There are few data on immune correlation of protection from breakthrough Omicron (B.1.1.529) infection in individuals who received booster vaccines. We thus compared a neutralizing antibody titers against Omicron within the first month after the mRNA booster at the time before omicron wave between healthcare works (HCWs) who experienced Omicron breakthrough infections and HCWs without Omicron infections.We enrolled HCWs without the history of SARS-CoV-2 infection who agreed with blood sampling 2 weeks after booster vaccination at Asan Medical Center, Seoul, South Korea, between November 2021 and December 2022 (Delta dominant era). We identified breakthrough infections by performing SARS-CoV-2 RT-PCR though nasopharyngeal swab specimen in HCWs who had COVID-19-related symptoms or had known exposure to confirmed SARS-CoV-2-infected patients, between 1 February and 25 April 2022 (Omicron dominant era). SARS-CoV-2 S1-specific IgG antibody titers were measured using enzyme-linked immunosorbent assay (ELISA). Plasma levels of live-virus neutralizing antibodies were measured using a microneutralization assay with SARS-CoV-2 omicron variants.Among 134 HCWs, 69 (52%) received two-dose ChAdOx1 nCoV-19 followed by BNT162b2, 50 (37%) three-dose BNT162b2, and 15 (11%) 3-dose mRNA-1273. Of them, 57 (43%) experienced breakthrough Omicron infection at median 121 days (IQR 99-147) after booster vaccination (breakthrough group), and the remaining 77 (57%) did not experience Omicron infection (non-breakthrough group). There was no significant different in “peak” SARS-CoV-2 S1-specific IgG level between breakthrough group (median 4484.4 IU/mL) and non-breakthrough group . In addition, there was no significant difference in “peak” neutralizing antibody titer (ID50) against Omicron between breakthrough group (median 2597.9) and non-breakthrough group .Serum samples were obtained from 134 healthcare workers 2 weeks after booster vaccination. Samples were analysed for SARS-CoV-2 S1-specific IgG antibody titers using enzyme-linked immunosorbent assay (ELISA) and plasma levels of live-virus neutralizing antibodies using a microneutralization assay with SARS-CoV-2 omicron variants. There was no significant difference in "peak" SARS-CoV-2 S1-specific IgG level (A) and “peak” neutralizing antibody titer (ID50) against Omicron (B) between breakthrough group and non-breakthrough group.We did not find the correlation of neutralizing antibody titers about several months before infection with breakthrough Omicron infections. These data suggest rapidly waning neutralizing titers to protect mild illnesses or asymptomatic Omicron infections several months after current booster COVID-19 vaccination in HCWs.All Authors: No reported disclosures.


I complexes have luminescent triplet charge-transferexcited states with diverse applications in photophysics and photochemistry,but for isoelectronic ZnII compounds, this behavior ismuch less common, and they typically only show ligand-based fluorescencefrom singlet π–π* states. We report two closelyrelated tetrahedral ZnII compounds, in which intersystemcrossing occurs with appreciable quantum yields and leads to the populationof triplet excited states with intraligand charge-transfer (ILCT)character. In addition to showing fluorescence from their initiallyexcited 1ILCT states, these new compounds therefore undergotriplet–triplet energy transfer (TTET) from their 3ILCT states and consequently can act as sensitizers for photo-isomerizationreactions and triplet–triplet annihilation upconversion fromthe blue to the ultraviolet spectral range. The photoactive 3ILCT state furthermore facilitates photoinduced electron transfer.Collectively, our findings demonstrate that mononuclear ZnII compounds with photophysical and photochemical properties reminiscentof well-known CuI complexes are accessible with suitableligands and that they are potentially amenable to many different applications.Our insights seem relevant in the greater context of obtaining photoactivecompounds based on abundant transition metals, complementing well-knownprecious-metal-based luminophores and photosensitizers.Many Cu The higher oxidation state of ZnII relative to CuI shifts MLCT excited states tohigher energies and usually leads to situations, in which ligand-based(or other) states are energetically lower and therefore dominate thephotophysical and photochemical properties.34 Moreover, ZnII complexes have a tendency to form polynuclearcompounds,36 many of which are labile andfor which proper characterization and speciation in solution are nottrivial because multiple species can exist in dynamic equilibriumwith each other.38 Thus, the lack of ligand fieldstabilization energy in the 3d10 valence electron configurationcan make it more challenging to obtain easily characterizable andsubstitution-inert mononuclear complexes compared to the above-mentioned3d6 or 3d8 configurations.Isoelectronic ZnII complexesuntil now is ligand-based fluorescence, for example, in many dipyrrin-basedcompounds,45 complexes with benzothiazole ligands,48 as well as with othernitrogen and oxygen donor ligands,50 or phosphines.51 Possible applications of fluorescent ZnII compounds in electroluminescence,54 including organic light-emitting diodes (OLEDs),57 circularly polarized luminescence,58 sensing,59 or cell imaging, have attracted interest.60 Similarly, aggregation-induced emission65 as well as thermally activated delayed fluorescence (TADF) receivedsubstantial attention with ZnII complexes.69The most frequentlyobserved emission type in ZnII compounds pursues the idea to introduce other emissiontypes than ligand-based fluorescence, for example, states with tripletand/or charge-transfer character in halide complexes.72 To date, this specific research thrust seems to concentrate largelyon the solid state, presumably because phosphorescence and charge-transferemission are trickier to obtain in fluid solution at room temperature.The solution phase is however very relevant for photoactive tripletand charge-transfer excited states, due to possible applications intriplet energy-transfer catalysis,73 triplet–tripletannihilation upconversion,74 photoinducedelectron transfer, and photoredox catalysis,75 including perspectives for solar energy conversion.76 In the course of our research program on photoactive complexesbased on abundant transition metals,77 we thereforeaimed to explore the possibility of obtaining ZnII compoundswith triplet charge-transfer excited states and furthermore wishedto assess their potential for usage in energy-transfer catalysis,upconversion, and photoinduced electron transfer.One of the most recent fundamental developments concerningphotoactiveZnII-based TADFemitters with (2-hydroxyphenyl)benzoxazole ligands,66 to which we attached di(anisyl)amino substituentsin an attempt to enhance the charge-transfer character between theelectron-rich phenolate ligand moiety and the electron-deficient benzoxazolesubunit  or in para-position (p-LH) to the benzoxazole unit were synthesized and fully characterized,as described in detail in the Supporting Information . The electronic coupling betweenthe tertiary amine donor and the benzoxazole acceptor subunits isexpected to be different in m-LH and p-LH, which in turn should lead to distinct properties of the anticipatedILCT excited states in [Zn(m-L)2] and[Zn(p-L)2]. Complexation was achievedby refluxing two equivalents of m-LH or p-LH with Zn(OAc)2 in tetrahydrofuran (THF) or tolueneovernight, following a previously published protocol for somewhatrelated ZnII compounds.66 Inour case, this led to products with ill-defined 1H-NMRspectra, and MALDI-TOF-MS furthermore indicated the formation of dinuclear[Zn2(m-L)3] or [Zn2(p-L)3] complexes with m/z = 1443, in addition to the anticipated signalsfor [Zn(m-L)2] or [Zn(p-L)2] at m/z = 938.Evidently, product mixtures were present, and therefore the yellowpowders were heated to 220 °C in a sublimation apparatus undervacuum overnight, which resulted in the deposition of free m-LH or p-LH ligand on the cooling finger.The remaining powders in the sublimation flask gave well-defined NMRsignals for pure [Zn(m-L)2] and [Zn(p-L)2], and the MALDI-TOF-MS signals for thedinuclear [Zn2(m-L)3] or [Zn2(p-L)3] complexes were no longerpresent. The identity and purity of the mononuclear target complexeswere furthermore confirmed by elemental analysis and HR-ESI-MS.Twonew ligands withdi(anisyl)amino substituents either in m-L)2] and [Zn(p-L)2] in toluene and dichloromethane . Wavelength differences of absorption bandmaxima between complexes and respective free ligands are minimal,but the molar extinction coefficients are roughly twice as high forthe complexes, due to the presence of two ligands per complex.The UV–vis absorptionspectra of [Zn2] has a distinct and comparatively weak absorption band in the visibleregion (with ε = 4550 M–1 cm–1 at the maximum at 417 nm in toluene), while the lowest absorptionband maximum of [Zn(p-L)2] is in the UV(366 nm in toluene) and features a substantially higher molar extinctioncoefficient at this wavelength . The emission band maximum of [Zn(m-L)2] is red-shifted compared to [Zn(p-L)2] (28 nm in toluene and 27 nm in CH2Cl2). DFT calculations  reproduce the experimentally observable maindifferences in UV–vis absorption spectroscopy. Specifically,distinct transitions at wavelengths longer than 400 nm for [Zn(m-L)2] are obtained 2], no such long-wavelength absorption transitionsare calculated 2], the HOMO is almost exclusively located on the bis(4-methoxyphenyl)amineunits (S44), whereas in [Zn(p-L)2], the HOMO is partly distributed over the 2-phenylbenzoxazoleunit . The LUMOs of both complexes aredistributed over the remaining ligand backbone without the bis(4-methoxyphenyl)amine.Based on the experimental UV–vis absorption and emission spectra,the 1ILCT energies are 2.65 eV (467 nm) for [Zn(m-L)2] and 2.89 eV (429 nm) for [Zn(p-L)2], whereas the calculations yield 2.46 eV for [Zn(m-L)2] and 3.19 eV for [Zn(p-L)2]. The experimental values for the 1ILCTenergies were estimated by normalizing the lowest-energy absorptionbands and the emission bands of the [Zn(m-L)2] and [Zn(p-L)2] complexes, andby determining the intersection of the corresponding absorption andemission bands . Attempts todetermine the respective 3ILCT energies by recording 77K emission spectra for [Zn(m-L)2] and[Zn(p-L)2] were unsuccessful because nophosphorescence was detectable. Instead, the 77 K emission spectraresembled those recorded at room temperature, indicating that the 1ILCT remains the only emissive excited state even at 77 K.Consequently, the 3ILCT energies were estimated from theTD-DFT calculations, and the energy-level scheme in The DFT calculations furthermoreconfirm the anticipated intraligandcharge-transfer (ILCT) character of the lowest-energy transitionsin the two Znomplexes 3. Specifon bands S4. Attem1ILCT and 3ILCT excited states (ΔEST) is substantially smaller for [Zn(m-L)2] (0.13 eV) than for [Zn(p-L)2] (0.73 eV). This can be qualitatively rationalized on thebasis of the clearer spatial separation between HOMO and LUMO in [Zn(m-L)2] in comparison to [Zn(p-L)2] (due to decreased electron–electron interaction),as discussed above 2] in deaerated toluene (p-L)2] at long delay times (>20ns) shows a very broad ESA band with a maximum at 450 nm along witha ground-state bleach at 390 nm. Evidently, [Zn(m-L)2] and [Zn(p-L)2] bothhave a fluorescent singlet excited state (1ILCT) and amuch longer-lived (nonemissive) triplet excited state (3ILCT), which causes the above-mentioned ESA bands. In aerated solutionsof [Zn(m-L)2] and [Zn(p-L)2], the 3ILCT excited states are efficientlyquenched by oxygen (S6), in line with their tripletcharacter. Based on time-resolved fluorescence experiments , the 1ILCT lifetimes(τsinglet) of [Zn(m-L)2] and [Zn(p-L)2] are 25 and 5.5 ns, respectively,in deaerated toluene at 293 K. The 1ILCT fluorescence decayof [Zn(m-L)2] is biexponential in bothtoluene and CH2Cl2; see SI page S23 for details. We speculate that the biexponentialnature of the excited-state decay for this compound stems from differentconformers that are present in solution. The τsinglet values reported for [Zn(m-L)2] in S23).The UV–vis transient absorption spectra recordedat shortdelay times (<20 ns) after the excitation pulses are dominatedby negative signals centered around 500 nm 5, which  toluene 5a is domy oxygen 5 and S6,1ILCT lifetime of [Zn(m-L)2] is longer (25 ns) than in CH2Cl2 (9 ns),in line with the energy gap law, though for [Zn(p-L)2] such a difference is not observed and the 1ILCT lifetime is 5.5 ns in both solvents (Table S3). The fluorescence quantum yield (Φfluo) of [Zn(p-L)2] 2] under identical conditions (50%). With 1ILCTlifetimes and luminescence quantum yields at hand, the rate constantsfor radiative (kr) and nonradiative excited-staterelaxation (knr) were calculated following eqs S5–S9. The intense fluorescence signalsand rapid intersystem crossing complicate the detection of the spectralsignatures of the 1ILCT states by transient absorption,but it seems plausible that the 1ILCT and 3ILCTstates have in fact similar spectroscopic signatures (S6).80 Based on their very different decaykinetics, the distinction between these two states is however clear-cut.Expectedly, the fluorescence lifetimes of the free ligands andcorresponding complexes are very similar to each other. In toluene,the solvents S3. The fgnatures 5 and S6.1ILCT to the (dark) 3ILCT state, relative actinometry experiments were performed.For this purpose, the molar quantities of triplet-excited anthraceneformed after selective excitation of [Zn(m-L)2] and [Zn(p-L)2] in the presenceof excess anthracene were determined by transient UV–vis absorptionspectroscopy (II complexes to anthracene is quantitative (which seemsreasonable given its high driving force) and given the known molarextinction coefficient of triplet-excited anthracene at 423 nm ,81 the concentration of triplet-excited [Zn(m-L)2] and [Zn(p-L)2] can beestimated in this manner. In a separate experiment, an aqueous solutionof [Ru(bpy)3]2+ with the same absorbance atthe respective excitation wavelength was irradiated using identicalinstrument settings. By monitoring the bleach of the correspondingMLCT absorption band at 455 nm with its known change in molar extinctioncoefficient ,82 the numberof photons absorbed by the individual solutions was determined; seeSI page S20 for full details. The intersystemcrossing quantum yield (Φisc) was then approximatedas the number of triplet-excited ZnII complexes dividedby the number of absorbed photons.To obtain quantitative information concerning the intersystem crossing(ISC) from the initially excited troscopy 6. Assumim-L)2], we obtain Φisc = 13%, whereas for [Zn(p-L)2], Φisc is substantially lower with merely 7.2%.This finding is compatible with the lower fluorescence quantum yieldfound for [Zn(m-L)2] (50%) relative to[Zn(p-L)2] (86%), and furthermore is inqualitative accordance with the smaller 1ILCT–3ILCT energy gap (ΔEST) in[Zn(m-L)2]  and the bleach recovery at390 nm in Figure S10), the lifetimes of the photoactive(but dark) 3ILCT excited states (τtriplet) of [Zn(m-L)2] and of [Zn(p-L)2] were determined. We find τtriplet = 38 μs for [Zn(m-L)2] and τtriplet = 62 μs for [Zn(p-L)2] in deaerated toluene at 293 K.By monitoringthe decay of the excited-state absorption band at350 nm in m-L)2] and [Zn(p-L)2] have appreciable intersystemcrossing quantum efficiencies (m-L)2] and [Zn(p-L)2] concentrationssuch that an optical density of 0.06 resulted at that wavelength,which implied substantially different complex concentrations (1.3vs 0.6 mol % relative to the substrates) due to their different molarextinction coefficients at 415 nm . With [Zn(m-L)2] asphotosensitizer, that photostationary state was composed of 81% cis-stilbene, whereas the [Zn(p-L)2] complex yielded a photostationary state with only 49% of cis-stilbene (trans-stilbene (2.14eV) and cis-stilbene (2.35 eV) .84 The 3ILCT energy of [Zn(m-L)2] (2.33 eV) liesbetween the triplet energies of trans- and cis-stilbene, and consequently this complex sensitizes the trans-to-cis photo-isomerization of stilbenefar more efficiently than the reverse reaction, resulting in the accumulationof the cis-isomer over time (3ILCT state of [Zn(p-L)2] (2.46eV) is energetically above the lowest triplet states of both trans- and cis-stilbene. Consequently,[Zn(p-L)2] sensitizes the cis-to-trans and the trans-to-cis photo-isomerization of this specific substrate equallywell, resulting in a photostationary state composed of roughly 1:1 cis- and trans-products (87 we have foundno evidence that the fluorescent 1ILCT excited states ofour zinc(II) complexes sensitize this reaction.Under these conditions,the photo-isomerization of t 60 min S22. Withm-L)2] photosensitizerleads to a photostationary state containingonly 30% of the cis-isomer, which is considerablylower than what is obtained in the case of stilbene (81%). This observationcan be understood on the basis of the higher triplet energy of trans-methyl cinnamate (2.37 eV)84 compared to trans-stilbene (2.14 eV).84 Consequently, [Zn(m-L)2] with its 3ILCT energy of 2.33 eV becomes a lessefficient sensitizer for the cis-to-trans isomerization of methyl cinnamate. When using [Zn(p-L)2] as a sensitizer for the methyl cinnamate substrate,a substantially higher cis photoproduct yield of49% is reached, due to the higher 3ILCT energy of thiscomplex in comparison to [Zn(m-L)2]. Forthe methyl cinnamate photo-isomerization reactions, we used an identicalphotosensitizer loading of 0.5 mol % in both experiments. When reducingthe catalyst load to 0.05 mol %, we determined a turnover number (TON)of 550 for [Zn(m-L)2] and 940 for [Zn(p-L)2].For the methylcinnamate substrate 2]and [Zn(p-L)2] is limited and furthermoreboth complexes suffer from lower stability in CDCl3 comparedto less polar solvents such as benzene or toluene. Conversely, thesubstrate is not sufficiently well soluble in benzene-d6 and toluene-d8. Consequently,only relatively modest decarboxylation yields of 8% when using [Zn(m-L)2] and 18% in the case of [Zn(p-L)2] were obtained . Upon irradiation of a solution of the substrate without ZnII photosensitizer, no decarboxylation product can be observed, which confirms thetriplet sensitization of the substrate by the [Zn(m-L)2] and [Zn(p-L)2] complexes.Compared to the previously used iridium complex, the ZnII sensitizers employed here have substantially lower triplet energies(2.33 and 2.46 vs 2.64 eV), which might help explain the lower photo-fragmentationyields observed here.88 Moreover, iminyl-radicalsformed in the photo-fragmentation reaction could potentially poisonthe catalyst because the ZnII complexes are expected tobe considerably more substitution-labile than cyclometalated IrIII compounds.To explore organic triplet reactivitybeyond photo-isomerizations,we focused on a decarboxylation reaction (m-L)2] and [Zn(p-L)2], it seemed meaningful to explore their ability to undergo photoinducedelectron transfer reactions. Toward this end, we identified 1,2,4,5-tetracyanobenzene(TCB) as a suitable reaction partner because this is a fairly strongelectron acceptor featuring a reduction potential of −0.74V vs SCE in MeCN,89 combined with a characteristicspectroscopic signature of its one-electron reduced form (TCB•–), which is readily identifiable by transientUV–vis absorption spectroscopy.Given thecharge-transfer character of the photoactive excited states of [Zn(m-L)2] at 420 nm in the presenceof excess TCB, the resulting UV–vis transient absorption differencespectrum (•– (91 and [Zn(m-L)2]+ (92 in line with the fact that the primary oxidation of [Zn(m-L)2] occurs on the triarylamine unit of thechelate ligand. For [Zn(m-L)2]+ the respective absorption band is observed near 750 nm (p-L)2]+, the analogous absorptionband is shifted to near 800 nm .After selective excitationof [Zn2]+ 7c. The lr 750 nm 7b, where1ILCT fluorescence of [Zn(m-L)2] and [Zn(p-L)2] by 2.5 mM TCB was observed, suggesting that photoinduced electrontransfer to TCB occurs from the long-lived 3ILCT statesof the ZnII complexes, not from the fluorescent 1ILCT states. Indeed, in laser flash photolysis experiments, quenchingof the 3ILCT states of [Zn(m-L)2] and [Zn(p-L)2] by TCB is detectableby monitoring the characteristic absorption bands and ground-statebleaches of the 3ILCT excited ZnII complexesat 350 and 390 nm , the Stern–Volmer-type plots in the insetsof S33b were obtained. Linear regression fits tothese data gave rate constants of 5.3 × 109 and 6.3× 109 M–1 s–1 forphotoinduced electron transfer from [Zn(m-L)2] and [Zn(p-L)2] to TCB, respectively.These values are relatively close to the diffusion limit in tolueneat 293 K (1.1 × 1010 M–1 s–1),84 in line with a high driving-forcereaction. In cyclic voltammetry, the first oxidation wave appearsat 0.32 V vs SCE for [Zn(m-L)2]  and at 0.80 V vs SCE for [Zn(p-L)2] . Given the 3ILCT energies in m-L)2]  and −1.66 V vs SCEfor [Zn(p-L)2] . Consequently, photoinduced electron transfer to TCB 89 is highly exergonic in both cases, which canexplain why nearly diffusion-controlled kinetics are observed.In time-correlated single photoncounting (TCSPC) experiments,no significant quenching of the d 390 nm 5, respecII complexes areboth rather strong photoreductants, but [Zn(m-L)2] is roughly 0.4 V more reducing in its 3ILCT excitedstate than [Zn(p-L)2]. This finding canbe traced back to substantially different ground-state oxidation potentials,which might have their origin in different extents of electronic couplingbetween the coordinating phenolate units and the triarylamine N-atoms.Specifically, in [Zn(m-L)2], the anionicphenolate unit stands in para-position to the triarylamineN-atom, which might enhance the electron density at the N-atom andcould lower the respective oxidation potential in comparison to [Zn(p-L)2], in which the phenolate unit stands in meta-position to the triarylamine N-atom.According to this analysis, the two Zn3ILCT excitedstates, both [Zn(m-L)2] and [Zn(p-L)2] should in principlebe able to sensitize triplet–triplet annihilation upconversion,which is an attractive process for the conversion of low-energy inputlight into higher