sequencing library preparation was performed by using Illumina’s Nextera XT DNA Sample Preparation Kit according to the manufacturer’s protocol. Amplicons were sequenced on Illumina’s MiSeq platform  by using the paired-end approach. The eight full segments of each H5N8 virus were assembled using CLC Genomics Workbench, version 21 .For sequence and phylogenetic analyses, genome sequences of H5Nx were aligned by MAFFT v4.787 , and theHaemagglutination inhibition (HI) assays were used to antigenically characterize the isolated viruses. The H5N1 AIVs were tested by using post-infection ferret antiserum raised against F.2015-7-A/duck/England/36254/2014 (H5N8), F.2017-13-A/chicken/Kumamoto/1-7/14 (H5N8), F.2016-16- A/gyrfalcon/Washington/410886/2014 (H5N8), and F.2015-48-A/Sichuan/26221/2014 (H5N6) of clade 2.3.4.4 viruses which were produced in Center of Excellence for Influenza Research and Surveillance at St. Jude . A panel of post-infection ferret antisera was treated with receptor-destroying enzyme II and heat-inactivated at 56 °C for 30 min and diluted to a final concentration of 1:10 in PBS and 0.5% chicken erythrocytes. The HI test was performed according to the WHO protocols [The nucleotide sequences of the H5N1 AIVs described in this study were deposited in the GenBank database with the accession numbers shown in Through surveillance, we isolated H5N1 viruses from one wild pintail duck and three domestic Pekin ducks, A/pintail/Egypt/RA19853OP/2021 in late 2021 and A/duck/Egypt/BA20360C/2022, A/duck/Egypt/BA20360OP/2022, and A/duck/Egypt/BA20361OP/2022 isolates in early 2022. The analysis of the complete HA gene segment showed that the HPAI H5N1 viruses belonged to phylogenetic clade 2.3.4.4b. The nucleotide sequence identities across all eight segments of the four viruses were 99.5–100%. As a representative virus, A/pintail/Egypt/RA19853OP/2021 (H5N1) had a high nucleotide identity (99–100%) to the HPAI A(H5N1) viruses of clade 2.3.4.4 from Europe and the Middle East . These iWe combined genome sequences generated in this study with all sequences of H5Nx viruses available in GenBank and the GISAID database (11). Phylogenetic analysis confirmed that the Egyptian A(H5N1) isolates are of clade 2.3.4.4b and clustered with the recent HPAIV A(H5N1) isolates from Europe, Africa, and the Middle East . The claThe time to the most recent common ancestor (tMRCA) was calculated to explain the emergence of the H5N1 viruses. Taking the intersection of the 95% highest posterior density (HPD) intervals of the tMRCA  suggestsThe receptor binding sites in the viral HA gene of the four viruses possess the conservative amino acid residues , which indicated that these viruses would preferentially bind to the α-2,3-sialic acid linkage, the avian-like receptors.The antigenic properties of H5N1 viruses were also assessed using ferret antisera against the World Health Organization’s candidate clade 2.3.4.4c H5N8 and clade 2.3.4.4a H5N6 vaccine viruses including A/gyrfalcon/Washington/41088-6/2014 (H5N8), A/duck/England/36254/2014 (H5N8), A/chicken/Japanese Kumamoto/1-7/2014 (H5N8), and A/Sichuan/26221/2014 (H5N6) . The preSeveral introductions of clade 2.3.4.4b viruses have been seen in Egypt. Those introductions are typically through wild migratory birds but eventually spill over to poultry. Some live bird markets in Northern Egypt sell both poultry and trapped wild birds for human consumption. Such an interface provides ample opportunity for cross-species virus spill-over. The viruses we detected were from such markets where the initial virus was detected in a migratory bird and then in domestic poultry. No human cases of clade 2.3.4.4b H5Nx infections were reported in Egypt but the mutations detected in analyzed viruses suggest that human infections can occur. A vigilant surveillance system at the human-wild bird-poultry interface is necessary.


CYP2E1) was characterized in DNA from blood mononuclear cells  and skin fibroblasts . Protein levels of CYP2E1 were analysed using Western blot in human cortex and knock-out mice brain samples. Results: We found 35 hypomethylated and 22 hypermethylated genes with a methylation M-value difference >0.5. Decreased methylation of cytochrome P450 2E1 (CYP2E1) was associated with increased protein levels in PD brains, but in peripheral tissues, i.e., in blood cells and skin fibroblasts, DNA methylation of CYP2E1 was unchanged. In CYP2E1 knock-out mice brain alpha-synuclein (SNCA) protein levels were down-regulated compared to wild-type mice, whereas treatment with trichloroethylene (TCE) up-regulated CYP2E1 protein in a dose-dependent manner in cultured cells. We further identified an interconnected group of genes associated with oxidative stress, such as Methionine sulfoxide reductase A (MSRA) and tumour protein 73 (TP73) in the brain, which again were not paralleled in other tissues and appeared to indicate brain-specific changes. Conclusions: Our study revealed surprisingly few dysmethylated genes in a brain region less affected in PD. We confirmed hypomethylation of CYP2E1.Background: Epigenetic factors including DNA methylation contribute to specific patterns of gene expression. Gene–environment interactions can change the methylation status in the brain, and accumulation of these epigenetic changes over a lifespan may be co-responsible for a neurodegenerative disease like Parkinson’s disease, which that is characterised by a late onset in life. Aims: To determine epigenetic modifications in the brains of Parkinson’s disease patients. Patients and Methods: DNA methylation patterns were compared in the cortex tissue of 14 male PD patients and 10 male healthy individuals using the Illumina Methylation 450 K chip. Subsequently, DNA methylation of candidate genes was evaluated using bisulphite pyrosequencing, and DNA methylation of cytochrome P450 2E1 ( Epigenetic factors, particularly DNA methylation, modify gene expression and have been shown to undergo dynamic changes throughout life and in differentiated neurons of the human brain ,2. MethySeveral huge GWAS in Parkinson’s disease found the SNCA gene as a highly significant genetic risk factor (reviewed in ). DNA meCYP2E1 hypomethylated, specifically in the cortex DNA from PD patients, associated with increased protein levels. In Cyp2e1-null mice, we observed decreased alpha-synuclein protein, suggesting that CYP2E1 in wild-type mice (and men) might contribute to increased alpha-synuclein expression.We here extended previous studies to a larger sample of cortical tissues from male PD patients and healthy controls, avoiding a confounding effect of imprinting by sex, using Illumina 450 K methylation bead arrays. Consistent with our preliminary data, we identified cytochrome P450 2E1 (CYP2E1) among the most intensely and repetitively hypomethylated genes. Independent techniques, i.e., bisulphite pyrosequencing (BPS) at single-base resolution, confirmed DNA was extracted from 24 cortex samples from 14 male PD patients (mean age ± SD: 77.7 ± 5.9 years) and 10 male neurologically healthy individuals (mean age ± SD: 76.0 ± 8.75 years) provided by the Brain Bank Munich and by Professor Schulz-Schaeffer. DNA extraction and bisulphite conversion were performed as described previously [Four hundred nanograms of bisulphite-treated DNA were analysed using the Infinium Human Methylation 450 K bead array according to the manufacturer’s instructions. The Ethics Committee of the Medical Faculty of the University of Bonn approved the study .