Applying these complexes in catalytic hydroboration reactions with representative aldehydes and ketones reveals that all are competent, however a definite donor substituent effect is discernible. The bifunctional nature of the complexes is also probed by assessing their performance in metallation of a triazole and phenylacetylene and addition across pyrazine. These results lead to an example of phenylacetylene hydroboration, which likely proceeds via deprotonation, rather than insertion as observed with the aldehydes and ketones. Collectively, the results emphasise that reactivity is strongly influenced by both the mixed‐metal constitution and mixed‐ligand constitution of the new aluminates.A series of heteroleptic monoamido‐monohydrido‐dialkylaluminate complexes of general formula [ Therefore, in a recent communication we reported a range of Lewis donor solvated lithium aluminates bearing two HMDS  and two hydride functionalities and established that lithium diamidodihydridoaluminates were able to function efficiently in hydroboration catalysis and metallation applications.n‐hexane at room temperature resulted in the immediate precipitation of a white powder 1 that so far has resisted recrystallisation thus ruling out X‐ray crystallographic authentication. However, NMR spectroscopic studies have revealed its general constitution. 1H and 13C spectra reveal the presence of two isobutyl groups and one TMP group and the hydride ligand. The 7Li spectrum displays a sharp resonance at δ=0.4 ppm albeit the 27Al spectrum does not contain any identifiable resonance, a common problem with quadrupolar aluminium centres in broadly unsymmetrical environments. The presence of an aluminium hydride was confirmed by performing a 1H{27Al} experiment which revealed a sharpening of the broad resonance at 3.18 ppm, and suggested formation of the expected cocomplex iBu2AlTMP(H)Li 1 in a 75 % isolated yield. Structural insight of the solution phase constitution of 1 was gained by performing a DOSY experiment in C6D6 solution,1 as dimeric with the formula [iBu2AlTMP(H)Li]2. Similar solvent‐free structures are known in the literature and contain the common [Li2H2] central square core depicted in 1.Cocomplexation between the commercially available aluminium hydride DIBAL(H) and LiTMP in 1 were obtained in high yield by addition of the appropriate ligand  into toluene solutions of 1, followed by introduction of n‐hexane and crystallisation at −30 °C  fragment is bonded, via the μ‐hydride, to a Li⋅donor fragment (donor=three THF in 2 and one PMDETA in 3). Both the Al−H and Li−H distances are similar in each molecule , with those to the group 13 metal systematically shorter by an average of 0.16 Å. Changing the Lewis donor from PMDETA to diglyme gives 4, which adopts a markedly different solid‐state structure. The main differences between these tridentate chelating ligands is a change from N to O donors, and importantly the latter only contains one terminal methyl substituent, reducing steric congestion when it ligates a metal atom. Charge‐separated ion pair structure 4 is best described as a lithium lithium‐dialuminate, since both cationic and anionic moieties contain lithium. The cationic moiety is a distorted octahedral lithium cation supported by two diglyme ligands. The anionic moiety is comprised of two peripheral iBu2AlTMP(H) units in distorted tetrahedral environments, that bond to a central lithium via μ‐TMP and μ‐hydride ligands. Furthermore the lithium ion displays a near square planar geometry (τ4=0.04),Me range: 2.946(7)–3.249(7) Å]. This arrangement can be described as an inverse Weiss motif. The Weiss motif is a surprisingly common structure found in dialkali‐metal “ate” complexes where the central, non‐alkali‐metal exists in a tetrahedral arrangement, with respect to four bridging ligands.4. Reaction of 1 with half an equivalent of DABCO affords 5 in which two symmetry equivalent iBu2AlTMP(H)Li subunits are connected by the bicyclic, binitrogen Lewis donor. As with 4, here the lithium ion coordination sphere is completed by μ‐TMP and μ‐hydride ligands. Employing a full equivalent of DABCO affords the stoichiometric variant 6, which differs from 5 by a terminal DABCO ligand, instead of the bridging mode observed in 5. In 2–6, despite the diversity of donor ligands, the Al−H and Li−H distances do not display any systematic differences across the series (Al−H range 1.61(3)–1.69(3) Å; Li−H range 1.76(4)–1.88(3) Å). The structures of 2–6 and the related lithium diamidodihydridoaluminates we reported previously,4 containing a range of either alkyl or amido groups in place of either two or three hydrogen atoms. Furthermore, these more substituted LiAlH4 modifications benefit from the synthetic advantages of enhanced solubility (soluble in hydrocarbon solvents), and easier to accurately weigh low loadings in catalytic applications.Next, a series of donor‐solvated derivatives of n‐hexane. After addition of a small amount of THF to the reaction mixture we were able to isolate a few crystals of (iBu2Alμ‐TMPμ3‐HNa)2⋅2THF, 7, that were found to be suitable for diffraction studies  Å and Na2−H2H 2.38(2) Å). The second sodium is also bonded to the hydride ligands [Na1−H1H 2.22(2) Å and Na1−H2H 2.19(2) Å] and two solvating THF molecules. Unfortunately, we were unable to reproducibly prepare 7 in acceptable yields and as such its characterisation remained restricted to this structural analysis.In the knowledge that alkali metal effects can profoundly influence structural morphology, reactivity and physical properties,s Figure . Crystal2–6 were also characterised in solution by 1H, 13C, 7Li and 27Al NMR spectroscopy and revealed the expected resonances, albeit as with 1, the 27Al spectra were of little diagnostic value. 1H and 13C spectra confirmed the presence of 2 iBu, 1 TMP, and an aluminium bound hydride ligand, as well as the appropriate donor resonance(s). Interestingly the 1H NMR spectrums of 5 and 6 are near identical, except for the chemical shift resonances of the DABCO CH2 protons. Moreover, in 6 the terminally bound DABCO ligand only contains one broad singlet instead of two triplets (vide infra). The 7Li NMR spectrum of 4 displayed one noticeably broad resonance at δ=−0.32 ppm, instead of the expected two resonances in accordance with the charged‐separated molecules in the crystal. Thus, a variable temperature 7Li NMR experiment was performed to investigate whether an exchange process is occurring 2 cation. The resonance at −0.36 ppm may be assigned to a contact ion pair resembling PMDETA analogue 3, which exhibits a sharp resonance at δ=0.38 ppm. We probed this exchange process further via a DOSY NMR study of 4 at room temperature in C6D6. The estimated molecular weights in this instance are 509 and 615 g mol−1] (note the Lewis donor resonances diffuse separately from the remaining resonances), which are intermediate between those of either the expected mass in the crystal (846 g mol−1) or a structure resembling 3 (424 g mol−1). In contrast, DOSY NMR studies of 3 indicate its greater robustness as no such exchange process occurs on the NMR timescale, where an estimated molecular weight (463 g mol−1) is in excellent agreement with the theoretical value (446 g mol−1).Complexes 1–6 represent a series of new monoamido‐monohydrido lithium aluminates, well defined in the solid and/or solution states. Key distinctions are 1 is donor free; 3 contains a solvating PMDETA ligand in the solid state and retains this arrangement in solution (via DOSY studies); 4 adopts an entirely novel solid‐state arrangement vide supra, and exhibits exchange of the diglyme ligands on the NMR timescale.In summary, complexes 1, 3, and 4, since 1 is donor‐free, while 3 and 4 contain broadly similar donor ligands, except for the donor atom identity (N or O), and the slightly reduced steric profile of diglyme which gives rise to a fluxional system in solution. Hydroboration catalysis using HBpin was selected to trial 1, 3 and 4, since it is an area currently attracting increasing interest in the main‐group arena, and the wider chemical audience.3⋅DABCO, DIBAL(H), LiAlH4)At the inception of these studies we sought to discover the answer to two key questions: (i) can these monoamido monohydrido lithium aluminates act as efficient catalysts in hydroboration applications; and (ii) does the structure, determined by the donor ligand play a role in any such catalytic performance? In this regard we initially selected 1 (2.5 mol % based upon a dimeric formula), 3 (5 mol %) or 4 (2.5 mol %) with benzaldehyde and pinacol borane in C6D6 solution resulted in fast and quantitative hydroboration in 15 minutes at room temperature in every case. Lowering the precatalyst loadings to 0.5 mol % of 1 or 4 and 1 mol % of 3 resulted in no loss of catalytic performance. Importantly these initial results demonstrate that 1, 3 and 4 are all suitable candidates for aldehyde hydroboration. Expanding the substrate scope to include dual‐functional cinnamaldehyde demonstrates catalyst selectivity with smooth and quantitative hydroboration occurring only at the carbonyl functionality. 1 and 4 achieve this transformation inside 30 min, whereas with 3 quantitative hydroboration occurs after 3 h. This demonstrates that subtle structural differences within these systems play a role in catalyst performance. This performance is broadly in line with previous aluminium‐based hydroboration catalysts. Roesky et al. achieved quantitiative hydroboration of cinnamaldehyde with his nacnacAlH2 complex in 6 h with 1 % loading, whereas we previously demonstrated 76 % conversion in 2 h with (HMDS)2AlH(μ‐H)Li(THF)3. Completing our studies into aldehyde hydroboration we screened complexes 2, 5 and 6 with cinnamaldehyde as a representative example. 2 contains a four coordinate lithium atom albeit bonded to three monodentate THF ligands, which are more likely to desolvate during the reaction, compared to tridentate PMDETA in 3, thus substrate access to lithium may occur more readily. 5 and 6 both have coordinately unsaturated three coordinate lithium atoms, and as such are readily accessible. Complexes 2, 5 and 6 all efficiently catalyse hydroboration of cinnamaldehyde in 30 min.Reaction of 1 (2.5 mol %) reaches 97 % conversion after 2.5 h at room temperature in C6D6. Using 3 as precatalyst, the reaction requires 65 h for quantitative hydroboration at room temperature, or 22 h at 70 °C. Using 4 this reaction takes 2 h for 90 % conversion, similar to the value using 1. Exploring the comparatively poor performance of 3 more thoroughly, 2,2,2‐trimethylacetophenone and 2,2,2‐trifluoroacetophenone were selected as substrates. The former shows enhanced steric features with respect to acetophenone, whereas the latter, approximately isosteric, is considerably more electron withdrawing. Hydroboration of 2,2,2‐trimethylacetophenone with 3 (5 mol %) occurs in 2.5 h at room temperature, implying that ligand steric features are not the only factor here in determining hydroboration efficiency. 