We previously found that NO inhibits catalases in Escherichia coli, stabilizing H2O2 around treated cells and promoting catastrophic chromosome fragmentation via continuous Fenton reactions generating hydroxyl radicals. Indeed, H2O2-alone treatment kills catalase-deficient (katEG) mutants similar to H2O2+NO treatment. However, the Fenton reaction, in addition to H2O2, requires Fe(II), which H2O2 excess instantly converts into Fenton-inert Fe(III). For continuous Fenton when H2O2 is stable, a supply of reduced iron becomes necessary. We show here that this supply is ensured by Fe(II) recruitment from ferritins and Fe(III) reduction by flavin reductase. Our observations also concur with NO-mediated respiration inhibition that drives Fe(III) reduction. We modeled this NO-mediated inhibition via inactivation of ndh and nuo respiratory enzymes responsible for the step of NADH oxidation, which results in increased NADH pools driving flavin reduction. We found that, like the katEG mutant, the ndh nuo double mutant is similarly sensitive to H2O2-alone and H2O2+NO treatments. Moreover, the quadruple katEG ndh nuo mutant lacking both catalases and efficient respiration was rapidly killed by H2O2-alone, but this killing was delayed by NO, rather than potentiated by it. Taken together, we conclude that NO boosts the levels of both H2O2 and Fe(II) Fenton reactants, making continuous hydroxyl-radical production feasible and resulting in irreparable oxidative damage to the chromosome.Immune cells kill invading microbes by producing reactive oxygen and nitrogen species, primarily hydrogen peroxide (H Mresponse .2O2 does so by diffusion, where it reacts with intracellular free iron (IF-iron) via Fenton chemistry (Fe(II) + H2O2→ Fe(III) +·OH + OH– to generate extremely reactive hydroxyl radicals, which can damage, among other molecules, chromosomal DNA  omal DNA , 7, 8. Domal DNA , 10. Speatalases , 12 and atalases , while Iatalases , 15 and ses , notabl2O2, when present simultaneously, kill WT E. coli at concentrations at which they are individually nonlethal . We have previously demonstrated that the nature of this phenomenon is not simply redundancy of two toxic treatments overwhelming antitoxicity mechanisms of the cells but potentiation of H2O2 toxicity by NO to cause catastrophic chromosome fragmentation (CCF) . NO, a radical species with one unpaired electron, can bind to other species with unpaired electrons, like transition metals    inc2O2 toxicity  to Fe(III), NO should be able to potentiate Fenton by cycling Fe(III) back to Fe(II), continuously feeding Fenton reaction with the disappearing key ingredient, reduced iron   . In contatalases , to stabA breaks . We alsotrations .2O2+NO killing. Is the catalase inhibition by NO and resulting H2O2 stability the cause of death or does it simply correlate with a concurrent respiration inhibition in WT cells? Another objective was to find additional cellular targets of NO, if they exist, that could potentiate H2O2 toxicity. Also, there could be mutants/conditions, in which NO would alleviate H2O2 lethality, as described for other organisms  with 2.5 mM H2O2 and 0.6 mM DEA NONOate (H2O2 + NO henceforth), two treatments that are bacteriostatic by themselves yet kill within minutes in combination, by precipitating CCF (A)  but could be stabilized by genetic inactivation of both catalases (C). The katE or katG single mutants also show reduced capacity to degrade H2O2 (C); however, unlike the sensitive katEG double mutant, both single mutants are resistant to H2O2-alone treatment . A. A2O2 ton 20 min B but couatalases C. The kaade H2O2 C; howeve2O2 in E. coli is similarly stabilized by NO (D and E). The unexpectedly rapid decline is because NO gas readily escapes from aqueous solutions, unless they are in bioreactors . For example, starting with 600 μM DEA NONOate, NO exposure is 24 μM x 20 min [this exposure inhibits catalases in Fig. 1D by ∼95% (and see below)], while starting with 60 μM DEA NONOate, NO exposure is reduced to 6 μM x 15 min, and the catalase inhibition is only ∼50% (see below). NONOate donors are known to yield actual NO concentrations much lower than the initial donor concentrations  NO-alone treatment (G). Additionally, the mutant is significantly less sensitive than WT to H2O2+NO treatment , which we also observed before for a similar H2O2+CN treatment , which is also well within the range for this mutant when grown in LB  by NO or as NO-inhibition of catalases. For example, the levels of IF-iron remain roughly the same in the katEG mutant (H and I), showing that catalase inactivation does not upset iron homeostasis in E. coli  to measure the concentration of IF-iron in our cells .2O2 toxicity . This increased iron makes the fur mutant only slightly sensitive to H2O2-alone treatment, yet significantly more sensitive than WT to H2O2+NO treatment (A). Thus, having more IF-iron available for Fenton makes cells vulnerable to H2O2, regardless of the presence or absence of NO.Previously, we showed that inactivation of catalases is necessary to explain NO potentiation of Htoxicity , but is r mutant ; in our cells do I. This ireatment A. Thus, fur mutant also accelerates the rate of chromosome fragmentation by H2O2+NO, leading to close to background levels of the remaining chromosomal DNA by 20 min of the treatment (B and C). In other words, intact chromosomal DNA all but disappears in the fur mutant treated with H2O2+NO. H2O2-alone treatment is even more interesting; coincident with the small drop in survival (A), there is a 25% loss of the chromosomal DNA in the first 10 min, which is then followed by a surprising resumption of label incorporation after 20 min (B and C). This fast ‘DNA recovery’ could be due to a faster scavenging of H2O2 in the fur mutant; however, contrary to this expectation, we found that the fur mutant actually scavenges H2O2 slower than WT cells (D), so that ∼1/3 of the original amount still remains after 20 min, at the time when essentially no H2O2 is detected in the medium of WT cells. A H2O2 scavenging defect has been reported previously in a fur mutant, wherein the sensitivity to H2O2 was attributed to low catalase activity rather than iron overload . However, regular ferritins (homologs of FtnA of E. coli) are known to release iron, if stimulated by chemicals like NO or CN . In particular, the ftnA inactivation alleviates H2O2+NO lethality (F), suggesting that FtnA depots provide a source of iron for the Fenton chemistry inside the cell. Contrary to this general idea and at the same time confirming the previous report (Woodmansee and Imlay 2003), we detected less IF-iron in WT cells treated with NO (H and I). It could be that, in the presence of NO, the ferritin-released iron is immediately bound by DNA or by other big molecules, like stable RNA, to become undetectable by EPR. We have observed something like this before in vitro with CN-complexed IF-iron, when the added plasmid DNA effectively sequestered iron away from CN complexes . Moreover, the double bfr dps mutant shows extreme sensitivity (F), suggesting that Dps and Bfr ferritins are redundant, and both are effective in sequestering iron when H2O2 is around. Thus, the H2O2+NO sensitivity of iron depot mutant confirms that in addition to H2O2 stabilization, NO potentiation does have a significant "iron dimension" to it, either releasing additional iron from iron depots FtnA or competing with IF-iron sequestration into Dps and Bfr.In contrast to the ferritin deficiency, both the reatment F. Moreovsitivity F, sugges2O2+NO sensitivity of ferritin mutants suggests a source of additional iron (FtnA) for promoting Fenton, but what is the mechanism of iron recruitment and keeping it reduced in the presence of H2O2? To state the problem differently, IF-iron will catalyze Fenton upon encounter with H2O2, but does it take multiple rounds of redox iron cycling to damage the chromosome beyond repair? To distinguish between the limited versus continuous Fenton, we inactivated the main Fe(III) → Fe(II) reduction enzyme, Fre. As the major siderophore reductase of E. coli, Fre catalyzes Fe(III) reduction to Fe(II) during iron acquisition from the environment (2O2 exposure: by reducing the oxidized Fe(III) back to Fe(II), Fre keeps Fenton reaction going as long as reducing equivalents and H2O2 are available (Increased or decreased Hironment . Howevervailable .fre mutant shows resistance to H2O2+NO (G) (2O2+CN (fre mutant has the same level of IF-iron as WT cells (E), indicating that (1) its decreased sensitivity to H2O2+NO is not due to a reduced levels of IF-iron and (2) no matter what the contribution of iron recruitment, continuous Fe(III) reduction back to Fenton-reactive Fe(II) is important for H2O2 toxicity in WT cells. Moreover, iron reduction by Fre seems to work in the same pathway as iron release from FtnA, as the H2O2+NO survival of the double ftnA fre mutant is not significantly different from those of single mutants (G). Overall, we conclude that the NO potentiation of the iron side of Fenton reaction significantly contributes to the overall lethality of the double treatment (H), whereas its complexity warrants further exploration, especially in comparison with the relatively straightforward NO stabilization of H2O2via inhibition of catalases.Indeed, the  H2O2+NO G 40), s, sfre mu , while in the combined treatment, H2O2 is stable due to catalase inhibition by NO (D). To make H2O2-alone and H2O2+NO treatments comparable, we used the katEG catalase-deficient background to ensure stability of H2O2 concentrations for the duration of experiment (C). Indeed, the katEG double mutant is equally sensitive to H2O2-alone and H2O2+NO treatments (katEG mutant has IF-iron levels comparable to that of WT cells (H), this sensitivity to H2O2-alone can be mostly attributed to the unscavenged H2O2.To probe the role of iron in the NO potentiation of Hatalases B, while on by NO D. To makperiment C. Indeedeatments ; at the WT cells H, this s2O2 is stable. To address it, we measured H2O2-alone and H2O2+NO sensitivity of the katEG fre mutant, whose catalase deficiency makes H2O2 stable (A), while the fre defect in iron reduction should restrict repeated cycles of Fenton. When confronted with H2O2-alone, the katEG fre triple