® software (version 2011.1) was used for the extraction of DNA methylation signals as raw signals without background normalization; methylation data were further processed via the Bioconductor lumi package using shift-scale colour bias adjustment and quantile normalization as further pre-processing steps implemented in the lumi package [p-value cut-off of 0.00001 was used to filter out signals below background, which left 320.898 CpG for testing.Illumina GenomeStudio package . A detecCYP2E1 PCR: PF1_GGGGTTGTTTTTGAGTAGGAGT and PR1_TCAATAAATCTCTTCCCCCTT C, Pyrosequencing: PS1_TTTTTATTTATGTTGAGG; C21orf55:PF1_TTATGAGGATGAG ATGATTTATTTG and PR1_Bio_CCCTAACTCCCTACTTCAATTAC; PS1_GAGATGAT TTATTTGTTGT; TP73: PF1_GGTTTAATAGGGAGTGGTAGTTATTTT and PR1_Bio_A CCCACCCTAACACTAACCA, PS1_AGTGGTAGTTATTTTAAAGG). Statistical analysis was performed using SPSS Statistical software 20.0 . Values are indicated as the mean ± SD, and comparisons between groups were performed using the Mann–Whitney U test. The level of significance was set at p < 0.05 and <0.01, respectively.A PyroMark Q24 System (Qiagen) was used for DNA methylation analysis  and 182 healthy controls  was analysed.The methylation of DNA derived from the pbms of 4 male de novo PD patients without prior l-DOPA medication (aged 63.2 ± 9.49) was analysed using pyrosequencing before and after pbms were exposed to 100 µM and 200 µM l-DOPA for 48 h.CYP2E1 in DNA from the blood of PD patients without prior l-DOPA intake , low l-DOPA dosage  and high l-DOPA dosage .In addition, we compared the methylation state of DNA from cell-cultured skin fibroblasts from 5 healthy probands  and 12 PD-affected patients  was used for pyrosequencing.β-actin  and rabbit polyclonal anti-CYP2E1  antibodies. Densitometric quantification and normalization to the β-actin levels were performed using LabImage 1D L340 .Western blot analysis was performed using 50 µg of protein per lane and mouse monoclonal anti-CYP2E1-null mice were described previously [eviously . We usedp-value adjustment.The epigenome-wide methylation analysis revealed 57 differentially methylated genes, but without reaching the level of significance after We found 35 hypomethylated and 22 hypermethylated in PD, with a methylation difference >0.5 of the M-value . The corCYP2E1 (5 CpGs hypomethylated in PD) and C21ORF56 (6 CpGs hypomethylated in PD). Large differences regarding the degree of methylation were found again in CYP2E1, but also in methionine sulfoxide reductase A  and cadherin 13   .CYP2E1 was significantly hypomethylated in the PD cortex at the CpGs sites investigated .To validate the array data, BPS of the respective genes of interest was performed, confirming that CYP2E1. Using these primer pairs, Jiménez-Garza et al. demonstrated the up-regulation of CYP2E1 methylation in blood from tannery workers exposed to toluene [CYP2E1 in all of the CpGs analysed .Hypomethylation of quencing B,C; howeCYP2E1 methylation might serve as a biomarker in accessible peripheral tissues, we analysed DNA from blood and skin fibroblasts. However, methylation of CYP2E1 was unchanged when comparing the control and PD in a cohort of 182 vs. 259 samples using primer pairs 1 and 2 .To determine whether  1 and 2 B,D. AddiBlood samples of another independent cohort were grouped according to the reported l-DOPA intake. In addition, to evaluate whether l-DOPA might exert an acute effect on methylation , a set oCYP2E1 DNA was not changed in PD patients treated with different L-DOPA dose ranges in vivo  nor in pbms treated with l-DOPA in vitro : control: 16 ± 2% vs. PD: 14 ± 3%; p-value = 0.8; p = 0.042; mean ± SD; Western blot analysis suggested increased expression of CYP2E1 protein in the brains of PD patients compared with those of controls , which is known to cause PD in particularly susceptible men, t manner J.CYP2E1 on SNCA protein levels was tested using CYP2E1-null and wild-type (WT) mice. In brain tissue, SNCA protein was significantly decreased in the null mice  gene and increased expression of CYP2E1 mRNA in the brains of Parkinson’s disease (PD) patients in an independent study using the 27 K micro bead array in a more heterogeneous group of tissues, suggesting that epigenetic variants of this cytochrome (CPY) might contribute to PD susceptibility [Cytochromes have been implicated in PD pathophysiology, primarily as a putative genetic risk factor . CYP2E1 CYP2E1, involved in the metabolism of xenobiotics such as trichloroethylene (TCE) and SNCA is interconnected and that increased expression of CYP2E1, and consequently SNCA, might cause neuron damage as a result of exposure to dietary or environmental toxicants that are activated by CYP2E1.Interestingly, the lack of CYP2E1 in the null mice was associated with the down-regulation of alpha-synuclein (SNCA), suggesting that SCNA in dopaminergic SN neurons after TCE exposure that might be mediated by CYP2E1 induction [These findings are in line with animal models demonstrating the up-regulation of nduction .The observed CYP2E1/SNCA interconnection might be induced by a CYP2E1-dependent increase of oxidative stress, known to cause DNA hypomethylation, including SCNA hypomethylation , which wCYP2E1 is mainly involved in the oxidative metabolism of rather few polar molecules, mainly ethanol and aldehydes . Except CYP2E1 has been implicated in PD pathophysiology at several levels. The induction of CYP2E1 generates reactive oxygen species (ROS) in the substantia nigra of rodents, and CYP2E1-null mice are protected from MPTP toxicity ,23. CYP2The hypomethylation of the CYP2E1 gene in the brain allows stronger expression of CYP2E1 in response to a given stimulus, and it was indeed shown that exposure to TCE causes PD in particularly susceptible men ,26,27 anIncreased CYP2E1 in humans typically results from ethanol consumption, but trichloroethylene (TCE) is another strong inducer (and preferred substrate) of CYP2E1 . We demoWe did observe hypomethylation of CYP2E1 in peripheral blood in the present study. In addition, in an earlier study of methylation in PD peripheral blood, no change was detected in a cohort of 221 vs. 227 human samples . In anotIt is uncertain whether the epigenetic dysregulation/hypomethylation of CYP2E1 observed here might represent the epigenetic remnant of an inherited change upon environmental exposure of anteceding generations to solvents , renderiMSRA gene body. Intragenic DNA methylation is generally correlated with increased transcriptional activity, and hypomethylation can decrease transcription [Significant differences were also detected in CpGs related to the MSRA, TP73, and CDH13 genes. MSRA plays a critical role in the antioxidant response, protecting cells from oxidative damage. Cells with increased levels of MSRA are resistant to oxidative stress. It efficiently reduces oxidized alpha-synuclein, which otherwise tends to form potentially neurotoxic protofibrils . The CpGcription . Thus, wTransactivating isoforms of p73 (Tp73) have p53-like pro-apoptotic activities . HypometCDH13) protects vascular endothelial cells from apoptosis due to oxidative stress. While the relevance of our observation to gene expression and its contribution to PD aetiopathogenesis remains unclear, CDH13 is known to be required for insulin release and thus contributes to the regulation of insulin secretion [Cadherin 13 (ecretion . Indeed,C21ORF56, chromosome 21 open reading frame 56, with several hypomethylated CpGs per gene. C21orf56 is an uncharacterized protein, and data regarding its gene function do not exist.Furthermore, we found CYP2E1 observed in the brain was not found in peripheral blood or skin fibroblasts, indicating the tissue specificity of CYP2E1 methylation. Furthermore, we found an association of the expression of SNCA and CYP2E1.The brains of PD patients and healthy individuals revealed different methylation patterns, including those of several candidate genes involved in PD pathology and oxidative stress . In part