2,2,2‐trifluoroacetophenone is quantitatively hydroborated in 15 min at room temperature. This fast reactivity may be expected since the presence of CF3 would significantly deplete the charge present at the ketone carbon atom making it more electrophilic and thus facilitate faster nucleophilic hydride insertion. One possible contribution towards the slow reactivity of 3 with acetophenone is that the presence of α‐hydrogen atoms may promote keto‐enol tautomerism under the reaction conditions. In this scenario, one may envisage a coordination of the substrate to the lithium ion, resulting in a sterically congested 5‐coordinate lithium ion, followed by tautomerisation and stabilisation of the enol form by H‐bonding to one nitrogen atom of the PMDETA ligand  than either 1 or 4 (30 min). Using cyclohexanone as substrate reveals that 3 is again slowest (3 h); whereas 4 performs marginally better than 1 (1.5 h versus 2 h) for this reaction. Finally, butan‐2‐one, an aliphatic ketone, is fastest using 1 as a catalyst (1.5 h), while both 3 and 4 are complete in 3.5 h at room temperature. Butan‐2‐one contains two sets of α‐hydrogens which may explain the slower reactivity with respect to 1. Hydroboration of acetophenone was also performed using 2, 5 and 6. All three complexes demonstrate essentially the same reactivity . This can be rationalised by considering the structural distinctions of the complexes. In each case the DABCO ligand is monodentate, rendering the lithium atom 3‐coordinate, instead of the fully saturated 4‐coordinate in 2–4, albeit the high lability of the solvating THF in 2 is likely to lower the coordination number as required during reaction. Therefore, one may anticipate that initial coordination of substrate to lithium, and thus subsequent insertion into the Al−H bond would occur more readily in these cases. Importantly this scenario validates the use and future exploration of bimetallic systems.Hydroboration catalysis of 1, 3, and 4 in these reactions reveal that 1, the donor free complex is the best catalyst and marginally outperforms 4. 3 in all cases is the least efficient catalyst in these processes. This reactivity can be related to the solid and solution phase structures. The structure of 3 in solution, via DOSY NMR studies, resembles that in the crystal structure. Thus, one expects the PMDETA group with its three donor atoms to remain tightly bound to lithium during the catalytic process. On the other hand, 4 can be considered a halfway‐house between 1 and 3. Spectroscopic studies indicate that part of the time in solution the complex resembles that of the solid‐state structure, which is donor free, akin to 1, (both diglyme ligands are involved in bonding to the separated lithium cation). The remainder of the time the complex likely resembles 3, and thus in general, delivers reactivity intermediate between that of 1 and 3. A lower lithium coordination number also plays a role in catalytic performance as indicated by reactivity of 2, 5 and 6 which hydroborate acetophenone faster than the other donor solvates studied. Once again, we turned to DOSY NMR studies to illuminate the solution constitution of these complexes to gain insight into the catalytic process. The estimated molecular weight of 2 is 486 g mol−1, in good agreement with the theoretical value (506 g mol−1), suggesting that 2 largely remains intact in solution. However, careful inspection of the spectrum reveals that the THF resonances diffuse slightly faster than the remaining resonances of 2 (MW 444 g mol−1), detailing that in solution some level of THF desolvation is in operation, lowering the metal coordination number. With 5 and 6 the solution constitution is ambiguous. Bridging DABCO complex 5 has an estimated molecular weight of 620 g mol−1, lower than the expected value of 691 g mol−1. Similarly terminally bound DABCO complex 6 has an estimated molecular weight of 514 g mol−1, higher than the expected value of 402 g mol−1. These intermediate values indicate that the solution constitutions may be in equilibria between the two crystallographically observed extremes, and moreover, account for the similarity in 1H NMR spectra of 5 and 6 (vide supra). In each case the DABCO resonances diffuse with the same coefficient as the remaining complex resonances, ruling out complete ligand desolvation, affording 1, on the NMR experiment timescale. Extending this argument one step further, we performed two representative reactions using bulk THF as reaction solvent. We selected 1 and 3 for these reactions and discovered that in each case quantitative hydroboration occurs within 30 min. These fast reactions can be tentatively attributed to, in the case of 1, breaking up of the dimeric aggregate into a monomeric solvent separated species, and in the case of 3 displacing the PMDETA ligand resulting in a similar more labile solution species, an arrangement that favours faster catalytic transformation. Corroborating this, the 1H NMR spectrum of 3 in [D8]THF solution reveals the presence of free PMDETA.The general trend observed between 1 or 3 with various substrate molecules. Unfortunately, we were unable to crystallographically characterise such a species, despite repeated attempts. However, the different hydroboration results, that is, the effect of the donor ligand leads us to surmise that hydroboration of aldehydes and ketones in C6D6 follows three basic elementary steps: (i) coordination of carbonyl substrate to lithium. Here, tentative evidence for the coordination step originates from the slow performance of 3 with acetophenone,  intermediate species.3 with 1‐methyl‐1,2,4‐triazole at room temperature for 1 h in a n‐hexane/toluene mixture, followed by cooling at −30 °C resulted in formation of colourless crystals suitable for X‐ray diffraction studies (Scheme Complexes 8 in a reasonable isolated yield (42 %). Heterotrileptic 8  and 1.58(2) Å. A lithium⋅PMDETA moiety is held in place by coordination from the triazolyl nitrogen atom of the aluminate moiety placed adjacent to the metallated carbon atom. Further, in one independent molecule  the X‐ray diffraction data revealed that the substrate has been metallated at the 5‐position, via amido basicity, giving 8 Figure  crystalliBu2AlTMP on its own can also metallate 1‐methyl‐1,2,4‐triazole. This reaction affords (iBu2AlC3H4N3)2, 9 in 30 % isolated yield, and is a rare example of a neutral bis‐alkylamidoaluminium compound acting as an amido base towards an aromatic C−H bond, albeit with hydrogen atoms in relatively acidic environments.9 were collected, these were of insufficient quality to justify publication (see Supporting Information).It is important to note that the aluminium reagent 2 unit. Using 3  we sought to discover whether deprotonation or addition would occur, and whether any deprotonation would derive from the more basic TMP unit or weaker hydride, and whether these complexes could then be implicated in subsequent catalytic hydroboration reactions. Reaction between 3 and phenylacetylene in a J. Young's NMR tube was carried out at room temperature. 1H NMR monitoring revealed the appearance of resonances corresponding to TMPH. Furthermore, the resonance corresponding to the acidic hydrogen of phenylacetylene disappeared, confirming that 3 reacts, once again, as an amido base  monohydrido lithium aluminate, iBu2AlHCCPhLi⋅PMDETA. Its structure closely resembles that of 3 via formal replacement of a TMP anion with a phenylalkynyl anion as was the case with the triazolyl anion in 8. Note similar structures are known in the literature, and Uhl reported dialkylaluminium alkynides adopting the bridging motif shown in 10, moreover with less bulky alkyl groups, a crystallographically authenticated pi interaction between aluminium and the triple bond was identified.Phenylacetylene was selected as a candidate metallation substrate because of its acidic CCH hydrogen and because of precedence from Roesky's research that implicated deprotonation as a key step in terminal alkyne hydroboration.e Scheme . The rea10 is similar to an in silico modelled active catalytic intermediate by Roesky et al.,  from which the B−H bond of pinacol borane adds across the triple bond, followed by addition of a second equivalent of phenyl acetylene to regenerate the active component. Since Roesky's neutral intermediate complex was found to be an excellent catalyst in alkyne hydroboration, the isolation of the bimetallic complex 7, by simple deprotonation, afforded the opportunity to learn about the role of anionic aluminates in this context (note that in this report we have two iBu ligands instead of a bulky β‐diketiminate).By extension to the present system, such an interaction between the triple bond and the carbophilic aluminium centre may prime the complex for insertion.1 (2.5 mol %) was dissolved in C6D6 to which phenylacetylene was added, followed by HBPin and the reaction monitored by 1H NMR spectroscopy. After heating for 18 h at 70 °C clean formation of the anti‐Markovnikov vinylboronate ester has occurred in 76 % yield, as referenced against an internal standard  proceeds in broad agreement to that of Roesky using a neutral NacNacAlH2 catalyst. Here, lithium aluminate 1, performs an initial deprotonation, then hydroboration occurs, followed by a deprotonation of a second phenylacetylene molecule, inducing catalytic turnover.This desirable outcome therefore suggests that well‐defined “ate” complexes can play an important role in hydroboration of a variety of unsaturated molecules. Scheme 6D6, followed by 1 (2.5 mol %) and HBpin. After heating the solution at 70 °C for 18 h the 1H NMR spectrum remained unchanged revealing that the addition of an Al−H across diphenylacetylene is not favoured, and gives credence to the reaction mechanism depicted pathway b in Scheme 10 with HBpin, we cannot completely discount pathway a.Next, we decided to attempt the hydroboration of an internal alkyne. Reasoning that since di‐phenylacetylene did not contain any hydrogen atom “primed” for deprotonation, that any hydroboration activity would, by necessity, occur via an insertion of the alkyne into the aluminium hydride bond, and further shed light on the likely reaction pathway. Thus, diphenylacetylene was added to a J. Young's NMR tube in C3 with pyrazine. Both metallation and addition across pyrazine has previously been observed depending on the specific reagent(s) employed,3, arising from the TMP ligand, or the nucleophilicity of the Al−H functionality would win out. A toluene/n‐hexane solution of 3 was stirred with one equivalent of pyrazine at room temperature for 2 h resulting in formation of a pale brown oil. 1H NMR spectra of the oil revealed clean formation of a single product, displaying four resonances between δ=7.3–4.0 ppm, in a 1:1:1:2 ratio consistent with hydride addition at the α‐carbon atom  that remain bound to the lithium atom in solution slow down hydroboration. On the other hand, the related tridentate ligand diglyme, displays exchange on the NMR timescale, and performs similarly to the donor free species. We suggest this is, at least in part due to the coordination saturation of the lithium ion. Using the Lewis donor ligand DABCO leads to a reduction of the solvation at lithium and leads to faster hydroboration in a representative hydroboration of a ketone. In these catalytic transformations, insertion of the polar carbonyl group into the Al−H bond is mooted as a key step. Heteroleptic dialkyl‐monoamido‐monohydrido complexes are also revealed to be capable of hydroborating phenylacetylene, however this activity is suggested to proceed via deprotonation of the substrate. The bifunctional activity of the complexes is also demonstrated stoichiometrically in metallation of substrates containing an acidic hydrogen atom, and in an addition reaction with pyrazine.Full experimental characterisation and synthetic procedures are described in the supporting information.