However, the interpretation of the Qunf δ18Oc remains in dispute. Here we provide a multi-proxy record from Qunf Cave and climate model simulations to reconstruct the Holocene local and regional hydroclimate changes. The results indicate that besides the Indian summer monsoon, the North African summer monsoon also contributes water vapor to southern Oman during the early to middle Holocene. In principle, Qunf δ18Oc values reflect integrated oxygen-isotope fractionations over a broad moisture transport swath from moisture sources to the cave site, rather than local precipitation amount alone, and thus the Qunf δ18Oc record characterizes primary changes in the Afro-Asian monsoon regime across the Holocene. In contrast, local climate proxies appear to suggest an overall slightly increased or unchanged wetness over the Holocene at the cave site.Qunf Cave oxygen isotope (δ Southern Oman speleothem oxygen isotope and multi-proxy data reveal diverse changes in the Afro-Indian summer monsoon circulations and local hydroclimate conditions during the Holocene, confirming climate model simulations. The Asian summer monsoon contains two large monsoon subsystems, the East Asian summer monsoon and the Indian summer monsoon. The speleothem δ18Oc records from both regions show a broadly similar pattern on the orbital scale, following Northern Hemisphere summer insolation (NHSI), nearly in phase at the precession band 10, which supports the conventional monsoon hypothesis11—the insolation hypothesis12. A large number of publications clearly show that the enhanced Asian summer monsoon from the precession minimum (Pmin) or NHSI maximum to the precession maximum (Pmax) or NHSI minimum, corresponds to the negative δ18Oc excursions in the speleothem records, and vice versa19. These data-model comparisons validate the interpretation that speleothem δ18Oc records from the Asian summer monsoon domain reflect regional Asian summer monsoon intensity or the scale of summer monsoon circulations on orbital scales10. However, the relation between the speleothem δ18Oc records and precipitation amount at the cave site may be complex3. A number of studies suggested that δ18Oc records from the Asian continent may partially reflect changes in moisture source, isotope fractionation at the source area, transport pathways, as well as the isotopic composition of the Bay of Bengal surface water22. When the Asian summer monsoon is strong, the spatial scale of the summer monsoon circulation expands, and more remote moisture is transported inland, resulting in lower precipitation oxygen isotope (δ18OP) values in continental regions and vice versa26. Theoretically, speleothem δ18Oc, as a proxy of the δ18Op at the cave site, is related to oxygen-isotope fractionations integrated from tropical oceans to the cave site along the moisture transport trajectory26. Besides, the influence of other factors on speleothem δ18Oc, such as the mixing of different moisture sources and rainfall amount, should not be precluded.Speleothem oxygen isotope (δmin (NHSI maximum) to Pmax (NHSI minimum). While a large set of Holocene speleothem records are available in the East Asian summer monsoon domain28, similar records in the Indian summer monsoon domain remain sparse and segmented. The well-known Indian summer monsoon record comes from Qunf Cave in Oman, southeastern Arabian Peninsula29. The Qunf δ18Oc record covers most of the Holocene and has been widely used as a typical Indian summer monsoon record. It has been highly cited during the past two decades and remains a unique Holocene δ18Oc record in the area up till now. Previous studies on the Qunf δ18Oc record, and additional speleothem records from Oman and Yemen, suggested that these records reflect changes in Indian summer monsoon precipitation amount during the Holocene32. The sharp decrease of the speleothem δ18Oc from 10.3 to 9.6 ka BP was interpreted to indicate a rapid northward shift of the summer position of the intertropical convergence zone (ITCZ) and the associated Indian summer monsoon rainfall, leading to a rapid increase in summer monsoon precipitation. After ~8 ka BP, the speleothem δ18Oc record shows an almost linear response to the orbitally-induced variation in NHSI at 30°N, which implies a gradual southward retreat of the ITCZ and gradual weakening of the Indian summer monsoon in response to a decrease in summer insolation29. This interpretation seems to be consistent with the conventional wisdom that increased NHSI enhances the land-sea thermal contrast and, thus, the summer monsoon intensity11. However, this classical notion of monsoon has been challenged by a number of recent modeling studies35.The Holocene, from ~11.7 ka BP  to the present, spans about half of a precession cycle, approximately from P38. A few marine upwelling records from the Arabian Sea show a consistent Holocene pattern with the speleothem records39. However, some marine upwelling records in the Arabian Sea suggest intensified Indian summer monsoon winds throughout the entire Holocene and show a significant lag to NHSI and the Indian summer monsoon intensity change inferred by speleothem δ18Oc records42. These observations were interpreted to be caused by the latent heat from the southern Indian Ocean and the global ice volume besides insolation43. Alternatively, the apparent lag to the NHSI is interpreted as the length of late summer and the effect of the Atlantic Meridional Overturning Circulation38.Marine records from the Arabian Sea near Qunf Cave, such as the upwelling-driven biological productivity, foraminifera assemblage, particle size, and oxygen minimum zone records were also interpreted as proxies of the Indian summer monsoon intensity44 were the first to show that their climate model actually reproduced this opposite response between land and ocean to NHSI at the precession band. This study was followed by multiple climate model simulations that confirmed the results from refs. 47. Later, ref. 2 coined the term “land-sea precession phase paradox” in the research forefront of the Asian summer monsoon, referring to the contrast between speleothem and marine proxy records. Also, Ruddiman48 argued that the Arabian Sea summer monsoon proxies are not tightly coupled to monsoon intensity but are influenced by other processes. Recently, refs. 46 explained the mechanisms for the opposing marine and terrestrial proxy response in the Indian summer monsoon domain. It appears that the upwelling-based proxies in the Arabian Sea are not linked to the Indian monsoon rainfall over India at precession timescales47. Changes in the low-level jet could cause a stronger wind stress curl during weaker summer insolation and lead to strong upwelling. This interpretation appears to largely reconcile the so-called “sea-land precession-phase paradox”, as the monsoon wind strength over the oceans is not necessarily coupled with monsoon precipitation