CYP11B2) expression by depolarization of adrenocortical cells. In this study, we found that YM750, an Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor, acts on adrenocortical cells to suppress CYP11B2 gene expression and aldosterone secretion. YM750 inhibited the induction of CYP11B2 gene expression by KCl stimulation, but not by angiotensin II and forskolin stimulation. Interestingly, YM750 did not inhibit KCl-stimulated depolarization via an increase in intracellular calcium ion concentration. Moreover, ACAT1 expression was relatively abundant in the zona glomerulosa (ZG) including these CYP11B2-positive cells. Thus, YM750 suppresses CYP11B2 gene expression by suppressing intracellular signaling activated by depolarization. In addition, ACAT1 was suggested to play an important role in steroidogenesis in the ZG. YM750 suppresses CYP11B2 gene expression and aldosterone secretion in the adrenal cortex, suggesting that it may be a potential therapeutic agent for PA.Primary aldosteronism (PA) is considered the most common form of secondary hypertension, which is associated with excessive aldosterone secretion in the adrenal cortex. The cause of excessive aldosterone secretion is the induction of aldosterone synthase gene ( CYP11B2) is the rate-limiting enzyme in aldosterone synthesis. Primary aldosteronism (PA), a condition characterized by excess production of aldosterone by the adrenal cortex, is a common cause of secondary hypertension [CYP11B2 was expressed [CACNA1D, ATP1A1, and ATP2B3 genes have been identified in the adrenal CYP11B2-positive regions, such as APNs/APMs  and APDH (formerly known as diffuse hyperplasia) [CYP11B2 expression and aldosterone secretion caused by cellular depolarization are linked to PA pathogenesis [Aldosterone is synthesized from cholesterol by the action of steroidogenic enzymes in the adrenal cortex. Aldosterone synthase CYP1B2 is thrtension . The cauxpressed . In partxpressed . Mutatiorplasia) ,7,8. CYPogenesis ,11,12,13KCNJ5 mutations have been reported [CYP11B2 inhibitors have also been developed [Drugs that act on the adrenal cortex and suppress aldosterone secretion are currently still under development; however, macrolides that suppress aldosterone secretion in APAs associated with reported . Additioeveloped . The receveloped  is usefuACAT is a cholesterol-metabolizing enzyme, expressed as ACAT1 in macrophages and adrenal glands and as ACAT2 in the liver . ACAT coACAT1 in APNs, APMs, and APDH has not been studied in detail.However, the relationship between ACAT activity and adrenocortical steroid synthesis remains unclear. Additionally, the expression status of ACAT1 in CYP11B2-positive aldosterone-producing cells.In this study, we demonstrated that YM750, an ACAT1 inhibitor, suppressed aldosterone synthesis, suggesting its potential as a novel therapeutic agent for PA. In addition, we reported the detailed expression status of CYP11B2 expression. The results showed that YM750 suppressed KCl-stimulated CYP11B2 expression but showed no effect on angiotensin II (Ang II)- and forskolin-stimulated CYP11B2 expression , including CYP11B2-positive cells.We examined and APDH . The resCYP11B2 expression and aldosterone secretion by inhibiting downstream calcium signaling and inducing expression of NURR1.To the best of our knowledge, this is the first study to demonstrate that YM750 suppresses CYP11B2 gene expression but showed no effect on Ang II- and forskolin-stimulated CYP11B2 expression. Additionally, YM750 did not suppress calcium ion influx into the cells. KCl stimulation depolarizes H295R cells and induces CYP11B2 expression via activation of intracellular calcium signaling [CYP11B2 expression via activation of transcription factors such as NURR1 and cyclic AMP response element-binding protein [CYP11B2 expression specifically.YM750 reduces the concentration of intracellular cholesteryl esters by inhibiting ACAT1 . The resCYP11B2 expression is regulated by transcription factors such as NURR1 and NGFIB [CYP11B2 and regulate gene expression [HSD3B2 and regulates gene expression [NURR1 and NGFIB expression at high concentrations. This suggests that the suppression of NURR1 and NGFIB. This suggests that ACAT1 is associated with suppressed CYP11B2 expression via repression of NURR1 and NGFIB. NURR1 primarily regulates CYP11B2 expression, while NGFIB regulates the expression of other steroidogenic genes [NURR1 expression as compared to NGFIB expression, thereby suppressing CYP11B2 expression specifically. In addition, TM750 suppressed CYP11B1 expression. It is known that H295R cells share common signal pathway related to the expression of CYP11B1 and CYP11B2 [CYP11B1 similarly responded to KCl-stimulated depolarization, compatible with inhibition of CYP11B1 expression by YM750. However, it is speculated that YM750 does not necessarily inhibit CYP11B1 in vivo because CYP11B1 expressed in the ZF but not in the ZG would be mainly regulated by ACTH-stimulated cAMP-PKA signaling pathway rather than potassium-stimulated depolarization. In addition, it remains unclear how YM750 effect on metabolism and cholesterol levels when it is clinically used as an aldosterone secretion inhibitor. Therefore, further studies are required for clarification in the future.It has also been reported that nd NGFIB . NURR1 apression . NGFIB bpression . YM750 sic genes ,23,24. T CYP11B2 . Therefohttps://www.proteinatlas.org/ENSG00000110048-OSBP#gene_information, accessed on 5 October 2022 [CYP11B2 expression via NURR1 and NGFIB expression [CYP11B2 expression via activation of OSBP, confirmatory evidence in support of this hypothesis is yet to be established  and suppresses the activation of extracellular signal-regulated protein kinase (ERK1/2) and mitogen-activated protein kinase (MAPK) signaling pathways . OSBP isber 2022 ) and mayablished . TherefoACAT1 is widely expressed in the adrenal cortex and is abundant in the ZG, including APNs. ACAT1 is also expressed in APAs and cortisol-producing adenomas [NURR1 is also widely expressed in the ZG [CYP11B2-positive cells were involved in aldosterone secretion to a greater extent as compared to APNs [ACAT1 expression plays an important role in excess aldosterone production in APAs, APMs, and APDH.In this study, we demonstrated that adenomas ,29, suggn the ZG , indicat to APNs . TherefoAngiotensin II  was dissolved in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin . Potassium chloride  was dissolved in water. Forskolin was dissolved in ethanol . YM750  was dissolved in dimethyl sulfoxide (Wako).H295R cells derived from human adrenocortical carcinoma were purchased from American Type Culture Collection and cultured in Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F-12 nutrient medium (Wako) supplemented with 10% fetal bovine serum , 1.25 mg/mL BSA (Sigma), 1× insulin-transferrin-selenium solution (Life Technologies), 5.35 mg/mL linoleic acid (Sigma), and 1× penicillin-streptomycin solution (Wako). For angiotensin II-, KCl-, forskolin-, and YM750-induced stimulation, cells were cultured in DMEM (Wako) containing 1% charcoal-treated FBS with penicillin-streptomycin. The culture and stimulation methods were based on previous studies ,32,33.