mutant showed a hybrid sensitivity pattern: until 30 min of the treatment, the mutant was almost as resistant as the completely resistant fre mutant, but by 45 min, it became almost as sensitive as the katEG mutant (B). Since H2O2 levels remain constant in the katEG fre mutant (A), the initial resistance of this mutant compared to the katEG (Fre+) strain must be due to the absence of iron cycling. Then, the eventual H2O2-alone toxicity in the katEG fre mutant must have a different nature—for example, because of a sudden availability of Fe(II) from an earlier unavailable source or a switch to a different mode of killing. In fact, the pattern of complete initial resistance with an eventual deep killing was reminiscent of the delayed mode-two killing of WT cells by 25 mM H2O2 in the presence of deferoxamine (DF)+NO +NO .Figure 3katEG fre mutant to H2O2-alone (C), meaning that both explanations apply to the late toxicity of H2O2 in the katEG fre mutant. Another evidence consistent with mode-two killing comes from chromosomal fragmentation: in contrast to the katEG mutant, where H2O2-alone induces CCF, fragmentation in the katEG fre mutant is much reduced (D and E). Between 10 and 30 min, there is a loss of 20% intact chromosomal DNA, but no associated loss in viability. However, between 30 and 45 min, another 20% loss in chromosomal DNA results in loss in viability by three orders of magnitude. Thus, the killing of the katEG fre mutant by H2O2-alone cannot be explained by only double-strand DNA breaks.These two possibilities could be distinguished by chelating iron: additional iron recruitment should be blocked by DF, while mode-two killing should be insensitive to DF. DF addition rescued, albeit partially, the late sensitivity of the O2-alone C, meanin reduced , D and Efre single mutant, which is partially resistant to the H2O2+NO treatment, the katEG fre triple mutant is initially resistant to H2O2+NO, but after 30 min again loses viability to reach survival titers similar to the katEG mutant (F). The same timing of viability loss in the katEG fre mutant during H2O2-alone (B) versus H2O2+NO (F) treatments indicates a significant metabolic switch in this mutant after 30 min of H2O2 exposure. In general, the overall similarity of the two sensitivity patterns means no further NO potentiation in the katEG fre triple mutant. This not only confirms that catalases are targets of NO inhibition but also implies that Fre controls an independent NO-potentiation pathway. Iron chelators completely save the katEG fre mutant from H2O2+NO treatment (C), again showing that NO blocks an unknown target of mode-two killing by H2O2. The chromosomal fragmentation and DNA disappearance during H2O2+NO treatment is again reduced in the katEG fre mutant relative to the katEG mutant (G and H). Moreover, at 45 min there is again no significant increase in either fragmentation or DNA loss to explain the precipitous loss in viability at this late time point (F).Similar to the G mutant F. The saO2-alone B versus  H2O2+NO F treatmereatment C, again G mutant , G and Hme point F.2O2 toxicity (I), as illustrated by the fact that minimizing iron reduction with the fre defect decreases drastically the density of double-strand breaks in the katEG fre mutant cells, saving them during the first 30 min of H2O2-alone or H2O2+NO treatments. In addition, the two-fold reduction of IF-iron in the NO-treated cells (H and I) further elevates the importance of procurement of Fe(II) for H2O2+NO killing.Thus, iron reduction offers at least two candidate activities, FtnA and Fre, for NO potentiation of Htoxicity I, as illed cells , H and I2O2 toxicity other than catalase inhibition could be revealed if lower bacteriostatic H2O2 concentrations for the katEG mutant could be again potentiated by NO. However, the static H2O2 concentrations for the katEG mutant in the range of 0.25 to 0.5 mM are not potentiated with 0.6 mM NO (A), implying that catalases are the only NO targets in our standard treatment conditions. Nevertheless, we found that lower NO concentrations, in the range of 0.06 to 0.15 mM, do sensitize the katEG mutant to 0.5 mM H2O2 treatment (B and C), indicating existence of secondary NO targets. Moreover, blocking Fe(III)→Fe(II) reduction in the katEG mutant by the fre defect all but eliminates the sensitivity of the triple mutant to this milder 0.5 H2O2 + 0.06 NO treatment (C), suggesting that the potentiation of the secondary NO targets is still via Fe(II) generation fueling multiple Fenton cycles. In fact, in the previous paper, we reported that, in contrast to H2O2-alone treatment, H2O2+NO-treated katEG mutant is completely saved by iron chelation with DF .In principle, NO pathways to potentiate H.6 mM NO A, implyireatment , B and Creatment C, sugges with DF . Potentichelator , H and IkatEG mutant (B), we decided to investigate the role of iron reduction in the chronic H2O2 toxicity with a gene candidate approach instead, using the standard conditions (2.5 mM H2O2 + 0.6 mM DEA NONOate), in order to align our readouts with previous results and to be able to also use catalase-proficient strains. Our objective was to verify the general metabolic process powering the FtnA/Fre-dependent IF-iron source that sustains Fenton in NO-treated cells. There was a strong candidate, as both iron recruitment from ferritins (2 (I). So, how are the free flavins reduced?Although we did find new NO-potentiating conditions for the G mutant B, we decerritins , 70 and erritins , 47 are itins . The resulting NO-mediated respiratory block leads to accumulation of NADH, which in stably growing cells comprises a few percent of the total NAD pool  to the Fenton-catalyzing Fe(II), using reduced flavins as electron donors  , which translates into 4.5× increase of NADH fraction in the total NAD pools  . Respiration in vivo is measured as cellular oxygen consumption (depletion of oxygen from the chamber containing the oxygen electrode), where plunging oxygen levels indicate normal respiration , while stable oxygen levels indicate inhibited respiration. Respiration in WT E. coli is expectedly inhibited by 3 mM CN and, unexpectedly and transiently, by a mere 0.25 mM H2O2 , the latter being a peculiar artefact of oxygen production by the catalase reaction (A). Indeed, respiration is unaffected by 0.25 mM H2O2 in the katEG mutant, even though higher H2O2 concentrations start inhibiting it in this mutant (B), probably reflecting mode-two killing , showing exquisite sensitivity of ubiquinol oxidases to NO (we will return to this point later).Woodmansee and Imlay proposed that the immediate target of NO inhibition is the electron transport chain of the aerobic respiration , specifiFig. 4NAD pool , and thiFig. 4A) , 40. IndAD pools . In the reaction A. Indeeds mutant B, probab killing . This miE. coli, Cyo, Cyd, and App, active during aerobic/oxic respiration (A). We found that eliminating any one of the three individual ubiquinol oxidases has no phenotype, in that the cyoB, or cydB, or appC single mutants are completely resistant to H2O2-alone treatment, while showing WT-like sensitivity to the H2O2+NO treatment (E). However, the double mutants in any two of the three cytochrome oxidases made the mutant growth impractically slow, so we decided to block the electron transport chain one step earlier, at the level of NADH dehydrogenases (NDH), Nuo and Ndh (A). Single ndh or nuo mutants again showed WT resistance to H2O2-alone and similar to WT sensitivity to H2O2+NO treatment (F) suggesting redundancy of the two enzymes under our growth conditions. It was the mutant lacking both NDHs that should be unable to respire and was expected to accumulate NADH, boosting the Fe(III)→Fe(II) reduction and thus sensitizing cells to H2O2. The ndh nuo double mutant is viable, even though slow-growing (A); the mutant indeed accumulates ∼10 times more NADH compared to the WT cells, and this NADH level does not further respond to the NO treatment . The IF-iron is increased about 2-fold in the ndh nuo mutant, to the levels between the WT and the fur mutant (D). The ndh nuo double mutant was extremely sensitive to H2O2-alone, dying ∼3 times faster than WT cells during H2O2+NO treatment, but eventually to the same final level of survival of ∼10−3 (E). The sudden plateauing of the survival by 10 min is because the ndh nuo mutant scavenges H2O2 faster than WT cells, within 3 min (F). The only catalase-proficient mutants known to be (slightly) sensitive to H2O2-alone were fur (A) and sodAB (G) (H and I)  promoting iron reduction.While WT cells consume O 3 mM CN C. The IFr mutant D. The ndof ∼10−3 E. The suin 3 min F. The onwere fur A and sodnd sodAB G 21), l, l2 withodAB (G) , H and IH and I) , so the H levels B promoti2O2 lethality of ndh nuo double after 20 min of treatment so that, by 45 min, survival became 10-5 (E). At the same time, in the first 20 min, NO slowed down H2O2 killing of the ndh nuo double, although the rate was still faster than in the WT cells (E). These two opposite effects created a peculiar "hybrid" sensitivity curve, with "NO-protection" during the first 20 min switching to NO potentiation after 20 min (E). Since the ndh nuo double mutant is killed by both H2O2-alone and H2O2+NO treatments, this generally confirms the previous idea  to suggest that NO changes the nature of H2O2 toxicity in the ndh nuo mutant, probably by complexing IF-iron, as reflected by its decrease in NO-treated cells (H and I).Addition of NO exacerbated the Hame 10-5 E. At theWT cells E. These r 20 min E. Since ous idea , 40 thatifferent E to sugged cells , H and I2O2-alone fails to cause fragmentation in WT cells (ndh nuo mutant (G and H); moreover, the kinetics of CCF induced in the ndh nuo mutant by H2O2+NO appears slower (G and H), again suggesting that NO actually retards the precipitous H2O2-alone toxicity in this mutant. Both the killing by H2O2-alone and by H2O2+NO in the ndh nuo mutant are blocked by iron chelation with DF (I), indicating exclusively  mode-one killing. The blocking effect of DF confirms that respiration inhibition poisons cells via increased iron reduction.While 2.5 mM HWT cells , 22, it o mutant , G and Hs slower , G and H with DF I, indicafre defect makes the ndh nuo fre triple mutant significantly less sensitive