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Sci., 2016, 7, 4428–4434.Correction for ‘Chemoselective dehydrogenative esterification of aldehydes and alcohols with a dimeric rhodium( The authors regret that the incorrect temperature was stated on p. 4432, left column, line 5, of the original paper. The corrected sentence, in which “at ambient temperature” has been corrected to “at 90 °C”, is as follows:1H NMR of the mixture resulting from treating 11 with excess NaOAc  in MeOH at 90 °C showed that 11 was fully converted into a small amount of 14 and a major new compound 16 (Scheme 6).”“The crude The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


Breakthroughs in cancer immunotherapies have demonstrated considerable success, though not without limitations. Non‐thermal plasma (NTP) for cancer therapy has been emerging as a potential adjuvant treatment via induction of immunogenic cell death (ICD). Cancer cells undergoing ICD stimulate a patient's immune system to mount an anticancer response. While promising, the underlying mechanisms of NTP‐induced ICD must be closely examined. Here, the interaction between non‐thermal plasma and cancerous cells is studied. The short‐lived reactive oxygen and nitrogen species  produced by plasma are the main effectors that elicit ICD in melanoma while, surprisingly, persistent species do not. This is demonstrated in vitro using a dielectric barrier discharge plasma system and is validated in a vaccination assay in vivo. Plasma generation of reactive species appears to be dictated by the total energy. Collectively, this work provides fundamental insight into plasma interactions with biological material. Furthermore, it lays the foundation for future development of NTP systems for clinical translation. The addition of plasma systems into the existing arsenal of cancer therapies opens the possibility for new combination strategies for safer and more robust control of cancer. ICD is a form of regulated cell death, characterized by the timely release of “danger signals” known as damage‐associated molecular patterns (DAMPs).NTP treatment of mice has been shown to reduce tumor burden and extend survival in different cancer types.+ and CD45+ immune cells into the tumor environment. In combination with a therapeutic vaccine, DBD plasma treatment also enhanced cancer‐specific T‐cell responses.Beginning in 2015, NTP has been investigated for its potential to induce ICD, and it has been reported to stimulate DAMP emission in multiple cancer cell lines in vitro.When plasma is generated, a complex environment of reactive species, charged particles, neutral molecules, ultraviolet radiation, and electric fields is present and can interact with the biological target. Reactive species produced by the DBD plasma in the presence of oxygen were reported as the major contributors for ICD induction, not the physical components.To address the above challenges in oncology and the underlying questions in plasma chemistry and biological interactions, we sought to delineate the RONS generated by DBD plasma that are responsible for plasma‐induced ICD. Knowing the species in plasma that are critical to ICD induction will lead to the development of an optimized clinical device. We have chosen to work with melanoma, a deadly disease when it reaches a regional or distant stage with 5‐year survival dropping to 63% and 17%, respectively.FigureIn this study, a thorough examination of the RONS generated by DBD plasma operated at ICD‐inducing regimes was performed. The emission of surface CRT on two melanoma cell lines was used as a surrogate marker of ICD in vitro. Plasma‐generated RONS were measured using electron paramagnetic resonance (EPR) spectroscopy, UV–vis spectrophotometry, and liquid chromatography–mass spectrometry (LC–MS). To further delineate the role of these species on ICD, solutions of RONS were prepared from commercially available sources, used to treat both cell lines, and assessed for their capacity to stimulate CRT. A vaccination assay was performed to validate our findings in vivo. Our results showed that alone, persistent RONS were not sufficient for DBD plasma–induced ICD and highlight the importance of the short‐lived species. Furthermore, we observed that generating the desired RONS with the plasma system is dependent on the plasma treatment energy and not on an individual treatment parameter . This study provides crucial information toward our fundamental knowledge of plasma–cell interactions that, through strategic optimization of DBD plasma parameters, would allow us to develop a clinical device for controlled delivery of RONS necessary for ICD Figure1.22.1FigureTo determine whether plasma can induce ICD, we first performed a screening for cell survival and expression of surface CRT in mouse (B16F10) and human (A375) melanoma cell lines. CRT exposure on the cell surface is one of several DAMPs associated with ICD, and has been identified as a critical determinant for anticancer immune responses.−1 mitoxantrone (MTX), a known chemotherapeutic ICD inducer, were used as a positive control.FigureCells were treated for 10 s with increasing pulse frequency to deliver higher‐energy treatments. Cells incubated for 24 h with 2 µg mLIt is important to note that surface CRT measured here is only analyzed on PI− cell populations. While dead or membrane‐compromised cells may have higher surface CRT expression after plasma treatment, they also have permeable membranes, resulting in intracellular staining of CRT on the endoplasmic reticulum. Since only surface‐exposed CRT increases immunogenicity and intracellular CRT does not,2.2During DBD plasma treatment of cells, PBS was removed from the well and plasma was generated directly onto melanoma cells. However, since the wells were not dried, there remains a residual layer of PBS Figure B, which 2.2.1•OH) and superoxide radical anions (O2•−) in PBS was assessed with the spin trap 5‐diethoxyphosphoryl‐5‐methyl‐1‐pyrroline N‐oxide (DEPMPO). A solution of DEPMPO (100 × 10−3m) was prepared in PBS  and treated with DBD plasma. •OH reacts with DEPMPO to form the DEPMPO–OH radical adduct, while O2•− would form DEPMPO–OOH.Figurex compounds) that decrease the stability of the adducts.2•− is not produced and/or not delivered to the liquid following DBD plasma treatment, •OH radical is present, but its dependence on pulse frequency and time cannot be determined.The concentration of hydroxyl radicals (anions O2•− in PBS•NO) was monitored in liquid solutions exposed to plasma by two methods. First, we used 200 × 10−6m solution of 2‐phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide (PTIO) in PBS. Nitronyl nitroxides such as PTIO react with •NO, forming imino nitroxides, in our case 2‐phenyl‐4,4,5,5‐tetramethylimidazoline 1‐oxyl (PTI).•NO in the liquid after DBD plasma treatment, but could only partially be used to assess the degree of frequency/time dependency of •NO production. Subsequently, we used a solution of a N‐methyl‐d‐glucamine dithiocarbamate (MGD)–iron(II) complex to detect •NO.2Fe2+–NO adduct can be formed from interaction of (MGD)2Fe2+ with NO2−,2 in concentrations corresponding to that created during plasma treatment. No signal was detected in this case, confirming that all radical adduct formed was due to the reaction with •NO. Large amounts of reducing agent must be added after plasma treatment due to the oxidation of Fe2+ to Fe3+, resulting in loss of paramagnetic nature of the adduct.2Fe2+–NO concentration at 500 Hz was approximately twice that at 250 Hz, suggesting the near‐linear frequency dependency, as was observed with all other RONS , singlet oxygen (1O2), and ozone (O3)N‐oxyl (TEMPO). The trends of their generation were observed from the intensity of the TEMPO EPR signal , though exact concentrations cannot be computed due to the semiquantitative nature of spin trapping.1O2 can be estimated by using a selective scavenger, NaN3.R2 = 0.900) with plasma treatment frequency  was prepared in PBS at 50 × 10y Figure E. Genera3 Figure E, which 2.2.2−) was detected using solutions of l‐tyrosine in PBS as described in past reports: 100 × 10−6m solutions of l‐tyrosine in 2× PBS were treated by DBD plasma, leading to formation of 3‐nitrotyrosine.