over the Asian continent2. However, in light of these recent discussions and data-model comparisons25, critical issues remain regarding: (1) What is the hydroclimate significance of Qunf δ18Oc variation? (2) What is the local hydroclimate variation pattern across the Holocene? (3) What is the relation between the Qunf δ18Oc record and NHSI? (4) Is precipitation at Qunf Cave sourced from single or multiple moisture sources? These questions call for further investigations, especially with speleothem multi-proxy data.Hsu et al.18Oc from the stalagmite Q5 from Qunf Cave, southern Oman, including (234U/238U)0, δ13C, trace elements, fluid inclusion δDfi, and Δ'17O records to reconstruct the local and regional hydroclimate history across the Holocene. We then compare the Qunf Cave record with climate model simulations to evaluate alternative interpretations of Qunf δ18Oc values.In this study, we provide multiple proxies in addition to δ29. The average summer  rainfall recorded at the Salalah GNIP station  is 1.65 mm d−1 (1988–1989), and the JJA rainfall of 1989 is 3.43 mm d−1 at Qairoon Hairiti GNIP station . The Somali jet brings considerable moisture to the Qunf Cave site during the summer, mainly from the tropical South Indian Ocean across the equator to the western Arabian Sea , from which stalagmite Q5 was collected, is located in southern Oman 50, δDfi values are more depleted, with a mean δDfi value of ~2.07‰. After ~6 ka BP, δDfi shows an increasing trend, with an average value of ~9.46 ‰ , which sits close to Qunf, also show a significant change in the moisture source, seasonality, and amount of rainfall above the cave around 6 ka BP49. Moreover, the slope of the ln(δ18Oc + 1)-ln(δ17Oc + 1) line shifts at ~6 ka BP from 0.510 to 0.523  consider that the Indian summer monsoon (or the Indian Ocean) was the sole moisture source in southern Oman51, our new data seem to imply an additional moisture source, yet unknown, that could be involved during the Holocene Humid Period49.Fluid inclusion δD6 ‰ Fig. . Consist17O is defined as the deviation of triple oxygen isotope data from a reference relationship between speleothem δ17Oc and δ18Oc52 (See supplementary text), which is related to distinct hydroclimate dynamics56. Speleothem Δ'17O is not sensitive to Rayleigh distillation from the ocean to the continent57 , re-evaporation of precipitation (depending on the re-evaporation rate and downdraft intensity), and convections62. Collectively, the process of stronger continental recycling and re-evaporation of precipitation may cause higher Δ'17O  during ~8-6 ka BP compared to early (−259 per meg) and late (−245 per meg) Holocene   Fig. . From ~663. The model simulations reveal a ~6% increase in relative humidity at the equatorial Atlantic, Arabian Sea and Qunf Cave site from 8 K to PI  hydrological conditions between 8 K (8 ka BP) and PI . Our model simulations indicate that during the 8 K period, the North African summer monsoon—characterized by the southwesterly wind extending across the continent and reaching the southern Arabian Peninsula was considerably stronger Fig. . Althoug PI Fig. . The ∆'1ene Fig. . The dir64. The North African summer monsoon has effective control over the Eastern Mediterranean deepwater ventilation on orbital time-scales through changing the discharge of the Nile River. This results in density stratification and breakdown of deepwater formation reflected by sapropel layers S1 in Ocean Drilling Program Site 968 from ~10.2 to 6.5 ka BP68. This strong North African summer monsoon period corresponds precisely with the lowest δ18Oc period in the Q5 record  that occurred from ~10.8 to 9 ka BP, followed by a relatively stable period (~ −2‰) between 9 and 6 ka BP, and then by a persistent increase of δ18Oc from −2‰ to ~ 0‰ till a hiatus from ~2.4 to 0.8 ka BP. After the hiatus, δ18Oc decreased from ~ 0‰ to ~−1‰ over ~400 years  waning process72. A significant mid-Holocene positive shift of δ18Oc at ~6 ka BP is also found in Hoti Cave in northern Oman 31.δars Fig. . δ18Oc s74. More broadly, the entire North African summer monsoon and Asian summer monsoon (Indian summer monsoon and East Asian summer monsoon) may be viewed as a large unified summer monsoon system. In this interpretative framework, the Qunf δ18Oc record shows the Holocene hydroclimate variation of this “super-monsoon” or Afro-Asian monsoon regime64, since the Qunf δ18Oc variations appear to follow the overall changes of the Afro-Asian monsoon intensity according to both empirical and model results as aforementioned.Taking a broader perspective, the monsoons over the African continent and the Indian Ocean exhibit remarkably similar hydroclimate trends during the middle to late Holocene. This suggests that they were sensitive to, or driven by, a common forcing mechanism on suborbital timescales18Oc record mostly reflects large-scale monsoon circulation changes, making local hydroclimate proxies an essential complement. Combining the (234U/238U)0, δ13C, and trace element records from Q5 0 values, as (234U/238U)0 is influenced by water flow rates and water–rock interaction times77.The Qunf δning the 34U/238U0 Q5 Fig.  enables 13C and trace elements ratio data. Lower summer temperature and slightly increased P-E could favor increased organic materials and reduced prior calcite precipitation (PCP), resulting in more negative δ13C values in the late Holocene80. Q5 δ13C values range from -6.4‰ to 1.1‰, with relatively stable values (~ −1‰) from ~10.8 to 6 ka BP and a decreasing trend from~−1‰ to ~−5‰ since ~6 ka BP 85, another founded on the moist static energy budget87, and a third that frames the monsoon as an extension of the zonal-mean ITCZ35. While these theoretical frameworks help explain certain aspects of modern monsoon variability, especially regarding monsoon rainfalls and thermodynamics, the interpretations of paleoclimate variations across various timescales are still in development89.The understanding of monsoons as an integral component of the global atmospheric circulation and hydroclimate is becoming prevalent18Op at the cave site, prior to the onset of the Asian summer monsoon, the moisture source is relatively proximate, resulting in heavier δ18Op. After the summer monsoon onset, the spatial-scale of monsoon circulation and moisture from remote sources increase dramatically 0, and δ13C].Physically, the summer barometric differentials and other boundary conditions on a large spatial-scale, regardless of their cause, drive the Asian summer monsoon circulation e.g., at 850 hPa)  is critical, reflecting both “pull” and “push” forcings from Northern and Southern Hemisphere that propel monsoon changes1. Thus, we use the interhemispheric differential in tropical insolation (30°N-30°S) as an integrated insolation forcing of the monsoon, consistent with previous evidence, such as the Asian summer monsoon variation pattern over the MIS 31. The Qunf