to the H2O2-alone killing , implying that the residual sensitivity of these mutants to H2O2-alone is primarily due to the higher initial IF-iron levels. In short, our investigation reveals some correlation between initial IF-iron levels and sensitivity to H2O2 but also demonstrates that the rate of iron reduction and the H2O2 stability are more important for the in vivo effects of Fenton chemistry.Limiting iron reduction with the  killing , demonstelevated D, implyikatEG mutant  flow in ndh nuo. In order to genetically test whether the ndh nuo versus the katEG defects sensitize cells to H2O2-alone by distinct pathways or the same pathway, we constructed a katEG ndh nuo quadruple mutant, in which increased iron cycling is combined with H2O2 stability. The quadruple mutant was not sensitive to NO-alone, showed a somewhat higher sensitivity to H2O2+NO, and was exquisitely sensitive to H2O2-alone treatment (A). This sensitivity of the quadruple mutant reflected the extreme rates of chromosome fragmentation after either treatment (B and C). Iron chelation with DF completely suppressed the sensitivity of the quadruple mutant to both H2O2-alone or H2O2+NO (D), as well as the CCF induced by both treatments (E and F), indicating that this fast killing is strictly mode-one. In other words, the unknown targets of H2O2 mode-two killing in the katEG mutant  the catalase-deficient G mutant  and (2) o mutant . Both coreatment A. This sreatment , B and C H2O2+NO D, as weleatments , E and FG mutant  are effi2O2-alone and H2O2+NO sensitivity curves of the four strains, WT, katEG, ndh nuo, and katEG ndh nuo, proved insightful. For the H2O2-alone treatment, to which WT cells were completely resistant, the effect of two pairs of H2O2-sensitizing mutations turned out to be additive. As a result, the quadruple mutant responded to the H2O2-alone treatment with a composite sensitivity curve, in which the early (fast) killing aspect of the ndh nuo mutant was combined with the late (deep) killing effect of the katEG mutant (G). Apparently, the early killing was due to the increased iron cycling and IF-iron levels (reflecting the ndh nuo defect), while the continuous later killing was due to the stability of H2O2 (reflecting the katEG defect). We conclude that ndh nuo and the katEG represent two independent pathways protecting E. coli against acute H2O2 toxicity.Comparison of the kinetics of HG mutant G. Appare2O2+NO treatment of the katEG ndh nuo quadruple mutant; compared with H2O2-alone, we observed ∼2.5-fold slower rate of killing (A), the effect that we have already observed with ndh nuo double mutant (B). In other words, even though NO potentiates H2O2 toxicity in WT cells, it clearly protects some H2O2-hypersensitive mutants from the same H2O2 concentrations. As a result of this "NO-buffering", in contrast to the different kinetics of killing of WT, katEG, ndh nuo, and katEG ndh nuo strains with H2O2-alone (G), the same four strains treated with H2O2+NO show, somewhat counterintuitively, similar initial rates ending with depth of killing differences within two orders of magnitude (H). We conclude that the nature of H2O2 toxicity, though masked by cellular resistance mechanisms in WT cells, is complex (mode-one + mode-two), while NO potentiation amplifies H2O2 toxicity even in WT cells, but also makes it mechanistically simpler, converting it to a slower mode-one in all mutant combinations (I).This point was further corroborated by H killing A, the efe mutant B. In othO2-alone G, the saagnitude H. We coninations I.2O2 toxicity, if any, we treated the katEG ndh nuo quadruple mutant with varying concentrations of H2O2-alone, looking for residual NO targets that potentiate H2O2 toxicity with two DEA NONOate concentrations, 0.06 mM and 0.6 mM (D). As controls, we tested WT, katEG double mutant, and ndh nuo double mutant with the same treatments as the quadruple mutant (A–C). As shown previously . In contrast, in the katEG mutant, 0.75 mM, 0.5 mM, and 0.2 mM H2O2 can be potentiated by 0.06 mM NO (corroborating 2O2 concentrations and even starts protecting katEG mutants at the highest H2O2 concentrations (B). This was earlier interpreted to mean mode-two killing in the katEG mutant, with NO switching it to mode-one, slower killing (I) (ndh nuo mutant (C), H2O2 was lethal by itself and was further potentiated by both 0.6 mM and 0.06 mM NO, similar to WT cells . Finally, in the katEG ndh nuo mutant, the lethality of the three intermediate H2O2 concentrations was reduced by both NO concentrations , suggesting no more targets for H2O2 potentiation by NO in this mutant of E. coli.In order to reveal the remaining pathways of NO potentiation of Hd 0.6 mM D. As cone mutant , A–C. Aseviously , WT cellg better A. In contrations B. This w killing I 22). I. I2O2 too mutant C, H2O2 w2O2] in the katEG mutant (B), the remaining ability to potentiate these [H2O2] with NO in the ndh nuo mutant (C) and finally, the disappearance of this potentiation with the removal of catalases in the katEG ndh nuo mutant (D) suggest that catalase inhibition by NO is a major cause of lethality during the 2.5 mM H2O2 + 0.6 mM DEO NONOate treatment. We suspected that this was because respiration, the other NO potentiation route, was more sensitive to lower NO concentrations that were still not enough to potentiate H2O2 toxicity. To test this idea, we measured respiration inhibition, both in vitro (E) and in vivo (F), by various low concentrations of NO and found that both processes are completely inhibited by 30 μM DEA NONOate (E and F), the concentration that inhibits catalases by less than 50% and causes ∼one order of magnitude of H2O2 killing  (G). In general, the shapes of the two curves were different enough to conclude that respiration inhibition cannot be the main reason for H2O2+NO lethality at the millimolar H2O2 concentrations used in this study. In particular, 15 μM of DEA NONOate was sufficient to inhibit 90% of respiration in vivo, while this NO exposure still was not enough to cause lethality in H2O2-treated cells (G). At the same time, as shown previously, the shape of H2O2 scavenging inhibition by various [NO] coincided with the H2O2+NO killing curve (G), strongly suggesting that it is the former that drives the latter  G. In gened cells G. At theng curve G, stronge latter .2O2, so the latter becomes lethal for WT E. coli by continually inducing multiple double-strand DNA breaks, which lead to catastrophic fragmentation of the chromosome the cells cannot repair , yet H2O2 entry into the cytoplasm will (presumably) instantaneously oxidize all IF-Fe(II) to Fe(III), self-limiting Fenton's damage. In addition, Dps mini-ferritin will use H2O2 to sequester all remaining IF-iron; however, these two challenges notwithstanding, NO somehow ensures a flow of Fe(II) even in the presence of H2O2, making Fenton continuous.We have previously systematically examined the earlier observations , 40 thatt repair . We alsontiation . However2O2-treated cells are the iron depots ferritins (FtnA), along with the previously identified Fre, and continuous free flavin reduction supported by the increased pools of NADH, resulting from the respiration block by NO. We modeled NO-inhibition of ubiquinol oxidases by genetically blocking the preceding step of NADH oxidation in the ndh nuo double mutant, deficient in NDH activity. The ndh nuo mutant not only accumulates NADH but also has increased IF-iron and is killed by H2O2-alone even faster than by H2O2+NO, confirming respiration inhibition as another route of NO potentiation of H2O2 toxicity. The katEG ndh nuo quadruple mutant, that keeps both the H2O2 and Fe(II) levels high, shows a remarkable sensitivity to H2O2-alone and instead of being potentiated by NO is actually saved by it, demonstrating that NO potentiation pathways are exhausted in the mutant. Finally, we show that respiration is inhibited at low NO concentrations, at which little lethality in the H2O2+NO treatments is observed. In contrast, lethality correlates well with catalase inhibition, which happens gradually and over a range of higher NO concentrations, elaborating our previous conclusion about H2O2 stability by complementing it with the nature of a continuous source of Fe(II).This work explored the nature of this continuous source of Fe(II) in NO-treated cells. We found that a potential system maintaining the pool of reduced iron in H2O2 lethal  reduction and had to reject this idea; eventually they found that the proximal Fe(III) reductant is a free flavin, FADH2, produced by Fre, in the reaction driven by NADH . They showed that cyo cyd double mutant (the one with significant growth defects to be usable under our conditions) is extremely sensitive to H2O2-alone treatment and that NO fails to increase this sensitivity further  by proposing that both CN and NO recruit additional iron from FtnA ferritin, via the same Fre-driven reduction dependent on the elevated NADH levels, while mini-ferritin Dps and bacterioferritin Bfr sequester iron during H2O2 treatment; (3) our ndh nuo double mutant behaves similar to their cyo cyd double mutant , confirming the importance of respiratory chain in providing reduced iron for Fenton; (4) that no additional NO targets, beyond catalases and respiratory terminal oxidases, contribute to NO potentiation of H2O2 toxicity; and (5) last but not least, that DNA damage during the H2O2+NO or H2O2+CN treatments takes the form of CCF (breaking the chromosome into at least 100 pieces), explaining why the cells cannot repair it.Our previous studies , 22 and 2O2+NO sensitivity of the bfr mutant, especially in the bfr dps double mutant combination, appears to be the first time that E. coli bfr mutant shows any phenotype, in contrast to the strong iron-accumulation defects of the bfr mutants in Pseudomonas, for example  the known low concentration of (presumably) dispersed IF-iron should produce enough OH· for only limited damage to DNA, because the oxidative impact will be similarly dispersed around the cytoplasm; (2) the resulting DNA damage should be all single-stranded   , would show better survival and less fragmentation after H2O2-alone treatment. In contrast to this expectation, H2O2-alone treatment kills the ndh nuo mutant fast and deep (E), apparently because