−, though other species that can yield the nitrated product may be present in our system . Therefore, the concentrations of ONOO− stated here are possibly an overestimation of the actual amount present. ONOO− increased near‐linearly as well with increasing plasma pulse frequency (R2 = 0.947) up to (3.4 ± 0.4) × 10−6m for 500 Hz  by plasma has been considered a key component of its anticancer properties.2O2 was measured with UV–vis spectrophotometry (400 nm) from the reaction with potassium titanium(IV) oxalate solution, and the addition of sodium azide (NaN3) .2O2 was undetected at 10 s treatments. H2O2 concentration increased near‐linearly with plasma treatment frequency at fixed treatment time (R2 = 0.965) , it confirmed that H2O2 did not saturate the liquid, and thus the increased exposure time can be used to assess concentrations at various frequencies. H2O2 concentration after 10 s treatment was extrapolated as 11.4 × 10−6, 31.2 × 10−6, and 67.8 × 10−6m for 100, 250, and 500 Hz treatments, respectively. At 50 Hz, H2O2 concentration was still below the detection limit even after 120 s, but is calculated to be 6.7 × 10−6m at 10 s.The generation of hydrogen peroxide (Hotometry 00 nm fro2−) and nitrate (NO3−) in PBS was determined using the Griess method.2− from NO3−, a nitrate reductase enzyme and cofactor were used to reduce NO3− to NO2− as described in the Experimental Section.3−: 1) NO3− is formed in the gas and enters the liquid (in ionic or HNO3 form), 2) NO3− is formed in the liquid from N2O5 that enters the liquid, and 3) NO2− enters the liquid and undergoes oxidation to NO3−. Therefore, although more NO3− was detected in the liquid following direct DBD plasma treatment, it is possible that the initial NO2− concentration was higher. This was taken into account in our cellular experiments with RONS solutions, where NO2− was used in amounts corresponding to total NO2− + NO3− concentrations measured here.The concentration of nitrite  are the main effectors of cell death, exogenous RONS solutions were prepared and added to both melanoma cell lines. Since RONS concentrations reported above were measured in 50 µL of PBS, while PBS is almost entirely removed from the wells prior to direct DBD plasma treatment of cells  H2O2 + NO3−/NO2−, which accounts for the concentration of all three species, and 2) H2O + NO2−, which assumes all NO3− originated from NO2−. Concentrations of RONS in solution can be found in the Experimental Section. We clearly see that both conditions did not significantly affect cell survival for the melanoma cell lines, as compared to direct DBD plasma treatment . The solution of ONOO− in PBS was prepared right before treatment and added to the cells immediately to prevent decay. Treatment of cells with exogenous ONOO− also did not affect cell survival , which further highlights that persistent RONS generated here by plasma are not the major effectors of cell death  was prepared immediately before treatment and 1 mL was added to the cells. The DBD electrode was then dipped into the solution and operated as before with the same parameters  from the high‐voltage DBD electrode. Although electric fields associated with DBD plasma alone do not affect cell death Figure A,B, whicP > 0.05) and the A375  melanoma cell lines . Again, there was no significant difference between the two conditions. The RONS solutions used for treatment were prepared as described above.P < 0.001; A375: 2.0 ± 0.8% vs 6.4 ± 1.1%, P < 0.001)  appeared to increase CRT slightly above untreated in both cell lines at nearly the same amount. Although this change was not statistically significant, it suggests that H2O2 may affect the immunogenicity of cell death, perhaps at higher concentrations. However, we note that it is not the major contributor to ICD in our DBD plasma treatment regime.Neither of the RONS solutions used to treat the melanoma cells elicited significant CRT Figure C,D. HoweTaken together, our data underline the essential role of short‐lived RONS for affecting ICD. While synergistic effects of PEF and RONS reduce cell survival, they do not elicit CRT required for ICD.2.5Our in vitro results relied on the detection of CRT as a surrogate maker for ICD, which is only one of several DAMP signals, including heat shock protein 70 (HSP70), HSP90, and HMGB1. However, some studies have shown that certain anticancer agents which are not ICD inducers, are also able to elicit DAMP emission.−6m of H2O2, 1770 × 10−6m of NO2−, and 35 × 10−6m of ONOO−), or MTX (2 µg mL−1) in vitro following the same treatment procedures as before. Untreated cells were used as a negative control. After treatment, cells were collected, resuspended in PBS, and incubated for 24 h at 37 °C with 5% CO2 to reduce the viability of cells in the suspension  and prevent subsequent tumor growth at the vaccination site. This procedure was selected after trying several different methods and optimization processes . Thirty‐two syngeneic C57BL/6J mice were vaccinated subcutaneously in the right flank (eight mice per group) and challenged with live B16F10 melanoma cells, 7 d later in the contralateral flank , PEF + RONS (RONS: 700 × 103) or when tumors began to ulcerate. Of the two mice that developed tumors at the vaccination site, one also developed a tumor at the challenge site, though it was small .Only 2 of the 32 mice (both from the untreated group) developed tumors at the vaccination site, indicating that the method of preparing the vaccine is relatively safe Figure B. This iP = 0.055), and also outperformed the positive control group (MTX) with six surviving mice compared to five, respectively. It is important to note that in the plasma group, there was still one mouse with a tumor that did not reach humane endpoints on day 50. All mice were sacrificed together when the final mouse with a tumor reached humane endpoints, and necropsies were performed to confirm complete tumor protection on the challenge site of the remaining mice . When mice were vaccinated with untreated cells, only 25% of the population was protected against tumor challenge . This is an attempt to eliminate tumor growth at the vaccination site, since if the fraction of living cells is too high, neoplastic lesions can develop at the vaccination site.bona fide ICD inducer for melanoma cells, but the short‐lived RONS are also crucial for stimulating ICD. Here we see that DBD plasma in this treatment regime is more than the sum of the persistent RONS and PEF.These data strongly suggest that not only is DBD plasma treatment a 3Immunotherapy, whereby the immune system is activated to attack cancer cells, is a major breakthrough in today's cancer treatment. It is generally believed that the cancer immunity cycle can be initiated with cancer cells that die in a way that activates the immune system, called immunogenic cell death.2O2, NO2−, NO3−, and ONOO− at concentrations equivalent to those generated by DBD plasma were prepared and used to treat melanoma cells to further delineate their role. We observed that none of these four species was able to elicit significant cell death or surface‐exposed CRT, a hallmark of ICD  and persistent RONS (lifetimes ≥1 s). Solutions composed of HD Figure . InteresD Figure A,B, but D Figure C,D. It iDetermining the importance of short‐lived species for desired biological effect is also critical for the development of clinical NTP devices. In addition to DBDs, another subset of NTP devices for medical applications is atmospheric pressure plasma jets (APPJs). In APPJs, the majority of the plasma is generated remotely, and plasma products are delivered to the biological target via a carrier gas or ionization waves.2O2, NO2−, NO3−, potentially ONOO−) are present in PTL. Therefore, its anticancer effect may be through a different mechanism than what we observed here. As stated above, we observed that RONS solution treatments containing equivalent amounts of H2O2  increased CRT slightly above untreated, though this change was not statistically significant . Liquid  is treated with NTP  to be enriched with RONS.t Figure . PerhapsTreatment of superficial cancers, such as melanoma studied here, is relatively straightforward, as NTP can be applied directly. However, tumors inside the body may become problematic and limit the use of NTP. One suggested approach is to use NTP in combination with intraoperative procedures. Following surgical tumor excision, NTP could be used to treat the tumor bed or surgical margins to eliminate remaining cancer cells. These studies still need to be performed, and for that, an ergonomic NTP device should be engineered for clinicians. Engineers and physicists are also designing different plasma source geometries for focused and minimally invasive treatment inside the body. This includes an endoscopic device (micrometer diameters) that can propagate plasma up to several meters in length.bona fide ICD induction that is relevant for melanoma, a disease with high potential for clinical implementation of NTP therapy, but this is also the first report demonstrating that NTP‐created vaccine can be safely prepared and offers complete protection. This could be further leveraged for plasma‐mediated control of nonsuperficial tumors. We encourage other research groups looking to perform the vaccination assay to follow our protocol, dictated in the Experimental Section and Supporting Information , for faster optimization, reduction of model animals used, and more comparative results.Although the vaccination assay has been performed previously in a CT26 colorectal cancer model,It is clear that several hurdles must be addressed before NTP becomes mature for clinical use, and understanding how NTP interacts with cancer cells will provide valuable insight into potential solutions. Ultimately, it is unlikely that NTP alone will be the solution to cancer therapy, but a combination of different therapies may be required. In the past, NTP has been combined with chemotherapy and even a therapeutic cancer vaccine.4bona fide immunogenic cell death of melanoma cells and the observed effect is not solely due to the persistent RONS generated . The short‐lived RONS produced by DBD plasma are required. As DBD plasma is highly tunable and treatment is localized, it may have unique advantages over current ICD inducers , and should be investigated further with other cancer types.In summary, we demonstrate that DBD plasma is able to induce 5Experimental Design: The overall objective of the study was to uncover the mechanism by which non‐thermal plasma elicits immunogenic cancer cell death by evaluating the role of RONS generated. This was accomplished by 1) determining an ICD‐inducing regime of plasma, 2) identifying the RONS generated in that regime, and 3) delineating their effect by comparing direct DBD plasma treatment and treatment with exogenously prepared RONS solutions. Our initial screenings of RONS effect on ICD were performed in vitro on two melanoma cell lines and validated in an in vivo vaccination assay using syngeneic mice.Cell Lines: The B16F10 murine melanoma cell line and the A375 human melanoma cell line were purchased from the American Type Culture Collection. Both cell lines were cultured in complete Dulbecco's modified Eagle medium containing 10% fetal bovine serum, 100 U mL−1 penicillin, 100 µL streptomycin, and 4 × 10−3m l‐glutamine. Cells were cultured in a humidified environment at 37 °C with 5% CO2.Microsecond‐Pulsed DBD Plasma Parameters for Direct Treatment: A microsecond‐pulsed power supply was purchased from Advanced Plasma Solutions, and a 1.25 cm diameter copper DBD electrode was used for treatment in 24‐well plates. The copper electrode was covered with a 0.5 mm fused‐silica dielectric  to prevent current arching. The microsecond‐pulsed power supply generated 17 kV pulses with ≈5 µs rise times and ≈1.5 µs pulse widths. The duty cycle was fixed at 100%.5 cells mL−1 (0.5 mL per well) 1 d prior to plasma treatment. On the day of plasma treatment, medium