δ18Oc record suggests an Indian summer monsoon waning trend from the middle Holocene to the present, broadly following SITIG. This observation is consistent with many other Indian summer monsoon records, such as the Sahiya and Mawmluh records from northern India92, the Tianmen record from the southern Tibetan Plateau93, and some of the upwelling records from the Arabian Sea and its adjacent area39  or nearly anti-phase to NHSI at the precession band42. Model simulations reveal contrasting hydroclimatic responses between land and ocean to NHSI changes at the precession band44. This phenomenon has been referred to as the “land-sea precession phase paradox”2. In the Indian summer monsoon regions, Jalihal et al.97 investigated the opposing marine and terrestrial responses, both empirically and theoretically. They discovered that, in addition to NHSI, precipitation changes over the ocean are influenced by changes in surface energy fluxes and vertical stability. This view largely reconciles the apparent discrepancy between the Qunf δ18Oc record and some previous marine upwelling records from the Arabian Sea in terms of the precession phase paradox.In the early 1990s, ref. 18Oc record and the Holocene hydroclimate history in the southern Arabian Peninsula. During the early to middle Holocene, corresponding to the Holocene Humid Period in southern Arabia, the North African summer monsoon, in addition to the Indian summer monsoon, may likely contribute water vapor to the Qunf Cave area as well. After ~6 ka BP, Qunf Δ'17O data suggest a possible reduction in moisture delivered by the North African summer monsoon, higher δ18Oc values suggest that only the Indian summer monsoon prevails with water vapor mainly derived from the proximate Arabian Sea like today. Our results are consistent with the interpretative framework that in addition to the amount effect, speleothem δ18Oc values are essentially related to oxygen-isotope fractionations integrated during the moisture transport from the source to the cave site. More broadly, the Qunf δ18Oc record may be viewed theoretically as an archive indicating the change of the vast monsoon regime, the unified North African summer monsoon - Asian summer monsoon system. Although the Qunf δ18Oc have lighter values and the precipitation amount might increase from ~10 to ~6 ka BP, Qunf (234U/238U)0, δ13C, and trace element ratio records  seem to suggest a lower P-E condition. This requires further investigation.Overall, our speleothem multi-proxy records and modeling results provide valuable insights into the interpretation of the Qunf δσ) and wettest (+1.5σ) June-July-August (JJA) years in our study area . FLEXPART simulations were performed during JJA in these years.To establish a modern source–receptor relationship for moisture transport in the study region, the Lagrangian trajectory model FLEXPART, driven by ERA-Interim reanalysis, was used. Based on ERA-interim precipitation (1979–2019), we created wet and dry composites from the three driest , where E-P for the total particles residing over A is given by:e-p is the rate of moisture change along the trajectory99. The simulation releases 50,000 particles, and K is the number of N particles that resides over A. This approach divides the whole atmosphere’s mass into small elements distributed homogeneously in the atmosphere according to the atmospheric mass distribution. These are then moved with the mass-consistent winds and are based on mass-consistent turbulence and convection parameterizations101. We calculated the mean net moisture flux based on E-P and backtracked elements that had a loss (E-P < 0) over the Qunf Cave region  for 12 days.The FLEXPART simulation was driven by ERA-interim through 6-hourly analyzes  and 3-hourly forecasts at intermediate times , with 1° × 1° resolution on 60 model levels230Th dates were obtained from stalagmite Q5. Powder sub-samples were hand-drilled parallel to the growth band near the stalagmite axis. The 230Th dating work was performed at Xi’an Jiaotong University Isotope Laboratory using multi-collector inductively coupled plasma mass spectrometers (Thermo-Finnigan Neptune-plus). We used standard chemistry procedures to separate U and Th for dating102. A triple-spike (229Th–233U–236U) isotope dilution method was employed to correct instrumental fractionation and determine U-Th isotopic ratios and concentrations. Details about the instrumental setup are provided by refs. 104. All ages are in stratigraphic order within dating uncertainties. The age model was constructed using the Constructing Proxy Records from Age (COPRA) program105. Q5 δ18Oc and δ13C records were adjusted to the new age model  immediately before analysis. Samples were run at average Ca concentrations of 200 ppm. Calibration was conducted offline using the intensity ratio method described by ref. 106. The results are shown by molar ratios of Mg, Ba, and Sr relative to Ca.The trace elements were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Thermo Scientific iCAP DUO 6300) in Instituto Pirenaico de Ecologia, Spanish Scientific Research Council (IPE-CSIC). The drilled powder (100–150 mg) was placed in tubes cleaned with 10% HCl, rinsed with MilliQ-filtered water, and dissolved in 1.5 mL of 2% HNO17Oc of Q5 was measured at Xi’an Jiaotong University Isotope Laboratory. After drilling the carbonate powder of the sample, we add in phosphoric acid  at 25 °C to extract CO2. Then the CO2 was equilibrated with an equal amount of the O2 gas for 30 min under 750 °C to reach a Pt-catalyzed equilibrium by an O2–CO2 Pt-catalyzed oxygen-isotope equilibration reaction system. In this system, the two post-equilibration gases were separated from each other cryogenically. Finally, δ17Oc was obtained through measurements of the resultant O2 and CO2 by a Thermo Scientific MAT 253 mass spectrometer60. The calculation of Δ'17O and the relative humidity at the moisture source follows the method from ref. 60 (See supplementary text).The δfi was analyzed at Xi’an Jiaotong University Isotope Laboratory. The Q5 calcite blocks were crushed at a temperature of ~120 °C. The liberated water was then transported to a wavelength-scanned cavity ring-down spectroscopy system . The analytical method is described in ref. 107, the maximum error on the reproducibility of crushed speleothem samples for this system is 2‰ for δDfi (1 SD). δDfi values are reported on the Vienna Standard Mean Ocean Water to SLAP2 (VSMOW2 – SLAP2) scale.Q5 δD108. The atmospheric model of EC-Earth is based on the IFS, including a land model H-TESSEL, developed at the European Centre for Medium-Range Weather Forecasts (ECMWF)98. In addition, the dynamic vegetation model LPJ-GUESS109 is coupled to the land component. The ocean component is the Nucleus for European Modelling of the Ocean (NEMO)110 and includes a sea-ice model LIM3111. We use the CMIP6 configuration of EC-Earth3-veg-LR, in which the