of the rapid fragmentation of its chromosomal DNA (G and H). Thus, DNA damage is mostly driven by a reason other than the preexisting levels of Fe(II) IF-iron.The deep, CCF-based lethality of oxidative damage is remarkable if we consider that its nature is based on Hlesions) ; (3) theA and C) , which hr mutant D, would and deep E, appareomal DNA , G and Hndh nuo mutants and the elevated NADH levels in the NO-treated cells (B). This continuous source of Fe(II) Fenton reactant explains not only the observed massive DNA damage but also why Fenton in H2O2+NO-treated cells is not self-limiting. Indeed, in the H2O2+NO-treated cells, fragmentation still continues 1 hour later, indicating that it depends not only on the stability of H2O2 in the presence of NO but also on the continuous source of Fe(II). An additional evidence for the importance of iron cycling over IF-iron levels is offered by the ndh nuo fre mutant, which has the same levels of IF-iron as its ndh nuo progenitor (D) but is much less sensitive to H2O2-alone treatment (fre defect in Fe(III) reduction.This reason, apparently, is continuous Fe(III) reduction to Fe(II), supported by high levels of NADH in the ed cells B. This cogenitor D but is reatment , becausendh nuo mutant suggests that rapid NADH oxidation by the electron transport chain in WT cells provides an effective shield against oxidative damage, while its inactivation puts Fenton chemistry in overdrive. It is also interesting to note that genetic inactivation of the ndh nuo pathway does more than to simply phenocopy NO-treatment. This is apparent for both WT background  and in the katEG background . The obvious explanation for the differences is that NO (at least at 0.6 mM DEA NONOate) does not cause the same level of NADH accumulation as the ndh nuo inactivation —and thus, the lower expected level of Fe(III) reduction. Perhaps there are minor ubiquinol oxidases (not inhibited by NO?) in E. coli yet to be characterized?Accumulation of NADH in the 2O2 + NO treatment in WT cells and since Fe(III), because of its higher charge, was expected to form complexes tighter than Fe(II) with big molecules like DNA (in vitro (H), as was also reported by others , implying the mode-two target is gone in the absence of NDHs. While the katEG mutants grow using aerobic respiration (C), the NDH mutants, ndh nuo and katEG ndh nuo, do not respire (C) and likely grow fermentatively. Thus, NO mimics the ndh nuo mutations and inhibits the fast mode-two killing, apparently by binding and protecting an undetermined target in the respiratory chain.Not only does NO potentiate Hcatalase , 77. As  present . In othemode-one D, implyipiration C, the ND respire C and likkatEG ndh nuo mutant with H2O2 will be similar to that observed with H2O2+NO in WT cells (compare A and D). However, the H2O2 lethality in the mutant is much higher than the H2O2+NO lethality in WT, revealing protective effects of NO in the WT cells. This is observed more clearly in the katEG ndh nuo survival of H2O2 challenge, where NO slows down cell killing considerably (A and D). In general, NO's effects in the cell vary, explaining contrasting effects of its combination with H2O2 in various mutants. We posit the two general ways NO could protect against H2O2 toxicity: (i) NO blocks a mode II target related to aerobic respiration (for example by binding heme iron) and (ii) NO acts as a general weak iron chelator.It isn’t only in the presence of chelators that NO shows its defensive side. Since NO targets both catalases and respiration, it could be expected conservatively that the loss in viability in the  compare , A and Diderably , A and D2O2 indirectly, via superoxide  were peroxynitrite a major contributor, the superoxide dismutase-deficient sodAB mutant would be more sensitive to H2O2+NO treatment, but in fact it is more resistant than WT (G); (2) peroxynitrite is toxic independently of iron (2O2 is blocked by iron chelation (via formation of peroxynitrite inside cells would at least affect WT cells and would kill the sodAB mutants—but it does not (A and G). Further experiments are needed to clarify any potential role of peroxynitrite formation in NO-potentiated H2O2 toxicity and its underlying chromosome fragmentation.In our experimental system, peroxynitrite contribution to the overall H than WT G; (2) pe of iron , 78, whehelation , 40; (3)does not , A and Gvia CCF. NO potentiation has two major routes, and both occur via its binding to heme-containing enzymes: (i) inhibition of catalases to make H2O2 stable and (ii) inhibition of respiration to boost iron recruitment and reduction in the presence of H2O2. In the future, it would be important to develop conditions with similar effects but utilizing more physiological low micromolar concentrations of H2O2 and NO. Due to its polyanionic nature, DNA binds iron avidly, creating a natural platform for Fenton chemistry. The resulting hydroxyl radicals should induce singly damaged sites including nicks in DNA, but their relationship to double-strand DNA breaks that fragment the chromosome is still unclear. Finally, it would be interesting to explore the interactions between ferritins and DNA in the presence of H2O2 and NO in vitro using plasmid-nicking assays.NO potentiates the intracellular Fenton reaction, causing lethality E. coli strains .Our  strains  are all  strains . Alleles strains . The mut2O2, deferoxamine mesylate, horseradish peroxidase, and o-dianisidine:2HCl were all purchased from Sigma. DEA-NONOate was from Cayman Chemical. A 60 mM stock solution of DEA-NONOate was prepared fresh each time by dissolving several milligrams of the chemical in 0.1 M NaOH. NAD+/NADH assay kit was from Abcam (ab65348).Catalase from bovine liver, H600 ∼ 0.3). At this point, the cultures were made 0.6 mM for DEA NONOate and/or 2.5 mM for H2O2 , and the shaking at 37 °C was continued. Viability of cultures was measured at the indicated time points by spotting 10 μl of serial dilutions in 1% NaCl on LB plates (LB medium above supplemented with 15 g of agar per liter). The plates were developed overnight at 28 °C, and the next morning, the pin-prick colonies in each spot with 10 to 200 colonies were counted under the stereomicroscope. All titers were normalized to the titer at time 0 (before addition of the treatment). For the iron chelator treatment, cultures grown as above were made 20 mM for deferoxamine mesylate 5 minutes before hydrogen peroxide treatment.To generate killing kinetics, fresh overnight cultures were diluted 1000-fold into modified lysogeny broth (LB) [10 g tryptone, 5 g yeast extract, 5 g NaCl, 250 μl 4 M NaOH per liter , bufferei, 0.1 mM EDTA, pH 7.8), kept ice-cold. Overnight cultures were diluted 1000-fold into LB8 medium and shaken for 3 h at 37 °C (A600 ∼ 0.3). At the desired timepoint after addition of H2O2 (±NO), 300 μl aliquots of cultures were withdrawn and cleared of the cells in a microcentrifuge for 1 min. Culture supernatant was diluted 1:10 into the potassium phosphate buffer. The diluted sample (667 μl) was mixed with 333 μl of the assay cocktail, and after 45 s at room temperature (∼20 °C), absorbance at 460 nm was measured.This follows our previous protocol . The 40 32P-orthophosphoric acid per milliliter of culture for 3 h at 37 °C (A600 ∼ 0.3) before addition of 0.6 mM NO + 2.5 mM H2O2 (or the indicated treatment). The reactions were stopped by addition of 312 μg of catalase (13 μl of 24 mg/ml stock), and aliquots of the culture were taken at the indicated times to make plugs. Cells of the aliquot were spun down, resuspended in 60 μl of TE buffer, and put at 37 °C. A total of 2.5 μl of proteinase K (5 mg/ml) was added, immediately followed by 65 μl of molten 1.2% agarose in the lysis buffer  held at 70 °C. The mixture was pipetted a couple of times before being poured into a plug mold and let solidify for 2 min at room temperature. The plugs were then pushed out of the molds and incubated overnight at 60 °C in 1 ml of the lysis buffer. Half-plugs were loaded into a 1.0% agarose gel in 0.5× Tris–borate–EDTA buffer and run at 6.0 V/cm with the initial and the final switch times of 60 and 120 s, respectively, at 12 °C in CHEF-DR II PFGE system (Bio-Rad) for 20 to 22 h. The gel was vacuum dried at 80 °C (on Whatman paper) and then exposed to a PhosphorImager screen (Fujifilm) overnight. The resulting signals were quantified with a PhosphorImager (Fuji Film FLA-3000).This generally follows our previous protocols , 83. All2O2 were measured using NO sensor ISO-NOP . Before calibration, the sensors were polarized in PBS  for over 12 h and 2 h, respectively. The NO sensor was calibrated by adding increasing concentrations of KNO2 to 0.1 M H2SO4+0.1 M KI. Changes in current (ΔpA) corresponding to increasing NO concentrations were measured to generate a standard curve. To measure NO released from DEA NONOate, LB or LB8 media were incubated at 37 °C on a temperature probe–controlled heated stir plate with stirring set at 170 rpm. The baseline current was recorded before DEA NONOate was added to the desired concentration, and the new current was recorded. The ΔpA calculated by subtracting baseline was used to determine the actual concentrations of NO. The H2O2 sensor was calibrated by adding increasing concentrations of H2O2 to PBS. Changes in current (ΔpA) corresponding to increasing H2O2 concentrations were measured to generate a standard curve (A). To measure H2O2 in cultures, 300 μl aliquots were withdrawn and cleared of the cells in a microcentrifuge for 1 min. The baseline current in PBS was recorded. Culture supernatant was diluted 1:10 into PBS, and the new current was recorded. The ΔpA calculated by subtracting baseline was used to determine the actual concentrations of H2O2.Actual concentrations of NO and H ISO-NOP , 84 and rd curve A. To meaA600 = 0.2 in LB8 as described above. Respiration was measured with a Digital Model 10 Clark-type oxygen electrode  at 37 °C, as described before , and lysed with French press. After pelleting debris by spinning for 20 min at 20,000g, supernatant was spun further for 2 h at 140,000g to collect the membrane pellet. Membranes were resuspended in 3 ml ice-cold potassium phosphate buffer by repeated pipetting and stored on ice at 4 °C. The NADH consumption assay