energy output radiation.96 In particular, upconversion from the visible to the ultravioletspectral region has received increasing interest lately,102 and our ZnII complexes with their relatively high 3ILCT energies seemed promising for this purpose. Previousupconversion studies with ZnII sensitizers focused on red-to-blueupconversion and on near-infrared sensitization of photochemical reactions.106 We identified the combination of [Zn(m-L)2] and (TMS)2napht  protected acetylenesin 1- and 4-positions has a triplet energy of 2.10 eV.98 The (TMS)2napht compound (2napht. TTET fromthe 3ILCT excited state of [Zn(m-L)2] to (TMS)2napht is thus expected to be exergonicby roughly 0.2 eV. This specific sensitizer-annihilator combinationfurthermore seemed beneficial because [Zn(m-L)2] absorbs less than [Zn(p-L)2]in the spectral region of the (TMS)2napht fluorescence, whichshould limit unwanted reabsorption phenomena. In the following, weaimed at a proof-of-principle experiment, to explore the applicationpotential of tetrahedral ZnII complexes for photochemicalupconversion.With their photoactive S)2napht 8e as paromplexes 4. The sucompound 8e differm-L)2] and 2 mM (TMS)2napht in deaerated toluene wasexcited at 430 nm with laser pulses of roughly 10 ns duration and16 mJ energy. At this wavelength, (TMS)2napht is essentiallytransparent and the light is absorbed exclusively by the ZnII complex. Emission spectra were then recorded in a time-gated manner,once immediately after the excitation pulses (blue trace in 1ILCT emission of [Zn(m-L)2] centeredaround 528 nm, yet between 350 and 400 nm, a weak luminescence band2] is absent in that case because the 1ILCTexcited state with its lifetime (τsinglet) of 25ns , and consequently, these UV emissions are attributableto the delayed (upconverted) annihilator fluorescence. This delayedfluorescence decays on a microsecond timescale 2] 2] under identical conditions,using a solution containing only the ZnII sensitizer (20μM) but no annihilator (black trace in 1ILCT emission intensity(Ifluo) and the integrated delayed annihilatorfluorescence (IUC) by comparing the blackand red traces in S48). Given a 1ILCT fluorescence quantum yield (Φfluo) of 50% (UC) of 0.73% . Given the incomplete intersystem crossingin [Zn(m-L)2] (Φisc =13%), the modest upconversion quantum yield is unsurprising. Normalizedto the amount of photogenerated triplet excited states on [Zn(m-L)2], the upconversion quantum yield is 5.6%,which compares favorably to many previously investigated systems.To estimate the upconversion quantumyield under the conditionsof this pulsed laser study, an additional reference experiment wasperformed, in which we recorded the prompt ) of 50% 1, this a1ILCT emission spectrain x1.82), whereas the prompt 1ILCT fluorescence shows the anticipatedlinear power dependence (inset of x1.03).Excitation power-dependent measurements were performed using a447 nm continuous-wave (cw) laser and yielded the series of upconversionfluorescence spectra shown in inset of 8c, x1.03II complexesfrom 3]2+, a well-known and widely usedbenchmark compound.110 For this purpose,deaerated solutions of [Zn(m-L)2], [Zn(p-L)2], and [Ru(bpy)3]2+ with essentially identical absorbance values (A0) of 0.05 at 405 nm were prepared (Table S4) and then irradiated at that wavelength with a cwlaser providing 0.526 W of output power. Following a previously publishedmethod (SI pages S43 and S44),112 we monitored the emission intensityof these three complexes as a function of irradiation time and estimatedtheir photodegradation quantum yields (Φdecomp) byconsidering the time period during which their photoluminescence intensitiesdecreased to 90% of the initial values at t = 0 (SIpage S43). Given the known  concentrationsof the complexes (c0) and the measurablenumber of photons absorbed within the above-mentioned time period, the Φdecomp values in Table S4). In comparisonto [Ru(bpy)3]2+, the inherent photostabilityof the two investigated ZnII complexes is an order of magnitudelower, which is perhaps unsurprising given the substantial structuraland electronic differences between these compound classes. In contrastto the 3d10 ZnII compounds, the octahedral 4d6 complex with RuII benefits from a high ligandfield stabilization energy, which decelerates ligand substitutionreactions in the electronic ground state, and furthermore might affectthe stability in excited states. The 3ILCT excited statesof the [Zn(m-L)2] and [Zn(p-L)2] complexes are considerably longer-lived 3]2+ (τtriplet <1 μs), which increases the likelihood of photo-decompositionin the ZnII compounds.Manyapplications require long-term photo-irradiation, and thus it seemedmeaningful to explore the photo-robustness of the Znprepared S4 and thprepared S4 and th3ILCT excitedstate of the ZnII compounds is quenched partially by trans-stilbene indicate that under conditions of TTET catalysis,[Zn(m-L)2] and [Zn(p-L)2] are considerably more photo-robust than in neat solventwithout any substrate present (p-L)2] in deaerated toluene degraded essentiallycompletely within 1.5 h of irradiation with a 415 nm LED providing7 W of output power 2] with a mixture containing 10 mM trans-stilbene(red traces in 3ILCT excited states is a major decompositionpathway in the [Zn(p-L)2] and [Zn(m-L)2] compounds, as suspected above  coordination compounds.40 Here, we demonstrate that intraligand charge-transfer (ILCT) excitedstates in tetrahedral ZnII complexes can display similarreactivity as metal-to-ligand charge-transfer (MLCT) excited statesin four-coordinate CuI compounds. In the latter, the photoactiveMLCT excited states are typically strongly distorted due to the transientformation of a Jahn–Teller susceptible CuII d9 metal species,113 opening up unwantednonradiative excited-state relaxation pathways. The ILCT excited statesof the ZnII complexes investigated herein do not sufferfrom this problem because the metal oxidation state remains unchanged.The ZnII cation presumably contributes to mediating intersystemcrossing from the initially excited 1ILCT to the 3ILCT states, from where typical triplet photo-reactivity becomesaccessible, including for example the photo-isomerization of olefinsand the sensitization of triplet–triplet annihilation upconversion.The 3ILCT energies of our ZnII complexes aresufficiently high to allow for upconversion from the blue to the ultravioletspectral range, and these 3ILCT excited states are furthermorevery reducing, making these ZnII compounds strong photoreductantseven in comparison to some of the well-established precious-metal-basedsystems.114 Since the intersystem crossingis not quantitative, fluorescence from the 1ILCT excitedstate remains observable in our ZnII compounds, in contrastto four-coordinate CuI complexes with photoactive MLCTstates, in which intersystem crossing is usually ultrafast. Thus,our study illustrates some clear analogies and differences betweentetrahedral ZnII compounds with photoactive ILCT statesand the more well-known four-coordinate CuI complexes.The application potentialof charge-transfer excited states inZnII complexes complement thedifferent types of photoactive excited states reported recently forfirst-row and other Earth-abundant transition-metal complexes,28 which includes the classical MLCT states ford6 complexes ,117 square-planar d8 compounds (NiII)118 and four-coordinated10 complexes (CuI),78 ligand-to-metalcharge-transfer (LMCT) states for TiIV, ZrIV,120 MnIV, FeIII and CoIII,126126 metal-centered (MC) states for VIII, CrIII and CoIII,132 as well as ligand-to-ligand charge-transfer (LLCT) excited statesfor two-coordinate CuI complexes.33 Given these findings, it seems reasonable to concludethat ZnII complexes with charge-transfer and triplet excitedstates would perhaps deserve greater attention in future studies aimingto discover new photophysics and photochemistry in first-row transition-metalcomplexes.The ILCT states of our ZnChemicals were obtainedfrom commercial suppliers in high purityand were used without further purification. Dry solvents were usedas purchased from commercial suppliers or from an Innovative TechnologyPureSolv micro multiunit solvent purification system.3 (7.26 ppm in 1H-NMR and 77.16 ppm in 13C-NMRspectroscopy), CD2Cl2 (5.30 ppm in 1H-NMR and 53.84 ppm in 13C-NMR spectroscopy), or DMSO-d6 (2.50 ppm in 1H-NMR and 39.52 ppmin 13C-NMR spectroscopy).133 The multiplicity of the signals is described with the followingabbreviations: s (singlet), d (doublet), t (triplet), q (quartet),p (pentet), m (multiplet), and combinations of these abbreviations.The coupling constants J are given in hertz (Hz).Nuclearmagnetic resonance (NMR) spectroscopy was performed usingeither a 400 MHz Bruker Avance III spectrometer or a 600 MHz BrukerAvance III spectrometer at 298 K. The latter instrument was equippedwith a direct observe 5 mm BBFO smart probe. Chemical shifts δare given in parts per million (ppm) and referenced to CDCl2Cl2) was evaporatedonto the sample plate and then the substrate was evaporated onto thematrix.High-resolution mass spectroscopy (HRMS) was performed by Dr. MichaelPfeffer on a maxis 4G QTOF EDI spectrometer from Bruker. MALDI-TOF-MSwas performed on a Bruker Microflex instrument operating in positivemode. The matrix  was performed by Sylvie Mittelheisserona Vario Micro Cube instrument from Elementar. The amounts of carbon,hydrogen, and nitrogen were determined. All values are given in percentages.Cyclic voltammetry was either performed in an MBraun glovebox underargon atmosphere using a Versastat4-200 potentiostat from PrincetonApplied Research, or under deaerated conditions using a Versastat3-200potentiostat from Princeton Applied Research.Photocatalyticreactions were performed in NMR tubes with tubecaps from VWR. The irradiation source was a SOLIS-415C high-powerLED from ThorLabs with a 380 or 400 nm cutoff filter.Photostabilitieswere measured using a SOLIS-415C high-power LEDfrom ThorLabs with a 400 nm cutoff filter or a Roithner LasertechnikGmbH 405 nm continuous-wave laser with an output power of 526 mW.All photophysical measurements were carried out at 293 K and thesolutions were purged with argon  for at least 5 minusing screw cap cuvettes. Steady-state optical absorption and UV–visspectro-electrochemical measurements were recorded using a Cary 5000spectrophotometer (Varian). Steady-state luminescence spectra weremeasured using a Fluorolog-3-22 instrument from Horiba Jobin-Yvon.Transient absorption and time-resolved absorption and emission measurementswere performed on an LP920-KS instrument from Edinburgh Instruments.The excitation source was a pulsed Quantel Brilliant B Nd:YAG laserequipped with an optical parameter oscillator (OPO) from OPOTEK ora Nd:YAG laser  witha beam expander (BE02-355 from Thorlabs). The transient absorptionspectra were detected on an iCCD camera (Andor), while kinetics ata single wavelength were recorded with a photomultiplier tube. Fluorescencelifetimes were measured on a LifeSpec II spectrometer (time-correlatedsingle photon counting technique) from Edinburgh Instruments usingpicosecond pulsed diode lasers for excitation at 405 nm.