atmosphere and land model has a T159 horizontal spectral resolution (roughly 1.125°–125 km) with 62 vertical levels. The NEMO and LIM have a nominal horizontal resolution of 1°, and NEMO has 75 vertical levels. The coupling between the atmosphere and ocean/sea ice is through the Ocean Atmosphere Sea Ice Soil coupler 112.The simulations based on the fully coupled Earth system model EC-Earth are used in this study. A consortium of European research institutions develops EC-Earth to build a fully coupled Atmosphere-Ocean-Land-Biosphere Earth system model for seasonal to decadal climate prediction and future climate projections113. The PI and 8 K simulations have the same boundary conditions except for the orbital forcing and greenhouse gas concentration differences  and a pre-industrial control simulation (PI) using EC-Earth3-veg-LR. The simulations follow the CMIP6-PMIP4 protocol for each experiment setup115. The simulation reaches the quasi-equilibrium after 300 years (meets the criteria global mean surface temperature trend <±0.05 K per century113, and the simulation continues running for another 700 years. The initial condition for the 8 K simulation is taken from the equilibrium state of PI simulation, with changed orbital forcing and Green House Gas concentration. For the 8 K condition it takes around 300 years to reach equilibrium, and we run for another 700 years. The last 200 years’ outputs are used to analyze both simulations.The initial condition for PI simulation with EC-Earth-veg-LR is taken from a previous PI run with EC-Earth3-LRSupplementary InformationPeer Review FileSource Data


However, how the temporal andspatial fluctuations of O2•– and ATP coordinate changes in HIRI and particularly how they synergisticallyregulate each other in the pathological mechanism of HIRI remainsunclear. Herein, we rationally designed and successfully synthesizeda dual-color and dual-reversible molecular fluorescent probe (UDP)for dynamic and simultaneous visualization of O2•– and ATP in real-time, and uncovered their interrelationshipand synergy in HIRI. UDP featured excellent sensitivity, selectivity,and reversibility in response to O2•– and ATP, which rendered UDP suitable for detectingO2•– and ATP and generatingindependent responses in the blue and red fluorescence channels withoutspectral crosstalk. Notably, in situ imaging with UDP revealed forthe first time synchronous O2•– bursts and ATP depletion in hepatocytes and mouse livers duringthe process of HIRI. Surprisingly, a slight increase in ATP was observedduring reperfusion. More importantly, intracellular O2•–—succinate dehydrogenase(SDH)—mitochondrial (Mito) reduced nicotinamide adenine dinucleotide(NADH)—Mito ATP—intracellular ATP cascade signalingpathway in the HIRI process was unveiled which illustrated the correlationbetween O2•– and ATPfor the first time. This research confirms the potential of UDP forthe dynamic monitoring of HIRI and provides a clear illustration ofHIRI pathogenesis.Hepatic ischemia–reperfusion injury (HIRI) ismainly responsiblefor morbidity or death due to graft rejection after liver transplantation.During HIRI, superoxide anion (O Traditional methods for the diagnosis of liver injury relyon the determination of serum alanine aminotransferase (ALT) and aspartateaminotransferase (AST),4 which are nonspecificbiomarkers of late-stage hepatocyte injury and are also influencedby other diseases5 and, therefore, failto reflect changes of targets in the liver. Imaging diagnostic techniquesfor the assessment of liver injury including magnetic resonance imaging(MRI), computed tomography (CT), and ultrasonic (US) imaging are onlysuitable for evaluating histological pathologies and liver functionalchanges due to the inherent low sensitivity and specificity.2 Liver biopsy is an invasive method for clinicaldiagnosis of liver injury. However, it provides limited diagnosticaccuracy due to observer variability and sampling bias,6 only provides static pathological status, andis unsuitable for the monitoring of the dynamic development of HIRI.Therefore, the development of a noninvasive and reliable method thatenables real-time and dynamic monitoring of HIRI is needed, whichwill facilitate monitoring of disease progression, formulate treatmentstrategies, and improve surgery outcomes for HIRI.Hepatic ischemia–reperfusion injury(HIRI) is a severe butunavoidable complication of liver resection and transplantation surgery,which involves two interrelated stages of partial ischemic insultand subsequent inflammatory-mediated reperfusion injury.8 As a major reactiveoxygen species (ROS), superoxide anion (O2•–) is produced excessively via an enzymatic reactioncatalyzed by xanthine oxidase and nicotinamide adenine dinucleotidephosphate (NADPH) oxidase during HIRI,9 and triggersabnormal oxidative/nitrative stress, hepatocytes death, and furtherlocalized inflammation, ultimately resulting in liver dysfunctionand failure.10 Meanwhile, as the ″molecularcurrency″ of energy transfer, adenosine-5′-triphosphate(ATP) depletion during ischemia is a critical hallmark in the processof HIRI.11 During HIRI, ATP depletion causesintracellular calcium ion overload, thereby leading to a cascade ofintracellular pathological events such as mitochondrial permeabilitytransition (MPT) pore opening, cytochrome C release, and eventualcell apoptosis.7 Therefore, O2•– and ATP are potential biomarkersfor the early detection of HIRI at the molecular level. While someprogress has been made in the field of detecting HIRI using O2•– or ATP as an oxidativestress marker or energy metabolism marker, respectively,14 how the temporal and spatial fluctuations of O2•– and ATP coordinate changes in the whole processof HIRI and particularly how they synergistically regulate each otherin the pathological mechanism of HIRI remain unclear, in a large partdue to the lack of reliable methods for the simultaneous and in situmonitoring of O2•– andATP in vivo during HIRI. Colorimetry and high-performance liquid chromatographyhave been used to measure endogenous O2•– or ATP levels in cell lysates and tissue homogenatesduring HIRI;17 unfortunately, the mechanisms for determining O2•– or ATP are different,independent, and generally incompatible with the detection of O2•– or ATP in living biologicalsystems. Therefore, to meet this demand, a new method enabling notonly real-time detection of O2•– and ATP, but also elucidation of their interrelationship and synergyin HIRI is of great importance, and could hopefully realize the earlydetection of HIRI at the molecular level and decipher the potentialetiology of HIRI.In the progressionof HIRI, enhanced oxidative stress and impairmentof energy metabolism synergistically interact, collectively playingcritical roles in liver injury.24 In particular, simultaneous imaging probes with multiple recognitionsites have attracted more and more attention thanks to their inherentadvantages