® Probe qPCR Mix  for analyzing CYP11B2 and HSD3B2 and KAPA SYBR FAST qPCR kit  for analyzing other genes. Primer sequences and TaqMan probes are listed in Total RNA was extracted using Sepasol-RNA I Super G  and cDNA was synthesized using the Prime Script RT Master Mix . Real-time RT-qPCR was performed using the Thermal Cycler Dice Real-Time System  along with the THUNDERBIRD4 H295R cells into 96-well plates, the cells were incubated with and without 10 mM YM750 for 24 h and 20 mM KCl for 6 h. Cell viability was measured using the Cell Counting Kit-8  following the manufacturer’s instructions.Two days after plating 2.5 × 105 H295R cells into 24-well plates, the cells were incubated with and without 10 mM YM750 and 20 mM KCl for 24 h. Aldosterone and cortisol concentrations in the media were measured using the Aldosterone ELISA Kit  following the manufacturer’s instructions.Two days after plating 3.0 × 104 H295R cells into 96-well plates, the cells were incubated with and without 10 mM YM750 for 24 h. The cells were then loaded with Fluo 4-AM  in the presence of 1.25 mM probenecid (Dojindo) and 0.04% Pluronic F-127 (Dojindo) for 1 h. Cells were then washed with PBS and the recording medium containing 1.25 mmol/L probenecid and 20 mM KCl was added to the media. Changes in intracellular calcium concentration were determined by measuring the fluorescence intensity .Two days after plating 2.5 × 10Three non-pathological adrenal glands  tissues), three multiple APMs (APM tissues), and three APDH (APDH tissues) were selected for immunohistochemical analysis based on information retrieved from the surgical pathology files from Tohoku University Hospital . NA tissues of nephrectomy cases due to renal carcinoma were harvested and subsequently evaluated to confirm the absence of neoplastic invasion, necrosis, and other histopathological abnormalities. The research protocols were approved by the Ethics Committee of Tohoku University Graduate School of Medicine . All patients read and signed informed consent forms that clearly stated the methodologies for the use of tissue samples and clinical data for diagnostic and scientific purposes in the present study. The research protocol was approved by the Institutional Review Board of the Tohoku University School of Medicine .Immunohistochemical analysis using hematoxylin and eosin stains was performed on 3 μm thick tissue sections prepared from formalin-fixed, paraffin embedded blocks. The protocols for immunohistochemistry used in this study are summarized in All data are presented as mean ± standard error of the mean. Statistical analyses were performed using Welch’s analysis of variance using GraphPad Prism 9.CYP11B2 expression and aldosterone secretion in H295R cells. Additionally, ACAT1 expression was confirmed in the ZG of normal adrenal glands and in non-neoplastic regions of PA. The results from this study suggest that YM750 may be a potential therapeutic agent for PA.In this study, we demonstrated that YM750, an ACAT1 inhibitor, suppressed


HFE in the Caucasians. We recently reported non-HFE mutations constitute the major cause of HH in Chinese. However, there is still a relatively high proportion of cases with primary iron overload from unexplained causes. We aimed to explore novel non-HFE mutations in Chinese patients with primary iron overload.Hereditary hemochromatosis (HH) is mainly caused by homozygous p.C282Y mutations in Whole exome sequence was conducted to screen mutations in novel HH-related genes in the 9 cases with unexplained primary iron overload. Then the representative candidate genes were screened for mutations in another cohort of 18 HH cases. The biological function of the selected genes and variants were analyzed in vitro.UBE2O p.K689R and PCSK7 p.R711W. Subsequent Sanger sequencing of the UBE2O and PCSK7 genes in the 27 cases with primary iron overload identified p.K689R in UBE2O, p.R711W and p.V143F in PCSK7 at frequency of 2/27,1/27 and 2/27 respectively. In vitro siRNA interference of UBE2O and PCSK7 resulted in down-regulated HAMP mRNA expression. Adenovirus generation of UBE2O p.K689R in cell lines resulted in increased expression of SMAD6 and SMAD7 and downregulation of p-SMAD1/5 and HAMP expression, and the reduction of hepcidin level.Whole exome sequencing of 9 cases with unexplained primary iron overload identified 42 missense variants in 40 genes associated with iron metabolism pathway genes such as HFE mutations in Chinese patients with HH. These may provide insights into the genetic basis of unexplained primary iron overload.Our study identified a series of novel candidate non-The online version contains supplementary material available at 10.1186/s13023-022-02349-y. HFE), hemojuvelin (HJV), hepcidin gene (HAMP), transferrin receptor-2 (TFR2), and its target FPN1  . TheHFE p.C282Y is the most common mutation, accounting for more than 90% of cases. Non-HFE-related HH accounts for a minority of the remaining cases. However, some patients with primary iron overload do not show mutations in the aforementioned genes. Studies with next generation whole exome sequencing (NGWES) found that mutations in the pro-peptide domain region of BMP6 and mutations in NMBR were new genetic factors predisposing to primary iron overload [In Caucasians patients with HH overload , 4.HFE genes including HJV p.Q312X, p.C321X [TFR2 p.I238M and G430R [SLC40A1 p.W158C [HFE genes in HH, including HJV p.E3D, p.H104R, and p.V274M, TFR2 p.A302E and p.L745R, and SLC40A1 p.Y333H [TMPRSS6 p.T331M, BMP4 p.R269Q, SUGP2 p.R639Q and DENND3 p.L708V [HFE [In Chinese HH patients the gene mutations are quite different from those in Caucasian patients , 6. In C p.C321X , 8 and pnd G430R , and SLC p.W158C , p.S209L p.W158C  were ide p.Y333H . We furtUBE2O), proprotein convertase subtilisin/kexin type 7 (PCSK7), then we analyzed the function and explored the mechanisms of representative genes and variants.However, there are still some cases with primary iron overload had undefined genetic causes . Therefore, in the present study, we explored novel HH-related genes by whole exome sequencing (WES) in a cohort of cases with primary iron overload with unexplained causes. We first identified mutations in a series of potential iron metabolism–related genes such as ubiquitin-conjugating enzyme E2 O . Informed and written consent was obtained from all patients.Genomic DNA was extracted from whole blood using a Genomic DNA Purification Kit . Quality control was performed by evaluating the 260/280 nM absorbance ratio and gel electrophoresis.HFE, HJV, HAMP, TFR2, SLC40A1) could not explain the severity of iron overload. Table WES was performed on DNA extracted from the peripheral blood of nine unrelated patients with primary iron overload, in which mutations in known HH-related genes . The Illumina HiSeq 2000 platform was used to generate paired-end 100 bp raw reads from each enriched library according to the manufacturer’s protocol. The 100 bp paired-end reads were aligned against NCBI build 37 of the human genome using Burrows Wheeler Aligner. Duplicate reads were marked, local indel realignment performed and base quality scores were recalibrated for each sample with the Genome Analysis Toolkit.Novel point mutations were identified using MuTect, while indel variants were identified using Somatic Indel Detector in the Genome Analysis Toolkit. The potential pathogenic variants were confirmed by Sanger sequencing.The criteria for the screening of the mutations in potential iron metabolism–related genes were as follow: in public data bases, the population frequency is less than 1%, no report or prediction as benign or likely benign, and predicted as disease causing by at least one of the prediction tools SIFT, Polyphen-2 and Mutation Taster.HFE mutations, 7 carried HJV mutations, 3 carried TFR2 mutations, 7 carried SLC40A1 mutations, 2 carried SUGP2 mutations, 1 patient carried TMPRSS6 mutation, and 1 patient carried BMP4 mutation. The clinical features of these 18 cases are shown in Additional file To investigate the novel candidate genes, we enrolled 18 unrelated patients with HH as the validation cohort. Among the 18 patients, 3 carried UBE2O and PCSK7 were PCR-amplified with associated boundary regions using specific primers  cell lines Huh-7 and HepG2 were obtained from the Cell Resource Center of the Chinese Academy of Medical Science . Huh-7 and HepG2 cells were cultured as described previously , 14.5 cells) were transfected with 20 nM siRNA using LTX reagent  in accordance with the manufacturer’s