Cellular senescence and cell competition are important tumor suppression mechanisms that restrain cells with oncogenic mutations at the initial stage of cancer development. However, the link between cellular senescence and cell competition remains unclear. Senescent cells accumulated during the in vivo aging process contribute toward age-related cancers via the development of senescence-associated secretory phenotype (SASP). Here, we report that hepatocyte growth factor (HGF), a SASP factor, inhibits apical extrusion and promotes basal protrusion of Ras-mutated cells in the cell competition assay. Additionally, cellular senescence induced by a high-fat diet promotes the survival of cells with oncogenic mutations, whereas crizotinib, an inhibitor of HGF signaling, provokes the removal of mutated cells from mouse livers and intestines. Our study provides evidence that cellular senescence inhibits cell competition-mediated elimination of oncogenic cells through HGF signaling, suggesting that it may lead to cancer incidence during aging. Ras mutations induce cell competition and cellular senescence to inhibit the proliferation of oncogenic mutated cells. Here the authors demonstrate that cellular senescence inhibits cell competition-induced elimination of oncogenic cells through HGF signalling. Although developmental studies on Drosophila were the first to report about cell competition, recent studies have shown that cells with oncogenic mutations compete with surrounding normal cells and are apically excluded from the epithelial layer in mammalian organs6. In contrast, cellular senescence is a phenomenon in which somatic cells display a finite replicative lifespan under in vitro culture conditions, which inhibits the proliferation of cancer-prone cells in precancerous lesions and benign tumors8.Somatic mutations in some oncogenes or tumor suppressor genes lead to cancer. At the earliest stage of cancer development, mutated damaged cells are selectively removed by several tumor-suppressive mechanisms, such as cell competition, cellular senescence, and various types of cell death pathways10. Skin homeostasis is maintained via epidermal stem cell competition, whereas epidermal stem cell aging leads to reduced cell competition, resulting in skin aging11. In addition, aging is a high-risk factor for cancer incidence. However, the effects of aging on cell competition as a tumor-suppressive mechanism remain unelucidated. Oncogenic mutations, including Ras signaling-related mutations, induce cell competition and cellular senescence. Therefore, they seem to occur simultaneously, inhibiting the proliferation of mutated cells as a barrier against tumor formation. Recent studies have exhibited the harmful side effects of cellular senescence that occurs during aging. Senescent cells accumulate throughout the body during the aging process14 and secrete proinflammatory factors, including cytokine, chemokine, modifiers of the extracellular matrix, and growth factors. This phenotype, termed as senescence-associated secretory phenotype (SASP), promotes cancer malignancy at the initiation or progression stages and contributes toward the development of numerous other age-related pathologies16.The functional decline of various tissues and organs due to aging leads to the development of multiple types of diseases. The status of cell fitness also changes during agingHere, we explored the function of a SASP factor, hepatocyte growth factor (HGF), in cell competition and discovered that HGF inhibits apical extrusion and promotes basal protrusion of Ras-mutated cells using in vitro and in vivo cell competition models. Furthermore, these data demonstrate that cellular senescence inhibits cell competition-induced elimination of oncogenic cells through HGF signaling. This mechanism may increase the risk of cancer development in the aging population.V12 expression. The induction of cellular senescence was confirmed via an observed decrease in LMNB1, an increase of senescence markers p16INK4a and SASP factors , senescence-associated β-galactosidase  staining with loss of Ki-67, a feature of senescent cell cycle arrest, and the formation of DNA damage foci  were treated with CM derived from control or RasV12-induced senescent IMR-90 cells for 3 days, followed by cell competition assay on the collagen gel-coated plate to mimic physiological tissues18. After treatment with CM derived from proliferating IMR-90 cells, similar to nontreated cells, MDCK-pTR GFP-RasV12 cells were extruded by surrounding MDCK cells. However, CM derived from RasV12-induced senescent IMR-90 cells promoted the nonextruded population   or oncogenic cells (MDCK-pTR GFP-RasV12 cells) in this model. To elucidate this point, a cell competition assay was performed using senescent CM-treated MDCK cells (parent) with nontreated MDCK-pTR GFP-RasV12 cells and vice versa  was prepared from human diploid fibroblasts, IMR-90 cells, which were subjected to senescence via H-Rasoci Fig. . In lineion Fig. , similarion Fig. , oncogenrsa Fig. . We founyer Fig. , indicatV12- or X-ray-induced senescent IMR-90 cells  analysis . It activates downstream signaling pathways and promotes carcinogenesis and cancer progression27. In the obese mice model, HFD induces senescence in mice hepatic stellate cells (HSCs) via deoxycholic acid (DCA) produced by the gut microbiota31. First, we checked the expression levels of HGF in human and mouse HSCs. Cellular senescence was induced by treatment with DCA and lipoteichoic acid (LTA), a major constituent of the cell wall of gram-positive bacteria, as previously described30. After DCA/LTA treatment, senescent HSCs exhibited a significant increase in HGF expression   and SASP factor (CXCL10) expression, DNA damage accumulation (53BP1), and SA-β-Gal activity V12-internal ribosomal entry sites (IRES)-luciferase expression in the livers of ND- and HFD-fed mice  and increased expression of mesenchymal marker protein (vimentin)  was performed to induce GFP-N-Rasice Fig. . Accordiice Fig. 26. In viion Fig. . In contays Fig. ; this fiice Fig. .Fig. 4CrLSL-RasV12-IRES-enhanced GFP (eGFP) and villin-Cre-ERT2 mice and its organoid exhibit Cre-dependent induction of RasV12 expression, which is evaluated through the simultaneous expression of eGFP18. Using this system, we examined the effect of cellular senescence on cell competition ex vivo and in vivo. Intestinal organoids established from cell competition mouse model were cocultured with nonsenescent or senescent IMR-90 cells. Coculturing organoids with senescent fibroblasts significantly inhibited apical extrusion and promoted basal extrusion of RasV12-transformed epithelial cells compared with organoids with nonsenescent fibroblasts   supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Sigma-Aldrich) at physiological oxygen conditions  at 37 °C. Early passage TIG-3 cells (<40 population doublings) were used as proliferating (control) cells, and late passage TIG-3 cells (>70 population doublings) that stopped proliferating were used as replicative senescent cells. For X-ray-induced senescence, IMR-90 cells were exposed to 10-Gy irradiation using a CP-160 X-ray machine (Faxitron X-ray Corporation). After X-ray irradiation, IMR-90 cells were plated at a density of 2500 cells cm−2. These cells were not passaged for 10 days after X-ray irradiation. Ras-induced senescence by 4-hydroxytamoxifen in IMR-90/ER: H-RasV12 cells was conducted as previously described44. CM was prepared by incubating proliferating (control) or senescent cells for 3 days in DMEM supplemented with 10% FBS. Proliferating (control) cells were plated at subconfluent density 1 day before starting CM preparation. CM was filtered through a 0.45-μm filter and then used for cell culture. MDCK (parent) and MDCK-pTR GFP-RasV12 cells were cultured according to the method described previously18. For human and mouse HSCs, senescence induction and cell culture were performed as described previously30. All cell lines used were negative for mycoplasma.TIG-3 cellsThe following reagents and recombinant proteins were used for cell competition assay: sodium dichloroacetate ; recombinant human IL-6 , IL-8 , GDF-15/IMC-1 , MCP-1 , HGF , TGF-β1 , CD105 protein , BSG protein , DPP4 protein , pentraxin 3/TSG-14 , and uPAR protein (84–95) .18 with minor modifications. In brief, after MDCK (parent) and MDCK-pTR GFP-RasV12 cells were treated with CM derived from proliferating (control) or senescent cells for 3 days (the details of which are provided in the “Cell culture” section), MDCK-pTR GFP-RasV12 cells were combined with normal MDCK (parent) cells at a ratio of 1:50 and cultured on type-I collagen gel-coated coverslips. After 16 h of tetracycline treatment to induce the expression of GFP-RasV12 in MDCK-pTR GFP-RasV12 cells, all cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and permeabilized in 0.5% Triton X-100 in PBS. After blocking the cells using 1% bovine serum albumin in PBS, cells were treated with Alexa Fluor 568 phalloidin . The nuclei were counterstained using 4′, 6-diamidino-2-phenylindole (DAPI), and coverslips were mounted using ProLong Diamond antifade mountant (Life Technologies). Images were obtained using an LSM710 confocal microscope (Zeiss). As shown in Fig. V12 cells were treated using 50 μM dichloroacetate or 250 ng/mL of each recombinant protein for 3 days and then cell competition assay with normal MDCK (parent) cells was performed as described above. For crizotinib treatment experiments, MDCK-pTR GFP-RasV12 cells were treated with 20 ng/mL recombinant HGF  cells as described above.Cell competition assay was performed as previously described−2 and were not passaged after X-ray irradiation. After 8 and 9 days of X-ray irradiation, HGF knockdown was performed (twice) as described above. Ten days after X-ray irradiation , IMR-90 cells were cultured in fresh DMEM supplemented with 10% FBS and incubated for 3 days. CM was filtered through a 0.45-μm filter and then used for cell culture as HGF-depleted senescent CM.HGF knockdown was performed via the transfection of siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. ON-TARGETplus human HGF siRNA  was used at a concentration of 25 nM for 2 days. Knockdown efficiency was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR). For the preparation of CM from IMR-90 cells with HGF knockdown, proliferating IMR-90 cells were exposed to 10-Gy irradiation using a CP-160 X-ray machine. These cells were plated at a density of 2500 cells cmmirVana™ miRNA Isolation Kit (Thermo Fisher Scientific). Then, the extracted RNA was subjected to reverse transcription using the PrimeScript RT Master Mix (TaKaRa). RT-qPCR was performed on a StepOne Plus PCR system (Thermo Fisher Scientific) using SYBR Premix Ex Taq II . The primers used for RT-qPCR are as follows: HGF (human): 5′-GCACTGAAGATAAAAACCAA-3′ (forward) and 5′-GTTTTCTCGCTTTATCAAAA-3′ (reverse), HGF (mouse): 5′-GGCCCACTCATTTGTGAAC-3′ (forward) and 5′-CATCCACGACCAGGAAC-3′ (reverse), p21 (mouse): 5′-CCTGGTGATGTCCGACCTG-3′ (forward) and 5′-CCATGAGCGCATCGCAATC-3′ (reverse)45, β-Actin (mouse): 5′-CGCCACCAGTTCGCCATGGA-3′ (forward) and 5′-ACAGCCCGGGGAGCATCGT-3′ (reverse), CDH1: 5′-CAGAAGATGACACCCGGGAC-3′ (forward) and 5′-GCCACATCATTGCGAGTCAC-3′ (reverse), ZO1: 5′-CCACACCGCTGGTTTTAAGC-3′ (forward) and 5′-TCTTCGGGTGGCTTCATCTG-3′ (reverse), Fibronectin (dog): 5′-AGCAAATGGCCAGAATCCGA-3′ (forward) and 5′-CTTGTAGTCAGTGCCGGGTT-3′ (reverse), ZEB1: 5′-GAAGGTGATCCAGCAAATG-3′ (forward) and 5′-CTTCCGCATTTTCTTTTTGG-3′ (reverse), β-catenin (dog): 5′-TGTATGGGTAGGGTAAATCAGGAAT-3′ (forward) and 