of eliminating false signals and improving sensitivityand accuracy, which can be applied to understand the relationshipbetween multiple bioactive molecules involved in disease progressionand diagnose diseases precisely.26 Although aseries of O2•–- orATP-responsive fluorescence probes have been reported,32 none of them has enabled the simultaneous and in situ visualizationof O2•– and ATP inbiological systems. If O2•–- or ATP-reactive fluorescent probes are used separately, the correlationbetween O2•– and ATPremains elusive due to the limitations of nonquantitative uptake,different spatial distribution, and spectral crosstalk. In addition,the existing O2•–-or ATP-responsive fluorescent probes are not suitable for dynamicmonitoring of O2•– andATP fluctuations in HIRI on account of their irreversible mechanisms.Therefore, we set out to develop a fluorescent probe capable of thesimultaneous and reversible detection of O2•– and ATP during HIRI, which can not only monitortemporal and spatial dynamics of O2•– and ATP in the HIRI process but also decipher thecorrelation and regulatory mechanism between O2•– and ATP in HIRI.Fluorescence imaging based on optical probesis emerging as anideal approach to monitor and understand various biological and physiologicalprocesses intuitively and effectively due to excellent sensitivity,high spatial and temporal resolution, visualization of biologicalevents at the molecular level, and noninvasive imaging.2•– and ATP in their independent blue and red fluorescence channels without spectral crosstalk. UDP exhibited up to 26-fold and204-fold fluorescence response upon activation by O2•– and ATP, respectively. UDP alsoexhibited high selectivity and reversibility toward O2•– and ATP. The synergistic effectbetween O2•– and ATPin hepatocytes stimulated by 2-methylestradiol  and oligomycin A was visualized. Meanwhile, real-time imagingof O2•– and ATP dynamicsin the process of HIRI in hepatocytes and mouse livers was realizedusing UDP, which was verified using commercial ROS and ATP assay kits.Notably, a combination of fluorescence imaging by UDP and biochemicaldata from commercial kits elucidated intracellular O2•–—succinate dehydrogenase(SDH)—mitochondrial (Mito) reduced nicotinamide adenine dinucleotide(NADH)—Mito ATP—intracellular ATP cascade molecularmechanisms in the process of HIRI for the first time.To meet the above requirements,rhodamine lactam skeleton combinedwith caffeic acid moiety was utilized to fabricate a dual-color anddual-reversible molecular platform (UDP) for the real-time detectionand dynamic imaging of O2•– andATP in vivo needs to satisfy three prerequisites. First, the responsegroups for O2•– andATP should be sufficiently sensitive and specific for the captureof O2•– and ATP incomplicated living systems. Second, the response to O2•– and ATP needs to be reversibleto enable dynamic monitoring of O2•– and ATP. Lastly, the fluorescence emission peaks withO2•–- and ATP-relevantfluorophores should be well-separated to avoid crosstalk for the simultaneousdetection of O2•– andATP, enabling the capture of authentic and reliable images of O2•– and ATP in organisms.The rational design ofa fluorescent probe capable of the simultaneous imaging and dynamicdetection of O2•–- and ATP-specific recognition groups and correspondingfluorophores, respectively, which were linked to diethylenetriamine selectively activated by O2•– is well separated fromthe red fluorescence channel of rhodamine , which is specifically openedby ATP, and the maximum fluorescence emission peak difference is ∼118nm. As such, spectral crosstalk was significantly avoided. UDP wassuccessfully synthesized (Scheme S1) andcomprehensively characterized by high-resolution mass spectrometry, 1H NMR, and 13C NMR . The purity of UDP was calculated to be 98%as determined by HPLC analysis .36To meet these criteria, inspired by our previous work, caffeicacid and rhodamine B were selected as Otriamine1A. Caffe2•– and ATP, the optical properties of UDP were investigated under simulatedphysiological conditions. First, the absorption spectrum and concentration-dependentfluorescence spectrum of UDP on the addition of various concentrationsof O2•– were measuredin PBS buffer solutions. As shown in 2•–, indicating that pyrocatechol was converted to benzoquinoneby O2•– oxidation.Under excitation at 380 nm, the blue fluorescence intensity at 470nm gradually enhanced with an increase of O2•– concentrations (0–65 μM), reachinga plateau when the O2•– concentrations were further increased from 65 to 80 μM . The blue fluorescence intensity at470 nm exhibited a good linear relationship with concentrations ofO2•– (0–65 μM),and the linear equation was F470nm = 89.99[O2•–](μM) + 318.72with a linear correlation coefficient of 0.997 , suggesting highly sensitive detection ofO2•– by UDP in vitro.We then evaluated the anti-interference performance of UDP towardO2•–. Only O2•– could trigger a significantenhancement of blue fluorescence without interference from other abundantROS, RNS, reducing substances, and metal ions found in organisms . In vitroexperiments indicated that blue fluorescence was enhanced after mixingwith O2•–, furtherconfirming that UDP could be utilized as a potential tool for thespecific detection of O2•– in complex biological systems.In order to demonstrate that UDP is a two-colorprobe in response to Oto 80 μM 1B. Notabt 470 nm S1. The bt 470 nm S1. The bFigure S3). The fluorescence intensity centered at 588 nm exhibiteda linear increase with ATP concentrations in the range of 0–22mM. The linear correlation equation was F588nm = 256.51[ATP](mM) + 41.13 with a linear correlation coefficientof 0.997 (S4). As such, the above in vitroexperiments confirmed that UDP had significant potential for ATP biosensing.The reactivity of UDP with ATP was then evaluated.When ATP wasadded, a maximum absorption peak at 563 nm accompanied by a flat absorptionpeak at 520 nm was observed due to the ATP-induced ring opening ofthe spirolactam of rhodamine 2C. In thof 0.997 1G, S4. A2•– and ATP and spectral crosstalk. 2•– could be effectively quenched by the reducingsubstance glutathione (GSH), and the subsequent addition of O2•– could restore the bluefluorescence. The reversible response of UDP with O2•– was stable for three repeated cycles.Similarly, the red fluorescence of UDP induced by ATP could also besignificantly suppressed by apyrase (ATP hydrolase), further demonstratingthe ability of UDP to monitor the dynamic changes of O2•– and ATP in vitro . The kinetics of UDP in response toO2•– and ATP were examinedto confirm the efficacy of UDP. As a result, when 65 μM O2•– was added, UDP immediatelyexhibited an enhanced emission intensity at 470 nm and rapidly achievedequilibrium . With the additionof 10 mM ATP, the fluorescence intensity at 588 nm increased graduallyand the intensity reached a stable maximum within 23 min . After reacting with O2•– or ATP, the fluorescence intensitiesat 470 and 588 nm were almost unchanged, which indicated that UDPexhibits good photostability. Taken together, these results confirmthat UDP exhibits high sensitivity, excellent selectivity, reversibility,and good photostability as well as resistance to pH interference withoutspectral crosstalk in response to O2•– and ATP, making UDP suitable for the simultaneousdetection of O2•– andATP in vivo during HIRI.We then investigated the reversible responses of UDP to Oin vitro 2D. Subserescence 2E. Howev2•– and ATP in vitro,the feasibility of using UDP for the simultaneous imaging and dynamicmonitoring of endogenous O2•– and ATP was investigated in hepatocytes. First, the potential biologicaltoxicity of UDP was comprehensively evaluated in hepatocytes. 