Oxidative stress mediated by reactive oxygen species(ROS) is akey process for adverse aerosol health effects. Secondary organicaerosols (SOA) account for a major fraction of fine particulate matter,and their inhalation and deposition into the respiratory tract causesthe formation of ROS by chemical and cellular processes, but theirrelative contributions are hardly quantified and their link to oxidativestress remains uncertain. Here, we quantified cellular and chemicalsuperoxide generation by 9,10-phenanthrenequinone (PQN) and isopreneSOA using a chemiluminescence assay combined with electron paramagneticresonance spectroscopy as well as kinetic modeling. We also appliedcellular imaging techniques to study the cellular mechanism of superoxiderelease and oxidative damage on cell membranes. We show that PQN andisoprene SOA activate NADPH oxidase in macrophages to release massiveamounts of superoxide, overwhelming the superoxide formation by aqueouschemical reactions in the epithelial lining fluid. The activationdose for PQN is 2 orders of magnitude lower than that of isopreneSOA, suggesting that quinones are more toxic. While higher exposurestrigger cellular antioxidant response elements, the released ROS induceoxidative damage to the cell membrane through lipid peroxidation.Such mechanistic and quantitative understandings provide a basis forfurther elucidation of adverse health effects and oxidative stressby fine particulate matter. Thisstudy provides mechanistic and quantitative understandingsof chemical and cellular superoxide release and oxidative stress inducedby secondary organic aerosols. Cellular release of •O2– can be triggered by a number of differentenzymatic systems such as mitochondrial oxidative phosphorylation,NAD(P)H [reduced nicotinamide adenine dinucleotide (phosphate)] oxidase,and xanthine oxidase.24 However, very littleis known about which •O2– generation mechanism is activated by inhaled PM. While •O2– can be converted into a less reactiveform  by SOD, excess •O2– is known to be cytotoxicas it can interfere with lipids, proteins, and DNA,25 but a specificconsequence of PM-triggered •O2– is yet to be identified. Several methods have been developed todetect cellular •O2– using spectroscopic,26 fluorescence,28 or luminescence30 assays. The Diogenes chemiluminescenceassay is suitable for monitoring cellular •O2– production as Diogenes is a very sensitive •O2– chemiluminescenceenhancer that is non-denaturing to living cells; however, the chemiluminescencereadouts represent light intensity without providing a quantitativemeasure for the production rate or concentration.30Macrophages are the first cellular responders of the innateimmunesystem that protect the lung from infection through bacteria, microbes,and pathogens by releasing 34 , are exposed to 9,10-phenanthrenequinone(PQN) and isoprene SOA. PQN is one of the most abundant quinones inatmospheric PM with cytotoxic effects both in vitro and in vivo.35 Quinonesare important components in anthropogenic SOA as they are generatedby the oxidation of polycyclic aromatic hydrocarbons and they canalso be directly emitted via diesel exhaust, tirewear, and biomass burning associated with soot and humic-like substances.37 Quinones are redox active and accept electrons from antioxidantsto form semiquinones,39 which can react with O2 to form •O2–, whichcan further be converted into H2O2.8 This pathway is a major source of H2O2 for naphthalene SOA.40 Isopreneis the most abundant biogenic VOC emitted from plants. The isopreneSOA represents a major component of biogenic PM,13 and it has been found to induce oxidative stress in human lung cells.41 Quinones and isoprene SOA can also generatesuperoxide by chemical processes, which we quantify using EPR witha spin-trapping technique and kinetic modeling. The phasor approachto fluorescence lifetime imaging (Phasor-FLIM) is a state-of-the-artcellular imaging technique that has been previously used to studycellular metabolism in detail.45 Here, we apply Phasor-FLIM for the first time tostudy NADPH oxidase activities as a potential mechanism of cellular •O2– release after PM exposure.In addition, we apply Laurdan FLIM and the third harmonic generation(THG) microscopy46 to investigate the impactsof PQN and isoprene SOA on cell membrane fluidity and lipids, respectively.Here, we develop a method to quantify superoxidegeneration bycombining the Diogenes chemiluminescence assay with electron paramagneticresonance (EPR) spectroscopy. RAW 264.7 macrophages, a widely-appliedand established macrophage cell line for studying the oxidative stressresponses of macrophages•OH photo-oxidation in a 19L potential aerosol mass (PAM) chamber47 . 100–500 μL of isoprene was placed in an open1.5 mL amber glass vial, which was kept inside a glass bottle. VOCgases were mixed with a carrier flow of 0.5 L min–1 of purified air from a zero-air generator and combined with humidified zero air prior to the PAM chamber inlet. •OH was generatedby UV radiation (185 nm) through the photolysis of water moleculeswith a relative humidity of ∼40%. The flow rate in the chamberwas 6.5 L min–1, resulting in a mean residence timeof approximately 3 min. While •OH concentrationsare higher (∼1010 cm–3) than ambientlevels (∼106 cm–3), the PAM-generatedSOA are found to be similar to ambient and chamber-generated SOA interms of the yield, oxidation state, hygroscopicity, and chemicalcomposition.50 Multiple PAM experiments were conducted, and theisoprene SOA had a mean particle diameter of 400 nm with a mass concentrationof particles of ∼100–1000 μg m–3, as measured using a scanning mobility particle sizer . The SOA particles were collected on pre-weighed47 mm polytetrafluoroethylene (PTFE) filters  at a flow rate of 5 L min–1 for 6 h. The filter samples were weighed and stored at −18°C for less than a week before analysis. The SOA samples wereextracted (with an extraction efficiency of 86 ± 3%) and dilutedin different volumes of the incomplete medium  to achieve various doses for cell exposure.A 5 mM PQN stock solution was prepared in dimethyl sulfoxide and keptin a freezer (−18 °C). The PQN solutions and SOA extractswere diluted in incomplete medium and kept at room temperature (roughly25 °C) for no longer than 2 h before cell exposure.Isoprene SOA particleswere generated with 4 cells/mLwith 200 μL per well into 96-well plates (Corning) and incubatedat 37 °C and 5% CO2 in an incubator for about 2 hfor cells to fully adhere to the bottom of the culture plate. Celldensity was kept the same for superoxide measurements and cell imagingand is within the typical ranges used for PM exposure studies in otherstudies (details in Table S1).Macrophage cells (ATCCTIB-71) were obtained and passaged in complete medium (DMEM supplementedwith 10% FBS and 1% penicillin streptomycin) until >80% confluent.Cells were then seeded at a density of 4 × 10Cellcytotoxicity was measured using the CellTox Green Cytotoxicity Assay,which measures the changes in membrane integrity that may occur asa result of cell death. In brief, after cells were cultured in 96-wellplates, the complete medium was replaced by 100 μL of incompletemedium and 50 μL of the CellTox Green reagent for each well.The CellTox Green reagent was produced according to the package directions.To facilitate dye/DNA binding, cells were incubated in the dark forat least 15 min before 50 μL of samples was added to initiateexposure. Fluorescence signals were then measured using a microplatereader  for 4 h with excitation and emission wavelengthsof 475 and 525 nm, respectively. The fluorescence signals were givenas the relative light unit (RLU).•O2– productionwith and without superoxide dismutase (SOD) pretreatments. The Diogenesprobe reacts with •O2– to emit flash a chemiluminescence signal that is proportional tothe •O2– productionrate. The output of the Diogenes chemiluminescence is in the RLU.To convert the RLU to •O2– production rates, the Diogenes chemiluminescence assay was calibratedwith EPR coupled with a spin-probe technique using the standardizedcell-free •O2– generationsystem—the hypoxanthine (HX) and xanthine oxidase (XO) system. •O2– production rates calculated from the EPR spinprobe method show a linear relationship with the RLU from chemiluminescence. Therefore, the slope from the linear regression was usedto convert the Diogenes chemiluminescence data to the •O2– production rate in the unit of micromolesper minute (see further details in the Supporting Information).A Diogenes chemiluminescenceassay combined with EPR spectroscopy was applied to quantify cellularand chemical •O2– measurements, after the cells were cultured in96-well plates, the medium was gently removed, and 50 μL ofthe Diogenes probe made in sterile water was added to each well toincubate for 2 min. Then, 100 μL of incomplete medium , and50 μL of the sample was added to start the exposure. For inhibitorexperiments that used Apocynin (Apo) as a specific NADPH oxidase inhibitor,51 10 μL of Apo (roughly 100 μM) wasadded before sample