was performed with 200 μM NADH and 20 μl membranes. After addition of CN or nitric oxide, when needed, the total reaction volume was made 1000 μl with potassium phosphate buffer.This was done as described . OvernigE. coli cells were prepared and processed as described  for 5 min. Cells were collected by filtration and resuspended in 700 μl ice-cold extraction buffer (from the Abcam kit). For NAD extraction, the resuspended cells were further diluted 50-fold in extraction buffer, lysed with 0.2 M HCl at 55 °C for 10 min, and neutralized to pH∼7.0 with NaOH. For NADH measurement, in the ndh nuo mutant, the filtered and resuspended cells were further diluted 10-fold in extraction buffer. For WT and NO-treated cells, the filtered and resuspended cells were used directly. Cells were lysed instantly with 0.2 M NaOH at 55 °C for 10 min and neutralized to pH∼7.0 with HCl. Cells were centrifuged at 12,000 rpm for 3 min to remove debris, and the collected supernatant was used directly in the Abcam NAD+/NADH assay protocol.Extracts from escribed . Brieflyet al.  and 20 mM DF (pH 8.0). The cells were incubated at 37 °C for 15 min with shaking at 220 rpm. The cells were washed twice with 2 ml of ice-cold 20 mM Tris-HCl pH 7.4 and then resuspended in 300 μl of ice-cold 30% glycerol, 20 mM Tris-HCl pH 7.4, transferred into an EPR tube and frozen on dry ice with ethanol. The A600 of the final cell suspension was measured after a 1:1000 dilution. Ferric chloride standards were prepared in the same Tris buffer containing glycerol. The spectrometer settings were the following: microwave power, 10 mW; microwave frequency, 9.05 GHz; modulation amplitude, 12.5 G at 100 KHz; time constant, 0.032; temperature, 15°K.The procedure generally follows the protocol by Sen et al.  with somAll data described in the manuscript are contained within the manuscript itself.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Ototoxicity is one of the main dose-limiting side effects of cisplatin chemotherapy and impairs the quality of life of tumor patients dramatically. Since there is currently no established standard therapy targeting hearing loss in cisplatin treatment, the aim of this study was to investigate the effect of nimodipine and its role in cell survival in cisplatin-associated hearing cell damage. To determine the cytotoxic effect, the cell death rate was measured using undifferentiated and differentiated UB/OC−1 and UB/OC−2 cells, after nimodipine pre-treatment and stress induction by cisplatin. Furthermore, immunoblot analysis and intracellular calcium measurement were performed to investigate anti-apoptotic signaling, which was associated with a reduced cytotoxic effect after nimodipine pre-treatment. Cisplatin’s cytotoxic effect was significantly attenuated by nimodipine up to 61%. In addition, nimodipine pre-treatment counteracted the reduction in LIM Domain Only 4 (LMO4) by cisplatin, which was associated with increased activation of Ak strain transforming/protein kinase B (Akt), cAMP response element-binding protein (CREB), and signal transducers and activators of transcription 3 (Stat3). Thus, nimodipine presents a potentially well-tolerated substance against the ototoxicity of cisplatin, which could result in a significant improvement in patients’ quality of life. Nimodipine belongs to the group of 1,4-dihydropyridines and exerts its effects by binding to the alpha1 subunit of L-type calcium channels, thereby decreasing calcium influx via negative allosteric inhibition . AlthougMentionable previous studies of our group showed a protective effect of nimodipine ,10,11 prCisplatin, a representative of platinum-based chemotherapeutics is still widely used in the antitumor therapy of many solid tumors, including testicular, ovarian, bladder, non-small cell lung carcinoma, and head and neck tumors ,18,19. DAs it is one of the main proteins nitrated and diminished expressed by oxidative stress induced by cisplatin, LMO4 has been linked to the main side effects ,23. TherThe murine cell lines UB/OC−1 a,c and Ubrain-specific homeobox/POU domain protein 3.1 (Brn3.1), Myosin 6 (Myo6), Myosin 7a (Myo7a), and also α9 acetylcholine receptor (α9AChR) gene expression was shown in both types of hair cells compared to its level in the undifferentiated state  and 10.5% ± 0.6%  at 100 µM cisplatin for UB/OC−1 cells  in cell culture supernatant was used for cytotoxicity measurement induced by 20 µM, 50 µM and 100 µM cisplatin. The results showed a nimodipine-dependent reduction in cell death rate for both UB/OC−1 and UB/OC−2 cells a,b. Withp < 0.05, p < 0.05) and at 50 µM cisplatin from 23.1% ± 2.3% to 13.5% ± 1.0%  and 12.6% ± 2.0% . At 100 µM cisplatin cell death decreased from 28.3% ± 2.5% to 17.0% ± 0.9%  and 11.9% ± 1.2%  in comparison to cells treated with EtOH. Further multiple statistical comparisons are listed in The analysis of UB/OC−2 cells after nimodipine pre-treatment and stress induction demonstrated a significant reduction in cytotoxicity at 20 µM cisplatin from 26.9% ± 2.5% to 16.8% ± 1.6%  and 14.1% ± 5.6% . For cells treated with 50 µM cisplatin and nimodipine a non-significant reduction in cell death from 46.3% ± 13.2% to 32.7% ± 12.7%  and 23.5% ± 7.1%  (p < 0.05) for UB/OC−1  a was det UB/OC−1 a after n UB/OC−1 b than thp < 0.05). Through nimodipine treatment, a decrease in cell death from 46.4% p < 0.05) and 22.6% p < 0.05) and from 69.8% p < 0.05) cells treated with 20 µM and 100 µM cisplatin was shown. However, only a lower reduction was measured from 18.4% No significant reduction was detected between EtOH treated cells and control cells. Cells without stress (0.9% NaCl) showed an increase in LDH level at both 10 µM and 20 µM nimodipine to 27.0% 7% n.s., b and 28.isplatin b. Immunoblot analysis showed a strong reduction in LMO4 after cisplatin treatment . Under 2Undifferentiated UB/OC−1 and UB/OC−2 cells treated with 10 µM and 20 µM nimodipine showed increased phosphorylation of Akt at serine residue 473 and phosphorylation of CREB at serine residue 133, without and during stress conditions a,b 6]. . 6]. IncIn the differentiated state, the immunoblots detected an increase in phosphorylation of Akt at serine residue 473 and CREB at serine residue 133 under 20 µM cisplatin, which increased after 10 µM and 20 µM nimodipine pre-treatment for both UB/OC−1 c and UB/p < 0.05) and 22.6% ± 11.2%  was observed at 0.9% NaCl, just as cells treated with 20 µM cisplatin to 57.5% ± 16.2%  compared with the solvent control. Treatment with 50 µM cisplatin resulted in a decrease to 83.9% ± 7.2%  and 63.5% ± 15.1% . In comparison to EtOH-treated cells, nimodipine-treated UB/OC−2 cells showed a decrease in the amount of intracellular calcium at 0.9% NaCl to 85.9% ± 9.8%  and 69.9% ± 2.9% , while nearly no reduction to 99.3% ± 8.8%  and 96.2% ± 4.6%  was detected at 20 µM cisplatin (p < 0.05). In both data analyses, it can be seen that the reduction in calcium by nimodipine after cisplatin treatment is less effective and even leads to an increase when looking at the data of UB/OC−2. Further multiple statistical comparisons are listed in The effect of different concentrations of nimodipine and cisplatin on the intracellular calcium concentration was investigated using the fluorescent dye Cal-520, which detects free calcium. The solvent control of nimodipine (EtOH) was set to 100% and compared with nimodipine-treated cells depending on the stress induction with cisplatin . In the isplatin b. The anCisplatin is widely used in antitumor therapy of solid tumors such as testicular, ovarian, bladder, non-small cell lung carcinoma, and head and neck tumors ,18,19. TThe formation of reactive oxygen and nitrogen species leads to the downregulation of the transcription regulator LMO4, during chemotherapy with cisplatin a 18,23,,23,26,32As in previous studies on neuronal and Schwann cells, nimodipine pre-treatment led to increased activation of Akt at the serine residue 473 as well as increased activation of the transcription factor CREB at serine residue 133 by phosphorylation . Both ceFurthermore, higher expression of LMO4 can lead via the stabilization of glycoprotein 130 to increased activation of its downstream target Stat3 via the Jak/Stat pathway. This is known to cause increased expression of anti-apoptotic genes and thus downregulation is associated with apoptosis and thus ototoxicity ,33. RosaThe maintenance of intracellular calcium homeostasis plays an essential role in a lot of important cell functions and cell survival and is regulated by a complex system ,42. NimoIn summary, it can be concluded that nimodipine reduces the cytotoxic effect induced by cisplatin accompanied by the upregulation of LMO4 and the associated activation of anti-apoptotic pathways in vitro .Clinical trials focusing on the treatment of ototoxic effect triggered by cisplatin have already been carried out numerous times in recent years and, since no standard therapy has yet been approved by the Federal Drug Administration, many studies with a potentially good outcome are currently underway . OtoprotBy reducing the side effects of cisplatin through nimodipine pre-treatment, a significant improvement in patients’ quality of life and better utilization of the chemotherapeutic effect could be achieved, if this protective effect can also be demonstrated in vivo. However, the most effective therapy against the side effects of cisplatin is not only the molecular inhibition of intracellular apoptosis mechanisms but also the optimization of the tumor-centered effect and blockade of the formation and impact of the toxic degradation products of cisplatin . In addiTM  and 50 Units/mL Interferon gamma  in undifferentiated state at 5% CO2 and 33 °C. For differentiation, cells were cultured as previously described [2 and 39 °C followed by verifying through gene expression analysis of the hair cell markers Brn3.1, Myo6, Myo7a and α9AChR after 14 days via quantitative polymerase chain reaction (qPCR). All experiments were performed in both differentiated and undifferentiated states  and UB/OC−2  were obtained from Ximbo  and have been established by Matthew Holly . Both were cultured in Eagle’s Minimum Essential Medium  supplemented with 10% fetal bovine serum , 100 U/mL penicillin, and 100 mg/mL streptomycin , 2 mM GlutaMAXescribed  with supv/v) served as control  diluted in EtOH . Cells treated with equal amount of EtOH  and cytotoxicity was measured by the activity of LDH as a marker for cell death using Cytotoxicity Detection Kit  according to manufacturer’s instructions. In brief, 100 µL cell culture supernatant in triplicates per sample and 100 µL reaction mix were incubated in the dark for 30 min. Absorbance was measured at 492 nm with Tecan Reader F2000 Pro  at four definite points of the wells. Absorbance of cells lysed with 2% Triton X-100  served as positive control (100% cell death) while medium signal without cells served as background signal. The calculation of the cell death rate was performed as described before [The 5 × 10d before .6 cells were seeded in Petri dishes  and treated as shown in TM Protease and Phosphatase Inhibitor Single-Use Cocktail . The proteins were extracted with 1x LDS Sample Buffer  and heated at 70 °C for 10 min, followed by protein concentration measurement performing bicinchoninic acid (BCA) assay using PierceTM BCA Protein Assay Kit  according to manufacturer’s instructions. After 5% β-mercaptoethanol  was added, the samples were again heated at 70 °C for 10 min. To separate protein, SDS-PAGE was performed using NuPAGETM 4-12% Bis-Tris Gels (1.5 mm × 10 well)  and 1× NuPAGE® MES SDS Running Buffer  followed by blotting onto 0.2 µm or 0.45 µm nitrocellulose membranes  depending on molecular weight of the proteins to be detected (0.2 µm < 20 kDa). After blocking with 5% skim milk  diluted in tris-buffered saline (TBS) with 0.1% Tween® 20 , membranes were incubated overnight at 4 °C with primary antibodies  and signals were detected with a CCD camera .The 2 × 10tibodies . Afterwatibodies  for at l4 cells were seeded in black flat 96-well plates . For measurement of intracellular calcium concentration Cal-520 No Wash Calcium Assay  was used. Five µM of the fluorescent dye Cal-520  were added to Hank’s Balanced salt solution  supplemented with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer , 0.02% PluronicTM F-127  and 1 mM Probenecid  from which 100 µL were given to each well. Afterward, incubating the plate for 90 min at 33 °C or 39 °C and adding 100 µL Probenecid dissolved in Minimum Essential Medium  intracellular calcium concentration was measured at Ex/Em = 490/525 nm with Tecan infinite F2000 Pro .The 1 × 10® RNA Plus  following manufacturer’s instructions. The RevertAid First Strand cDNA Synthesis Kit  was used to transcribe 2 µg of RNA into cDNA. 20 ng cDNA, 5 µM specific primer  in a total volume of 20 µL were prepared respectively. qPCR was performed with Rotor-Gene Q .RNA was isolated with NucleoSpinc primer , and 1× p values were <0.05. Data were expressed as the mean ± S.D.Statistical analysis of three independent replicates was performed with unpaired, two-sided student’s t-test and one-way ANOVA followed by Tukey’s post hoc test . Significance was accepted if Nimodipine not only acts via vasodilation as a calcium channel antagonist but also shows a protective effect against cell death induced by cisplatin in auditory cells. This offers the possibility to extend the application of vasospasm prevention after subarachnoid hemorrhages to the treatment of prevention of cisplatin-mediated side effects or auditory cell damage associated with degenerative diseases.The transcriptional regulator LMO4 and the accompanied increased activation of anti-apoptotic pathways via Akt, CREB, and Stat3 seemed to be associated with the otoprotective effect. Future studies should aim to prove the obtained results in vivo in order to enable future application in patients.


The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highly abundant and central source of reactive oxygen species. NOX2 is a key enzyme of the innate immune system involved in antibacterial response, but excessive NOX2 activity is involved in oxidative stress and inflammation in many diseases. Inhibition of NOX2 has great potential as a therapeutic strategy. An intriguing pharmacological approach for inhibiting NOX2 is to target the p47phox subunit and thereby block the protein-protein interaction with p22phox, whereby assembling and activation of NOX2 is prevented. However, the shallow binding pocket of p47phox makes it difficult to develop drug-like p47phox/p22phox inhibitors. Recently, the small molecule LMH001 was reported to inhibit the p47phox/p22phox interaction, reduce endothelial NOX2 activity, and protect mice from angiotensin II-induced vascular oxidative stress. These noteworthy results could have significant impact on the field of NOX2 pharmacology, as specific and efficient inhibitors are scarce. Here, we synthesized and tested LMH001 to have it available as a positive control. We established a robust synthetic route for providing LMH001, but subsequently we experienced that LMH001 is chemically unstable in aqueous buffer. In addition, neither LMH001 nor its breakdown products were able to inhibit the p47phox/p22phox interaction in a non-cellular fluorescence polarization assay. However, LHM001 was a weak inhibitor of NOX2 in a functional cell assay, but with same low potency as one of its breakdown products. These findings question the activity and suggested mechanism of LMH001 and constitute important information for other researchers interested in chemical probes for studying NOX2 biology. NADPH oxidase 2 (NOX2) is a multi-subunit enzyme complex and a main source of reactive oxygen species (ROS). In phagocytes, NOX2 produces ROS in relation to respiration burst as a defense mechanism against bacterial infections . In otheDue to its central role in disease, NOX2 is an attractive drug target. However, developing high-quality chemical probes  and precOne way of reducing NOX2 activity is to target the intracellular organizer subunit p47phox with small molecules. This approach inhibits the protein-protein interaction (PPI) between p47phox and p22phox , thereby1 (2 (1 inhibits the binding between a fluorescently-labelled p22phox peptide and p47phoxSH3A−B in a cell-free fluorescence polarization (FP) assay. However, despite binding specifically to the target, p47phox, cell-based activity data are lacking for both 1 and 2. In contrast, CPP11G and CPP11H block translocation of p47phox to the membrane and diminish the subsequent NOX2 assembling and activity in cells -tagged p47phoxSH3A−B and a fluorescently FITC-labelled p22phox peptide.Recently, LMH001  was presLMH001 could provide significant progress to the field, considering the lack of reliable and potent chemical probes for studying p47phox/p22phox inhibition. Thus, incited by the results in Fan et al., we synthesized and tested LMH001 with the aim of using it as a positive control in future studies.1H NMR, 13C NMR), 2D NMR , and liquid chromatography–mass spectrometry (LC−MS). The synthetic procedures are described in LMH001 was obtained as white solid in high purity (>95%) by chemical synthesis in nine steps. The final product was thoroughly characterized by 1D NMR  between the fluorescent peptide probes (cyanine 5 (Cy5)-p22phox149−162 and carboxytetramethylrhodamine (TAMRA)-p22phox151−162) and p47phoxSH3A−B  were added to a fixed concentration of the p22phox probe  in a final DMSO concentration of 2%. Assay plates were incubated in darkness for 2 min (explorative method) or 20 min (standard method) at room temperature before measuring the FP levels. The Cy5- and TAMRA-probes were measured at excitation/emission values of 635/670 and 530/585 nm, respectively. Kd values were derived by fitting the FP values to the one-site specific binding equation: Y = Bmax × X/(Kd + X), with Bmax being the maximal FP value, X is the p47phoxSH3A−B concentration, and Y is the variable FP values.First, saturation experiments were performed to determine the binding affinities (SH3A−B and the fluorescent peptide probes in 1×HBST (2% DMSO). The non-fluorescent p22phox151−162 peptide (Sequence: Ac-PPTNPPPRPPAE-NH2) was used as a control. The assay was performed by adding increasing concentrations of the test compounds  to a fixed concentration of p47phoxSH3A−B  and peptide probes  using the same conditions as described above. FP values were fitted to the equation Y = Bottom + (Top-Bottom)/{1 + 10^[(LogIC50-X)*HillSlope]}, where X is the logarithmic value of the compound concentration. Hereby, the IC50 value was obtained, which together with the Kd value, probe and p47phoxSH3A−B concentrations was used to calculate the theoretical competitive inhibition constant, the Ki value. An additional explorative experiment of testing the activity of LMH001 was done in water instead of 1×HBST buffer. Here, 5 and 8 µM of p47phoxSH3A−B concentration was used for Cy5-p22phox149−162 and TAMRA-p22phox151−162, respectively.Synthesized compounds were then tested for their ability to inhibit the interaction between p47phox2O/acetonitrile (MeCN)/formic acid, 95:5:0.1 v/v%) to buffer B  and a flow rate of 1 mL/min was used. Injection volume was 5 µL. Total sample run time was 6 min, and the percentage of buffer B increases from 0% to 100% during 0.25–4.25 min, maintains at 100% until 5 min, then decreases to 0% at 5.10 min, and stays at 0% until 6 min. A 10 mM DMSO stock of LMH001 was diluted into 100 µM in different assay buffers including MeCN, water, 1 × PBS , HBSS , 100 mM PPB , 1×HBST, Hepes , Tris , and a 63.5:36.5:0.1 v/v% mix of H2O/MeCN/trifluoroacetic acid (TFA) and incubated for 5 and 30 min before analysis by LC−MS. The percent LMH001 remaining over time was calculated relative to 5 min of LMH001 in MeCN.The stability of LMH001 was tested by electron spray ionization (ESI) LC−MS using an Agilent 6130 mass spectrometer instrument coupled to an Agilent 1200 high-performance liquid chromatography system installed with a C18 reversed-phase column  and a diode array detector. A binary solvent system of buffer A  in buffer (1×HBST) or water for 15 min before analysis by LC−MS. LC−MS spectra were obtained using the same LC–MS system, solvents, and gradient as for the chemical stability assay, but here the LC–MS was installed with a Poroshell C18 reverse-phase column . Injection volume was 10 µL and the protein eluted at 3.2 min.LMH001 was tested for covalent reactivity towards p47phox ebselen . 