was removed and cells were washed twice with PBS to remove serum and other organics from cell culture medium. PBS from the second wash was left in the well until right before plasma treatment. For DBD plasma treatment, PBS was removed and the DBD electrode was lowered into the well and positioned 1 mm above the cells with a z‐positioner , a chemotherapeutic used as a positive control, was diluted to a 5 mg mL−1 stock solution and prepared into a working solution of 2 µg mL−1 in complete medium. Cells were incubated with mitoxantrone for 24 h before collection and analysis.Both cell lines were seeded into 24‐well plates at 3 × 10Treatment with Pulsed Electric Fields: On the day of treatment, cells were washed with PBS and 1 mL of PBS or RONS solution  was added to each well immediately before treatment. The DBD electrode was then submerged into the liquid and operated at 17 kV and 500 Hz for 10 s, 1 mm above the cells. This method was to subject the cells to PEF generated from the microsecond‐pulsed power supply and DBD electrode without the production of plasma  was added into a 24‐well plate and distributed evenly across the bottom. The DBD electrode was positioned 1 mm above the surface of the liquid using the z‐positioner , sodium nitrite (NaNO2) , and potassium nitrate (KNO3)  dissolved in PBS . ONOO− was prepared from NaOONO  dissolved in PBS. Four different RONS solutions were prepared1)2O2/NO2−/NO3−—H2O2: 700 × 10−6m; NO3−: 410 × 10−6m; NO2−: 1360 × 10−6mH2)2O2/NO2−—H2O2: 700 × 10−6m; NO2−: 1770 × 10−6mH3)−—35 × 10−6mONOO4)2O2/NO2−/ONOO−—H2O2: 700 × 10−6m; NO2−: 1770 × 10−6m; ONOO−: 35 × 10−6mH2 for 24 h until further analysis. This procedure most closely mimics the process of direct DBD plasma treatment and is most realistic to the concentration of RONS generated by plasma and experienced by the cells.On the day of treatment, the cells were washed twice with PBS, to follow identical handling procedures with plasma treatment. PBS from the second wash was removed immediately before treatment and 50 µL of RONS solution was added into the well and rocked to ensure even distribution on cells. 45 µL was removed and the remaining 5 µL was left on the cells for 10 s. Following treatment, 500 µL of fresh, complete medium was added to the well and cells were incubated at 37 °C with 5% COCell Survival Assay: Cell survival was quantified with a trypan blue exclusion test. Following 24 h incubation, cell supernatant was collected. Cells were then washed with 0.5 mL of PBS and detached with 200 µL of accutase. PBS from the wash was also collected with the cell supernatant. The cell suspension was collected, pooled with their supernatant and PBS wash, and homogenized by pipetting. A 50 µL sample was acquired and equal parts 0.4% trypan blue  was added to the sample. Cell counts were performed using a TC20 Automated Cell Counter (Bio‐Rad). The live cell concentration was recorded, and data were represented as a normalization to control.CRT Expression from Cell Lines: CRT was measured using dual staining of PI and a monoclonal CRT antibody. Following 24 h after incubation, cells were washed with PBS, detached with 200 µL of accutase, and washed twice with 2 mL of FACS buffer  + 2 g bovine serum albumin  + 1 g NaN3  in 100 mL H2O). Each sample was split into two vials and one was stained with monoclonal primary rabbit anti‐CRT antibody  while the other was stained with rabbit IgG, monoclonal isotype control  for 40 min at 4 °C. Cells were then washed once with FACS buffer. 0.5 µL of PI was added to each sample immediately before being quantified with a flow cytometer. Fifteen thousand events were collected and only the PI− cells were analyzed for surface CRT expression. Data were analyzed and gated using the FlowJo software . Data were expressed as percent CRT positive after accounting for nonspecific binding with their corresponding isotype. The gating strategy is described in detail in Figure S9 in the Supporting Information.Mice and Antitumor Vaccination Assay: Thirty‐two 8‐week‐old female C57BL/6J mice were purchased from Charles River and housed in a pathogen‐free room at the Animal Center of the University of Antwerp. The sample size of this study (eight mice per group) was chosen using information in the literature−6m of H2O2, 1770 × 10−6m of NO2−, and 35 × 10−6m of ONOO−), or 3) mitoxantrone (2 µg mL−1) in vitro, while untreated cells were used as a negative control. After treatment, cells were collected, washed twice with PBS, and resuspended in PBS at 106 cells mL−1. Cell suspension was incubated for 24 h at 37 °C with 5% CO2 to reduce the viability of the cells and prevent subsequent tumor growth at the vaccination site.The vaccines for this assay were prepared from B16F10 melanoma cells exposed to 1) DBD plasma (500 Hz), 2) PEF + RONS (RONS: 700 × 105 cells in ≈100 µL) on the right dorsal side. After 7 d, each mouse was injected with 104 live B16F10 cells (in ≈100 µL) on the left dorsal side. Tumor size and growth were followed up to day 50 as defined prior to the start of the experiment. Three orthogonal diameters were measured using a digital caliper, and volumes were calculated using (4/3π)r1 × r2 × r3. The humane study endpoint was set to when the total tumor volume exceeded 1500 mm3 or if tumors began to ulcerate. All animal experiments were approved by the University of Antwerp Animal Research Ethical Committee (ECD‐dossier 2017‐53).On the day of vaccination, mice were shaved with electric clippers  and injected with vaccine  solution in H2O and H2SO4 . Concentration of H2O2 in plasma‐treated samples was determined by UV–vis measurements performed on a Genesys 6 (Thermo Fischer) spectrophotometer with quartz cuvettes . Titanium(IV) reagent  was prepared by dissolving 0.354 g of potassium bisoxotitanate(IV) dihydrate in a mixture of 2.72 mL of sulfuric acid and diluted to 10 mL with Milli‐Q water. 50 µL of plasma‐treated sample was added to the cuvette and diluted with 150 µL of PBS. 50 µL of sodium azide (NaN3)  was added to the cuvette and thoroughly mixed. Afterward, 50 µL of Ti sulfate solution was added and homogenized. Air bubbles in the cuvette were eliminated with a sonicator (Branson 3200 ultrasonic bath) and water droplets were wiped from the cuvette before reading at 400 nm.2− and NO3−Detection of NO: A nitrate/nitrite colorimetric assay kit  was used according to the provided protocol. To detect NO2− only, 50 µL of Griess reagent 1 (sulfanilamide) was added to each sample in a 96‐well plate, and 50 µL of Griess reagent 2 (N‐(1‐naphthyl)ethylenediamine) was immediately added afterward. The absorbance wavelength was read with a microplate reader Infinite 200 Pro (Tecan) at 540 nm. To detect NO3− and NO2−, a nitrate reductase mixture  and an enzyme cofactor mixture  were added to each sample prior to the addition of Griess reagents. This allowed for the conversion of nitrate into nitrite. The absorbance was measured in duplicates and the samples were prepared in triplicates. The concentrations were calculated based on the obtained calibration curve.EPR Spectroscopy Analysis: 50 µL capillaries (Ringcaps) were used to collect plasma‐treated samples, and a MiniScope MS200 spectrometer (Magnettech) was used to perform the analysis. After each plasma exposure experiment, the samples were immediately placed into a capillary tube. The overall time between exposure and analysis was 1 min. The general EPR parameters were as follows: frequency 9.4 GHz, power 3.16 mW (31.6 mW in case of (MGD)2Fe2+–NO), modulation frequency 100 kHz, modulation amplitude 0.1 mT, sweep time 30 s, time constant 0.1, and sweep width 15 mT. The simulated spectrum was double integrated to determine the concentrations reported here. Simulations were performed using hyperfine values obtained from the literature in the Spin Trab Database . EPR calibration was performed using solutions of 4‐hydroxy‐TEMPO  as reported elsewhere.Table1O2/O3Detection of O/: TEMP spin trap was dissolved in PBS (50 × 10−3m) to detect O/1O2/O3 following DBD plasma treatment . TEMP reacts with these oxygen species to form the spin adduct TEMPO, which can be detected by means of EPR spectrometry. To determine the contribution of O/O3, 100 × 10−3m sodium azide (NaN3)  was added to the TEMP solution before plasma treatment to quench 1O2. Therefore, the collected spectrum of TEMPO was a result of the remaining oxygen species.•OH and O2•− with EPR SpectroscopyDetection of : DEPMPO spin trap  was dissolved in PBS (100 × 10−3m) to detect •OH and O2•−. DEPMPO reacts with O2•− to produce DEPMPO–OOH while it reacts with •OH to produce DEPMPO–OH. Experiments using 50 Hz pulse frequency were performed once with treatment times of 10 s or more.•NO with EPR SpectroscopyDetection of : The spin probe PTIO  was dissolved in PBS (200 × 10−6m) to detect •NO from DBD plasma treatment. •NO reacts with PTIO to form PTI that can be detected through EPR spectroscopy. When pulse frequency was varied from 50 to 500 Hz, treatment time was fixed at 50 s in order to generate detectable levels of •NO.•NO. MGD was dissolved in deionized water (20 × 10−3m) and combined with Fe(II)SO4·7H2O (4 × 10−3m) . This solution was treated with DBD plasma and Na2S2O3 (100 × 10−3m in deionized water degassed with argon)  was immediately added to the sample and collected for EPR analysis. When pulse frequency was varied from 50 to 500 Hz, treatment time was fixed at 120 s in order to generate detectable levels of •NO.The MGD spin trap  was also used to detect Detection of ONOO−with LC–MS: Solutions of 100 × 10−6m l‐tyrosine  and 100 × 10−6m diethylenetriaminepentaacetic acid  in 2 × PBS were exposed to plasma for a given period of time, as described by Wende et al.