exposure. In order to prevent cross-well interferences,black sealing films  were usedto seal the bottom of the plates. Continuous luminescence measurementswere conducted using the plate reader every 3 min for 4 h to obtainthe •O2– productionrate  macrophagesrelease •O2– at ∼0.06μM min–1 with a total production of ∼14μM , which represents •O2– production from anormal metabolism regulated by mitochondrial respiration.6 Cell-free wells were also used as “acellularcontrols” to capture any background signals, which were subtractedfrom the data with cells when necessary.For cellular ionrate 1A,B.Theoduction1C,D was •O2– production followed the same protocolas that of cellular •O2– measurements exceptcells were absent and the reaction time started when PQN or isopreneSOA particles were dissolved or extracted in incomplete medium (pH7.4). Accounting for the time it took to prepare samples, chemical •O2– measurements usuallystarted at 15 min.The measurements of chemical 16 was used to estimate the chemical •O2– production from PQN. KM-SUB-ELFtreats mass transport and chemical reactions involving ROS, antioxidants,surfactants, and redox-active compounds in the epithelial lining fluid.The model considers the redox cycling reactions between ascorbateand quinones to generate •O2–.25 The model simulations were conducted with PQN dosesranging from 0 to 10 μg mL–1. Initial ascorbateconcentrations of 10 and 40 μM52 wereused to obtain total •O2– production with a reaction time of 4 h. The model results from 10and 40 μM ascorbate correspond to the upper and lower boundsof the KM-SUB-ELF model in A kinetic multi-layer model of thesurface and bulk chemistry in the epithelial lining fluid (KM-SUB-ELF)•O2– production from the isoprene SOA was estimated usinga SOA aqueous kinetic model from our previous work.14 The kinetic model includes chemical reactions of SOA components,ROS coupling reactions, and radical–radical reactions. SOAchemistry includes decomposition of organic hydroperoxides (ROOH),generating •OH, •OH oxidationof primary and secondary alcohols (ROH), and the subsequent reactionwith O2 to form α-hydroxyperoxyl radicals, whichcan decompose to generate the hydroperoxyl radical HO2•, and •OH oxidation of SOA components.The model simulations were conducted with initial SOA concentrationsranging from 0 to 1000 μg mL–1, 50% of ROH,and 1–4% ROOH of total SOA mass. Model uncertainties are associatedwith uncertainties in the relative abundance of ROOH and ROH groupsin the SOA.Chemical •O2– in phagocytosis as activated bybacterial products and cytokines.53 Theactivation of NADPH oxidase generates •O2– through the reaction: NADPH + 2O2 →NADP+ + H+ + 2•O2–.55 Here, we utilize the auto-fluorescenceof NADPH measured using FLIM combined with experiments using NADPHoxidase activator PMA56 and inhibitor Apocyninto study the NADPH oxidase activities. It should be noted that NADH(nicotinamide adenine dinucleotide) has identical fluorescence propertiesto NADPH,57 and thus, FLIM cannot differentiatebetween NADH and NADPH, and we denote the FLIM signals as NAD(P)H.Note that NADH reacts with oxygen to produce either water or H2O258 or produces significantlylower •O2– than NADPH.59 NAD(P)H expresses in two forms inside cells,bound and free states. Each state has its own fluorescence lifetime,and in the Phasor-FLIM method, the NAD(P)H fluorescence lifetime isvisualized in polar coordinates by calculating the Fourier sine andcosine transformations of the FLIM photon histogram curve.43 Since oxidized NAD(P)+ does not emitauto-fluorescence, the conversion of NAD(P)H to NAD(P)+ reduces the fraction of bound NAD(P)H and causes a shift from thebound end to free end of the NAD(P)H free-bound trajectory.61 Therefore, the relative locations on the trajectory can be usedto obtain the bound NAD(P)H fractions, and the decrease of bound NAD(P)Hfractions indicates the activation of NADPH oxidase activities (seefurther details in the Supporting Information).TheNADPH oxidase complex is an important source of 4 cells mL–1 and incubated overnightbefore exposure to PQN and isoprene SOA for imaging. PQN and SOA inmedia in the absence of cells produced low fluorescence backgroundsignals. Cells were imaged in a 37 °C and 5% CO2 environmentbefore exposure and at different time points after the addition ofsamples (see Table S2). Since NAD(P)H mainlyresides in cell membranes and cytoplasm62 and since the fluorescence lifetimes of nucleus do not change significantlybefore and after sample exposure , the nucleus was cropped out, that is, only the fluorescence signalsfrom the cell membranes and cytoplasm were included in calculatingthe bound NAD(P)H fractions. The bound NAD(P)H fractions from alldata points for each sample were averaged from multiple cells (N = 12–23) and are shown in Supporting Information and Figure S6).63 To avoid the lifetimeof phosphorescence interfering with the NAD(P)H fluorescence lifetime,we used PBS buffer to wash cells after 10 min of incubation. Afterreplacing with fresh incomplete medium, cells were loaded back tothe FLIM system for imaging. Using this method, the lifetime phasorsof PQN fall onto the NAD(P)H free-bound trajectory, as shown in Figure S5.For FLIM imaging, cells were seeded at a density of 4 ×10exposure S5, the n64 Laurdan  is a fluorescentmembrane marker used to investigate membrane fluidity. Shorter Laurdanfluorescence lifetimes correspond to increases in membrane fluiditydue to lipid peroxidation. After seeding cells in an eight-well plate,complete medium was replaced with incomplete medium, and Laurdan dyewas added to each well to achieve a final concentration of 5 μMand incubated under the cellular condition for 30 min before exposureto the control, PQN, PMA, and isoprene SOA. After some period of exposure,the cells (4 × 104 cells mL–1) wereloaded to the FLIM system for imaging at 800 nm excitation wavelength(see further details in the Supporting Information and Table S2).The FLIM Laurdan imaging technique was used tomeasure cell membranefluidity as a result of sample exposure.46 The excitation of the THG signal requires laser wavelengths of upto ∼1000 nm, which conventional tunable Titanium Sapphire laserscould not achieve. The THG imaging was carried out in the deep imagingvia emission recovery (DIVER) system from the LFD, which uses SpectraPhysics Insight DS + femtosecond laser tunable in the range of 68–1300nm. The actual focal depth difference was found to be ∼0.75mm for different wavelength excitations, which for NAD(P)H is 740nm and for THG is 1050 nm. The THG signal is generated at the interfacebetween media with the difference in the third-order nonlinear susceptibility,refractive index, and dispersion and can be used to detect lipids.The THG images were taken before and after 10 min exposure to isopreneSOA and PQN. Note that THG microscopy is not affected by long-lifetimephosphorescence from PQN.The THG imaging techniquewas applied to detect the accumulation of lipids inside the cellsafter exposure to isoprene SOA and PQN.–1) performedon different days shows that no significant difference was determinedbetween the mean of data (p > 0.1). The time profileof superoxide production and total production are presented as themean of the triplicate measurements with error bars representing theuncertainties propagated from the standard deviations of triplicatemeasurements of samples and controls. For imaging, all data pointsfor each sample were averaged from multiple cells (N > 10). Results were analyzed by using unpaired student t-tests. The results were considered statistically significantata of p-value ≤ 0.05 for all exposure groupscompared to control groups.For Diogenes chemiluminescencedata, for each dose, triplicate measurements were performed on thesame experimental day along with vehicle and acellular controls, whichwere used to correct the experimental data. Experiments with somedifferent doses were performed on different days. One-way ANOVA followedby Tukey’s post-hoc test on the total superoxide productionfrom the same dose of PQN  due to the relatively fast onset of aqueous reactions.Note that a previous study by Tong et al.