200 µM 2 culture flasks in RPMI-1640 medium  containing 10% foetal bovine serum and penicillin/streptomycin (50 U/mL) at 5% CO2/95% air in a humidified atmosphere at 37°C. PLB-985 cells (2 × 105 cells/mL) were differentiated into granulocyte-like cells by addition of DMSO  for 4–5 days. DMSO stocks (100 mM) of LMH001 and its breakdown products, compounds 13 and 15, were diluted 10-fold in PBS and transferred to a 384-well plate (5 µL per well). The assay was performed in a volume of 50 µL per well in transparent non-binding surface 384-well plates (Corning #3640). Differentiated PLB-985 cells (106 cells/mL) were resuspended in HBSS buffer (GIBCO #14025050) supplemented with 1.6 mM WST1  and dispensed into the 384-well plate containing serial dilutions of compounds for 10 min. NOX2 was activated by addition of phorbol-12-myristate-13-acetate PMA . Absorbance (OD440) was measured with a Spectramax Paradigm reader (Molecular Devices) for 3 h. To graph the data, the rate of superoxide was calculated for the first hour. The flavoprotein inhibitor diphenyleneiodonium (DPI) and the NOX2 small molecule inhibitor GSK2795039  protection group of the aromatic alcohol of the acid intermediate was cleaved off during the saponification and a re-protection step had to be introduced  with a reference to the authors’ patent application describing LMH001 and other bi- and tri-aromatic compounds as NOX2 inhibitors . The pattroduced . Insteadtroduced . The synescribed . LMH001 ohydride .Ki value. We here tested LMH001 against peptide probes (Cy5-p22phox149−162 and TAMRA-p22phox151−162) based on the PRR of p22phox and their interaction with the tandem SH3 domain of p47phox (p47phoxSH3A−B), as done previously   . To inve.054 µM) , which i.054 µM) . We ther.054 µM) . The sam.054 µM) . Interes.054 µM) . Finally.054 µM) .13 and 15 for LMH001  . Also, Lrmediate . Howeverin water .13 and 15, we performed concentration-dependent measurements of the impact of the compounds on NOX2-dependent superoxide generation by PLB-985 cells stimulated with the phorbol ester PMA (50 = 54 µM), approximately 200 times less potent than the activity observed by Fan et al. in COS-phox cells (0.24 µM). Interestingly, the breakdown product 15 showed an inhibition pattern (IC50 = 63 µM) globally overlapping with LMH001, while the other breakdown product 13 displayed no activity. Overall, this suggests that the weak measured NOX2 inhibition of LMH001 is due to the breakdown product 15.In order to evaluate a potential inhibitory effect of LMH001 and the two breakdown products ster PMA . LMH001 Chemical compounds that activate or inhibit the activity of disease-relevant proteins are valuable tools for target validation during the drug discovery process and can be used as lead compounds. However, the value as a probe for studying biological events depends, among other things, on the reliability of the data and specificity of the compound . Many NOSH3A−B/p22phox PPI in FP , but this was not the case. Instead, considering that LMH001 contains substructures shared by PAINS, such as a catechols and phenolic Mannich bases, we tested if LMH001 covalently reacted with p47phoxSH3A−B. Indeed, LMH001 led to partial conversion into a modified protein with an increased molecular weight (MW) corresponding to a Mannich base-like reaction via a quinone methide intermediate of LMH001. Although, this covalent reaction apparently did not functionally affect the interaction between p47phoxSH3A−B and the p22phox peptide probe in our FP assay, it may induce effects in assays using other p47phox protein constructs or in cells.Unfortunately, our FP results clearly showed that LMH001 was unable to inhibit the p47phoxhed data . FurtherThere could also be other explanations for the disagreement between our FP data and those of Fan et al. Perhaps LMH001 was not made in pure form in the original study and an impurity was responsible for the apparent activity. Of notice, chemical characterization data for compound identity and purity are lacking in the paper by Fan et al. Alternatively, a very specific buffer might be needed for obtaining chemical stability of LMH001 and activity in the FP assay. Fan et al. did not specify the assay buffer. Notably, molecular docking used by Fan et al. to design LMH001 cannot count as evidence for binding , and theSH3A−B pocket, or activity via other proteins than p47phox or even NOX2. However, since LMH001 is a chemically unstable molecule, any pharmacological effect it may have is likely due to degradation products or metabolites. In Fan et al., a very short half-life of 0.042 h (2.5 min) was reported after iv injection in mice, supporting this notion. Fan et al. observed that LMH001 was >200-fold more potent in inhibiting superoxide produced by NOX2 in cells activated by PMA (IC50 = 0.24 µM), compared to antioxidant quenching of superoxide produced in a cell-free xanthine oxidase assay. In our cell assay, however, LMH001 only demonstrated weak NOX2 inhibitory activity (IC50 = 54 µM), which is in the potency-range of the antioxidant effects reported by Fan et al. Interestingly, the catechol breakdown product 15 showed similar inhibitory activity as LMH001. This indicates that the weak NOX2 inhibition of LMH001 was in fact due to the compound degrading to 15 causing antioxidant activity.Despite the lack of evidence for LMH001 being able to bind p47phox, its instability in buffers, and the ambiguous FP data, LMH001 might still have genuine pharmacological effects. Alternative mechanisms could be antioxidant properties, inhibition of NOX2 by other ways than binding into the p47phox13 and 15 is the structural similarity to apocynin  or even its trimer (in vitro (via some of the mechanisms reported for apocynin and its analogues.Another interesting aspect of LMH001 and its breakdown products apocynin . Apocynis trimer . Apocynis trimer . Numerouin vitro . Similarin vitro . NOX2 acin vitro . Vanilliin vitro . An altein vitro , is thatin vitro , and thein vitro . OverallSH3A−B or inhibits the p47phoxSH3A−B/p22phox PPI. Furthermore, LMH001 is highly unstable in standard buffers and is thereby in practice an untestable compound. The main degradation products 13 and 15 were also inactive in FP. Consequently, the reported pharmacological activity of LMH001 (SH3A−B pocket by either LMH001 or its breakdown products. Weak NOX2 inhibition was observed for LMH001 in cells, but this was likely caused by degradation into 15 and resulting antioxidant activity. We also noticed the structural similarity of LMH001 and the degradation products 13 and 15 with apocynin–a well-known but pharmacologically very complicated and unspecific molecule. Based on this, we hypothesize that LMH001 might work under some biological conditions as a prodrug for apocynin-like compounds. Overall, LMH001 is in our view not a useful chemical probe for investigating NOX2’s role in biology until its mechanism of action is elucidated in details.In conclusion, we were not able to reproduce the FP data reported by Fan et al. Our data suggest that LMH001 does not bind p47phoxf LMH001  cannot b


Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze–thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of their normal physiology, including hormonal regulation. These organoids may provide an environment that mimics the in vivo equine oviduct and facilitate improved in vitro embryo production in equids. The oviduct  is not merely a tube for the transport of sperm, oocytes, and embryos. The oviduct is critical for successful fertilization as the final maturation of oocytes and maintenance of the sperm reservoir to facilitate sperm capacitation as well as fertilization and early embryo development occur in this organ . Both seIn mares, a conventional in vitro fertilization (IVF) protocol has not been reliable as an artificial reproductive technique (ART), most likely due to the limitations of the extant culture conditions that fail to support equine sperm capacitation . IntracyOrganoids are three-dimensional (3D) cell clusters that are generated from either stem cells or organ progenitor cells that self-assemble and remain genetically and phenotypically stable during a long-term cell culture (months) ,5. OrganEquine oviductal organoids may provide an in vitro environment that is more similar to conditions in vivo, which may allow improvements in ARTs such as IVF or ICSI. Additional applications include addressing knowledge gaps in the understanding of normal physiological processes in the mare that are challenging to evaluate in vivo  because the direct visualization of the oviduct in vivo is not straightforward. Our objective was to develop a reliable and repeatable protocol to establish viable organoids in the long term using oviductal tissue from mares. Here, we describe the establishment of mare oviductal organoids utilizing fresh tissue and the re-establishment of oviductal organoids following cryopreservation and thawing. This protocol was developed by adapting and modifying a combination of previously reported culture protocols for human and mouse oviductal organoids and human, mouse, and mare endometrial organoids ,12,13,14No. 20 scalpel blade 100 × 15 mm tissue culture dishes 35 × 10 mm tissue culture dishes 48-well culture plates 2 mL cryovials 50 mL conical tubes Microcentrifuge tubes 12.5, 20, 200, and 1000 µL pipette ClipTip tips 40 µm cell strainers 100 µm cell strainers 20 mL syringes 0.22 µm syringe filters IcePhosphate-buffered saline MEM Eagle with Earle’s salts RPMI 1640 DMEM/F12 without phenol red with glutamax HEPES Penicillin–streptomycin Sodium pyruvate Glutamax Bovine serum albumin Fetal bovine serum B27 Plus N2 Insulin–transferrin–selenium Nicotinamide Recombinant human EGF Recombinant human FGF-10 Recombinant human Noggin A83-01 N-acetyl-L-cysteine Y-27632 SB202190 Dimethyl sulfoxide Collagenase V Dispase II Matrigel or Cultrex UltiMatrix  Cell Recovery Solution or Cultrex Organoid Harvesting Solution 4% paraformaldehyde 2% agarose prepared with water 70% ethanol.Surgical scissors Thumb forceps Biosafety cabinet ThermoMixer C shaking–heating block 10, 20, 200, and 1000 µL ClipTip pipettes Inverted microscope TMS Sorvall Legend Micro 21R Microcentrifuge  2 incubator Heracell VIOS 160i COCool Cell Liquid nitrogen and storage tanks Extra-long forceps Cryo gloves IsoTemp water bath .Thaw Matrigel/Cultrex in 4 °C before starting (can thaw overnight).Warm a 48-well plate to 37 °C.Prepare the handling medium  and digestion solution (5 mL per oviduct).Transport the equine oviduct in sterile PBS (room temperature for immediate processing or maintained overnight at 4 °C).Place oviduct in a 100 × 15 mm Petri dish with 5 mL handling medium.Cut the oviduct open lengthwise with scissors and scrape the lumen of the oviduct with a scalpel blade to release the cells into the handling medium.Transfer the handling medium with the scraped cells into the microcentrifuge tubes. Discard the remaining oviductal tissue.g for 6 min at room temperature (20–22 °C). Remove and discard the supernatant.Centrifuge at 600× Add the digestion solution (see Reagents Setup below) to the cell pellets (1 mL per tube with approximately 5 tubes per oviduct).Place the tubes in the shaking–heating block  with intermittent pipetting using a 1000 μL pipette for 10–20 min. Periodically check aliquots on an inverted microscope (approximately every 5 min) for dissociation of the oviductal glandular epithelium, which appear as worm-like structures.CRITICAL STEP Do not over-digest. The oviductal glandular epithelium should be as intact as possible whilst removing the stromal cells and fibrous tissue. 