instructions.For the siRNA-mediated transient knockdown of gene expression, Huh-7 and HepG2 cells , PCSK7 siRNA (ID siG000009159), and the negative control siRNA (ID siN0000001-1–5) .Gene knockdown was performed using UBE2O and UBE2O p.K689R sequences were cloned into the pADV-EF1-mScarlet-CMV-MCS-3xFLAG vector, and Huh-7 and HepG2 cells were infected with the adenovirus.The The isolation of total RNA from cell lines and real-time PCR assays were conducted as described previously . The priImmunofluorescence analysis was conducted as described previously , 14. CelWestern blot analysis was performed as described previously , 14. MemThe human hepcidin detection kit  was used to determine the concentration of hepcidin in cell culture supernatants according to the manufacturer’s protocol.P < 0.05 was considered to indicate statistical significance.All experiments were carried out at least three times. The Fisher’s exact test was used to determine the difference of mutation frequency between patients and healthy subjects using SPSS v21.0 software. UBE2O p.K689R, PCSK7 p.R711W , one case carried p. R711W  and two cases carried p. V143F  in PCSK7, and with allele frequency of 0.0002, 0.0012, 0.0020 in gnomAD database  by Western blot. Higher expression of Smad6 and Smad7 was observed in UBE2O-knockdown HCC cells. Moreover, we found higher ratio of pSmad1/5/tSmad1 in Huh-7 cells, but lower ratio of pSmad1/5/tSmad1 in HepG2 cells Fig. . The resUBE2O p.K689R as a representative for initial mechanism study. Results from qRT-PCR showed that mutant UBE2O (p.K689R) down-regulated HAMP mRNA level to a greater extent than wild-type UBE2O in HepG2 cells and Huh-7 cells , blocking its association with activated type I receptor BMPRI and resulting in increased BMP/SMAD signaling . We usedlls Fig. A. Consisays Fig. B, C.Fig.UBE2O p.K689R HCC cells than wild-type HCC cells  is encoded at the 17q25 region. As an E2/E3 hybrid ubiquitin-protein ligase , UBE2O dHepcidin deficiency is the common feature in HH and is responsible for iron overload in HH. Hepcidin acts in close connection with ferroportin to regulate iron metabolism. When plasma or hepatocyte iron concentrations increase, signaling pathways including ERK/MAPK and BMP/SMAD pathways are activated and induce hepcidin mRNA expression, leading to increased plasma hepcidin. Hepcidin then interacts with ferroportin, resulting in decreased duodenal iron absorption and a decreased release of iron from the spleen coming from erythrophagocytosis . The BMPUBE2O-knockdown cells, and the level of p-SMAD1/5 decreased in UBE2O-knockdown HepG2 cells. However, the decreased p-SMAD1/5 level was not observed in UBE2O-knockdown Huh7 cells. Previous studies have shown that the expression of p-SMAD1/5 is always inconsistent with that of HAMP or hepcidin in certain cell types. Specifically, the level of hepcidin could be affected by a variety of factors in addition to p-SMAD1/5, such as inflammation and stress. In UBE2O p.K689R–expressing cells, the expression of SMAD6 and SMAD7 increased and the expression of p-SMAD1/5 decreased, along with reduced expression of HAMP mRNA. This suggests that UBE2O may influence iron metabolism by regulating the BMP/SMAD pathway. We speculate that UBE2O regulates the BMP/SMAD pathway through ubiquitination of SMAD6 and SMAD7, and we will explore this hypothesis in future research.In the present study, we found increased expression of Smad6 and Smad7 and decreased expression of HAMP mRNA in PCSK7 variations in patients with primary iron overload. PCSK7 is a family member of nine secretory serine proteases related to bacterial subtilisin and yeast kexin (PCSK1–PCSK9) [PCSK7 by a genome-wide association study [HAMP mRNA was decreased in PCSK7-knockdown HCC cells, further indicating that PCSK7 may be a candidate gene or modifier gene causing iron overload involved in HH.We also identified high frequency of 1–PCSK9) . Oexle eon study . Guillemon study . Our stuPCSK7 needs further study. Finally, the genetic association needs further validation in a larger cohort of patients with primary iron overload.This study has several limitations. First, this study lacked in vivo data, and animal models are needed to clarify the effect of UBE2O on iron metabolism. Second, the mechanisms by which PCSK7 impact hepcidin expression are unknown, and thus the molecular signaling involving hepcidin regulation by HFE mutations in Chinese patients with HH. which may provide insights into the genetic basis of the unexplained primary iron overload.In conclusion, our study identified a series of novel candidate non-Additional file 1. Table S1. Clinical characteristics of validation cohort with primary iron overload.Additional file 2. Table S2. Primers for the Sanger sequencing of UBE2O, PCSK-7 gene.Additional file 3. Table S3. Quality control of the whole exome sequence of the 9 cases with primary iron overload.Additional file 4. Mutations identified in known iron metabolism–related genes or genes associated with iron metabolism pathway by NGWES.Additional file 5. Table S4. Missense variants in genes associated with iron metabolism pathway identified in patients with primary iron overload by NGWES.Additional file 6. Unknown potential iron metabolism–related genes identified with mutations in at least two of the nine cases by NGWES.


Increasing evidence has demonstrated that circular RNAs (circRNAs) are implicated in cancer progression. However, the aberrant expression and biological functions of circRNAs in clear cell renal cell carcinoma (cRCC) remain largely elusive.Differentially expressed circRNAs in cRCC were filtered via bioinformatics analysis. Aberrant circPOLR2A expression was validated in cRCC tissues and cell lines via qRT-PCR. Sanger sequencing was used to identify the backsplicing site of circPOLR2A. In vitro and in vivo functional experiments were performed to evaluate the role of circPOLR2A in cRCC malignancy. RNA pull-down, mass spectrometry, RIP, FISH and immunofluorescence assays were used to identify and validate the circPOLR2A-interacting proteins. Ubiquitination modification and interaction between proteins were detected via Co-IP and western blotting. The m6A modification in circPOLR2A was validated by the meRIP assay.Bioinformatics analysis revealed that circPOLR2A was highly expressed in cRCC tissues and metastatic cRCC tissues. CircPOLR2A expression was associated with tumor size and TNM stage in cRCC patients. In vitro and in vivo functional assays revealed that circPOLR2A accelerated cRCC cell proliferation, migration, invasion and angiogenesis, while inhibiting apoptosis. Further mechanistic research suggested that circPOLR2A could interact with UBE3C and PEBP1 proteins, and that UBE3C could act as a specific ubiquitin E3 ligase for the PEBP1 protein. The UBE3C/circPOLR2A/PEBP1 protein-RNA ternary complex enhanced the UBE3C-mediated ubiquitination and degradation of the PEBP1 protein which could inactivate the ERK signaling pathway. Rescue experiments revealed that the PEBP1 protein was the functional downstream target of circPOLR2A. Furthermore, m6A modification in circPOLR2A was confirmed, and the m6A reader YTHDF2 could regulate circPOLR2A expression.Our study demonstrated that circPOLR2A modulated the UBE3C-mediated ubiquitination and degradation of the PEBP1 protein, and further activated the ERK pathway during cRCC progression and metastasis. The m6A reader, YTHDF2, regulated circPOLR2A expression in cRCC. Hence, circPOLR2A could be a potential target for the diagnosis and treatment of cRCC.The online version contains supplementary material available at 10.1186/s12943-022-01607-8. Renal cell carcinoma (RCC), arising from genetic changes, accounts for approximately 2% of all adult malignancies . Among dCircular RNAs (circRNAs) are covalently closed