(•O2– by the isoprene SOAin the surrogate lung fluid (pH of 7.4) using the EPR spin trappingtechnique. We have very recently found that the EPR spin trap methodis not ideal to measure •O2– in neutral conditions due to the low trapping rate of •O2–,65 whichlikely explains the difference between Tong et al. and this work.Similar temporal trendsare observed for cellular rene SOA 1B, whileconstant 1B and S7ng et al. did not •O2– productionas obtained from the integration of control-corrected •O2– production rates over exposure time.The measured chemical total •O2– production increases with an increase of dose, and it plateaus at∼10 μM. PQN generates •O2–via redox-cycling reactionswith antioxidants,16 while the isoprene SOA yields •O2–via a series of aqueous reactions including decomposition of α-hydroxyperoxylradicals and organic hydroperoxides as well as OH oxidation of primaryor secondary alcohols.14 We applied kineticmodels that include these chemical mechanisms to simulate chemical •O2– production,16 producing consistent results as the measurements within model uncertainties(shaded areas). While the model does not perfectly reproduce the observedtemporal evolution of the •O2– production rate , this agreementis still remarkable, given that the reaction system is very complicatedwith numerous reactions involved;14 thediscrepancies may be due to incomplete chemical mechanisms, untreatedspecies contained in media, and the inexplicit treatment of pH thatcan modulate ROS formation.66The dose–response relationships areshown in •O2– releaseinduced by PQN and the isoprene SOA shows inverted U shapedose–response distributions. Under low doses, macrophages release •O2– under regular cellularmetabolisms with a control level of ∼14 μM , which is higher than the chemical •O2– production (<0.05μM). Once the dose reaches a threshold, macrophages are activated,and cellular total •O2– production increases sharply, further dominating over chemical •O2– production. The onsetdose of cell activation to release an excess amount of •O2– for PQN (∼0.2 μg mL–1) is much lower than for the isoprene SOA (>40μgmL–1), indicating that macrophages are more sensitiveto PQN than to the isoprene SOA in producing ROS. This suggests thatquinones are intrinsically more toxic than the isoprene SOA, consistentwith previous studies that show that anthropogenic aromatics havehigher toxicity compared to biogenic aerosols.69Interestingly,cellular •O2– productioninduced by PQN and isoprene SOA then decreases at higher doses, surpassedby chemical •O2– production(PQN at ∼5 μg mL–1 and SOA at ∼450μg mL–1). The decreases of cellular •O2– release at higher doses  to that of filter blanks(fresh filter without particle collection), which were much lowerthan those from the positive control lysed cells  , confirmingthat cells are alive during the whole course of exposure. Instead,the decreased cellular release of •O2– is most likely due to redox homeostasis by cellsupregulating the antioxidant response elements to scavenge ROS forprotection against proinflammatory effects.70 Previous studies have found that antioxidant enzymes can be upregulatedby the transcription factor Nrf2 when oxidative stress is inducedupon exposure to ambient and diesel exhaust PM containing polycyclicaromatic hydrocarbons and quinones.72 Based on the kinetics of cellular •O2– releases  S8, confivs oral, tidal volume, and frequency),individual differences in the lung anatomy, the airflow patterns inthe lung airways, and the presence of deposition hot spots in lung.73 Phalen et al. (2006) demonstratedby dosimetry model predictions that the surface PM deposition canreach up to 85.5 μg cm–2 but vary by up to3 orders of magnitude in the tracheobronchial region.74 With a bottom area of 0.32 cm2 and a workingvolume of 0.2 mL of a 96-well plate, the surface doses for the SOAand PQN in the current study are 5–412 and 0.02–5.3μg cm–2, respectively. Combining all the uncertaintiesand variabilities (up to 4 orders of magnitude) and also consideringthat the surface area of the alveolar region is about 200 times largerand the deposition in the alveolar region is up to 3 folds higherthan in the tracheobronchial region,75 theapplied surface doses are within relevant ranges for real-life exposurescenarios and consistent with doses applied in previous submergedcell exposure studies (details in Table S1).The conversion of doses to ambientPM concentrations is highlycomplex as respiratory deposition is largely affected by the natureof breathing were used for exposure (Table S2). Thephasors for cell membranes and cytoplasm mainly fall along a metabolictrajectory represented by a line joining positions of free NAD(P)Hand protein-bound NAD(P)H on a phasor plot (45 The relative locationson the trajectory were used to obtain the bound NAD(P)H fractionsH is observed upon exposure to phorbol 12-myristate13-acetate (PMA), commonly used as an inducer to activate NADPH oxidaseand increase endogenous •O2– production . Additionally,apocynin (or Apo), a specific NADPH oxidase inhibitor, results inhigher bound NAD(P)H fractions (Table S2), the bound fractions in •O2– production substantially upon exposure to PQN,the isoprene SOA, and PMA (•O2– production mainly through activating the NADPHoxidase. We observe that the bound NAD(P)H fraction for controls withApo is higher than isoprene SOA and PMA with Apo. The higher fractionin controls may be explained by the increase of NADH bound fractionsfrom the increase of mitochondrial oxidative phosphorylation (OXPHOS),as a recent study showed that Apo can enhance ATP production and mitochondrialmembrane potential, which are determined by the regulation of OXPHOS.76 This may further suggest that the exposure toPQN and the isoprene SOA induced a certain level of mitochondrialdysfunction, although future dedicated studies are necessary to furtherinvestigate this aspect.We then applied cellular imaging techniques to study the mechanismof cellular sor plot 2A.43,45 ractions2B. Macrooduction S9. Additoduction S9. Additoduction S9. AdditFigure S10. Since Laurdan dye is incorporatedinto the hydrophobic phase in the membrane,64 only the fluorescence signals from cell membranes were selectedfor fluorescence lifetime calculation. A total of 15 cells from eachsample were averaged and are presented as violin plots in Figure S11), a key event suggestedto involve the activation of NADPH oxidase observed in previous studies.78 The doses for PQN and the isoprene SOA are 1.74 and 305 μgmL–1, respectively, and cellular •O2– dominates chemical production. Therefore,with the revelation of increased membrane fluidity and laden lipidsaround the cell membranes, we suggest that macrophages are undergoinglipid peroxidation caused by •O2– or other ROS produced thereafter. Note that the Diogenesprobe can diffuse through the cell membrane, but SOD is membrane-impermeant.The addition of SOD largely eliminated the chemiluminescence intensity,suggesting that the observed cellular •O2– signals were primarily from extracellular •O2–. This is consistentwith NADPH oxidase being a transmembrane protein that regulates redoxsignaling by reducing extracellular O2 to •O2–.53 Thisis also consistent with the observed oxidative stress induced by PQNand the isoprene SOA, which occurred mainly on the cell membrane bylipid peroxidation. Liu et al. (2022) suggested thatthe diffusion of chemically formed H2O2 intocells is a main contributor to intracellular H2O2 upon exposure to the naphthalene SOA.23 As the chemical lifetime of •O2– is much shorter than that of H2O2, •O2– is not expectedto readily pass though the cell membranes. Although the penetrationof •O2– through themembrane could still occur with the assistance of anion channels,79 our study indicates that this pathway is notsignificant. Note that the dismutation of extracellular •O2– can occur and the product H2O2 can diffuse across the membrane into the cytoplasmto cause oxidative damage intracellularly.To investigate theimpact of PQN and the isoprene SOA on cell membranes, we quantifiedthe lifetime changes of a solvatochromic probe (Laurdan) and the THGimages of cells after exposure. The images of FLIM-Laurdan are shownin •O2– is mainly produced from normal cellular metabolism via mitochondrial respiration with minor contributions from chemicalreactions. After a threshold dose to macrophages, NADPH oxidase activitiesare upregulated for respiratory burst, releasing massive amounts of •O2–, which can cause oxidativestress by increasing cell membrane fluidity through lipid peroxidation.Further increases of doses lead to the activation of antioxidant responseelements, reducing the net cellular •O2– production. At very high doses and long exposuretimes, chemical •O2– production becomes comparably important or dominant if the escalationof oxidative stress leads to cell death. Given that cellular •O2– release mostly dominatesover its chemical production by PM reactive and redox-active components,widely-applied acellular assays that measure oxidative potential andROS activity may need to be interpreted with caution.82 While the current work is based on the standard submergedcell culture method, this classical condition should be extended torepresent more realistic conditions with multiple types of cells includingmacrophages and epithelial and endothelial cells to simulate syntheticinteractions between cell populations83 with an application of the air–liquid interface for simulatingexposure and respiratory deposition of aerosol particles.67The mechanistic understandings obtained in thisstudy provide abasis for further elucidation of adverse health effects and oxidativestress upon respiratory deposition of PM. Future studies should beextended to include other chemical compounds that have high oxidativepotential and redox activity such as transition metals; the presenceof metals in ambient PM might enhance the importance of chemical ROS.