5′-TGTATGGGTAGGGTAAATCAGGAAT-3′ (reverse), and β-Actin (dog): 5′-GGCACCCAGCACAATGAAG-3′ (forward) and 5′-ACAGTGAGGCCAGGATGGAG-3′ (reverse). p16, IL-6, IL-8, CXCL10, β-actin (human), p16, CXCL10, and GAPDH (mouse) were detected as previously described30. The quantity of all samples was acquired using the standard curve method and was normalized to the housekeeping gene ACTB and GAPDH according to the manufacturer’s protocol.Total RNA was extracted using the Cell pellets were lysed in lysis buffer (0.1 M Tris-HCl (pH 7.5), 10% glycerol, and 1% sodium dodecyl sulfate (SDS)), boiled for 5 min, and then centrifuged for 10 min at 15,000 rpm. All protein concentrations were determined using BCA Protein Assay Reagent (Pierce). Each cell lysate was electrophoresed using SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore). After blocking with 5% skim milk (Megmilk) or 5% bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween 20 (TBST), the membrane was treated with primary antibodies against Ras , lamin-B1 , GAPDH , and phospho-Met (Tyr1234/1235) (D26)  overnight at 4 °C in a blocking buffer. Membranes were then washed thrice in TBST and incubated with enhanced chemiluminescence (ECL) anti-mouse IgG, horseradish peroxidase-linked whole antibody  or ECL anti-rabbit IgG, and horseradish peroxidase-linked whole antibody  for 1 h at room temperature. After washing the membrane thrice with TBST, the signal was resolved using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and imaging was performed using a FUSION imaging system (Vilber-Lourmat).CM from proliferating (control) and X-ray- or Ras-induced senescent IMR-90 cells was used for performing ELISA and cytokine array. The ELISA and cytokine array were performed using Quantikine ELISA Human HGF Immunoassay  and Proteome Profiler Array Human XL Cytokine Array Kit , respectively, according to the manufacturer’s instructions. In the cytokine array, signal intensity was detected using a FUSION imaging system (Vilber-Lourmat) and quantified using NIH Image J software. The signal intensities of six reference spots in each membrane were measured and defined as 100%.30. Biopsies of mouse liver were fixed in 10% formalin for 24 h, progressively dehydrated through gradients of alcohol, and embedded in paraffin. Samples were sectioned using a microtome (3-μm thick) for antibody staining, deparaffinized in xylene, rehydrated, and then exposed to heat-induced antigen retrieval for 20 min in 1× Target Retrieval Solution . After washing with PBS, the sections were incubated in 1× Power Block  for 10 min at room temperature. After blocking and washing with PBS, the sections were incubated with primary antibodies overnight at 4 °C. The primary antibodies used for mouse samples were as follows: α-SMA , vimentin , desmin , HGF , CXCL10 , 53BP1  and phalloidin . Alexa Fluor 488 goat anti-mouse , Alexa Fluor 594 goat anti-rabbit , and Alexa Fluor 594 donkey anti-goat antibodies  were used as secondary antibodies. SA-β-Gal activity with Ki-67 protein was detected using a Cellular Senescence Detection Kit and SPiDER-β-Gal  with recombinant anti-Ki-67 antibody (SP6) , respectively, according to the manufacturer’s instruction.Immunofluorescence analysis was conducted using antibodies against γ-H2AX  and phospho-(Ser/Thr) ataxia telangiectasia mutated (ATM)/ ataxia telangiectasia and Rad3-related (ATR) substrate  as previously describedV12 cells that were treated with CM derived from proliferating (control) and X-ray- or Ras-induced senescent IMR-90 cells (the details are provided in the “Cell Culture” section) for 3 days using mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer’s protocols. Microarray analysis was conducted using the Plant and Animal Gene Expression platform (Agilent). Labeled cRNA was prepared from 1 to 5 μg of total RNA using Agilent’s Quick Amp Labeling Kit. Following fragmentation, 1.65-μg cRNA was hybridized using the Agilent expression microarray according to the manufacturer’s protocols. Arrays were scanned using the Agilent Technologies G4900DA SG12494263. Array data export processing and analysis were conducted using Agilent Feature Extraction v11.0.1.1. GO analysis was performed using DAVID46.Total RNA was extracted from MDCK-pTR GFP-RasV12-IRES-EGFP-Luc plasmids were constructed as shown below: human G12V N-Ras (N-RasV12)47 and IRES-EGFP with luciferase12 sequences were cloned into a pSBbi-Hyg plasmid . The IRES-EGFP sequence was based on a pIRES2-EGFP plasmid (Invitrogen).pSBbi-Hyg-EGFP-Luc (control) and pSBbi-Hyg-N-Ras42 with minor modifications. In brief, 27-μg pSBbi-Hyg-EGFP-Luc or pSBbi-Hyg-N-RasV12-IRES-EGFP-Luc plasmid and 3-μg pCMV(CAT)T7-SB100 plasmid   were injected into the tail vein using TransIT-EE Hydrodynamic Delivery Solution (Mirus) according to the manufacturer’s protocols. Mice were orally administered with 50 mg/kg crizotinib  at time points indicated in Fig Male C57BL/6 mice (CLEA Japan Inc.) of age 7–8 weeks were maintained under specific pathogen-free conditions and fed ND  or a high-fat diet  for 3 months. The room was maintained at a controlled temperature (~25 °C) and humidity (~50%) and 12 h light–dark cycles. HTVi was conducted as previously described27. For in vivo experiments, 6–10 weeks old Villin-RasV12-GFP mice were given a single intraperitoneal injection of 2 mg of tamoxifen in corn oil , and were then sacrificed days after Cre activation. For some experiments, the senolytic drug ARV825 31 was intraperitoneally administrated for 5 consecutive days . For culturing intestinal organoids, isolated crypts from the mouse small intestine were entrapped in Matrigel  and plated in a non-coated 35-mm glass-bottom dish as previously described48. The crypts embedded in Matrigel were covered with Advanced DMEM/F12 supplemented with N2 , B27 , 50 ng ml−1 EGF , 100 ng ml–1 Noggin , 1.25 mM N-Acetylcystein , and R-spondin conditioned medium collected from 293T-HA-Rspol-Fc cells kindly provided by Dr. Calvin Kuo (Stanford University). After 96 h of culture, organoids were incubated with tamoxifen  for 24 h to induce transgenes. Subsequently, tamoxifen was washed out, and organoids were cultured for 24 h for analyses.In Fig. In situ hybridization–immunohistochemistry codetection was performed according to the standard workflow with some modifications. In brief, immunohistochemical detection was first performed via a standard polymer detection method using nestin polyclonal antibody , and the immunohistochemical signal was detected using the DAB chromogen. After immunohistochemical detection, the samples were fixed in 10% neutral-buffered formalin for 30 min, and then in situ hybridization was performed using RNAscope Probe Mm-Hgf-1  and RNAscope 2.5 HD Reagent KIT-RED.t test, or one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s multiple comparisons posthoc test using the R software for statistical computing (64-bit version 3.6.1). Nonparametric statistical analyses were conducted using the Wilcoxon rank-sum or chi-square test using the R software for statistical computing. A P-value of <0.05 was considered statistically significant.Parametric statistical analyses were conducted using the unpaired two-tailed Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


OsPUB16 generated through CRISPR/Cas9 system exhibited the markedly enhanced water-deficit tolerance, while OsPUB16 overexpression lines were hypersensitive to water deficit stress. Moreover, OsPUB16 negatively regulated ABA and JA response, and ospub16 mutants produced more endogenous ABA and JA than wild type when exposed to water deficit. Mechanistic investigations revealed that OsPUB16 mediated the ubiquitination and degradation of OsMADS23, which is the substrate of OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE 9 (SAPK9) and increases rice drought tolerance by promoting ABA biosynthesis. Further, the ChIP-qPCR analysis and transient transactivation activity assays demonstrated that OsMADS23 activated the expression of JA-biosynthetic gene OsAOC by binding to its promoter. Interestingly, SAPK9-mediated phosphorylation on OsMADS23 reduced its ubiquitination level by interfering with the OsPUB16-OsMADS23 interaction, which thus enhanced OsMADS23 stability and promoted OsAOC expression. Collectively, our findings establish that OsPUB16 reduces plant water-deficit tolerance by modulating the ‘SAPK9-OsMADS23-OsAOC’ pathway to repress ABA and JA biosynthesis.Ubiquitin-mediated proteolysis plays crucial roles in plant responses to environmental stress. However, the mechanism by which E3 ubiquitin ligases modulate plant stress response still needs to be elucidated. In this study, we found that rice PLANT U-BOX PROTEIN 16 (OsPUB16), a U-box E3 ubiquitin ligase, negatively regulates rice drought response. Loss-of-function mutants of  Plants face various adverse environmental stimuli during their growth, such as drought, salinity and extreme temperatures. Different from animals, plants cannot move, so they must endure abiotic stresses, which greatly limit the distribution of plants, alter their growth and development, and reduce crop productivity. To adapt to the major adverse environmental conditions, plants have developed multifaceted strategies at different levels. Among these, protein post-translational modifications play crucial roles. However, how protein post-translational modifications in response to environmental stress hormones modulate plant stress response still needs to be elucidated. Here, our results showed that the two types of post-translational modifications, phosphorylation and ubiquitination, regulate the balance between plant growth and stress response. Furthermore, there is the close link between the two types of post-translational modifications, which was affected by environmental stress hormones. The research will help us understand the mechanisms underlying the “trade-off” between plant growth and stress response, and engineer stress-resistant and high-yielding crops. S proteasome-mediated proteolysis. Briefly, JA is perceived by F-box protein CORONATINE INSENSITIVE1 (COI1), which recruits the E3 ubiquitin ligase to form complex SCFCOI1 to target JAZ proteins, resulting in SCFCOI1-dependent ubiquitination and degradation of JAZ proteins through the 26S proteasome [Drought stress inhibits plant growth and severely limits crop production . Phytohooteasome ,14. The oteasome ,16.OsAOC’ pathway to synergistically inhibit rice seed germination [JAZ proteins modulate seed germination through the interaction with ABA-signaling transcription factors ABSCISIC ACID-INSENSITIVE 3 (ABI3) or ABI5 and thus repress their transactivation activity [A series of studies have shown that JA signaling can interact with ABA signaling to synergistically modulate multiple physiological processes including seed germination and biotic/abiotic stress response in plants –20. In rmination . In Arabactivity . As a neactivity . AlthougPopulus [The ubiquitin-proteasome system plays important roles in plant growth and adaptation to environmental stress . ProteinPopulus . DespiteOsAOC’ module by mediating ubiquitination and degradation of OsMADS23. OsMADS23 binds to the promoter region of JA-biosynthetic gene OsAOC to regulate JA biosynthesis, and SAPK9 enhances OsMADS23 protein stability as well as the transactivation activity by interfering with OsPUB16-OsMADS23 interaction via phosphorylation in an ABA-dependent manner. Thus, our findings provide a mechanistic understanding of how OsPUB16 modulates both ABA and JA signaling pathways that are integrated by the ‘SAPK9-OsMADS23-OsAOC’ module during the water-deficit response in rice.Our previous study demonstrated that SAPK9-mediated phosphorylation on OsMADS23 conferred rice water-deficit tolerance in an ABA-dependent manner . In thisOsPUB16 expression in response to MV (methyl viologen), an oxidative stress inducer in plants, as well as ABA and MeJA, which are two well-recognized environmental stress hormones. We found that the mRNA levels of OsPUB16 were rapidly, strongly induced by MV and the two hormones  were generated for investigating the biological functions of OsPUB16. The mutant ospub16-1 harbored a one-nucleotide (T) insertion and ospub16-2 harbored a 25-nucleotide deletion in the first exon of OsPUB16, respectively, which might result in frameshift mutations and thus early termination of protein translation   exhibited the enhanced growth (The CRISPR/Cas9-mediated knockout mutants (nslation . Knockouhibition . Notablyd growth . We then-2 (+/-) , which ispub16-1 . Togethed growth .ospub16 mutants and OsPUB16-OX plants was examined. Here, given that ospub16-2 (-/-) mutants were lethal, the ospub16-2 (+/-) mutants were used for the evaluation of water-deficit tolerance. After water withholding and rewatering, the survival rates of ospub16-1 and ospub16-2 (+/-) were 51.3% and 19.2%, respectively, which were significantly higher than that of WT (5.4%), whereas few OsPUB16-OX plants survived  showed WT phenotypes, indicated by the reduced water deficit tolerance , survived . To furtnce Figs  and S4.  