−1: 0–1.0 min 2% B, 1.0–4.0 min 2% to 18% B, 4.0–5.0 min 18% to 97% B, 5.0–6.0 min 97% B, 6.0–7.0 min 97% to 2% B, and 7.0–9.0 min 2% B. The parameters used in electrospray ionization tandem mass spectrometry in positive mode were as follows: capillary, 0.5 kV; cone, 22 V; extractor, 3 V; source temperature, 150 °C; desolvation temperature, 600 °C; desolvation gas flow, 1000 L h−1; cone gas flow, 0 L h−1; collision gas flow, 0.15 mL min−1; collision energy, 2 V.The separation and detection of 3‐nitrotyrosine was done by a Waters ACQUITY ultraperformance liquid chromatograph (UPLC) coupled to a Waters triple quadrupole mass spectrometer (Xevo TQ MS). The used column was a Waters ACQUITY UPLC HSS T3 2.1 mm × 100 mm column (1.8 µm particles), heated to 40 °C. The 9 min gradient was used for separation with A) water containing 0.1% formic acid and B) acetonitrile containing 01% formic acid, at a flow rate of 0.6 mL minm/z 182–136) and 3‐nitrotyrosine (transition m/z 227–181). For calibration of 3‐nitrotyrosine, eight standard solutions were made ranging from 0 to 10 × 10−6m and analyzed using the MRM method. The samples were diluted to a starting concentration of 10 × 10−6m tyrosine in 95% water and 5% acetonitrile containing 0.1% formic acid.A multiple reaction monitoring (MRM) method application was optimized for the detection of tyrosine . The fixed effect was the treatment, and the random effects included were the different dates the experiment was performed and the flasks the cells used were split from. The interactions between the treatment and the date as well as interactions between the treatment and the flasks were tested. The random slope model was used when the interactions were significant (P < 0.05) and the random intercept model was used in all other cases. The fixed effect tests determine whether there was a significant difference between treatments (P < 0.05). When the difference is significant, the Dunnett's test for statistical significance was used to calculate adjusted P value compared to the control. A P value of <0.05 was considered statistically significant. For all in vitro experiments, treatment conditions were performed in duplicates on the same day and repeated on three separate days as a minimum. The total number of observations for each treatment group is defined in the figure or figure legend. The survival curve of the vaccination study was prepared in Graphpad Prism and compared using the log‐rank (Mantel–Cox) test. A P value of <0.05 was considered statistically significant. All figures were prepared in Graphpad Prism (Graphpad Software). For all chemical species, a nonlinear regression was used to determine the best‐fit line and R2 value with a y‐intercept constraint at zero. Analysis was performed and figures were prepared in Graphpad Prism (Graphpad Software). No data were excluded.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


It has been evidenced that Fe–salen as a molecular precursor facilitates the confined‐growth of metal hydroxides, while Co2+ plays a critical role in catalyzing the transformation of organic ligand into nanocarbons and constitutes an essential component for CoFe hydroxide. The resulting Co1.2Fe/C hybrid material requires an overpotential of 260 mV at a current density of 10 mA cm−2 with high durability. The high activity is contributed to uniform distribution of CoFe hydroxides on carbon layer and excellent electron conductivity caused by intimate contact between metal and nanocarbon. Given the diversity of molecular precursors, these results represent a promising approach to high‐performance carbon‐based water splitting catalysts.Metal–salen complexes are widely used as catalysts in numerous fundamental organic transformation reactions. Here, CoFe hydroxide/carbon nanohybrid is reported as an efficient oxygen evolution electrocatalyst derived from the in situ formed molecular Fe–salen complexes and Co Electrochemical water splitting is as a promising approach for sustainable hydrogen production.2+, Fe3+, and 2‐mercapto‐5‐nitrobenzimidazole (MNBI) in N,N‐dimethylformamide (DMF). MNBI as the carbon source was presumed to interact with metal cations though a precise coordination chemistry in solution is unclear.To alleviate these disadvantages, metal (hydr)oxides have been hybridized with carbon materials such as graphene,−2, favorable reaction kinetics and outstanding long‐term durability.To fully realize this concept and develop a facile method for preparing electrocatalyst, we report here the solvothermal fabrication of a bimetallic CoFe hydroxide/carbon composite (CoFe/C) with an easily‐accessible molecular Fe–salen ethylenediamine) complex as precursors at a temperature as low as 160 °C. The resulting hybrid material exhibited high OER performance in alkaline media with a low overpotential of 260 mV for achieving a current of 10 mA cmMetal–salen complexes are famous for their widespread application in a variety of catalytic reactions such as polymerization and oxidation.SchemeN,N′‐bis‐‐o‐phenylenediamine , was simply synthesized by aldehyde‐ammonia condensation reaction (see the Supporting Information). Fe,Co hydroxides decorated nanocarbon  electrocatalyst was fabricated by an one‐step, in situ solvothermal reaction of salen‐1, Fe(NO3)3 and Co(NO3)2 at a ratio of 1:1:2 in DMF at 160 °C. The as‐prepared product was obtained as a black precipitate.The preparation process is illustrated in FigureThe as‐prepared product was characterized by scanning microscopy (SEM). As shown in Figure S4a,b (Supporting Information), irregular hydroxide nanoparticles were found to embed in amorphous and interconnected carbon matrices. The energy‐dispersive X‐ray spectroscopy (EDX) analysis in Figure S4c (Supporting Information) confirms the coexistence of Co, Fe, C, O, and N elements in the sample. Transmission scanning electron microscopy (TEM) and high‐resolution microscopy (HRTEM) in 1.2Fe/C. XPS spectrum in Figure S6a (Supporting Information) shows same element compositions as determined from EDX measurements. The surface molar ratio of Co/Fe in sample was 1.21:1 based on the XPS measurement, which is consistent with the result of ICP analysis. In Figure3+.3/2) and 796.8 eV (Co 2p1/2), and the corresponding satellite peaks at 786.1 and 803.1 eV are consistent with the presence of Co2+.X‐ray photoelectron spectroscopy (XPS) was carried out to investigate the composition and chemical oxidation states of the constituent elements in CoIII]+ complex in the stock solution. Evidently, salen‐1 exhibits two intense absorption bands at 285 and 334 nm in DMF due to the π → π* and n → π* transition of —C=N— motif,2+ and Fe3+ cations to the above solution (DMF)]+, suggesting preferential coordination of Fe3+ with salen‐1 3]2+, [CoII(DMF)4]2+, and [CoII(DMF)2(NO3)]+ were identified from MS spectrum, pointing to the solvated Co2+ ions  with a mass loading of 0.17 mg cm−2, Hg/HgO was employed as the reference electrode and Pt mesh as the counter electrode. To investigate the effect of carbon hybridization, Co1.2Fe‐layered double hydroxide (Co1.2Fe–LDH) was also prepared by a coprecipitation method .Figure−1, whereas an overpotential of 260 mV was required by Co1.2Fe/C to launch a current density of 10 mA cm−2, which is negatively shifted by 50 and 65 mV over that of commercial RuO2 and Co1.2Fe–LDH. Furthermore, a lower Tafel slope for Co1.2Fe/C (45.18 mV dec−1) in comparison with Co1.2Fe–LDH (90.84 mV dec−1) implied rapid reaction kinetics ,ƞ10 = 340 mV),2O4/PANI–MWCNTs (ƞ10 = 310 mV),2O–plasma exfoliated CoFe LDHs (ƞ10 = 290 mV),ƞ10 = 266 mV) .The electrochemical performance of the in situ synthesized Co1.2Fe/C at a constant current density of 10 mA cm−2 was evaluated by chronopotentiometric measurement. Over a period of 15 h, only slight increase in the potential was observed, demonstrating an outstanding long‐term stability for Co1.2Fe/C  indicate the valence states of +2 for Co and +3 for Fe, and CoFe hydroxides were still the main phase in all resultant Co, Fe/C samples. As demonstrated in Figure S10 (Supporting Information), the highest OER activity was achieved at a Co2+/Fe3+ ratio of 2:1, consistent with an optimal structure of Co1.2Fe/C. The ligand effect on performance was investigated by using a series of salen derivatives . These ligands were classified into two categories with one category having two phenolic hydroxyl groups  and another having four phenolic hydroxyl groups . The catalysts derived from H4salen turned out to be more active than those derived from H2salen. On the other hand, replacing the phenylenediamine bridge moiety of salen‐1 with an ethylenediamine bridge, or changing the substitutions on the phenoxyl ring were found to impose negligible effect on the final activities. Although the formation of formal coordination bond between Co2+ and —PhO− was not evidently supported, multiple phenolic hydroxyl groups in H4salen are expected to weakly interact with free Co2+ ions. These pre‐positioned Co2+ might facilitate the subsequent carbonization step.The OER performance of CoFe/C hybrid was tuned by optimizing the ratios between Co1.2Fe/C and Co1.2Fe–LDH at an overpotential of 300 mV. As shown in Figure 1.2Fe/C evidenced higher conductivity and faster charge transport of the carbon‐hybridized material.In order to understand the factors contributing to the high OER activity of metal carbon hybrids, electrochemical impedance spectroscopy (EIS) was carried out for both Co1.2Fe–LDH, the Co 2p3/2 XPS peak for Co1.2Fe/C is positively shifted from 780.8 to 781.2 eV and the Fe 2p3/2 XPS peak for Co1.2Fe/C is negatively shifted from 712.1 to 711.6 eV . The difference in XPS spectra implies partial electron transfer from carbon to metals (Co and Fe) caused by their intimate contact.Besides high electron conductivity, a synergetic effect between CoFe hydroxides and nanocarbons also plays a role in improving catalytic activity. In comparison with CoCdl), Co1.2Fe/C was estimated to have a electrochemical active surface area (ECSA) of 131 cm−2 and Co1.2Fe–LDH has a ECSA of 214 cm−2 . The ECSA normalized current density (jECSA) for Co1.2Fe/C is much higher than that for Co1.2Fe–LDH . Assuming that all Co and Fe ions participate in water oxidation reaction, Co1.2Fe/C produced oxygen with a turnover frequency (TOF) of 0.26 s−1 at an overpotential of 350 mV. This value exceeds the TOF of Co1.2Fe–LDH (0.004 s−1) by a factor of 65 at the same overpotential. The above results suggest that the remarkable OER activity for Co1.2Fe/C arises from the intrinsic activity of carbon‐supported CoFe nanoparticles rather than the fluctuation on ECSA.Based on the double‐layer capacitance (2+ ion. Due to the catalysis effect of Co2+ ion in conversion of salen ligand to amorphous nanocarbons, the hybrid catalyst was fabricated at relatively low temperature, which is a notable advantage over conventional synthetic methods for metal–carbon OER catalysts. The resulting Co,Fe/C showed ligand‐dependent OER activity with the optimized performance obtained by H4salen at a Co/Fe ratio of 2:1. Based on TEM and EIS measurements, the superior OER activity shown here can be dedicated to the following two aspects. First, molecular precursors lead to confined growth of metal hydroxide in carbon matrix, efficiently inhibiting the aggregation of the active sites. Second, the strong coupling between CoFe hydroxides and graphitic carbons improves electron transport in electrode. These results provide a promising molecular approach to rational design of heterogeneous water splitting catalysts under mild conditions.In summary, we have developed a simple, one‐pot method for preparation of electrocatalyst consisting of integrated CoFe hydroxides and nanocarbons from the in situ formed molecular Fe–salen complex and CoThe authors declare no conflict of interest.SupplementaryClick here for additional data file.