Delayed cerebral ischemia (DCI), a complication of subarachnoid hemorrhage (SAH), is linked to cerebral vasospasm and associated with poor long-term outcome. We implemented a structured cerebral microdialysis (CMD) based protocol using the lactate/pyruvate ratio (LPR) as an indicator of the cerebral energy metabolic status in the neurocritical care decision making, using an LPR ≥ 30 as a cutoff suggesting an energy metabolic disturbance. We hypothesized that CMD monitoring could contribute to active, protocol-driven therapeutic interventions that may lead to the improved management of patients with SAH.Between 2018 and 2020, 49 invasively monitored patients with SAH, median Glasgow Coma Scale 11 (range 3–15), and World Federation of Neurosurgical Societies scale 4 (range 1–5) on admission receiving CMD were included. We defined a major CMD event as an LPR ≥ 40 for ≥ 2 h and a minor CMD event as an LPR ≥ 30 for ≥ 2 h.We analyzed 7,223 CMD samples over a median of 6 days (5–8). Eight patients had no CMD events. In 41 patients, 113 minor events were recorded, and in 23 patients 42 major events were recorded. Our local protocols were adhered to in 40 major (95%) and 98 minor events (87%), with an active intervention in 32 (76%) and 71 (63%), respectively. Normalization of energy metabolic status (defined as four consecutive samples with LPR < 30 for minor and LPR < 40 for major events) was seen after 69% of major and 59% of minor events. The incidence of DCI-related infarcts was 10% (five patients), with only two observed in a CMD-monitored brain region.Active interventions were initiated in a majority of LPR events based on CMD monitoring. A low DCI incidence was observed, which may be associated with the active interventions. The potential aid of CMD in the clinical decision-making targeting DCI needs confirmation in additional SAH studies.The online version contains supplementary material available at 10.1007/s12028-022-01492-5. Aneurysmal subarachnoid hemorrhage (SAH) accounts for 5% of all strokes and has a peak incidence between 50 and 60 years of age , 2. Desp2) [The detection of DCI is complex, especially in patients with poor-grade SAH, as they often require sedation and mechanical ventilation. Thereby, advanced multimodal neuromonitoring in the NCC unit is required to adjust cerebral and systemic physiology to prevent the development of manifest cerebral infarcts , 23. Bes2) , 25. In 2) , 26.CMD is an invasive neuromonitoring bedside technique using a catheter with a semipermeable membrane probe placed in brain parenchyma, typically in the frontal lobe. By pumping a fluid, the perfusate, that mimics cerebrospinal fluid, the CMD membrane will allow small molecules such as lactate, pyruvate, and glucose to enter the perfusate and to be analyzed at fixed time intervals. Because the returning perfusate, named dialysate, mimics the extracellular fluid of the brain parenchyma, changes in dialysate concentration of selected molecules reflects alterations of brain physiology , 28. LacCMD can detect energy metabolic changes up to 16 h prior to DCI, potentially enabling the clinician to provide interventions , 31. To Since late 2017, our local clinical protocols have been refined to apply the CMD surveillance data for real-time clinical decision making as a part of the multimodal neuromonitoring of patients with SAH. Hourly measurements of CMD LPR and glucose are performed and an LPR warning system with a 24/7 protocol for potential active interventions to be applied at elevated CMD LPR levels has been implemented. The aim of this study was to evaluate the use and clinical efficacy of CMD-based interventions in the NCC of patients with aneurysmal SAH and to assess the prevalence of DCI-related infarction.The study was approved by the ethical review board in Lund (2017/4069) and conducted in accordance with the ethical standards given in the Helsinki Declaration.This retrospective study included all patients with aneurysmal SAH monitored with CMD and treated in the NCC unit at Skåne University Hospital, Lund, Sweden between January 2018, and December 2020. This 3-year period was chosen to evaluate the recently implemented protocol for CMD-based interventions, based on the expected yearly number of patients with SAH treated in our unit. According to local protocols, patients with a Glasgow Coma Scale (GCS) motor score ≤ 5 or with an imminent risk of deterioration due to hydrocephalus received an external ventricular drainage (EVD) for ICP monitoring and cerebrospinal fluid (CSF) drainage. Each patient who received an EVD also received a CMD catheter. Patients were monitored for ICP, CPP, central venous pressure and mean arterial pressure (MAP), although not autoregulatory indices such as the pressure reactivity index (PRx). For details of our vasospasm protocol, see Supplementary Figure 1.2 at 5.3 kPa was aimed at. In the event of brain swelling, a slightly lower PaCO2  could be considered; in the event of vasospasm, a slightly higher PaCO2 could be used (up to 6.0 kPa). Low-dose dobutamine was the initial choice to enhance cardiac output, while norepinephrine was also frequently added. A cardiac output monitor was not routinely used.Patients were to be kept normovolemic, normothermic and treated with oral nimodipine (primarily 60 mg q4h). The colloid of choice for volume expansion was albumin (5 or 20%), and crystalloid  was used to correct fluid deficits. Blood glucose was to be maintained between 5 and 10 mmol/L. Hb ≤ 8 g/dL, or when vasospasm was suspected Hb ≤ 10 g/dL, was treated with transfusions. Blood oxygen saturation aim was > 95% and normoventilation was used. For patients on a ventilator normoventilation with PaCOComputed tomography angiography (CTA) or digital subtraction angiography (DSA) was performed to identify the cause of the SAH, and open neurosurgical clipping or endovascular coiling was typically performed within 24 h of SAH onset should an aneurysm be detected. In addition to ICP and CPP monitoring, frequent although not systematic TCD evaluations were performed.Cerebral vasospasm was suspected if the patient (1) deteriorated neurologically ; (2) if TCD measurements showed increased blood flow velocities in the middle cerebral arteries ; or (3) if two consecutive CMD samples showed elevated LPRs (vide infra) .Relevant patient data including age, sex, smoking habits and diagnosed hypertension were collected Table . SAH staPatients who had CMD monitoring for ≤ 72 h were excluded. CT images obtained during the time of CMD monitoring were reviewed to determine CMD probe placement. Patients with inadequate CMD probe placement were excluded. Patients with no CT scan during days of CMD monitoring were excluded.A CMA-70 catheter  with a 10 mm, 20 kDa molecular weight cut-off semipermeable membrane tip was implanted free hand via a separate burr hole placed lateral to the EVD burr hole in the nondominant (right) frontal lobe targeting the watershed area between the vascular territories of the middle and anterior cerebral arteries. In some patients treated with microsurgical clipping, the CMD was placed through the craniotomy targeting the same vascular area. Artificial CSF pumped at a rate of 0.3 μL/h was used as perfusate fluid. The fluid was collected in microvials and analyzed bedside every hour using enzymatic techniques . Samples collected the first 2 h after implantation were discarded.Microdialysis raw data were collected, processed, and analyzed to calculate the LPR. Based on previous reports , normal A major LPR event was defined as ≥ 2 consecutive values ≥ 40. Two additional serial LPR values ≥ 40 following a major event were considered a part of the same major event if presented within 4 h. A minor LPR event was defined as ≥ 2 consecutive values ≥ 30 not fulfilling the criteria of a major LPR event. Minor events were considered as such, even if they later transitioned into major events. LPR values between 30 and 40 following a major event were not considered as separate minor events. Thus, minor events could give rise to separate major events, but major events were not considered to give rise to minor events as these values were seen as part of the LPR normalization process. LPR normalization was defined as four consecutive samples < 30 for minor, and < 40 for major events .CMD data were analyzed continuously to identify major and minor events. At each event, it was determined whether an active or conservative approach was taken with regard to the local protocols. For any event without active intervention, it was noted whether this course was argued in the medical record and these conservative events were either termed “argued for” or “quiet.”The CMD protocol measures to counteract DCI