RNA molecules originating from precursor mRNA via noncanonical backsplicing in eukaryotes , 10. ThePhosphatidylethanolamine Binding Protein 1 (PEBP1) is a member of the phosphatidylethanolamine-binding protein family . InitialN6-methyladenosine (m6A), an epigenetic modification of RNA, is involved in RNA metabolism, including transcription splicing, translation and degradation . It was In the present study, we screened out a circRNA originating from POLR2A, hsa_circRNA_092437 (circBase ID: hsa_circ_0000741), named circPOLR2A, by analyzing the expression profiles of circRNAs in cRCC. Higher expression of circPOLR2A was verified in cRCC compared to matched adjacent normal tissues. We demonstrated that circPOLR2A promoted cRCC progression by mediating the ubiquitination of PEBP1 and activating the Raf1/MEK/ERK pathway. In addition, we further revealed that low expression of YTHDF2 contributed to upregulation of circPOLR2A in cRCC, which indicated that the expression of circPOLR2A was associated with m6A methylation modification. Our work suggested that circPOLR2A could act as an oncogene in cRCC progression and a potential target in the treatment of cRCC.All methods and materials are described in the p value < 0.01 and log2(fold change) > 3  repository: GSE100186, determining the expression profiles of circRNAs in 4 pairs of cRCC and nontumorous tissues ; GSE1378Next, we determined the 9 circRNAs in 32 pairs of cRCC samples and adjacent normal samples via quantitative reverse transcription PCR (qRT-PCR) with divergent primers. As shown in Fig. circPOLR2A is generated from exons 9–10 of the POLR2A gene on chromosome 17p13.1  Western blot indicated that no AGO2 protein was detected in the precipitates of RNA pull-down. (b) No significant change in PEBP1 mRNA expression was determined after circPOLR2A knockdown or overexpression.  Bioinformatics analysis of the CPTAC database suggested that PEBP1 protein levels were associated with cRCC tumor size (c), pathologic T stage (d) and Fuhrman grade (e) in the cRCC cohort. (f) Kaplan-Meier method and log-rank test confirmed that the PEBP1 protein level was a favorable prognostic factor in the cRCC cohort from CPTAC database.Additional file 3: Supplemental Fig. 3. Statistical analysis on the tube formation assay. (a) Total number of junctions in the tube formation assay for circPOLR2A knockdown or overexpression. (b) Total vessels length in the tube formation assay for circPOLR2A knockdown or overexpression.  Total vessels length in the tube formation assay for rescue experiments.Additional file 4: Supplemental Fig. 4.  Bioinformatics analysis suggested that YTHDF2 expression was associated with cRCC grade (a) and stage (b) in the cRCC cohort from TCGA database. (c) Western blotting detected the YTHDF2 level after circPOLR2A knockdown or overexpression. (d) The 4 putative m6A motifs of circPOLR2A predicted by the SRAMP prediction server. (e-h) The expression of METTL3 and YTHDF2 in cRCC cells transfected with siNC, siMETTL3 or siYTHDF2. 10e, 10f, METTL3 expression; 10 g, 10 h, YTHDF2 expression.  The schematic diagram illustrated the structure of circPOLR2A-WT (i) or circPOLR2A-MUT (j).Additional file 5: Supplemental Table 1.Additional file 6: Supplemental Table 2.Additional file 7: Supplemental Table 3.Additional file 8: Supplemental Table 4.Additional file 9: Supplemental Table 5.Additional file 10: Supplemental Table 6.Additional file 11. Supplemental Methods and Materials.


PubMed database to search papers on pharmacogenetics of ISDs in adults or pediatric patients of any gender and ethnicity receiving immunosuppressive therapy after kidney transplantation. We utilized as search term: “cyclosporine or tacrolimus or mycophenolic acid or sirolimus or everolimus and polymorphism and transplant”. Our data showed that polymorphisms in CYP3A5, CYP3A4, ABCB1, and UGT1A9 genes could modify the pharmacokinetics of immunosuppressants, suggesting that patient genotyping could be a helpful strategy to select the ideal ISDs dose for each patient.Immunosuppressant drugs (ISDs) are routinely used in clinical practice to maintain organ transplant survival. However, these drugs are characterized by a restricted therapeutic index, a high inter- and intra-individual pharmacokinetic variability, and a series of severe adverse effects. In particular, genetic factors have been estimated to play a role in this variability because of polymorphisms regarding genes encoding for enzymes and transporters involved in the ISDs pharmacokinetic. Several studies showed important correlations between genetic polymorphisms and ISDs blood levels in transplanted patients; therefore, this review aims to summarize the pharmacogenetics of approved ISDs. We used  Allograft transplantation is the best treatment for organ end-stage failure, and a subsequent immunosuppressant drugs (ISDs)-based therapy is routinely used to prevent graft rejection. ISDs, however, are drugs with a narrow therapeutic window. Large inter/intra-patient variability both in pharmacokinetics (PK) and pharmacodynamics (PD) may lead to severe toxicity or lack of efficacy . AmongstTacrolimus (TAC), also known as FK-506, is an immunosuppressant that belongs to the class of calcineurine (CNI) inhibitor, and it is available in two formulations: the immediate-release formulation (given twice daily) and the prolonged-release Tac (given once daily). In blood, the main reservoir for TAC are erythrocytes, but the percentage of the drug associated with these cells varies widely, and this variability seems to be due to inter-patient differences in hematocrit, the drug-binding capacity of erythrocytes, and concentration-dependent distribution of the drug between blood and plasma . The metTypocladium inflatum, and together with TAC, it belongs to the class of CNI-inhibitor. After entering the small-bowel epithelial cells, part of CsA is transported back into the lumen by an active mechanism of transport mediated by membrane-bound P-glycoprotein 1. CsA is metabolized in the liver mainly by CYP3A4 and in a lesser extent by CYP3A5; more than 30 metabolites have been described, and more than 90% of these metabolites are excreted in the bile [Cyclosporine (CsA) is a lipophilic cyclic peptide formed by 11 amino acids produced by the fungus Mycophenolic acid (MPA) is a potent, selective, reversible, and non-competitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) type II, an enzyme implicated in de novo guanosine nucleotide synthesis, which is necessary for lymphocyte proliferation . TherefoEverolimus (EVE) and sirolimus (SIR) act by inhibiting the response to IL-2, blocking the activation of T and B cells. This is obtained by their binding the cytosolic protein FK-binding protein 12 (FKBP12) forming a complex that inhibits the mammalian target of rapamycin (mTOR) pathway by directly binding the mTOR Complex1 (mTORC1) . The metThe inter- and intra-individual variability observed in transplant recipients treated with ISDs can be due to multiple factors, such as age, body weight, hematocrit, hepatic and renal function, interaction with other drugs, comorbidities, and polymorphisms in genes involved in the metabolism or transporter of these drugs ,9. In paThe most important class of enzymes involved in the metabolism of ISDs is the CYP3A subfamily. In humans, there are four different CYP3A isoenzymes: CYP3A4, CYP3A5, CYP3A7, and CYP3A43 . For ISDIn regards to CYP3A4, the main polymorphism implicated in the metabolism of ISDs occurs at position 392 and is an A>G substitution producing a variant allele (CYP3A4*1B) with increased enzymatic activity . In the Another class of enzymes involved in inter-patient variability are the microsomal electron transfer flavoprotein Cytochrome P450 oxidoreductase (POR), which have been seen influencing CYP3A