CO2), which is the substrate for photosynthesis, and ozone (O3), a damaging oxidant. How plants respond to changes in these atmospheric air pollutants, both directly and indirectly, feeds back on atmospheric composition and climate, global net primary productivity and ecosystem service provisioning. Here we discuss the past, current and future trends in emissions of CO2 and O3 and synthesise the current atmospheric CO2 and O3 budgets, describing the important role of vegetation in determining the atmospheric burden of those pollutants. While increased atmospheric CO2 concentration over the past 150 years has been accompanied by greater CO2 assimilation and storage in terrestrial ecosystems, there is evidence that rising temperatures and increased drought stress may limit the ability of future terrestrial ecosystems to buffer against atmospheric emissions. Long‐term Free Air CO2 or O3 Enrichment (FACE) experiments provide critical experimentation about the effects of future CO2 and O3 on ecosystems, and highlight the important interactive effects of temperature, nutrients and water supply in determining ecosystem responses to air pollution. Long‐term experimentation in both natural and cropping systems is needed to provide critical empirical data for modelling the effects of air pollutants on plant productivity in the decades to come.Human activities result in a wide array of pollutants being released to the atmosphere. A number of these pollutants have direct effects on plants, including carbon dioxide ( Cumulative anthropogenic emissions of CO2  are dominantly responsible for global warming and can be divided in two main sources: land‐use change and fossil fuel consumption . Over the second half of the 20th century, fossil fuel and industrial CO2 emissions rapidly increased with an average growth rate of 1.1% per year in the 1990s and 3.3% per year in the 2000s. There are three major sinks for CO2 emissions: oceans, land and the atmosphere . Future CO2 emissions over the 21st century will vary with socio‐economic development, population growth and climate policy, as explored by the Intergovernmental Panel on Climate Change (IPCC). To limit global warming to 2 °C above pre‐industrial levels, which is the target of the 2015 UN Paris Agreement, either rapid development and adoption of negative emission technologies is required, or total global emissions should not exceed 1000 Pg of CO2 from 2011 to 2100 (Anderson Anthropogenic activities have been affecting the atmospheric concentration of greenhouse gases (GHG) arguably over the past 8,000 years, but most profoundly since the Industrial Revolution. Today, our atmosphere has the highest atmospheric concentrations of CO3) is a secondary pollutant formed as a by‐product of the photochemical oxidation of NOx in the presence of carbon monoxide (CO), methane (CH4) and non‐methane hydrocarbons  with a smaller contribution from stratospheric exchange  over the Pacific Ocean to more than 100 ppb down‐wind of large industrial emitting sources  in 1997 , and will either remain at current levels or increase under the other scenarios  have played on a global scale . Grasslands, agricultural lands and deciduous forests tend to show larger O3 fluxes and annual deposition rates than tundra and coniferous forests . Oliver et al. (2018) examined the impact that increased concentrations of O3 have had on European vegetation at the regional scale using a land surface model. Their simulations suggest that rising [O3] from 1901 to 2001 decreased European gross primary productivity by 3–9% on average, with some regions showing up to 30% decreases in gross primary productivity. Using a fully coupled chemistry–carbon–climate model, Yue et al.  facilities  and ability to accommodate interactive treatments has improved understanding of the interactions of rising [COr et al.  used an 2] has been emphasised in recent studies of experiments in grassland ecosystems. At the biodiversity, [CO2] and N (BioCON) experiment on a perennial grassland in Minnesota, USA, elevated [CO2] failed to stimulate total biomass of plants when water supply was reduced by ~40% and N supply was limiting. However, if N, water or both were provided at ambient or elevated levels, then elevated [CO2] stimulated biomass by ~30%  content in the grains of crops exposed to elevated [CO2] (Myers et al. 2], recent studies that investigated the combination of rising [CO2] and warming reported that elevated temperatures can restore mineral concentrations to ambient conditions in soybean (Köhler et al. et al. 3], rising [CO2] and climate change on yield quality.The FACE experiments in cropping systems have quantified the effects of elevated [COe.g. Aspinwall et al. et al. et al. 2] and [O3], implying that genotypes that are most productive today will continue to be most productive in a high [CO2], high [O3] future (Resco de Dios et al. et al. 2 responsiveness or O3 tolerance has not been found (Ainsworth et al. 2] and/or [O3] (Yendrek et al. 2] and/or [O3] along with higher temperature and reduced water availability is not yet feasible, long‐term studies have enabled identification of genotypes that are consistently CO2‐responsive or O3‐tolerant across variable growing seasons (e.g. Sanz‐Sáez et al. et al. 2 fertilisation effects (e.g. Hsiang et al. The FACE experiments are now moving towards experimentation aimed at adaptation of cropping and forest systems to future climates by harnessing genetic variation and transgenic approaches (2 and O3 cycles have greatly intensified over the past 100 years. Today, terrestrial ecosystems take up nearly one‐third of the CO2 emitted from anthropogenic sources and one‐fifth of the tropospheric O3 produced by chemical production. In recent decades, rising CO2 has likely contributed to the greening of the planet, while O3 pollution has likely reduced terrestrial net primary productivity. The capacity of ecosystems to continue to filter atmospheric pollutants has important consequences for future climate and provisioning of ecosystem goods and services. However, recent studies suggest that the ability for terrestrial ecosystems to buffer anthropogenic emissions will diminish with rising temperature and drought stress. Thus, it is critical that we develop deeper understanding of the effects of rising [CO2] and [O3] pollution on net primary productivity, ecosystem C storage and crop production in agro‐ecosystems, and the interactive effects of rising temperature, variable water supply and nutrient availability.The global CO


The cause of colorectal cancer (CRC) is multifactorial, involving both genetic variants and environmental risk factors. We systematically searched the MEDLINE, EMBASE, China National Knowledge Infrastructure (CNKI) and Wanfang databases from inception to December 2016, to identify systematic reviews and meta‐analyses of observational studies that investigated gene–environment (G×E) interactions in CRC risk. Then, we critically evaluated the cumulative evidence for the G×E interactions using an extension of the Human Genome Epidemiology Network's Venice criteria. Overall, 15 articles reporting systematic reviews of observational studies on 89 G×E interactions, 20 articles reporting meta‐analyses of candidate gene‐ or single‐nucleotide polymorphism‐based studies on 521 G×E interactions, and 8 articles reporting 33 genome‐wide G×E interaction analyses were identified. On the basis of prior and observed scores, only the interaction between rs6983267 (8q24) and aspirin use was found to have a moderate overall credibility score as well as main genetic and environmental effects. Though 5 other interactions were also found to have moderate evidence, these interaction effects were tenuous due to the lack of main genetic effects and/or environmental effects. We did not find highly convincing evidence for any interactions, but several associations were found to have moderate strength of evidence. Our conclusions are based on application of the Venice criteria which were designed to provide a conservative assessment of G×E interactions and thus do not include an evaluation of biological plausibility of an observed joint effect. AICRAmerican Institute for Cancer ResearchCIconfidence intervalCNKIChina National Knowledge InfrastructureCRCcolorectal cancerCTNNB1cadherin‐associated protein β1CUPcontinuous update projectCYP24A1cytochrome P450 family 24 subfamily A member 1G×Egene–environmentGATA3GATA binding protein 3GECCOGenetics and Epidemiology of Colorectal Cancer ConsortiumGWASgenome‐wide association studyHIATL1hippocampus abundant transcript‐like 1MINK1misshapen like kinase 1NAT2N‐acetyltransferase 2NSAIDnonsteroidal anti‐inflammatory drugORodds ratioPI3Kphosphatidylinositol‐4,5‐bisphosphonate 3‐kinasePIK3C2Gphosphatidylinositol‐4‐phosphate 3‐kinase catalytic subunit type 2 gammaPTCHD3patched domain containing 3RRrelative riskSHMT1serine hydroxymethyltransferase 1SNPsingle‐nucleotide polymorphismSOCCSScottish Colorectal Cancer StudyTCF7L2transcription factor 7 like 2WCRFWorld Cancer Research Fund InternationalColorectal cancer (CRC) is the third most common cancer worldwide, with 746,000 new cases in men and 614,000 new cases in women.CRC risk is determined by a complex interplay of both genetic variants and environmental exposures. It has been speculated that their interactions ‐ known as gene–environment (G×E) interactions should also be important determinants of CRC risk. To date, genome‐wide association study (GWAS) have shown that up to 50% of CRC heritability can be explained by common and rare variants included in popular genotyping arrays.A number of systematic reviews, meta‐analyses and genome‐wide G×E interaction analyses that explored interaction effects in CRC have been published. We recently collected and evaluated the evidence across existing meta‐analyses of observational studies in dietary factors and gene‐diet interactions for the 5 most common cancers.We systematically searched the MEDLINE, EMBASE, China National Knowledge Infrastructure (CNKI) and Wanfang databases from inception to December 2016. The search strategy and Medical Subject Headings terms are displayed in Supporting Information Table S1. All identified publications went through a 2‐step review before being included. Titles were reviewed by 1 investigator (TY). Abstracts and full texts were reviewed by 2 investigators (TY and MT). Any discrepancies were resolved by discussion.Three types of studies were eligible for this umbrella review: (i) systematic reviews of observational studies assessing interaction effects between genes and environmental exposures in CRC; (ii) meta‐analyses of candidate gene‐ or SNP‐based studies and analyses combining individual level data from multiple studies exploring G×E interactions and (iii) genome‐wide investigation of G×E interactions on CRC risk within GWAS consortia. We excluded reviews without explicit systematic literature searches; and systematic reviews or meta‐analyses of observational studies that explored associations between CRC risk and genes or environmental exposures only.p value for interaction and the p value (or I2) for heterogeneity.One investigator (TY) extracted data which were then checked by a second investigator (ZM). For each eligible article, we extracted the first author, year of publication, the dietary and genetic risk factors examined, study design and the number of studies included. For meta‐analyses, we extracted the summary study‐specific relative risk estimates  along with the corresponding 95% confidence intervals (CIs), the number of cases and total