were in the following order: (1) to immobilize the patient in a horizontal bed position to potentially increase cerebral blood flow, to intensify neurological assessments, to control and normalize blood oxygen saturation and plasma sodium concentrations, to control volemic status and treat hypovolemia with crystalloid or colloid boluses, to control Hb and transfuse to Hb > 10 g/dL; (2) to increase CPP by 20% by either lowering the level of CSF drainage to decrease ICP, or by increasing MAP with vasopressors or inotropic drugs, preferably dobutamine; and (3) to perform CTA and based on the results consider DSA and angioplasty, for instance intraarterial verapamil administration .The primary outcome of this study was DCI-related infarcts defined as cerebral infarcts on CT or MRI scan within 6 weeks post SAH onset, not present during the first 24–48 h after aneurysmal treatment and without apparent cause such as aneurysm occlusion [Statistical analyses were performed using IBM SPSS version 26 . Values are expressed as median and range or interquartile range. Patient age and day of LPR event occurrence were expressed as means and standard deviations (SD).n = 33) or with inadequate (n = 8) or unknown (n = 7) CMD probe placement were excluded. Hence, 49 patients were included  women and 10 (20%) men. The mean age was 63 years (SD 10.9). Most patients were WFNS grade IV or V (63%) on admission. Modified Fisher grade was III–IV in 45 patients (92%). Eight patients had dilated pupils and seven patients had pupils sluggish or unresponsive to light. Thirteen patients had focal neurological deficits and 12 had seizures prior to admission (Table The median (range) time from symptom onset until EVD/CMD insertion was 6 (2–36) hours. Five patients had unknown symptom onset as they were found unresponsive in their homes. Thirty-six patients received endovascular treatment, and the remaining 13 underwent craniotomy and microsurgical clipping. The median (range) time from symptom onset to aneurysm treatment was 15 (4–36) hours for coiling and 8 (2–20) hours for clipping.The median (range) NCC admission time was 16 (4–30) days, and patients were monitored by CMD for a median of 6 (3–16) days.During a total monitoring time of 8,127 h, 7,223 CMD samples in 49 patients were analyzed. Of these, 637 samples were excluded. Hence, 6,586 samples could be included of which 1,734 had an LPR ≥ 30 mmol/L and 448 samples an LPR ≥ 40 mmol/L. Of the included 49 patients, 41 had 113 minor events spanning 1,277 h. All but three of these patients also had at least a single LPR value ≥ 40 mmol/L, yet only 23 patients had ≥ 2 consecutive hours of LPR ≥ 40. In total, 42 major events spanning 395 h were recorded  and 67 minor events (59%). The median (range) MCA flow rate was 69.5 (49–211) cm/s for major events, and 70 (26–203) cm/s for minor events. At major events, TCD values indicated vasospasm in two cases and severe vasospasm in one case based on our local protocol. At minor events, TCD values indicated vasospasm in ten cases and severe vasospasm in one case.An active approach was taken in 32 of 42 major events (76%). The remaining 10 events were conservatively managed without any specific intervention initiated. In eight of these, the conservative approach was argued for, whereas in two cases no note of the event was made in the medical records. At minor events, an active intervention was initiated in 71 out of the 113 events (63%). Of the 42 conservatively managed minor events, the approach was argued for in the medical records in 27  and 23 active minor events (32%). Per protocol, an important strategy at LPR elevation was to increase CPP by at least 20%, which was the aim in 20 major events (48%). For minor events, action to actively increased CPP was taken in 39 events (35%)  and 42 minor events (59%) with an active approach. In 19 cases (27%), the minor event transitioned into a major event despite active interventions. In eight active major events (25%) and four active minor events (6%) the outcome was unknown due to either missing values or CMD dysfunction Fig. .Fig. 3FlThe major events managed actively had a median (interquartile range) duration of 7 (3–14) hours, whereas minor events with active approaches had a duration of 8 (3–16) hours. In contrast, the conservatively managed argued major events lasted 3 (2–5) hours and the minor event 2 (2–8) hours.n = 8) or MRI (n = 2), were diagnosed in ten patients (20%). Of these, five developed within the first day post SAH onset and/or were caused by the aneurysm-securing procedure. Thus, the total incidence of DCI-related infarcts was 10% (observed in five patients).New cerebral infarcts, detected by CT  was observed, considerably lower than in other reports. A recent meta-analysis reported that the risk of DCI development in modified Fisher grade III and IV patients with SAH was 30% and 42%, respectively . In a prAccording to the protocols, after ≥ 2 consecutive hours of increased LPR values an active approach should be initiated, which was performed in a majority of events. Only active measures recorded in the medical record were considered. In the critical care record system that was used, infusion pumps, ventilator settings and vital parameters were logged automatically every hour. In addition, NCC nurses and assistant nurses manually registered other clinical findings of importance in defined intervals. Thus, the collected data should be considered complete regarding both action taken and time of action. We argue that the microdialysis LPR must be interpreted considering the full clinical context and other monitoring parameters. In an otherwise clinically stable, conscious SAH patient with an isolated LPR elevation and no sign of neurological deficit or deterioration on other neuromonitoring, it may be justified to closely observe, without other active treatment measures. Thus, a conservative approach might be advocated and argued for in the medical journal. However, firm conclusions of the undocumented “quiet” LPR events cannot be drawn. The lack of initiated interventions and documentation for an LPR event could either be due to the LPR event being missed, or that the event was noted, and a conservative approach was chosen on the basis of clinical evaluation without being documented.The study has some limitations. The CMD only monitors a limited area of the brain tissue at risk, and in some patients the CMD monitoring time may have been too short to enable the detection of delayed cerebral energy metabolic changes. Thus, the frequent occurrence of CMD probe dysfunction resulting in gaps in the monitoring for secondary insults in some patients is a limitation. The cause of the CMD dysfunction is at present unknown. However, we have attempted to prevent dysfunction by refining the surgical implantation technique.2 monitoring is not routinely performed in patients with SAH at our institution, and is not included in our current protocol. Arguably, several reports have suggested an important role for PbtO2 in the multimodal neuromonitoring of patients with SAH. PbtO2 monitoring could have aided in differentiating ischemic from nonischemic causes of energy disturbance. However, here we used the increased LP ratio as an indicator of energy metabolic disturbance without differentiating between a failure of oxygen delivery (ischemic hypoxia) or mitochondrial dysfunction [Although positron emission tomography may have provided more details to the extent of energy metabolic perturbation , it was function .ICP is in our department most commonly monitored using a combined intraventricular and intraparenchymatous monitor, ensuring correct ICP readings despite the EVD being opened for CSF drainage. By CSF drainage, intracranial hypertension is uncommon in these patients. Although CMD and ICP were not correlated in the present study, a contribution of ICP crises to our present findings is unlikely. At the time of many LPR events, TCD investigations were not available, nor were CT-perfusion imaging studies , and we The study focused on CMD as a well-incorporated part of the clinical decision making and multimodal monitoring in patients with SAH. Although only 10% of the patients included in the study developed DCI-related infarcts, LPR events indicating energy metabolic disturbance of the monitored brain tissue were commonly observed. Adherence to the new local protocols may be associated with a decreased DCI incidence by promoting active management and investigations at time of LPR events. Future studies in larger cohorts of patients with SAH are needed to validate these findings.Supplementary file1 (DOCX 69 kb)Supplementary file2 (DOCX 13 kb)Below is the link to the electronic supplementary material.