than WT , which ster loss . The finOsPUB16 knockout or overexpression on the stomatal development in rice leaves. Interestingly, we found that the stomatal density was increased in ospub16-1, but reduced in OX-4 leaves, in comparison with that in WT  strengthened its interaction with His-OsPUB16, while the mimicked phosphorylation (GST-OsMADS23T20D S36D) reduced their interaction  and their corresponding wild type through the primary root elongation inhibition assay. We found that, similar to their response to exogenous ABA [OsMADS23-overexpressing lines were more sensitive but osmads23-1 mutant was more tolerant to MeJA than their corresponding wild type . To further verify the regulation of OsAOC by OsMADS23, we performed a transient transactivation assay in N. benthamiana leaves through the luciferase reporter assay. The promoter region of OsAOC (pOsAOC) was fused to the LUC gene to generate the reporter, while the effector construct contained OsMADS23 driven by the CaMV 35S promoter . ospub16-1 could grow, but ospub16-2 (-/-) was lethal. As to this, there might be two reasons. The large DNA fragment deletion in ospub16-2 genome caused earlier termination of protein translation than that in ospub16-1, which might be responsible for the lethality of the ospub16-2 mutant. It has been reported that, in addition to causing frameshift mutations, CRISPR/Cas9-mediated sequence alterations in the coding region of the target gene may also result in altered splicing of the respective pre-mRNA, and such altered splicing products also give rise to aberrant protein [ospub16-1 mutant, some kind of functional mRNA of OsPUB16 might be expressed, perhaps by the altered splicing, which ensures its survival.Plants tightly control intracellular protein levels via various strategies to adapt to various adverse conditions. Emerging evidence indicates that the E3 ubiquitin ligase-mediated degradation pathway is one of the most important mechanisms maintaining the homeostasis of intracellular proteins . The U-b protein –55. In tospub16 mutants  reduced its interaction with OsPUB16, and thus was less ubiquitinated by OsPUB16 (T20D S36D) was more stable, while the dephosphorylated OsMADS23 (OsMADS23T20A S36A) was labile than the native form  cv Nipponbare was used in this study. The knockout mutants of OsPUB16 were generated by CRISPR-Cas9 system from Nipponbare. For generating OsPUB16-overexpressing lines, the open reading frame (ORF) of OsPUB16 fusion with GFP (green fluorescence protein) driven under the 35S promoter was cloned into pCAMBIA1301, and then transformed into rice calli by Agrobacterium tumefaciens-mediated transformation [OsPUB16 coding sequence driven by its promoter region  was cloned into the binary vector pCAMBIA1301, and the resulting construct was introduced into ospub16-1 mutant. The seeds of homozygous transgenic plants were used for the research. T-DNA insertion mutants of OsMADS23 and its overexpression lines were used [-2s-1) with 60% humidity. Primers for vector construction and transformant identification are listed in Rice  was calculated from the decrease in weight at indicated time points compared with time zero, based on the following formula WLR (%) =  / initial weight × 100%. The average survival rate and water loss rate were calculated from three independent experiments.The stomatal movement observation was performed as described previously . Two-weeOsActin1 as the internal control. Relative changes in gene expression level were quantified based on three biological replicates via the 2-ΔΔCt method [Total RNA was extracted from 2-week-old seedlings using TRIzol reagent (Takara). The qRT-PCR analysis was performed with t method . PrimersOsPUB16 fused with GFP was cloned into pCAMBIA1301 .The ORF of n system . After iOsPUB16 was cloned into pGBKT7 vector, and JAZs or OsMADS23 was cloned into pGADT7 vector, respectively. Then the corresponding plasmids were co-transformed into the yeast strain Y2HGold for two-hybrid assay. The transfected yeast cells were plated on synthetic defined (SD)/-Leu-Trp (-LW) or SD/-Ade-His-Leu-Trp (-LWHA) medium, and incubated at 28°C for 2 days. Primers for these constructs are listed in We used the Matchmaker Gold Yeast Two-Hybrid System (Clontech) to perform the yeast two-hybrid assay, according to the manufacturer’s instructions. The ORF of OsPUB16 was cloned into pFGC-cYFP and OsJAZ9 or OsMADS23 was cloned into pFGC-nYFP. The generated cYFP and nYFP constructs were transformed into Agrobacterium GV3101, respectively, and then co-infiltrated into 4-week-old N. benthamiana leaves with an efficient agroinfiltration expression system [N. benthamiana grew at 25°C for 48–72 hours, and YFP and RFP fluorescence signals were visualized using the confocal microscope (Leica SP8). The primers used for these constructs are listed in BiFC vectors pFGC-nYFP and pFGC-cYFP  were usen system , with HYN. benthamiana leaves as described previously [OsPUB16 or OsMADS23 was fused with the N- and C-terminal parts of the luciferase reporter gene LUC (nLUC and cLUC), respectively, to generate the constructs OsPUB16-nLUC and cLUC-OsMADS23. The resulting constructs were transformed into N. benthamiana leaves with the agroinfiltration expression system. After 36 hours, the leaves were sprayed with 0.32-mg/ml D-Luciferin potassium salt in 0.1% (v/v) Triton X-100, and the LUC signal was observed under low-light cooled CCD imaging apparatus . The primers used for BiFC assay are listed in JW771 and JW772 vectors were used, and the LCI assay was performed in eviously . BrieflyOsPUB16 was cloned into pGEX-4T-1 and transformed into DE3 to produce GST-OsPUB16, and OsMADS23 or OsJAZ9 into pET-28a to produce His-OsMADS23 or His-OsJAZ9  or IgG. Then the immunoprecipitated DNA was purified with phenol/chloroform after reverse crosslinking and proteinase K treatment. The immunoprecipitated DNA was used for qPCR analysis. The primers for ChIP-qPCR used are listed in The EpiQuik Plant ChIP Kit (Epigentek) was used for ChIP assays. Briefly, 10-day-old OsMADS23-GFP seedlings were harvested and fixed in 1% formaldehyde, and chromatin was isolated from 2 T20D S36D or His-OsJAZ9 for 5 hours, in the presence of 50 μM MG132, and then the immunoprecipitation was performed with anti-GST or anti-His beads. The immunoprecipitated proteins were detected with anti-Ubi antibody  and anti-GFP antibody, respectively. The immunoblot analysis of Histone H3.1 (H3.1) with anti-H3.1 antibody  was used as a loading control of protein lysates. Three independent experiments were repeated, and representative pictures were shown.Briefly, the total proteins were extracted from 10-day-old wild type or OsPUB16-OX plants by using the NP40 lysis buffer . Then the protein extracts were incubated with GST-OsMADS23, GST-OsMADS232, 5 mM DTT). Purified recombinant GST-OsMADS23, OsMADS23T20D S36D, OsMADS23T20A S36A or His-OsJAZ9 was incubated with equal amount of protein extracts for different time, in the presence of 10 mM ATP. Anti-His antibody  or anti-GST antibody  were used to detect recombinant protein level by immunoblot analysis.Cell-free degradation assays were performed as previously described . Two-weeOsPUB16 (LOC_Os01g66130), OsMADS23 (LOC_Os08g33488), SAPK9 (LOC_Os12g39630), OsAOC (LOC_Os03g32314), OsLOX1 (LOC_Os03g49380), OsAOS2 (LOC_Os03g12500), OsLOX3 (LOC_Os03g49260), OsJAZ5 (LOC_Os04g32480), OsJAZ6 (LOC_Os03g28940), OsJAZ7 (LOC_Os07g42370), OsJAZ8 (LOC_Os09g26780), OsJAZ9 (LOC_Os03g08310), OsJAZ10 (LOC_Os03g08330), OsJAZ11 (LOC_Os03g08320), OsJAZ12 (LOC_Os10g25290), OsJAZ13 (LOC_Os10g25230).S1 Fig(A) The phylogenetic analysis of a subset of PUB proteins from different plants. (B) Alignment of OsPUB16 with other plant U-box proteins. The solid red line indicates the conserved U-box domain, and solid block line indicates the ARM repeats domain. The accession numbers are as follows: OsPUB16 (LOC_Os01g66130.1), OsPUB15 (LOC_Os08g01900.1), SB03g379800 (Sobic.003G379800.1), SB07g011400 (Sobic.007G011400.2), ATPUB4 (AT2G23140.1), SlU-box9 (Solyc01g014230.2.1), SlU-box62 (Solyc12g100000.1.1), MDP000221363, MDP0000411998, StPUB40 (PGSC0003DMT400046296), GrPUB55 (Gorai.008G079700.1), GrPUB53 (Gorai.007G219900.1). (C) Schematic representation of the U-box (amino acids 269 to 332) domain and the ARM repeats near the C- terminus in OsPUB16 protein.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(A) Schematic presentation of the gene structure of OsPUB16 and CRISPR-cas9 editing site. White boxes: untranslated regions; Black boxes: exons; black line: intron. TGG: PAM (protospacer adjacent motif). The CRISPR-cas9 target site and mutation were shown in homozygous mutants of ospub16. The letter in red represents the nucleotide insertion, and the red dots represent nucleotide deletion. (B) Frameshift mutations in ospub16 mutants in (A) resulting in early termination of protein translation. (C) Two-week-old wild type  and ospub16 mutants. Scale bars, 2 cm. (D) Shoot length measurement for 2-week-old seedlings. Data are means ± SD (n = 10). (E) Three-month-old WT and ospub16 mutants. Scale bars, 10 cm. (F) Plant height measurement for 3-month-old plants. Data are means ± SD (n = 10). (G) Analysis of OsPUB16 by quantitative real-time PCR analysis. The transcript levels in wild type were defined as “1”. Error bars indicate SD . OsActin1 was used as the internal control. (H) Analysis of OsPUB16 by semi-quantitative PCR analysis. The red arrows are the positions of primers for quantitative real-time PCR and semi-quantitative PCR analysis. (I) Four-month-old WT and OsPUB16 overexpression  plants. Scale bar, 10 cm. (J) Expression analysis of OsPUB16 independent OsPUB16 overexpression transgenic lines . The transcript level in the wild type (Nip) was defined as “1”. Data are means ± SD (n = 3). (K) Plant height measurement for 4-month-old plants. Data are means ± SD (n = 10). In (D), (F), (G), (J) and (K), the significant difference between transgenic plants and wild type was determined by Student’s t test. *p < 0.05, **p < 0.01 or ***p < 0.001.