participants, the For systematic reviews, we performed descriptive analyses and presented the authors’ main conclusions. The evaluation process is described in detail in Supporting Information methods.p value for interaction <0.05, or for G×E interactions reaching genome‐wide significance threshold, we used an extension of the Human Genome Epidemiology Network's Venice criteriaFor meta‐analyses of G×E interactions with a 2‐sided amount of evidence, the extent of replication and the protection from bias . Each G×E association was graded based on the as Table . On the et al.,Second, we established a prior score category (expected) for the G×E interactions using a framework presented in Boffetta Third, we examined the overall plausibility of each interaction by combining the prior score and the strength of the observed evidence. Higher weight was given to the observed evidence in case of conflicting results between the observed evidence and the prior scores.p value for interaction <0.05) that were identified from the candidate meta‐analyses or for interactions that were concluded as suggestive by the authors of the systematic reviews, we tested the interactions in the Scottish Colorectal Cancer Study (SOCCS)Finally, for the statistically significant G×E interactions : N‐acetyltransferase 2 (NAT2) and processed meat intakeNAT2 and red meat intakeSHMT1) C1420T polymorphism and folate intakeSHMT1 C1420T polymorphism and folate intake and rs6983267 and aspirin use in the SOCCS,α = 0.05) in our data (data not presented) or in the GECCO (data not presented). Each identified G×E interaction is described in detail in Supporting Information results.Twenty articlesp value for interaction from each genome‐wide G×E interaction analysis are presented in Supporting Information Table S7). The following G×E interactions were identified that reached genome‐wide significance threshold specified by the authors of the original publication: rs4143094 (10p14) and processed meat intake(PTCHD3) at 10p12.1, misshapen like kinase 1 (MINK1) at 17p13.2 and NSAID useEight articlesp < 10−5; Fig. Here, we present the evidence for the identified G×E interactions in relation to CRC risk with main genetic effects ; p = 7.8×10−11; I2 = 91.1%],16) in a meta‐analysis including 13,348 cases and 26,438 controls of European ancestry Aspirin use was associated with a reduced risk of CRC on the basis of a meta‐analysis of 39 studies with 151,367 cases  and the association was classified as class IV (weak)  CRC reportsk) Table  main genp > 10−5; Fig. Here, we present the evidence for the identified G×E interactions in relation to CRC risk with no main genetic effects  due to the high heterogeneity between the studiesp > 10−5) in a meta‐analysis of 7 GWAS from Europe including 8,749 cases and 18,245 controls PTCHD3, 17p13.2/MINK1 and NSAID use could not be properly evaluated due to the missing elements of the extension of the Venice criteriaIn a meta‐analysis of 10 studies including 8,634 cases and 8,553 controls, regular use of aspirin and/or NSAIDs, compared to non‐regular use, was associated with lower risk of CRC The RR for use of estrogen plus progestogen therapy on CRC risk was 0.74  (Table p > 10−5) of 18,299 cases A meta‐analysis of 8 prospective studies in the latest CUP CRC reportsNAT2 based on the weak (Weak – 3) prior score and the weak observed score  prior score and a weak observed score (Tables No evidence was found for the interaction between k Weak –  prior scNo evidence was found for the interaction between rs1944511 11q23.3) and overweight based on a weak (Weak – 3) prior score and a weak observed score . In particular, the benefit of regular aspirin use on CRC risk was confined to individuals with T allele of rs6983267, which has been associated with impaired binding of cadherin‐associated protein β1 (CTNNB1)/ transcription factor 7 like 2 (TCF7L2) and lower expression of MYC.CTNNB1 expression in colon cancer cell lines.CTNNB1/TCF7L2 binding is not constitutively active is necessary for the susceptibility to the effects of aspirin on the Wnt/CTNNB1 pathway.Based on the prior and observed scores, only the interaction between rs6983267 (8q24) and aspirin use was found with a moderate overall plausibility score and a main genetic effect (p > 10−5), even though they may provide clues to discovering novel CRC susceptibility loci that have not been readily detected in GWAS by their marginal effects of genetic factors.Moderate overall plausibility scores were also found for the interactions between rs4143094 (10p14) and processed meat intake, rs9409565 (9q22.32) and light‐to‐moderate alcohol drinking (1–28 g/day), rs964293 (20q13.2) and use of estrogen plus progestogen therapy, as well as rs2965667 (12p12.3), rs16973225 (15q25.2) and aspirin and/or NSAID use. However, these interactions are regarded as tenuous due to the lack of main genetic effects (PIK3C2G) locus, rs16973225 (15q25.2/interleukin 16) and aspirin and/or NSAID use on CRC risk. PIK3C2G gene encodes a protein of the phosphatidylinositol‐4,5‐bisphosphonate 3‐kinase (PI3K) family,PI3K.Interleukin 16 may stimulate monocyte induction of proinflammatory cytokines associated with tumorigenesis,interleukin 16 gene may be associated with the production of inflammatory cytokines that modify the chemopreventive effect of aspirin and/or NSAIDs on CRC.Little is known about the underlying molecular mechanisms of the interactions between rs2965667 at the 12p12.3/ phosphatidylinositol‐4‐phosphate 3‐kinase catalytic subunit type 2 gamma (CYP24A1) is biologically plausible. CYP24A1 is greatly expressed in malignant colon tumours,CYP24A1 have been associated with CRC risk.p <0.001].p = 0.037).CYP24A1 may only be a metabolizing enzyme for progestrogens but not estrogen, since an interaction effect was only found with the use of combined estrogen‐progestogen therapy and not with estrogen‐only intake.The interaction between use of estrogen plus progestogen hormone preparations and the rs964293 variant in the 20q13.2, known as cytochrome P450 family 24 sub‐family A member 1 (GATA3) region, on the association between processed meat intake and CRC risk is even less clear. GATA3 has been described as a master regulator of T‐helper 2 cell differentiation in mature CD4 (+) T cells and has been associated with T cell development.GATA genes or silencing of expression can increase CRC risk.The mechanism of the modifying effect of the rs4143094 variant at the 10p14 locus, near GATA binding protein 3 ((HIATL1) locus and CRC risk has not been understood. HIATL1 is a member of the solute carrier group of membrane transport, which makes the move of substances  directly into or out of cells possible.HIATL1 that interact with alcohol on CRC risk through genome‐wide G×E interaction analyses can also impact HIATL1 expression,HIATL1 on CRC risk through its influence on HIATL1 expression levels.Furthermore, the mechanism of the modifying effect of alcohol consumption on the association between rs9409565 at the 9q22.32/ Hippocampus Abundant Transcript‐Like 1 Studies of G×E interactions require much larger sample sizes than main effect analyses due to small effect sizes, multiple testing, misclassification due to imperfect measures of environmental exposures and more model parameters.The strengths of umbrella reviews have been described elsewhere.I2 to assess the heterogeneity in the evaluation, however, it has been reported that I2 represents what proportion of the observed variance would remain if we could eliminate the sampling error rather than how much the effect size varies.Our study has limitations. First, interaction effects that have not yet been assessed through meta‐analyses or systematic reviews would not have been included, since umbrella reviews do not focus on individual studies. Second, we did not use an established tool to assess the risk of bias in the included observational studies, because available tools such as Q‐Geniep value thresholds are typically used to evaluate the creditability of findings. Candidate gene studies with liberal p value thresholds might be biased toward false positive findings, while the genome‐wide approach is prone to false negative observations, because true interactions may not reach a stringent genome‐wide significance threshold. This, as well as other issues such as genotyping and imputation problems, could be a reason why none of the interactions identified in the candidate gene studies were replicated in genome‐wide G×E studies.We combined systematic literature reviews, candidate and genome‐wide G×E studies together. The main reason for this was to provide a comprehensive overview of the existing literature on G×E interaction in relation to CRC risk. However, an issue is that in each of these types of studies, different criteria and The extension of Venice criteria used here has been applied in the past to assess joint effects of environments and genes on risk of multiple cancers.Our assessment maps the status of evidence on the associations between G×E interactions and CRC risk. Despite the identified studies exploring a wide variety of G×E interactions on CRC, we conclude that we did not find highly convincing evidence for any interactions, but several associations were found to have moderate strength of evidence using our set of guidelines.Though most of the evaluated G×E interactions in our study were with no main genetic effects, it has been suggested that such kind of risk loci that have not been readily detected in traditional GWAS may be identified by testing for interactions between SNPs and environmental risk factors, even though there is no strong evidence for a G×E interaction.None declared.Not required.No additional data available.Study design: MT and ET; Literature search: TY; Study selection: TY and MT; Data extraction: TY and ZM; Data analysis: TY and MT; Study draft and revision: TY, MT, ET, XL, ZM, JL, SMF, JPAI, MGD and HC; Article guarantor: Dr. Maria Timofeeva and Dr. Evropi Theodoratou.Appendix S1: Supplementary methodsClick here for additional data file.Figure S1 Categories for the credibility of cumulative epidemiological evidence. The 3 letters correspond (in order) to amount of evidence, replication and protection from bias. Evidence is categorized as strong, when there is A for all 3 items, and is categorized as weak when there is a C for any of the 3 items. All other combinations are categorized as moderate .Table S1. Keywords and Search Strategies for Meta‐analyses of G × E Interactions used in the Umbrella Review.Table S2. Score Categories for Credibility of an Interaction Between an Environmental Exposure and a Genetic Variant Based on the Strength of Evidence for a Main Effect of Each of Them  .Table S3. Search Strategy Used for the Identification of Main Environmental Effects for Colorectal Cancer Risk.Table S4. Search Strategy Used for the Identification of Main Genetic Effects for Colorectal Cancer Risk.Table S5. G × E Interactions in Relation to CRC Risk That Were Reported From Meta‐analyses of Candidate Gene‐ or SNP‐based Studies.Table S6. General Characteristics of 33 Genome‐wide G × E Interaction Analyses in GWAS Consortia.Table S7. SNPs With the Smallest P for G × E Interactions in Relation to CRC Risk That Were Reported From Each Genome‐wide G × E Interaction Analysisa.Table S8. General Characteristics and Main Findings of the Systematic Reviews of Observational Studies. Suggestive Associations for G × E Interactions in Relation to CRC Risk That Were Identified by the Authors of the Original Systematic Reviews are Shown in Bold.Click here for additional data file.