11.Stop the digestion by transferring the tube contents to 50 mL tubes containing RPMI 1640 with 20% fetal bovine serum .12.Place a 100 µm cell strainer in a new 50 mL tube and transfer the cell suspension through the cell strainer. Repeat ~4 times with the same strainer.CRITICAL STEP Discard the fibrous tissue. This can be removed with thumb forceps.13.Thoroughly rinse the inverted cell strainer into a 35 × 10 mm Petri dish with 2 mL RPMI 1640.14.Place a 40 µm cell strainer into the 50 mL tube and transfer the same diluted cell solution through the cell strainer. Repeat ~4 times with the same strainer. .15.Rinse the inverted cell strainer using the same RPMI 1640 medium that was used for the 100 µm cell strainer.16.Transfer the glandular suspension from the 35 × 10 mm Petri dish to the microcentrifuge tubes.17.g for 6 min at room temperature (20–22 °C). Remove and discard the supernatant.Centrifuge at 600× 18.Combine the pellets for each oviduct into one centrifuge tube and resuspend in 1 mL DMEM/F12.19.g for 6 min at room temperature (20–22 °C). Remove and discard the supernatant.Centrifuge at 600× 20.Place the cell pellet on ice.21.Add 20x Matrigel/Cultrex and mix carefully.CRITICAL STEP Ensure the Matrigel/Cultrex is at 4 °C. CRITICAL STEP Avoid generating bubbles whilst mixing.22.Dispense 25 μL droplets into the center of each well in the pre-warmed 48-well plate . 23.Place the plate with the droplets into a 37 °C incubator for 30 min.24.2).Add 250 μL organoid medium to each well and place the 48-well plate in an incubator .Warm a 48-well plate to 37 °C.Transfer the well contents to microcentrifuge tubes (4 wells per tube).g for 6 min at room temperature (20–22 °C). Remove and discard the supernatant.Centrifuge at 600× Add 200 μL DMEM/F12 to the pellet. Pipette 500 times through a 200 µL pipette tip set at 150 µL.OPTIONAL STEP Pool the pellets at this stage if desired.6.Add an additional 800 µL DMEM/F12.7.g for 6 min at room temperature (20–22 °C). Remove and discard the supernatant.Centrifuge at 600× 8.Add 200 µL DMEM/F12 to the pellet. Pipette 300 times through a 200 µL pipette tip set at 150 µL.9.Add an additional 800 µL DMEM/F12.10.g for 6 min at room temperature (20–22 °C). Remove and discard the supernatant.Centrifuge at 600× 11.Add Matrigel/Cultrex and plate as described above.Remove the culture medium from the wells and replace with 250 µL Cell Recovery Solution (Corning) or Organoid Harvesting Solution (Cultrex) and place the plate on ice for 45–60 min.Transfer the well contents to the microcentrifuge tubes (4 wells per tube).g for 6 min at room temperature (20–22 °C). Remove and discard the supernatant.Centrifuge at 600× Add 1 mL DMEM/F12 to the cell pellet.g for 6 min at room temperature (20–22 °C). Remove and discard the supernatant.Centrifuge at 600× Combine 4 wells of organoids per 0.25 mL of freezing media (see Reagents Setup below) in a cryovial. Add the freezing medium to the cell pellet dropwise.Place the cryovial into Cool Cell and cool to −80 °C overnight.Transfer the vials to liquid nitrogen the following day and store the cryovials in liquid nitrogen.Remove the cryovial from the liquid nitrogen tank.Thaw the vial for 1 min in air and then 1 min in a 25 °C water bath .Add 5 mL of a thawing medium (see Reagents Setup below) dropwise and gently mix for 10 min.Transfer the cell suspension to a 15 mL centrifuge tube.g for 10 min at room temperature (20–22 °C).Centrifuge at 100–200× Remove the supernatant and discard it.Add Matrigel/Cultrex and plate as described above.Remove the culture media from the wells leaving only the Matrigel droplets.Add 250 μL Cell Recovery Solution (Corning) or Organoid Harvesting Solution (Cultrex) to each well. Place the 48-well plate on ice for 45–60 min.Transfer the contents of the wells to the microcentrifuge tubes.g for 6 min at room temperature (20–22 °C). Remove and discard the supernatant.Centrifuge at 600× Resuspend the pellet in 1 mL PBS.OPTIONAL STEP Pool the pellets at this stage if desired.6.g for 6 min at room temperature (20–22 °C). Remove and discard the supernatant.Centrifuge at 600× 7.Add 500 μL of 4% paraformaldehyde without disrupting the pellet. Incubate at room temperature (20–22 °C) for 30 min.8.Remove and discard the paraformaldehyde.9.Add 50 μL of warm 2% agarose to the cell pellet.10.Pick up the whole pellet in agarose with a 200 μL pipette tip and transfer to a Petri dish to solidify as a droplet.CRITICAL STEP Cut the tip off the pipette before transferring the agarose and the cell pellet.CRITICAL STEP Move quickly as the agarose will begin to cool.11.Transfer the solidified agarose droplet to a microcentrifuge tube containing 70% ethanol until processing for embedding.12.Routinely embed in paraffin wax, section, and stain with hematoxylin and eosin or a periodic acid-Schiff reagent and hematoxylin.Remove the culture media from the wells leaving only the Matrigel droplets.Add 250 μL Cell Recovery Solution (Corning) or Organoid Harvesting Solution (Cultrex) to each well. Place the 48-well plate on ice for 45–60 min.Transfer the contents of the wells to the microcentrifuge tubes.g for 6 min at room temperature (20–22 °C). Remove and discard the supernatant.Centrifuge at 600× Resuspend the pellet in 1 mL PBS.OPTIONAL STEP Pool the pellets at this stage if desired.6.g for 6 min at room temperature (20–22 °C). Remove and discard the supernatant.Centrifuge at 600× 7.Fix the organoid pellet in 500 μL of 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer prior to routine TEM sample preparation and imaging.The above methods resulted in organoid generation, utilizing: (i) fresh equine oviducts collected post mortem during a transitional stage between winter anestrus and cycling utilizing cells from the length of the oviduct ; and (ii) frozen–thawed organoids at passage 1, day 8 (P1D8), then culturing for an additional four weeks to P3D14 A. The orThe generation of oviductal organoids has been previously reported utilizing oviductal tissue from mice and women ,14. DescMouse oviductal organoids demonstrated a variability in organoid growth among the segments with the infundibulum displaying the greatest growth rate . In the ®, which may affect the direct contact of the gametes and/or embryos with the organoids. An alternative method to improve ARTs may utilize extracellular vesicles (EVs) secreted by oviductal organoids. A method for isolating secretions from the lumen of human endometrial organoids has been reported that utilized centrifugation to disrupt the organoids without affecting the cellular viability [Although the co-culture of equine oviductal organoids with gametes and/or embryos may improve ARTs, most organoids are generated utilizing an extracellular matrix such as Matrigeliability , which cExtracellular vesicles are nanoparticles that contain nucleic acids and proteins within a lipid bilayer and are naturally secreted by cells to facilitate intercellular communication . EVs proHowever, the collection of in vivo-derived EVs is not practical, sustainable, or repeatable. Therefore, an in vitro source of oviductal cell-derived EVs that is physiologically similar to the in vivo oviductal environment—such as EVs produced by oviductal organoids—would facilitate advancements in many ARTs. A novel approach may be to isolate the EVs that the organoid cells produce for co-incubation with equine gametes and/or embryos. Although additional research is required to evaluate the functional outcome, EVs secreted by equine oviductal organoids may provide an in vitro environment that can closely simulate the in vivo oviduct. This may facilitate improvements in ARTs, including IVF in equids.The 3D organoid culture methods described here provide a roadmap to improve our understanding of equine oviductal physiology and the potential to make significant advances in equine ARTs to improve equine reproductive efficiency.For all items below, combine the components and then filter-sterilize using syringe filters.MEM with Earle’s salts.HEPES (25 mM).Penicillin (100 U/mL).Streptomycin (0.1 mg/mL).Pyruvate (0.1 mM).Glutamax (2 mM).v/v).Bovine serum albumin (5% Handling medium :MEM witRPMI 1640.Collagenase V (0.4 mg/mL).Dispase II (1.25 U/mL).Digestion solution :RPMI 16DMEM/F12 without phenol red with glutamax.Penicillin/streptomycin (1%).B27 Plus (2%).N2 (1%).Insulin–transferrin–selenium (1%).Nicotinamide (1 mM).Recombinant human EGF (50 ng/mL).Recombinant human FGF-10 (50 ng/mL).Recombinant human Noggin (100 ng/mL).TGFβ/Alk inhibitor A83-01 (0.5 µM).N-acetyl-L-cysteine (1.25 mM).SB202190 (10 µM).Y27632 (10 µM).Organoid medium:MEM with Earle’s salts.Dimethyl sulfoxide (10%).HEPES (25 mM)Penicillin (100 U/mL).Streptomycin (0.1 mg/mL).Pyruvate (0.1 mM).Glutamax (2 mM)v/v).Fetal bovine serum .Penicillin (100 U/mL).Streptomycin (0.1 mg/mL).Pyruvate (0.1 mM).Glutamax (2 mM).v/v).Fetal bovine serum (20% Thawing medium :MEM wit