Further experimental results showed that the Y(II), qP, and ETR, which reflect plants’ photochemical efficiency, increased significantly with CaCl2 treatment. These results indicated that the drought tolerance of the mutant was decreased, and the exogenous application of calcium ions could effectively promote the drought tolerance of the dorn1 mutant. Transpiration loss controlled by stomata is closely related to drought tolerance, further, we examined the transpirational water loss in dorn1 and found that it was greater than wild-type (WT). Besides, the dorn1 mutant’s stomatal aperture significantly increased compared with the WT and the stomata of dorn1 mutant plants tend to close after CaCl2 treatment. Taken together, our results show that DORN1 plays a key role in drought stress tolerance in plants, which may depend on calcium and calcium-related signaling pathways.Water shortages caused by climate change seriously threaten the survival and production of plants and are also one of the major environmental pressures faced by plants. DORN1 was the first identified purinoceptor for the plant response to extracellular ATP. It has been established that DORN1 could play key roles in a series of biological activities in plants. However, the biological roles of DORN1 and the mechanism remain unclear under drought stress conditions in plants. Here, DORN1 was targeted for knockout by using the CRISPR/Cas 9 system. It was found that the loss function of DORN1 resulted in a significant decrease in the effective quantum yield of PSII [Y(II)], the photochemical quenching coefficient (qP), and the rate of photosynthetic electron transport through PSII (ETR), which reflected plants’ photochemical efficiency. Whereas Y Y(NO) is considered an indicator for PSII photodamage. Under normal conditions, the rate of repair of PS II is balanced with the rate of damage. Drought stress reduced the rate of repair and disturbed the dynamic balance, which led to the decreased activity of PS II. Therefore, it is also worth investigating whether overexpression of DORN1 can promote drought tolerance in plants. Furthermore, CaCl2 could reduce the increased Y(NO) caused by drought stress in the dorn1 mutant. This finding suggested the dorn1 mutant exhibited more severe photo or oxidative damage under drought conditions, and this damage is also closely related to [Ca2+]cyt.Although no obvious phenotype was observed in the immunity . These feostasis , especiaeostasis . Plants eostasis . We foun2+]cyt as a specific second messenger through the plasma membrane DORN1 receptor in plants cyt signal could propagate along with tissues [2+ level in the dorn1 mutant was significantly lower than that in the wild type after ATP treatment [2+]cyt is critical for stomatal movement, application of exogenous Ca2+ to plant tissue induced stomatal closure cyt.-induced stomatal closure, which may provide a link between the DORN1-associated eATP signaling pathway and plant drought stress tolerance.We show that DORN1 regulates drought avoidance via modulating [Ca−2 s−1 light of long-day conditions with a 16 h light/8 h dark cycle, then plants transferred to nutrient soil after one week of growth. For the CaCl2 treatment, seedlings were grown on nutrient soil for the indicated number of days, and then 1 mM CaCl2 was sprayed on the leaves once per day (1 mL per plant).All Arabidopsis plants used in the current study were in the Columbia-0 background. The seeds were surface sterilized with 75% ethanol containing 0.05% Tween-20. The seeds were placed on plates that contained 1/2 MS medium  supplemented with 0.6% agar  and 1% sucrose , and then stratified at 4 °C for 2 days in darkness. The plates were then placed for seed germination and plant growth in a growth chamber set at 22 °C under 110 ± 5 μmol mhttps://www.arabidopsis.org/, accessed on 20 December 2017). Synthetic guide RNA (AT5G60300-sgRNA:5′-GATATTGACCACAATCATGT-3′) targeting the first exon was designed by the E-CRISP tool . The oligomer of designed sgRNA was synthesized and cloned into the pKSE401 binary vector carrying the Cas9 gene (Addgene plasmid # 62202).Nucleotide sequence encoding for DORN1 (AT5G60300) was retrieved from the Arabidopsis Information Resource .Genomic DNA was extracted from the positive Tv/Fm, the PSII maximal photochemical efficiency was defined as (Fm − F0)/Fm, where Fm is the maximum fluorescence emission from the dark-adapted state measured with a pulse of saturating light and F0 is the minimal fluorescence from the dark-adapted state. Y(II), the effective quantum yield of PSII photochemistry was defined as (Fm’ − Fs)/Fm’, where Fm’ is the maximum fluorescence emission from the light-adapted state measured with a pulse of saturating light and Fs is the steady-state level of fluorescence emission at 110 μmol m−2 s−1. qP, was defined as (Fm’ − Fs)/(Fm’ − F0’) according to Lazár [(II) represents the number of electrons transferred per photons absorbed by PSII, the ETR  × PAR × 0.5 × 0.84 as per Genty [(NO), the quantum yield of non-regulated energy dissipation of PSII, and Y(NPQ), the quantum yield of regulated energy dissipation of PSII were also defined by Kramer [Chlorophyll fluorescence parameters were measured using Image-PAM with a MAXI measuring head , as described previously by Demmig-Adams . Fv/Fm, to Lazár . As Y(IIer Genty . Y(NO), y Kramer .dorn1-c3/c6 were detached from plants cultivated in the growth chamber and quickly weighed to obtain fresh weights (W0). The shoots were then placed in filter paper under the light intensity of 110 ± 5 μmol m−2 s−1 at 25 °C and 50% relative humidity. The weight was recorded every 30 min , and the loss of fresh weight  was used to indicate the transpirational water loss rate.Water loss was measured as described previously with minor modifications . Shoots nih.gov).The leaves from the same position within the rosette were preserved in the wash solution  at room temperature for 3–5 h, washed with distilled water, and placed in the clear solution  overnight or for a few days . Four imp < 0.05.The results were expressed as the mean ± standard deviation. The data were statistically evaluated by one-way ANOVA between the different groups and Tukey’s multiple comparisons test; The difference was considered to be statistically significant when


Substantial stable carbon isotope fractionation of aqSOA molecules provides robust evidence for extensive aqueous-phase processing. Contrary to the paradigm that these aqSOA compounds are largely biogenic, radiocarbon-based source apportionments show that fossil precursors produced over one-half of the aqSOA molecules. Large fractions of fossil-derived aqSOA contribute substantially to the total water-soluble organic aerosol load and hence impact projections of both air quality and anthropogenic radiative forcing. Our findings reveal the importance of fossil emissions for aqSOA with effects on climate and air quality.Incomplete understanding of the sources of secondary organic aerosol (SOA) leads to large uncertainty in both air quality management and in climate change assessment. Chemical reactions occurring in the atmospheric aqueous phase represent an important source of SOA mass, yet, the effects of anthropogenic emissions on the aqueous SOA (aqSOA) are not well constrained. Here we use compound-specific dual-carbon isotopic fingerprints (δ Isotope fingerprinting is used to track precursor sources and formation pathways of aqueous SOA, such as oxalic acid, finding that fossil fuel precursors contributions have largely been underestimated. The majority of OA is secondary (SOA), formed in the atmosphere from the oxidation of precursor gases2. Most SOA are composed of oxygenated and hygroscopic compounds, thereby, have harmful impacts on respiratory health and large, and uncertain effects on atmospheric radiative forcing3. However, current global models typically underpredict SOA magnitude, distribution, and dynamics, suggesting a limited understanding of their sources and formation processes4. An increasing number of modeling and experimental studies point toward aqueous chemical reactions occurring in cloud droplets and wet aerosols as an important missing pathway for SOA formation8. Aqueous SOA (aqSOA) formation often involves mixed effects of anthropogenic and biogenic emissions3. The extent to which aqSOA is controllable or “natural” is an important subject of ongoing research for both air quality and climate issues3. However, the poorly understood aqueous chemical processes and the complexity of anthropogenic-biogenic interactions renders any firm conclusion hard to come by2.Organic aerosol (OA) is the dominant component of atmospheric fine particulate mass8. Fossil precursors are noticed to be less polar and less hydrophilic than biogenic precursors, thus diminishing the possibility of partitioning to the aqueous phase, and subsequent aqSOA formation9. However, a few recent studies in East Asia suggest that fossil precursors may substantially contribute to the formation of aqSOA. For example, fast aqueous-phase oxidation of polycyclic aromatic hydrocarbons emitted from fossil fuel combustion was observed in Beijing winter haze and may explain the observed SOA10. Radiocarbon-based estimations attributed ~50% of water-soluble OA to fossil sources in the East Asia outflow12, compared to <30% in Europe, U.S., and South Asia18. Nevertheless, on aqSOA formation, a quantitative estimation of the fossil contribution remains challenging, largely due to the lack of specific and reliable technical tools in tracking the sources and formation mechanism of aqSOA in ambient aerosols.In model simulations, aqSOA are put to largely derive from the natural biogenic precursors, such as the oxidation products of isoprene13C−Δ14C) of aqSOA molecules19 to quantify and characterize aqSOA sources and atmospheric chemical processes. The molecular-level Δ14C contents of aqSOA tracers will provide direct constrain on their origins19, whereas δ13C fingerprints