activity . Human PHowever, inter- and intra-patient variability cannot be entirely explained with the polymorphisms in genes encoding for these two classes of enzymes, and therefore, other genes must be involved .Thus, polymorphisms in transporters could be associated with variations in ISDs levels. Glycoprotein , which belongs to the family of the ATP binding cassette (ABC) membrane transporter, is encoded by multidrug-resistance gene 1 (MDR1), which is a very polymorphic gene, with about 100 SNPs identified in the coding region , and amoFinally, polymorphisms in IMPDH2 genes that encodes for the target protein of mycophenolic acid and in UGT1A9 mainly involved in the metabolism of MPA may be responsible for the intra- and inter-individual variability of MPA levels ,24.For all these reasons, the aim of this review was to investigate the effects of several polymorphisms in genes involved in metabolism or transport of ISDs and their influence on the levels of these drugs.PubMed, and the following the search terms were used: “cyclosporine and polymorphism and transplant”; “tacrolimus and polymorphism and transplant”; “mycophenolic acid and polymorphism and transplant”; “sirolimus and polymorphism and transplant”; and “everolimus and polymorphism and transplant”. Papers on adults or pediatric patients of any gender and ethnicity receiving immunosuppressive therapy after transplant were included. Studies were excluded if: (i) immunosuppressant were used to treat other diseases, (ii) articles were written in a language other than English, (iii) papers were about unspecified genotypes, (iv) transplanted organs were other than kidney, and (v) primary outcomes were other than ISDs’ PK. The summary of the literature search is shown in The database used to retrieve the papers was p < 0.05) [0 in CYP3A5 expressers than in non-expressers (p < 0.01). However, no significant differences were found at 3 and 6 months post transplantation [0 (4–8 ng/mL) after initial dose was achieved less amongst expressers in comparison with non-expressers, and CYP3A5 non-expressers presented higher C0 (>8 ng/mL) 3 months post transplantation. A lower C0 in CYP3A5*1 carrier was also found in other studies carried out in different ethnic groups [In the context of immunosuppressive therapy after solid organ transplant, several studies have explored the role of SNPs in cytochromes. In particular, tacrolimus pharmacokinetics is mainly affected by polymorphisms in CYP3A4 and CYP3A5 genes. In this regard, Cheung et al. studied a population of 86 adult Chinese kidney transplant patients and found that CYP3A5 expressers needed a higher TAC dose compared with the nonexpressers . Similar < 0.05) . Moreoveantation . Furtherc groups ,38,39,400/D), showing that it was lower in CYP3A5*1 carriers compared to wild-type [In addition, numerous papers evaluated the through concentration/dose ratio  was significantly more frequent in PM than EM at 5–7 days after transplant (p = 0.01), and about 30% of EM had subtherapeutic exposure (C0 < 5 ng/mL) 5–7 days post transplant (p = 0.001) [0–24 or Cmin during administration of either IR-Tac or LCPT. They also found that tacrolimus Cmax with IR-Tac or LCPT was 33% and 11% higher in CYP3A5 expressers than non-expressers [0 < 20 ng/mL) or 20.8% toxic concentrations (C0 ≥ 20 ng/mL). On the contrary, 25% of the heterozygote carriers showed overexposure contrary to none of the 1/*1 carriers. When new TAC starting doses of 0.10, 0.20, and 0.30 mg/kg/d were chosen for CYP3A5*3/*3, CYP3A5*1/*3, and CYP3A5*1/*1 genotypes, respectively, authors found that TAC overexposure was reduced in the CYP3A5*3/*3 group (p = 0.038), and none of the heterozygous patients presented toxic TAC C0 [Furthermore, Lloberas et al. stratified 272 renal transplant recipients into poor metabolizers , intermediate metabolizers , and extensive metabolizers . Their results showed that dose-adjusted C= 0.001) . Min et = 0.001) . Similar= 0.001) . Furtherpressers . Moreovec TAC C0 .However, a few studies did not find any association between CYP3A polymorphisms and tacrolimus PK, as demonstrated by Shuker et al., who showed how adapting the tacrolimus starting dose to the different CYP3A5 genotypes does not increase the number of patients achieving therapeutic tacrolimus exposure early after transplantation and does not improve the clinical outcome in a population with low immunological risk . Similar0/D) TAC concentration. The authors observed a significant higher C0/D in carriers of the ABCC2 1249A allele and a lower C0/dose in patients with the ABCC2 3972T allele; on the other hand, they did not find any association between ABCB1 and ABCG2 genotypes and the C0/dose TAC concentration [Nevertheless, inter- and intra-patient variability cannot be entirely explained with the presence of SNPs in genes encoding for cytochromes, and therefore, other genes must be involved, such as genes encoding for transporters. In this context, Ogasawara et al. investigated the influence of polymorphisms in ABCB1, ABCC2, and ABCG2 on dose-normalized . Moreover, an IM pattern resulted, as an independent predictor of number of tacrolimus blood levels out of therapeutic range (p = 0.015), while RM pattern was inversely related to the TAC administered dose (p = 0.006) [Furthermore, a recent retrospective study assessed the variability in tacrolimus blood concentrations in 75 transplanted patients and investigated if tacrolimus blood levels were correlated with the compresence of several genetic polymorphisms: CYP3A5*1 (G6986A), CYP3A4*1B (A392G), CYP3A4*22, ABCB1 , and SLCO1B1 (T521C). Based on the effect of their genotypes, patients were stratified into three groups: reduced tacrolimus metabolism (RM), increased metabolism (IM), and transporters polymorphisms (TM). Results showed that the percentage of patients with TAC levels out of therapeutic range was significantly higher in the IM group when compared with the WT or the TM group (= 0.006) .In addition to evaluate the effects of SNPs on pharmacokinetics and pharmacodynamics parameters, we reported the results of several studies that also considered the clinical outcomes of the polymorphisms. In this context, Anutrakulchai et al. found that the genotype-guided group had more patients with tacrolimus concentrations in the therapeutic range and had also a lower proportion of over-therapeutic concentration. Surprisingly, they observed that delayed graft functions (DGFs) were more frequent in the genotype-guided group, whilst there were no significant differences of glomerular filtration rates and of graft or patient survivals during a median 37-month follow-up period . Thervet0 levels and C0/D ratio; this suggests that a pharmacogenetic pre-evaluation could be useful for prescribing the most appropriate drug dose according to the patient’s genetic profile  [0 and C0/D were significantly higher in patients carrying CYP3A5*3/*3 genotype than in patient carriers of A/A or G/A genotypes (2/D) between CYP3A4*1/*1 or CYP3A4*1/*18B carriers compared to the CYP3A4*18B/*18B group, while no difference was found in C0/D among the three genotypes [p = 0.03) [0) of CsA than G/G and G/T carriers (p = 0.001) in the early post-transplant period. Authors also found a significantly higher CsA C0 in ABCB1 3435 T/T carriers compared to C/C and C/T carriers (p = 0.002). In addition, significantly higher CsA C0/D was observed in patients with the ABCB1 1236TT-2677TT-3435TT haplotype compared to patients with other genotypes (p = 0.001) [0 in CYP3A5*1/*1 carriers was lower than CYP3A5*1/*3 and CYP3A5*3/*3 carriers during the early period post transplant. In addition, patients wild-type homozygotes for MDR1 C3435T had a slight but significantly lower dose-adjusted C0 than heterozygotes [0 or C2 levels were not associated with CYP3A5, CYP3A4, and