(TIF)Click here for additional data file.S4 Fig(A) Construction of the complementary vector. The coding sequence of OsPUB16 driven by its own promoter was cloned into pCAMBIA1301. (B) Analysis of OsPUB16 by quantitative real-time PCR analysis in WT, ospub16-1 mutant and complementary lines. The transcript levels in WT were defined as “1”. Error bars indicate SD . OsActin1 was used as the internal control.(TIF)Click here for additional data file.S5 Fig Seed germination of WT and ospub16-1 mutant on 1/2MS medium without or with different concentrations of ABA (A) or MeJA (B) at 4 DAG (day after germination). Bars = 1 cm.  Seed germination rate on 1/2MS medium without or with 5 μM ABA (J) or 3 μM MeJA (L) at 4 DAG. Data are means ± SD with biological triplicates . In (B) and (D), the significant difference between the treated and untreated plants was determined by Student’s t test. *p < 0.05.(TIF)Click here for additional data file.S6 Fig(A) Representative pictures showing the cell-free degradation assay for recombinant His-OsJAZ9, in the presence of 10 mM ATP. Recombinant His-OsJAZ9 was detected by anti-His antibody. The Coomassie blue–stained ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Rbc L) was used as a loading control. (B) Relative recombinant protein levels of His-OsJAZ9 at indicated time points in the cell-free degradation. Data are means ± SD . The significant difference between the treated and untreated was determined by Student’s t test. ns, not significant.(TIF)Click here for additional data file.S7 FigYeast two-hybrid assays to test the interaction of OsPUB16 with OsMADS23 (A) or SAPK9 (B). Yeast cells were grown on synthetic defined (SD)/-Leu-Trp (-LW) medium and SD/-Ade-His-Leu-Trp (-LWHA) medium. The AD-T + BD-P53 was used as a positive control, and AD-T + BD-lam as a negative control.(TIF)Click here for additional data file.S8 FigYeast cells were grown on synthetic defined (SD)/-Leu-Trp (-LW) medium and SD/-Ade-His-Leu-Trp (-LWHA) medium.(TIF)Click here for additional data file.S9 FigNicotiana benthamiana with or without the presence of SAPK9. Strong YFP fluorescent was detected when OsPUB16-cYFP and OsMADS23-nYFP were co-expressed, but the fluorescent signals were reduced in the presence of SAPK9-FLAG, not FLAG alone. ABA treatment further reduced the YFP fluorescent signals in the leaves co-expressing OsPUB16-cYFP and OsMADS23-nYFP with SAPK9-FLAG, but not in the leaves containing OsPUB16-cYFP and OsMADS23-nYFP. HY5-RFP was used as a nuclear-localized marker. YFP and RFP fluorescence signals were visualized using the confocal microscope (Leica SP8). YFP, fluorescent channel in yellow and RFP fluorescent channel in red.Confocal images showing the interference SAPK9 with the OsPUB16-OsMADS23 interaction, particularly in the presence of ABA. OsPUB16-cYFP and OsMADS23-nYFP were co-expressed in the leaves of (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Overall, our results provide support for employing ACNs as drug delivery materials with therapeutic potential to control inflammatory disorders.Although 3-aminopropyl functionalized magnesium phyllosilicate nanoparticles  are well-known nanomaterials employed as drug carriers, their effects on immune cells remain unclear. To address this issue, we explored murine dendritic cells (DCs) as these cells belong to the innate arm of the immune system and function as antigen-presenting cells to elicit adaptive immune responses. We examined the in vitro effects of ACNs on DCs isolated from B6 mice. ACN treatment significantly down-regulated the expression of inflammasome-related markers, including NLRP3, caspase-1, and IL1β. The ACNs-induced anti-inflammatory DC phenotype was further confirmed by down-regulation of the AKT/mTOR/HIF1α signaling pathway. Such anti-inflammatory effects of ACNs on DCs occurred independently of DC subtypes. To document the effects of ACNs on DCs more clearly, we examined their anti-inflammatory effects on lipopolysaccharide (LPS)-activated DCs. As expected, excessive inflammatory responses  of LPS-activated DCs were dramatically attenuated by ACN treatment. Furthermore, ACNs down-regulated IFNγ production by antigen-specific CD4 A drug delivery system (DDS) is essential for efficient drug delivery to the desired target location and is required to elicit optimal drug efficacy. Aminoclay nanoparticles (ACNs), one of the drug carriers that can be orally administered, are synthetic organic–inorganic layered materials delaminated to water-soluble cationic nanosheets in water . ACNs wi+ T cells that mediate adaptive immune responses, such as antibody production against pathogens [+ T cells, but mDCs have a high intrinsic capacity for MHC class II-mediated antigen presentation. Furthermore, based on their functional differences DCs are classified into two groups, IRF8+ DC1 driving Th1 responses and IRF4+ DC2 driving Th2 responses [Most studies on ACNs have focused on their delivery effects as nanocarriers but little is known about their physiological effects in recipients. In particular, the effects of ACNs on the immune system remain unexplored. Among immune cells, dendritic cells (DCs), as innate antigen-presenting cells (APCs), play an essential role in initiating adaptive immune responses. They take up antigens and present them to CD4athogens . In addiathogens ,10. Uponathogens . Compareathogens . During athogens ,14. DCs esponses ,16.In this study, we employed murine DCs to investigate the in vitro immunological effects of ACNs. For this purpose, we examined whether ACN treatment modulates DC phenotypes  and exhibits divergent effects on distinct DC subtypes. To test the modulatory effect of ACNs on LPS-activated DCs, we analyzed the production of inflammatory cytokines , the expression of DC1 and DC2 markers , and the production of mtROS by DCs upon LPS challenge. We next examined the effect of ACNs on the capacity of fluorescently labeled LPS to bind to the cell surface of DCs. Furthermore, we investigated whether ACNs can affect antigen-specific adaptive immune responses using the ovalbumin (OVA)-specific DO11.10 T cell receptor (TCR) transgenic (Tg) mouse system and KJ1-26 mAb specific to DO11.10 TCR-expressing T cells.ACNs are layered materials composed of magnesium phyllosilicate functionalized with aminopropyl groups ,2 FigurA. Althou+CD11b− pDCs, B220−CD11b+ mDCs, and B220−CD11b− cDCs [It has been reported that the AKT/mTOR/HIF1α signaling pathway is involved in the inflammatory processes of DCs ,18. Thus1b− cDCs ,16  and a marker of TCR signaling strength  in KJ1-26+CD4+ T cells using flow cytometry  are organic cationic compounds with more than two amino groups. It has been previously reported that polyamines possess immunomodulatory properties. For example, putrescine treatment suppressed the expression of TNFα, IL6, and IL8 in intestinal porcine epithelial cell lines after LPS stimulation in vitro . In addi+ T cell polarization [Although IL4-producing immune cells  play a crucial role in Th2-mediated immune diseases such as atopic dermatitis (AD) ,34,35,36rization . Furtherrization . In addiIn conclusion, our results show that ACN treatment directly attenuates inflammatory responses in LPS-induced DCs. However, what molecules (or receptors) are responsible for interacting with ACNs remains unclear. Thus, it will be interesting to investigate the receptors expressed on the DCs involved in these anti-inflammatory immune responses mediated by ACNs via binding with negatively charged bioactive molecules such as ROS.This study was designed to determine the effect of ACNs on LPS-induced DC activation. To address this issue, magnetically activated cell sorting (MACS)-purified DCs were treated with ACNs and LPS for 16 h and, subsequently, DCs were harvested and further analyzed using flow cytometry.Escherichia coli (serotype 0111:B4) were purchased from Sigma-Aldrich . MyD88 inhibitor (T6167923) was purchased from Toronto Research Chemicals . OVA peptide323–339 (ISQAVHAAHAEINEAGR) was synthesized by Peptron Inc. .WT B6 mice were purchased from Jung Ang Lab Animal Inc. . IL12p40 reporter (Yet40) B6 mice were provided by Dr. R. Locksley . DO11.10 OVA-specific TCR Tg mice used in this study are of the Balb/c genetic background. These mice were maintained at Sejong University and were used for experiments at 6–12 weeks of age. They were maintained on a 12 h light/12 h dark cycle in a temperature-controlled barrier facility with free access to food and water. Mice were fed a γ-irradiated sterile diet and provided with autoclaved tap water. Age- and sex-matched mice were used for all experiments. The animal experiments were approved by the Institutional Animal Care and Use Committee of Sejong University (SJ-20190301). Unlabeled LPS and FITC-labeled LPS derived from ACNs were synthesized as described in a previous report . In brie+ DCs were isolated from B6 mice using a MACS system , following the manufacturer’s instructions [+ DCs were enriched >94% after MACS. Primary cells were cultured in RPMI 1640  culture media supplemented with 10% FBS, 10 mM HEPES, 2 mM l-glutamine, 100 units/mL penicillin-streptomycin, and 5 μM 2-mercaptoethanol.Splenic CD11cructions . CD11c+ Yet40 B6 mice were intraperitoneally (i.p.) injected with 2 mL of 4% thioglycolate broth  in deionized water. Five days later, peritoneal macrophages were harvested from peritoneal lavage fluid and were subsequently plated on 24-well plates to select adherent macrophages. Two hours later, the nonadherent cells were removed via washing with warm RPMI medium.The following monoclonal antibodies (mAbs) were obtained from BD Biosciences : fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-Cy7-, or allophycocyanin (APC)-conjugated anti-CD11c (clone HL3); FITC-, PE-Cy7-, or APC-conjugated anti-CD3ε (clone 145-2C11); FITC-, PE-Cy7-, or APC-conjugated anti-CD4 (clone RM4-5); FITC- or PE-Cy7-conjugated anti-CD11b (clone M1/70); FITC- or PE-Cy7-conjugated anti-B220 (clone RA3-6B2); PE-conjugated anti-IL12p40 (clone C15.6); and PE-conjugated anti-CD86 (clone GL1). The following mAbs from Thermo Fisher Scientific  were used: FITC-, PE-Cy7-, or APC-conjugated anti-MHCII (clone M5/114.15.2); FITC- or PE-Cy7-conjugated anti-DO11.10 TCR (clone KJ1-26); PE-conjugated anti-IL1β (clone NJTEN3); PE-conjugated anti-Phospho-mTOR (Ser2448) (clone MRRBY); PE-conjugated anti-IRF4 (clone 3E4); PE-conjugated anti-IRF8 (clone V3GYWCH); PE-conjugated anti-Nur77 (clone 12.14); and PE-conjugated anti-IFNγ (clone XMG1.2). The following mAb from LifeSpan BioSciences  was used: PE-conjugated anti-caspase-1 . The following mAbs from R&D systems  were used: PE-conjugated anti-NLRP3 (clone 768319); PE-conjugated anti-Phospho-AKT (Ser473) (clone 545007); and PE-conjugated anti-HIF1α (clone 241812). Cells were harvested and washed twice with cold 0.5% BSA-containing PBS (FACS buffer) for staining surface markers. For blocking Fc receptors, the cells were incubated with anti-CD16/CD32 mAbs (clone 2.4G2) on ice for 10 min and subsequently stained with fluorescently labeled mAbs. Flow cytometric data were acquired using a FACSCalibur flow cytometer  and analyzed using FlowJo software .For intracellular staining, splenocytes were incubated with brefeldin A, an intracellular protein transport inhibitor (10 μg/mL), in RPMI medium for 2 h at 37 °C. The cells were stained for cell surface markers, fixed with 1% paraformaldehyde, washed once with cold FACS buffer, and permeabilized with 0.5% saponin. The permeabilized cells were then stained for an additional 30 min at room temperature with the indicated mAbs  [Splenic DCs were cultured with ACNs in the presence of LPS (1 μg/mL) for 16 h, followed by incubation with 1 μM Dihydrorhodamine 123 (DHR 123)  at 37 °C for 30 min. ROS fluorescence intensity was determined using flow cytometry.Cells were fixed in pre-warmed Fix Buffer I (BD Phosflow™ Cat. No. 557870) for 10 min at 37 °C. Immediately after washing with cold PBS, permeabilization was performed with cold PhosflowPermBuffer II (BD Phosflow™ Cat. No. 558050) for 30 min on ice. Next, the cells were washed twice with staining buffer (1× PBS with 2% FBS) for 10 min and subsequently stained with PE-conjugated anti-Phospho-mTOR (Ser2448) and anti-Phospho-AKT (Ser473) mAb in staining buffer for 30 min at room temperature (RT). More than 5000 cells per sample were acquired using the FACSCalibur and analyzed with the FlowJo software package.t-test was performed to compare two groups . Two-way ANOVA analysis was carried out using the VassarStats (http://vassarstats.net/anova2u.html) (accessed on 22 February 2022) .Statistical significance was determined using Excel . Student’s