2•– and ATP signaling via two distinguishable blue and red channels,spectral crosstalk was first examined between the O2•– and ATP channels. Blue fluorescencechannel  for O2•– and redfluorescence channel  for ATP were selected, plus channel 3  to excludespectral crosstalk. As shown in Figure S9, the blue fluorescence and red fluorescence in normal hepatocytesrepresented endogenous O2•– and ATP levels, respectively, whereas channel 3 showed negligiblegreen fluorescence. 2-ME promoted O2•– production by inhibiting the activity of superoxidedismutase (SOD).37 After incubation with2-ME and ATP in hepatocytes, both blue fluorescence and red fluorescencewere enhanced, while the green fluorescence in channel 3 was negligible,which implied that there was no spectral crosstalk between the blueand red channels or interference from fluorescence resonance energytransfer (FRET).Having confirmed the excellent performance of UDP in response toO2•– andATP levels in hepatocytes stimulated by exogenous substances usingconfocal fluorescence microscopy. As shown in 2•–-associated blue fluorescence enhancement and a 0.37-fold ATP-relatedred fluorescence reduction in comparison to the control group. However,after pretreatment with Tiron (O2•– scavenger) and subsequent incubation with 2-ME,38 the blue fluorescence was reduced and the redfluorescence was conversely elevated . Hence, these results unequivocallydemonstrated that UDP could function as a powerful tool for the real-timevisualization and reversible monitoring of endogenous O2•– and ATP variations in hepatocytes.The intimate correlation between oxidative stress and energy metabolismin hepatocytes was confirmed via the differentiated blue fluorescencesignal for O2•– andred fluorescence signal for ATP.We then turned our attention to the imagingof endogenous Oelevated 3C. We noeostasis 3B. Oligoatocytes 3D. Howev2•– and ATP fluxes in living HIRI cells. Existing evidence suggeststhat the pathological mechanism of HIRI is intimately linked withoxidative stress and energy metabolism.41 EndogenousO2•– and ATP can beapplied as promising, in situ hallmarks that are closely related tooxidative stress and energy conversion, respectively.13 Hence, we attempted to simultaneously detect HIRI with the aid ofUDP in response to O2•– and ATP.We next turned our attention tothe dynamic imaging of O42 which was validated by a series of hepatocyteinjury markers such as ALT, AST, tumor necrosis factor-α (TNF-α),and lactate dehydrogenase (LDH).44 As depictedin Figure S11, the levels of ALT and ASTwere significantly elevated in HIRI hepatocytes in comparison withcontrol hepatocytes. Interestingly, increases of TNF-α and LDHrelease were observed in the HIRI group rather than in control group.Collectively, these results revealed significant injury in HIRI hepatocytes.In order to trace O2•– and ATP in the whole process of HIRI in detail, hepatocytes weredivided into five groups to monitor O2•– and ATP levels in the respective phases of ischemiaand reperfusion, including control group, 20 min of ischemia group,40 min of ischemia group, 40 min of ischemia followed by 20 min ofreperfusion group, and 40 min of ischemia followed by 40 min of reperfusiongroup. The five groups were stained with UDP, and the O2•–-related blue fluorescencesignal and ATP-representative red fluorescence signal were captured.As illustrated in 2•– .First, a HIRI model in hepatocytes was establishedby glucose-serum-oxygen deprivation and subsequent reperfusion,2•– and ATPin HIRI hepatocytesand the reliability of the variations of O2•– and ATP during HIRI, commercial ROS and ATP assaykits were used to evaluate ROS and ATP levels in the whole processof HIRI. 2′,7′-dichlorodihydrofluorescein diacetate(DCFH-DA) could be oxidized by ROS to produce fluorescent 2′,7′-dichlorofluorescein(DCF), which was used to image ROS in HIRI.50 We found that ROS tended to rise gradually during ischemia, whilea large explosion of ROS was observed during reperfusion (S13). ATP concentrations decreased sharply from 13 to 3.3nmol/mgprot in cells after 40 min of ischemia, whereas ATP levelsincreased slowly from 3.3 to 4.9 nmol/mgprot in hepatocytes that receivedreperfusion for 40 min  has been reported as an effective drug commonly used to protectthe liver from HIRI by directly removing ROS.51 For HIRI hepatocytes pretreated with 0.5 mM NAC for 1 h, the bluefluorescence was clearly reduced, while the red fluorescence was significantlyrestored . These results indicated that UDPcould serve as a robust tool for reversible detection of the changesin O2•– and ATP inthe livers of mice. Afterward, the performance of UDP-mediated fluorescencevisualization of O2•– and ATP in mouse livers throughout the HIRI process was systematicallyinvestigated in a HIRI mouse model. HIRI models were established inmice according to the previously reported method, in which microvesselclamps were used to block the hepatic artery and portal vein bloodsupply, resulting in approximately 70% blood flow deprivation in theliver, followed by the removal of the microvessel clamps for the reperfusionprocess.50 The determination of serum ALT,AST, and TNF-α levels were investigated. It was discovered thatALT, AST, and TNF-α levels were significantly increased in theserum of mice subjected to HIRI, indicating the HIRI mice sufferedsevere hepatic injury . Inorder to image O2•– and ATP fluctuations for the whole process of HIRI, mice were dividedinto five groups consisting of control group, 20 min of ischemia group,40 min of ischemia group, 40 min of ischemia followed by 20 min ofreperfusion group, and 40 min of ischemia followed by 40 min of reperfusiongroup. The five groups of mice were then injected with UDP throughthe tail vein, and the blue and red fluorescence signals in the liverswere collected  staining of the major organs were performed toevaluate the biocompatibility of UDP. The UDP-treated group of miceshowed no apparent difference with the trend of body weight for thecontrol group which received saline . After injection with UDP, no obvious damage was observed in theheart, liver, spleen, kidney, and lung tissues, indicating