ABCB1 genotype. Moreover, the incidence of biopsy-proven acute rejection (BPAR) between the different ABCB1 genotype groups was comparable, and no significant difference in the incidence of BPAR was found between CYP3A5 expressers and nonexpressers or between CYP3A4*1 homozygote versus CYP3A4*1B carriers [Cyclosporine is one of the most used immunosuppressive agents after organ transplant, and several studies have explored the role of SNPs in cytochromes and transporters genes that might influence its pharmacokinetics. Cyclosporine PK is mainly affected by polymorphisms in CYP3A4, CYP3A5, and MDR1 genes. In fact, a study conducted by Żochowska and colleagues showed that patients with at least one functional CYP3A5*1 or CYP3A4*1B allele need significantly greater dosages of cyclosporine to reach target drug levels than patients with the CYP3A5*3 or CYP3A4*1 allele (< 0.218) . Meng et< 0.218)  and Qiu < 0.218)  showed tenotypes . In anotenotypes . Another = 0.03) . Moreove = 0.03) . In anot= 0.001) . Moreoveozygotes . Yates eozygotes . On the ozygotes  and Sienozygotes  reportedcarriers .0 and C0/D ratio levels. Instead, numerous other papers including a total of 384 patients showed that the presence of CYP3A4*22 allele, ABCB1 2677 T/T, and ABCB1 3435 T/T genotypes as well as the ABCB1 1236TT-2677TT-3435TT haplotype was associated with higher cyclosporine concentrations (C0/D and C2/D ratio), suggesting that a pharmacogenetic pre-evaluation could be useful for a targeted treatment of transplanted patients . In addition, a correlation between the presence of UGT1A9-440C>T allele and the inter-individual variability in peak concentrations was observed in the same population (p < 0.05) [0–12h/MPA-D when compared with UGT1A9-275T carriers. Instead, MRP2 and UGT1A9 genotypes did not show significant differences in MPA C0, MPA-D, or MPA C0/D in 104 pediatric renal transplant recipients [0–12 h compared with IVS1 + 985AA carriers (p = 0.002). They also found a significantly higher dose-adjusted MPAG AUC0–12 h in patents with the UGT1A9-1818CT rather than patients with UGT1A9-1818CC (p = 0.002). Moreover, UGT1A9-440C>T and -331T>C mutant carriers were correlated with an increase in MPAG AUC0–12 h rather than wild-type (p = 0.028). In addition, carriers of UGT1A8*1/*1 had higher MPAG AUC0–12 h compared to carriers of UGT1A8*1/*1 and UGT1A8*2/*2 (p = 0.004). Finally, patients with the UGT1A7 622TT genotype had lower MPAG AUC0–12 h than UGT1A7 622CC (p = 0.012) [p = 0.046 and p = 0.021, respectively) [In solid organ transplant, mycophenolic acid (MPA) is used mainly as a CNI/sparing agent in order to diminish the dosage of these drugs and, by doing so, prevent their side effects. The most studied SNPs for the MPA are those involving ABCB2 and UGT family genes. In this regard, Fukuda et al., in 32 pediatric renal transplant recipients, observed that those heterozygous for MRP2-24T>C who also had UGT1A9-440C>T or UGT2B7-900A>G and MRP2-24T>C wild-type patients who carried both UGT1A9-440C>T and UGT2B7-900A>G presented a 2.2- and 1.7-times higher MPA dose compared to carriers of no UGT-SNPs ( < 0.05) . Moreovecipients . These rcipients  Table 1p < 0.001= 0.012) . Furtherctively) . Furtherctively)  and by SOn the other hand, only one study by Yang et al. that investigated several polymorphisms in different genes such as UGT1A8, UGT1A9, UGT2B7, ABCB1, ABCC2, ABCG2, SLCO1B1, and SLCO1B3 in 191 adult kidney-transplanted patients did not show an obvious impact of these genetic polymorphisms in metabolic enzymes and transporters on the PK of MPA .In summary, some papers involving a total of 372 patients demonstrated that the presence of UGT1A9 T-275A polymorphism was associated with lower MPA concentrations, while a paper including 125 patients showed that the UGT1A9 1399 T/T genotype was related to higher MPA through blood concentrations and lower MPA doses; however, considering that few pharmacogenetic studies in transplanted patients are present in the literature, the role of SNPs affecting MPA pharmacokinetics should be further investigated. p = 0.003 and p = 0.019, respectively). Regarding ABCB1 polymorphisms, sirolimus levels were higher in ABCB1 3435C>T with C/T genotypes than C/C and T/T in the late period (p = 0.038) [p < 0.0001) [0/D ratio in subject carriers of at least one CYP3A5*1 allele compared to patients with a homozygous CYP3A5*3 genotype (p < 0.05) in 85 Chinese renal-transplanted patients. On the other hand, no significant differences in SRL C0/D ratios were found between patient carriers of ABCB1 1236C>T, 2677G>T/A, and 3435C>T genotype compared to wild-type. In addition to this, haplotype analysis  within the ABCB1 gene, including ABCB1 1236C>T, 2677G>T/A, and 3435C>T SNPs, showed that carriers of CGC/CGC diplotype had a mean SRL C0/D about 30% lower than carriers of CGC/TTT or TTT/TTT diplotype regardless of their CYP3A genotype (p < 0.05). These findings suggested that the haplotype of ABCB1 might be a better indicator for the prediction of SRL blood concentration than single SNP [0/D was lower in CYP3A4 rs2242480, and both TAC and SRL C0/D were higher in CYP3A5 rs15524 TT carriers compared with CT and CC carriers. Moreover, patients with ABCB1 rs1045642 SNP and TT genotype had lower SRL C0/D at only 15 months after transplant [Pharmacogenetics studies involving mTOR inhibitors have primarily focused their attention on the effects of SNPs in CYP3A4, CYP3A5, and ABCB1 genes . A study= 0.038) . Moreove 0.0001) . Furtherngle SNP . Furtherngle SNP ,87 (Tablansplant .0 and C0/D ratio levels, while the effects of SNPs in CYP3A4 and ABCB1 genes in transplanted patients under mTOR-inhibitor therapy should be further investigated (In summary, some papers including a total of 272 patients demonstrated that the presence of CYP3A*1 allele was associated with lower sirolimus Cstigated .Since the introduction of CsA in 1983 and Tac in 1989 for immunosuppressive therapy in transplanted patients, great progress in terms of graft survival has been made [In this work, we reviewed the studies on the genetic polymorphisms associated with PK and PD variability in kidney-transplanted patients. Our results suggest that some SNPs are associated with the blood concentrations of immunosuppressant drugs in transplanted patients. Specifically, it has been observed that higher tacrolimus and cyclosporine doses are needed for patients carrying the CYP3A5*1 and/or CYP3A4*1B allele. Moreover, high cyclosporine blood levels were related to the presence of the CYP3A4*22 allele, ABCB1 2677 T/T and ABCB1 3435 T/T genotypes, as well as at ABCB1 1236TT-2677TT-3435TT haplotype . In acco0 and C0/D ratio levels, while the effects of SNPs in CYP3A4 and ABCB1genes should be further investigated for these drugs.Furthermore, our results showed that the presence of CYP3A*1 allele was associated with lower sirolimus CConcerning the mycophenolic acid, it has been observed that the UGT1A9 T-275A polymorphism was associated with lower blood concentrations, while the UGT1A9 1399 T/T genotype was related to higher MPA through blood concentrations and lower MPA doses even if few pharmacogenetic studies were conducted in transplanted patients under MPA treatment, and therefore, the role of SNPs affecting MPA pharmacokinetics should be further investigated . DespiteIn conclusion, several studies showed that SNPs in enzymes and transporters influence ISDs’ pharmacokinetics; therefore, a genetic pre-evaluation of kidney-transplanted patients could be useful for prescribing a targeted treatment in order to improve the efficacy of the therapy and, at the same time, minimizing overexposure and toxicity. Nonetheless, further pharmacogenetics studies should be conducted to confirm the role of genotyping as a standard practice in transplantation.