of molecular tracers can differentiate various atmospheric processes/reactions20. The technique was applied to purified oxalic acid and other similarly abundant organic acids . Oxalic acid, likely represents the highest oxidation state of OA, has frequently been used as a proxy for aqueous processing in both laboratory and field studies23. Organic acids that have high O/C ratios (around 1−2), are among the most abundant SOA components and are key end products in the aqueous-phase photochemical oxidation of various volatile organic compounds (VOCs) and intermediates such as glyoxal (Gly) and methylglyoxal (MeGly) in clouds or wet aerosols25. Therefore, these organic acids can serve as signature compounds to trace the aqSOA formation pathways26.Here we leverage the recent advent of compound-specific dual-carbon isotope fingerprint  air masses ” and “continental outflow (n = 21)”  and major components of PM2.5 during the continental outflow were 2-5 times higher than during the coastal background (Supplementary Table anth)  are significantly higher in the coastal background samples (1.5 ± 0.4) than the continental outflow (0.9 ± 0.3) (Supplementary Table 28. The ratio of water-soluble organic carbon (WSOC) to organic carbon (WSOC/OC) is another indicator of atmospheric photochemical aging29. For example, the WSOC/OC ratios increased from ~40% in South Asian source region to ~70% in the Indian Ocean receptor site due to the photochemical aging of OC during long-range atmospheric transport29. However, in this study, the WSOC/OC ratios of the continental outflow samples (44 ± 12%) cannot be distinguished statistically from the coastal background (39 ± 10%)   mass fractions in OC observed in the continental outflow samples than the coastal background  during atmospheric reactions/processing15. Secondary aerosols stemming from the oxidation of precursor gases due to the KIE typically lead to lower δ13C values in particulate products compared to the gaseous reactants, whereas aerosol aging  can lead to a release of isotopically lighter carbon such as in CO2 and CO, leaving residual (aged) aerosols enriched in 13C29. Figure 13C values for oxalic acid are on average −24.6 ± 2.7‰ in the continental outflow samples and substantially more enriched in 13C in the coastal background samples (−19.9 ± 2.3‰). This clear contrast can likely be attributable to different emission sources and/or atmospheric processing.The δ13C composition of oxalic acid in this study. First, the δ13C compositions of azelaic acid (C9 diacids), an oxidation product of biogenic unsaturated fatty acids33, in the coastal background samples (−34.1 ± 2.8‰) and continental outflow (−34.7 ± 2.6‰) are very close 34, while excluding marine phytoplankton and C4 plant sources that both instead are more enriched in δ13C36. This is consistent with the limited marine-biogenic contributions as indicated by the mass ratios of nss-SO42- to SO42- (99 ± 1%) and nss-K+ to K+ (94 ± 4%) in all samples29. Given contributions from marine and C4 plants sources are mostly excluded at the Heshan site, the potential precursors of oxalic acid would then be anthropogenic VOCs  and C3 plant-derived biogenic VOCs , of which the δ13C values are similar , followed by partitioning of SVOCs to wet aerosols or cloud/fog droplets, with subsequent aqueous-phase transformation to pyruvic acid (Pyr) and glyoxylic acid (ωC2) and finally oxidized to oxalic acid26. In this case, oxalic acid-C carries a depleted δ13C signal as the oxalic acid is an oxidation product, as opposed to a residual C pool. Our dataset displays a significant correlation of oxalic acid with ALW , which stands for the aqueous-phase pathway.Oxalic acid is an SOA end product and can be formed by two pathways. The first pathway is a stepwise photochemical breakdown of longer-chain aliphatic diacids13C values of oxalic acids and relevant SOA species can help to explore the formation pathway. First, the higher homologs aliphatic diacids from C3 to C9, in contrast to C2, show no significant statistical difference  processes. Note that the source mixing contribution is minimal as mentioned above.The compound-specific δ13C signatures of oxalic acid against an increase in WSOC concentrations . Atmospheric aqueous-phase processing would simultaneously cause a decrease in the δ13C signature of reaction products and an increase in the WSOC mass concentrations. This is precisely what was observed during this campaign. Overall, the results indicate that aqSOA formation processes may be a significant route to form water-soluble OA in the atmosphere of South China and similar settings.This aqueous reaction effect on isotope fractionation may also apply to water-soluble OA, which is a cocktail of primary OA  and SOA species. Figure 13C signature, which undergoes strong isotope fractionation during atmospheric processing, radiocarbon isotopic fingerprint (Δ14C) is conservative and highly precise in distinguishing fossil from biogenic/biomass sources19. Individual water-soluble diacids and related compounds  were isolated by preparative capillary gas chromatography techniques (Methods). It is possible to harvest a sufficient mass of C2 oxalic acid from aerosol samples, while the other isolated compounds had to each be pooled per month to obtain sufficient carbon mass for the 14C measurement. The determined Δ14C compositions reported as the “fraction of modern carbon” (Fm) are shown in Supplementary Table Fm signatures into fractional contribution from fossil (ffossil) versus non-fossil  sources  from anthropogenic activities. For the continental outflow samples, the ffossil−C2  are even higher than biogenic/biomass source contributions. Back trajectory analysis reveals that during the latter period the receptor site was downwind from highly polluted continental source regions, which thus apparently is a source of oxalic acid in the regional aerosol  was one of the most abundant anthropogenic diacids accounting for 5% of total diacids  is often used to discriminate the relative significance of biogenic versus anthropogenic source contribution to diacids. Here, C6 adipic acid is the photochemical oxidation product of cyclic hexene emitted from the fossil fuel combustion sources43, whereas C9 azelaic acid is a photochemical degradation product of biogenic unsaturated fatty acids33. Lower C6/C9 ratios (0.37 ± 0.13) were observed in the coastal background samples than continental outflow (0.76 ± 0.21). Furthermore, C6/C9 ratios correlates well with the parameter of ffossil−C2  and its aqueous processing precursors (ωC2 and MeGly)  and methylglyoxal . Therefore, a dominance from fossil fuel sources to their aqSOA formation in the continental outflow is obvious.The fossil contents of oxalic acid in the continental outflow samples are compared with its presumed precursors of longer-chain diacids (Cly) Fig. . The res3 and succinic C4 acids are believed to be partly produced from aqueous processing26, there is for these C3 and C4 a somewhat lower but still large contribution from fossil sources . This demonstrates that C3 and C4 diacids instead were mainly originated from the stepwise photochemical breakdown of biogenic unsaturated fatty acids even in highly polluted continental outflow air mass. The large contrast of fossil fraction among C2 diacids and its higher homologs  further indicate that substantial fossil precursors contribute to the formation of C2 through aqueous-phase processing, which results in a much higher fossil content of C2 than of its higher homologs.Although malonic C14C-derived concentrations of oxalic acid from non-fossil sources (bio/bb−C2) span from 40 to 417 ng m−3 (207 ± 88 ng m−3), whereas from fossil source (fossil−C2) are 21 to 650 ng m-3 (230 ± 168 ng m−3)  for the coastal background samples, suggesting that the bio/bb−C2 in low biomass burning scenario was formed mainly from biogenic precursors. These independent lines of evidence are suggestive of and consistent with that background C2 were biogenic, while bb−C2 likely represent additional non-fossil C2 concentrations. We therefore assume that, when there is little biomass burning contribution (levoglucosan ≈ 0), the concentration of bio/bb−C2  accounted for 78% of total C2 in the continental outflow samples, whereas its contribution is 39% in the coastal background . By extension, the contribution of anthropogenic emissions in aqSOA and gasSOA are likely to be consistent under ambient conditions with high ALW content. Our observation-based isotope constraints question the paradigm, largely based on model simulations, which instead have held that aqSOA, which may contribute as much mass as gasSOA to SOA budget, are largely formed from biogenic emissions4. Previous study considered that fossil precursors may be more hydrophobic than biogenic precursors, and thus are likely to form SOA preferentially through gas-phase processing9. However, the oxidation products of fossil precursors are largely water-soluble and thus their chemical reaction in the aqueous phase constitutes an unneglected pathway, which may be competitive to SOA formation via the gas-phase chemistry.The anthropogenic Cund Fig. . Note th13C and 14C signatures of oxalic acid. Lower oxalic acid concentrations were found in the coastal background samples, with higher non-fossil contributions, and more enriched in 13C. Higher oxalic acid concentrations in the continental outflow samples are characterized by higher fossil contributions and more depleted in 13C. The hygroscopic particles and precursors in continental outflow air masses facilitate the aqueous-phase chemical processes, which results in lighter δ13C values and higher fossil contribution to oxalic acid.Figure 13C values of oxalic acid  of oxalic acid would surpass its aqueous-phase formation, which is evident in the increasing δcid Fig. . The intcid Fig. , of whic48, while much less attention has been paid to fossil/anthropogenic precursors . Fossil precursors have been thought