UDP exhibitedgood biocompatibility . Furthermore,after intraperitoneal injection with UDP, H&E staining of majororgan tissues in the control group and HIRI groups of mice was conductedto identify the histological changes. As a result, there was no evidentinjury in the heart, liver, spleen, kidney, and lung tissues of thecontrol group, whereas in the liver tissues of HIRI mice, the cytoplasmwas lightly stained, and there were a few lymphocytes and granulocytesclustered near the portal area, demonstrating liver injury occurredin HIRI mice  to ubiquinol(UQH2).58 As a central hub connectingoxidative phosphorylation with the TCA cycle, SDH is indispensablein regulating intracellular ATP levels.60 Thus, we speculatedthat excess O2•– inHIRI may regulate intracellular ATP levels by affecting the activityof SDH. An SDH activity kit was used to assess SDH activity of HIRIhepatocytes and normal hepatocytes. As illustrated in 2•–. In order to provethat O2•– is responsiblefor inactivation of SDH, we used 2-ME to stimulate the productionof O2•–, and foundthat the activity of SDH was downregulated, while the activity ofSDH was distinctly enhanced in hepatocytes pretreated with Tiron.These results confirmed that excessive O2•– inactivated SDH in HIRI hepatocytes.Under physiological conditions, ATP in the liveris mainly generated through oxidative phosphorylation in the electrontransport chain of mitochondria, which jointly coordinates with thetricarboxylic acid (TCA) cycle to constitute the bioproductive activitiesof mitochondria.60 Intracellular ATP levelswere evaluated using UDP. The results indicated that the ATP-relatedred fluorescence was significantly reduced in the normal cells treatedwith 3-NPA compared with normal cells, indicating that the inactivationof SDH could lead to a depletion of intracellular ATP. Furthermore,it was observed that the ATP level in HIRI cells was low, and the3-NPA treated HIRI cells showed a lower ATP level , which is an inhibitor of SDH 7E.59,60 TP level 7F. As su2•–, proteomic analysisanalyzed by LC–MS was conducted on SDH to explore the post-translationalmodification of SDH caused by O2•–. As shown in 2•– were identified.62 Therefore, we speculated that excess O2•– oxidized histidine, cysteine, methionine, andtryptophan residues in SDH during HIRI, resulting in the inactivationof SDH.In orderto investigate the specific inactivation mechanism of SDH by O2•–. The TCA cycle in which SDH participatesis a key metabolic pathway for the production of the electron donorNADH.63 Electrons from NADH and succinatein the mitochondrial inner membrane pass through an electron transportchain to O2 and drive the synthesis of ATP.63 We hypothesized that SDH regulated intracellularATP levels by influencing mitochondrial NADH contents, and then, weexplored whether the activity of SDH could regulate the levels ofmitochondrial NADH. Herein, we first isolated mitochondria from hepatocytesfrom different treatment groups using a mitochondria isolation kitand then detected NADH concentrations of the mitochondria in eachgroup using an NADH assay kit  production duringHIRI. After exposure of 1 mM NADH to normal cells for 1 h, the additionalNADH could increase mitochondrial NADH contents from 0.90 to 1.8 nmol/mgprot with high sensitivity and excellent selectivityin response to O2•– and ATP was developed. UDP can react with O2•– and ATP synchronously and reversibly, and independentlymonitor fluorescence in the blue and red channels without interferencefrom spectral crosstalk. The excellent performance of UDP in responseto O2•– and ATP invitro enabled the simultaneous imaging and dynamic monitoring of endogenousO2•– and ATP in hepatocytes.UDP was then used to visualize in situ O2•– and ATP dynamics in hepatocytes during HIRI anddrug treatment in real-time via the well-separated blue and red signals.More importantly, UDP realized the visualization of O2•– and ATP in mouse livers duringHIRI. Finally, the intracellular O2•–—SDH—Mito NADH—Mito ATP—intracellularATP cascade-mediated signaling pathways in HIRI was uncovered forthe first time. In summary, this research provides a fluorescent probeable to investigate the correlation and synergy between O2•– and ATP in HIRI and can provideinsight into the relationships of interlinked active molecules inthe progression of HIRI.To achieve the simultaneous detection ofO


The binding of small molecules to the double helical structure of DNA, through either intercalation or minor groove binding, may significantly alter the stability and functionality of DNA, which is a fundamental basis for many therapeutic and sensing applications. Here, we report that small-molecule DNA binders can also be used to program reaction pathways of a dynamic DNA reaction, where DNA strand displacement can be tuned quantitatively according to the affinity, charge, and concentrations of a given DNA binder. The binder-induced nucleic acid strand displacement (BIND) thus enables innovative technologies to accelerate the discovery and characterization of bioactive small molecules. Specifically, we demonstrate the comprehensive characterization of existing and newly discovered DNA binders, where critical parameters for binding affinity and sequence selectivity can be obtained in a single, unbiased molecular platform without the need for any specialized equipment. We also engineer a tandem BIND system as a high-throughput screening assay for discovering DNA binders, through which 8 DNA binders were successfully discovered from a library of 700 compounds. The binding of small molecules to the double stranded DNA may significantly alter its stability and functionality, which is the basis for many therapeutic and sensing applications. Here, the authors report that DNA binders can be used to program reaction pathways of a dynamic DNA reaction, where DNA strand displacement can be tuned quantitatively according to the affinity, charge, and concentrations of a given DNA binder. Recent advances in designing, screening, and characterizing DNA binders have not only promoted our understanding of their essential roles in critical biological processes, such as DNA replication10, DNA damage12, and transcriptional regulation13, but also enabled exciting applications ranging from guiding the differentiation of pluripotent stem cells14 to epigenetic regulation15 and to inducing trinucleotide-repeat contraction16. Beyond their important roles in biological systems, DNA binders have also found increasing applications in artificial systems for constructing and controlling DNA origami assembly and conformation17, stabilizing DNA nanostructures against low-magnesium buffers and nuclease degradation19, modulating the bond strength of DNA-nanoparticle superlattices20, and constructing DNA-based rectifiers and excitonic circuits22. To date, most applications of DNA binders rely on their static interactions with either dsDNA or more complex