NE efficientlycleaved S-protein, rendering exposure of amyloidogenic segments andaccumulation of the amyloidogenic peptide 194–203, part ofthe most amyloidogenic synthetic spike peptide. NE is overexpressedat inflamed sites of viral infection. Our data propose a molecularmechanism for potential amyloidogenesis of SARS-CoV-2 S-protein inhumans facilitated by endoproteolysis. The prospective of S-proteinamyloidogenesis in COVID-19 disease associated pathogenesis can beimportant in understanding the disease and long COVID-19.SARS-CoV-2 infectionis associated with a surprising number ofmorbidities. Uncanny similarities with amyloid-disease associatedblood coagulation and fibrinolytic disturbances together with neurologicand cardiac problems led us to investigate the amyloidogenicity ofthe SARS-CoV-2 spike protein (S-protein). Amyloid fibril assays ofpeptide library mixtures and theoretical predictions identified sevenamyloidogenic sequences within the S-protein. All seven peptides inisolation formed aggregates during incubation at 37 °C. Three20-amino acid long synthetic spike peptides  fulfilled three amyloid fibril criteria:nucleation dependent polymerization kinetics by ThT, Congo red positivity,and ultrastructural fibrillar morphology. Full-length folded S-proteindid not form amyloid fibrils, but amyloid-like fibrils with evidentbranching were formed during 24 h of S-protein coincubation with theprotease neutrophil elastase (NE)  Recently emerged SARS, MERS, andsince 2019 also SARS-CoV-2 result in severe disease. Although coronavirusinfections are common, not before COVID-19 has such a wide distributionof complex symptoms involving organs other than the respiratory tractbeen reported. COVID-19 pathogenesis is multifactorial and complex.2 Severe COVID-19 includes acute respiratory distresssyndrome (ARDS) from innate immune system inflammatory reactions resultingin lung damage;3 cytokine storm;4 heart damage, including heart muscle inflammation;kidney damage; neurological damage; damage to the circulatory systemresulting in poor blood flow. Long-COVID-19 symptoms include persistentemotional illness and mental health conditions resembling neurodegenerativediseases.2 What could be the basis forthis pathogenesis?Coronaviruses use the homotrimericsurface spike protein (S-protein) to attach to human cells. Each SARS-CoV-2S-protein subunit comprises 1273 amino acids.5 Neurotropiccolonization and cross-seeding of S-protein amyloid fibrils to induceaggregation of endogenous proteins has been discussed in the contextof neurodegeneration.6 Notably, blood clottingassociated with extracellular amyloidotic fibrillar aggregates inthe bloodstream has been reported in COVID-19 patients.7 Hypercoagulation/impaired fibrinolysis was demonstratedin blood plasma from healthy donors experimentally spiked with S-protein.7Amyloidosis from several culprit proteinsmanifests as systemicand localized disorders with many phenotypes overlapping with reportedCOVID-19 symptoms. It has been proposed that severe inflammatory diseaseincluding ARDS in combination with SARS-CoV-2 protein aggregationmight induce systemic AA amyloidosis.9 FXIIKallikrein/Kinin activation, and thromboinflammation,10 suggesting potential links between S-protein amyloidogenesisand COVID-19 phenotypes. We therefore hypothesized a potential molecularlink between S-protein and amyloid formation. Inspired by previoushypotheses about human and viral protein amyloids and interactionsbetween them,13 in particular SARS-CoV spike proteins,15 we asked the question: Is SARS-CoV-2S-protein amyloidogenic?Amyloidosis is associated with cerebralamyloid angiopathy, bloodcoagulation disruption, fibrinolytic disturbance,Supporting Information). In vitro amyloid fibrils were formed in both peptide subpools. Encouragedby the results, we generated 20-AA peptides from the full-length SARS-CoV-2S-protein. We aimed to address the most amyloidogenic sequences andused the WALTZ  prediction algorithm16 to identify suchsegments (Supporting Information).We obtained a 316 peptide pool library(divided into two subpools)from a peptide scan through the entire SARS-CoV-2 S-protein (ProteinID:P0DTC2) .All but one (Spike365) of the predicted sequences are in β-sheetconformation in the SARS-CoV-2 Spike cryo-EM structure in its closedstate.1 The C-terminal part of the protein(Spike1166) is not resolved in the structure.Seven amyloidogenic sequences distributedover the entire S-proteinwere identified and named according to the starting position in theS-protein , and negative stain transmissionelectron microscopy (TEM).Solubilized peptides were monitoredfor S3C).Fibrils from most of the syntheticpeptides were detected withina few hours by at least one assay 1. Spike1Tm > 50 °C)17 and would not readily denature spontaneously. Furthermore,sucha large protein with complex folding will not easily misfold intoan amyloid state. However, proteolysis is an obvious candidate mechanism.What would be a plausible mechanism for S-protein fibrilformationduring SARS-CoV-2 infection? SARS-CoV-2 S-protein is fairly stable, British and Danish dementia (ABri/ADan), andFinnish familial amyloidosis (AGel). Proteolysis of full-length proteinsis also evident in many other amyloid disease deposits from ATTR,ALys, AA, and ASem1.20 and is further proteolyzed during inflammation.SARS-CoV-2 S-protein isproteolyzed during infection by host furin-likeenzymes and by serine proteases such as the transmembrane protease,serine 2 (TMPRSS2), at the cell surface21 Neutrophils act by phagocytosis of opsonized pathogens and by extracellularrelease of enzymes such as neutrophil elastase (NE). NE is a serineprotease coupled to obstructive lung diseases such as cystic fibrosis,chronic obstructive pulmonary disease,22 and alpha-1-antitrypsin deficiency.23Neutrophilsare the dominating class of leukocytes and a firstresponder during acute inflammation. Neutrophils are recruited tothe bronchoalveolar space of patients infected with a range of differentrespiratory viruses, including SARS-CoV-2.in silico proteolytic cleavage by NE using ExpasyPeptidecutter. One of the resulting peptides, Spike194–213, closelymatched Spike192, only frame shifted by two amino acids (Supporting Information), implying a testablehypothesis.The amino acid sequence of SARS-CoV-2 S-protein was subjectedto in vitro. S-protein showed a complexthermal unfolding trajectorywith multiple transitions around 45–65 °C and a majorunfolding transition with a midpoint of denaturation (Tm) of 79 °C (S4A) with differentialscanning fluorimetry (DSF) (S4), similar to literaturevalues for full-length S-proteins,24 confirmingfolded protein at starting point. S-protein refolded upon coolingalbeit noncooperatively (Tm 59 °C) (We subjected full-length SARS-CoV-2 S-protein toNE cleavage ry (DSF) 2 and S4,ratively 2A. NE unm 59 °C) 2B. Co-inm 59 °C) 2C. Mass m 59 °C) 2D–F.S5).Most importantly, we discovered amyloid-like fibril formationuponproteolytic cleavage using TEM. Neither NE nor SARS-CoV-2 S-proteinincubated alone formed fibrils 2G–H. FibTable S1) fromthe S-protein structure and classified these into three groups: (i)formed after 1 min (Table S2). Three peptides containing segmentsfrom our seven spike peptides (S5) are likely composed of a mix of fibrils initiated by anamyloidogenic peptide seed recruiting nicked S-protein for elongationand branching.We then performed LC-MS/MS analysisof peptides formed after 1min and 6 h of digestion at 2:1 excess of NE over S-protein. We identified98 NE cleavage peptides , demonstrating thatremoval of this amino acid will alter the amyloidogenic propertiesof the peptide. Fibril “shaving” is known for severalamyloidogenic proteins and peptides.25 Itis possible that Y204 is cleaved after S-protein aggregation.We performed fibrillation experimentsidentical to that for theother spike peptides also on the short fragment 194–203 4A, B. Th26 We performeda thrombin induced fibrinogen to fibrin conversion followed by plasminogentPA assay27 in the presence and absenceof Spike peptide fibrils (Supporting Information).It is known that S-protein affects the formation of persistentamyloid-like microclots in human blood, a potential pathological causeof long COVID-19 symptoms.Figure S3) during fibrinformation decreased the fibrinolysis . As a translational strategy, PET imaging may henceserve as an option for clinical studies to complement liquid biopsiesto assess amyloid microclots.26We tested two fluorescent analogues of positron emission tomography(PET) amyloid tracers, CN-PiB  and DF-9 , known tobind to neurological Aβ amyloid and cardiac AL, AA, and ATTRamyloid and found strong binding with concomitant fluorescence responsetoward Spike192 fibrils Inconclusion, we herein proposed a simple molecular mechanismfor how SARS-CoV-2 S-protein endoproteolyzed by NE can form amyloidogenicS-peptides, such as segment 194–203, and lead to exposure ofmultiple amyloidogenic segments in proteolytically nicked S-protein.in vivo. We found that all common coronaviruses infectinghumans contain amyloidogenic sequences . Nonetheless, the magnitude of diverse COVID-19 symptomswas not previously reported. The segment 194–213 is uniquefor SARS-CoV-2  which, in combinationwith acute inflammation and neutrophil recruitment known to be moreprevalent in COVID-19 compared to other viral infections, could explainthe putative COVID-19 associated amyloid formation. It should be mentionedthat amyloidosis is rather common in the elderly population18 and its associations with viral infections isa matter of discussion.13 Recent studiesdemonstrate that COVID-19 recovered patients have an increased riskof type II diabetes, an amyloid associated disease.29 While our study is limited to in vitro findingsof pure preparations of peptides and proteins, the results proposetaking S-protein amyloidogenesis into account when studying COVID-19and long COVID-19 symptoms.It is possible that other amyloidogenic peptides and S-proteinnicked by other proteases could be involved if this process occurs


The FDA approval of venetoclax in combination with hypomethylating agents (azacitidine or decitabine) or low-dose cytarabine has offered renewed hope for elderly/unfit patients with newly diagnosed acute myeloid leukemia (AML). In the pivotal Phase III VIALE-A and VIALE-C studies, complete response rates were superior with venetoclax combination therapy compared to azacitidine 66% vs 28%), and low-dose cytarabine alone 48% vs 13%); moreover, overall survival was prolonged at 14.7 and 8.4 with venetoclax plus azacitidine and low-dose cytarabine, respectively . Fur. Fur8].