to consist mostly of hydrophobic components, and thus fossil-derived SOA have been expected to be less soluble in water9. Smog chamber experiments show that water-soluble organic aerosols exhibited more similarities to biogenic SOA than fossil SOA50. In fact, low fossil contributions to WSOC (0−29%) have been reported in Europe, North America, and South Asia across different seasons18, and 14C analyses of aerosol OC and its sub-fractions have been interpreted such that the majority of fossil-derived OA was water-insoluble52. Hence, these earlier field and laboratory studies show that water-soluble OA is only to very limited extent stemmed from fossil sources. In contrast, the results from the current study, using an extensive set of molecular 13C/14C data in China, strongly point to anthropogenic-fossil emissions as an important source of water-soluble OA. This is also supported by a large contribution of fossil sources to WSOC (30−60%) reported for both Chinese urban areas55 and a receptor station in the southeast Yellow Sea intercepting a large regional footprint12. Taken together, these two sets of findings imply a significant missing pathway responsible for the enhancement in fossil fuel-derived water-soluble OA in China.Many studies have investigated the formation of aqSOA from biogenic/biomass precursors 13C of oxalic acid is used as the proxy for aqSOA formation) simultaneously cause an increasing in the WSOC mass concentration, suggesting that aqSOA constitute a substantial water-soluble OA source , which reveals a dominant fossil source to aqSOA  with more negative δ13C values and more fossil contributions than the bulk WSOC pool.To test the spatial extent of the findings, we retrieved aerosol samples from five emission hotspot megacities of China Fig. . The difely Fig. . Althoug13C and 14C than the WSOC pool in Guangzhou, Wuhan and Shanghai. As shown in Fig. 57, which results in oxalic acid enriched both in δ13C and Δ14C. The differing isotopic signatures of different emission sources would also contribute to the enrichment of carbon isotopes of oxalic acid. Note here that biogenic contributions to oxalic acid can be expected to increase during summer due to it being the growing season.On the contrary, in summer, oxalic acid was more enriched in 58. As for oxalic acid, its interactions with inorganic salts in the aqueous phase may modify the CCN activity of aerosol particle61.The large contribution of fossil-derived carbon to aqSOA highlighted in this study has important implications for aerosol climate forcing and regional air quality. Aqueous-phase processing of fossil precursors, competitive to gas-phase pathway, could amplify the impact of anthropogenic emissions on water-soluble OA, alter the chemical property and hygroscopicity of aerosols. The derived hygroscopic SOA affect not only direct radiative forcing, but also cloud condensation nuclei (CCN) activity, and the subsequent cloud-mediated effect of anthropogenic aerosols on the climate system62. Our year-round observation at the Heshan receptor site witnesses a positive feedback loop among ALW, inorganics, as well as aqSOA compounds. The dataset supports that the inorganic particles induce ALW growth  mixing model64, the effect of anthropogenic oxalic acid on aerosol water accounts for 3−38% (average: 10%) of the ALW contributed by organic compounds 41. A model study predicted that 92% of increased ALW in eastern China by the year 2100 can be attributed to the increase in anthropogenic nitrate aerosol30. Here, the average nitrate concentration in the continental outflow samples was nearly 20 times higher than coastal background  are strongly warranted to better constrain the chemical mechanism responsible for fossil-derived aqSOA formation.In this study, the δ64. An absence of accounting for such aqueous processing incurred by fossil precursors could lead to an underestimation of the anthropogenic contribution to organic aerosols. Understanding the role of anthropogenic emissions in aqSOA formation and the chemical mechanisms involved are of critical concern for future climate and air quality projections, and within the context of energy needs and choices.Overall, compound-specific dual carbon isotopic evidences from field observations show that fossil anthropogenic precursors contribute to the formation of aqSOA to a much higher extent than expected. Atmospheric aqueous-phase processes are expected to increase due to the enhanced evapotranspiration in a warmer world68. During the winter northeast-monsoon season, this specific site well intercepts high anthropogenically dominated outflow airmass from Chinese continental. The strength of anthropogenic impact decreases in the summer southeast/southwest monsoon, with the arrival of clean air mass originating from the Western Pacific/Indian Ocean. Field experiments started in June 2017 and ended in May 2018. PM2.5 aerosol samples (n = 32) were collected onto pre-combusted quartz-fiber filters by using a high-volume sampler at an air-flow rate of 1 m3 /min for 48 h.The regional receptor site was positioned in Heshan Atmospheric Environmental Monitoring Superstation , a rural site located at 50 km southwest to the megacity of Guangzhou in the central PRD. The surrounding area of the sampling site is dominated by farmlands and forests and is far from local anthropogenic emissions2.5 samples were additionally studied in five emission hot spot megacities of China , which were collected continuously every 24 h for 1 week in winter (January 2018) and in summer (July 2018). Continuous sets of 7 filters collected in one week were combined to represent the seasonal characteristic of each urban site. The sample filters were stored in darkness at −20 °C prior to analysis. Field blank filters were collected for each campaign.PMhttp://ready.arl.noaa.gov/HYSPLIT.php). The back trajectories are grouped into two major transport pathways with vastly different source regions during the sampling campaigns.Data of atmospheric concentration of meteorological parameters and gaseous pollutants were obtained from the hourly monitor by Heshan Atmospheric Environmental Monitoring Superstation. To characterize different geographical sources of air masses encountered at Heshan, 3-day isentropic backward air mass trajectories were computed at an elevation of 100 m for every 12 h using the HYSPLIT model (version-4) and meteorological datasets from the NOAA air resources laboratory  in a hot water bath at 100 °C for about 1 h. The derivatives were extracted with n-hexane and finally quantified using GC-MS (Agilent 7890 A gas chromatography and Agilent 5975C mass spectrometry). The concentrations reported here are corrected for field blank, but are not corrected for recovery efficiencies.The concentration of typical aqSOA species  was measured following earlier protocols+, NH4+, K+, Mg2+, Ca2+, Cl−, NO3−, and SO42−), biogenic SOA tracers; and sugar compounds are provided in the Supplementary Method Relevant details of the analysis method for aerosol carbonaceous components , water-soluble inorganic constituents  is taken as the sum of water associated with individual aerosol chemical components, with the assumption that the particles are internally mixed13C values of diacids derivatives were determined using GC ISOLink2/IRMS (Thermo Fisher Scientific). The δ13C values of free diacids and related compounds were then calculated by an isotopic mass balance approach, based on the measured δ13C values of derivatives and the derivatizing agent . The isotopic fractionation during the derivatization step has been reported to be less than 0.73‰ in our earlier publications19. Each sample was measured in duplicate and the mean δ13C values were reported. The difference in δ13C of diacids  in replicate analyses was generally <1‰, though the analytical accuracy was < 2‰ for other compounds .The δ14C compositions builds on our previously publications19. In brief, the filter was extracted with the Milli-Q water and derivatized with BF3/1-butanol. Microgram quantities of C2, C3, C4, ωC2, and MeGly were isolated and collected by preparative capillary gas chromatography (pcGC) through around 50 consecutive runs (5 μl per injection), which was sufficient for off-line natural abundance radiocarbon analysis. The pcGC isolates were combusted at 920 °C and the resulting CO2 was finally converted to graphite via hydrogen reduction method. Measurements of 14C were performed using Accelerator Mass Spectrometry 19. In all cases, 14C results are reported as “fraction of modern carbon” (Fm) normalized to a common δ13C value of −25‰. The Fm values were further converted into the fraction of non-fossil source (fbio/bb) with a conversion factor of 1.06 to compensate for the excess 14C produced by nuclear bomb testing in the 1950s–1960s. The concentration of diacids in blank filters was <0.06% of the real samples, therefore, no filter blank subtraction was performed for the isotope analysis in this study. Sample results were corrected for procedural blanks using a pair of processing standards with modern or fossil radiocarbon composition. An isotopic mass-balance approach was adopted to correct the carbon contribution of butanol groups  added in the derivatives of diacids, where appropriate. Calculations for blank correction, Fm results and error propagation are shown in Supplementary Method The analytical method for the isolation of individual diacids to determine Δ13C and Δ14C composition of WSOC builds on ultrasonication extraction protocols were reported in Supplementary Method 13C composition of WSOC were performed on a Flash 2000 elemental analyzer connected to a Thermo Scientific Delta V isotope ratio mass spectrometer. Measurements of the Δ14C composition were performed using accelerator mass spectrometry facility  of the Guangzhou Institute of Geochemistry of the Chinese Academy of Sciences (GIGCAS).The analytical method for the measurements of δSupplementary InformationPeer Review File