DNA structures through intercalation or minor groove binding.Interactions between small-molecule binders and double-stranded DNA (dsDNA) are the fundamental basis of many anticancer and anti-infection pharmaceuticals24. Mechanistically, TMSD can be considered an SN2 reaction, which is initiated at a short complementary single-stranded DNA (ssDNA) toehold domain and progresses through a branch migration process to displace a prehybridized DNA strand. In this reaction pathway, metal cations, such as Mg2+, play critical roles in stabilizing the initial duplex as well as the triplex intermediate against the repulsion caused by the dense negative charges of phosphorate backbones , it is possible to switch the dominant strand displacement reaction pathway to SN1, where the duplex is first dissociated because of the strong charge repulsion and then exchanged with the invader strand  has become the most widely used elementary reaction in dynamic DNA nanotechnologynes Fig. 25. By pland Fig. 26.Fig. 1N1 reaction pathway by stabilizing the duplex reactant through specific DNA-binder interactions and thus reduces the yield of the overall strand displacement in a concentration-dependent manner , binding site size (n), cooperativity (ω), enthalpic (ΔH) and entropic (−TΔS) contributions, as well as sequence selectivity, can be accurately determined without the need for any specialized equipment. We also introduce a tandem BIND reaction system and engineer it into an HTS assay, through which eight new DNA binders were successfully discovered from a library of 700 compounds.In this work, we demonstrate that reaction pathways of TMSD under low metal cation conditions can be further programmed using small molecular DNA binders. We name this binder-responsive dynamic DNA reaction as binder-induced nucleic acid strand displacement (BIND). Specifically, the addition of low concentrations of dsDNA binders suppresses the Sner Fig. . Upon fuway Fig. . More imN1 reaction pathway  with a certain DNA binder for 5 min, followed by a strand displacement reaction driven by an invader strand I  as a model DNA binder, we observed sharp decreases in reaction yields against increasing concentrations of SG-I until a critical binder concentration (CBC) at which the CP duplex was fully occupied  and displacement (SN2) reaction pathways were further confirmed using kinetic analysis  as a positively charged nonbinder, we also confirmed that BIND is highly binder-specific in both the SN1 and SN2 reaction pathways. Therefore, four classes of distinct BIND profiles can be established for small molecules with varying combinations of affinities and charge conditions: strong positively charged binders      Fig. , strong  0) Fig. , weak po 0) Fig. , and non 0) Fig. . Here, wnil Fig. , echinomnil Fig. , Ru and the binding site size (n) were determined by fitting the binding curves at the SN1 region against the lattice-ligand model developed by McGhee and Von Hippel  and n (2.6 vs. 2.4 nM), confirming the feasibility of BIND for profiling the binding properties of small-molecule DNA binders  and charge condition , and the results were highly consistent with reported values determined under similar conditions  is another important parameter to characterize how the binding of one binder affects the binding of the next. A binder of positive cooperativity is likely to attract free binders to fill the empty neighboring slots on the dsDNA, which is featured by a sharp binding curve with a narrower dynamic range than that of a noncooperative binder. Interestingly, when CBC was plotted against the Kd values of the 16 binders, all noncooperative binders formed a line, as shown in Fig. Kd to a higher numeric value and thus are positively cooperative. To test this hypothesis, we measured ω for Pico Green and Eva Green, which were clearly distributed in the space of noncooperative binders (on the line) and that of positively cooperative binders (in the lower space) in Fig. Kd of Eva Green was 25 times greater than that of Pico Green 28.In addition to een Fig. . We furtKd values of 15 binders using BIND at 27 °C, 30 °C, 32 °C, 35 °C, and 37 °C and fit the results using Van’t Hoff’s equation , enthalpy (ΔH), and entropy (ΔS), without the need to melt the DNA over wide temperature ranges. We measured the .94 Fig. .Fig. 4Pr29. For example, BIND revealed a favorable enthalpic contribution of −14.3 kcal/mol for a classic intercalator, thiazole orange, indicating that the main driving force for binding is base stacking and the formation of hydrogen bonds  of −11.6 kcal/mol with a slight enthalpic penalty of +1.9 kcal/mol  from 0 to 100 . A linear relationship was then established when plotting CT against the ratios between sink and BIND probes  assays , which binds selectively to AT-rich sites in the minor groove Fig. 31,32. BI100 Fig. . We thenbes Fig. . We defiays Fig. 32.αAT/αGC), we were able to determine and rank the sequence selectivity of all nine binders  with the rank order netropsin > DAPI > berenil > Hoechst 33258. Another four binders were found to be selective for GC-rich sequences (αAT/αGC < 1) with the rank order actinomycin D > doxorubicin > thiazole orange > ethidium bromide. Netropsin was found to be highly selective to the AT-rich sink and showed no binding to the GC-rich sequence even when the sink was 100 times in excess  and GC-rich (5’-GCGCC-3’) sink probes  and negative control (N.C.), respectively, throughout the screening. Using these two controls, each tested 10 times, a Z’ factor of 0.64 was determined, suggesting that the tandem BIND was of excellent assay quality for HTS Fig. . This wa34. A threshold of 0.25 (μS + 3 σS) was set, and 8 hits were successfully returned 36, isothermal titration calorimetry (ITC)37, and thermal melting analysis38, allow the accurate characterization of binding affinities but can seldom provide information on sequence selectivity. Moreover, these techniques often require specialized equipment and procedures that are not widely available in many biochemical laboratories. Although FID is a simple assay that allows the determination of sequence selectivity of DNA binders, it relies heavily on the relative binding strength between a given binder and an indicator  and thus may introduce significant measurement bias upon usage38. BIND enables a simple, unbiased assay that allows the comprehensive characterization of all critical thermodynamic properties of DNA binders on a single platform. Using 16 well-studied DNA binders, we demonstrated that BIND not only led to highly consistent numeric values of the dissociation constant, binding site size, and enthalpy contribution of each binder with previous studies using varied characterization techniques but also offers quantitative information on the binding cooperativity and sequence selectivity through unique BIND profiles.BIND also provides a molecular platform for the comprehensive thermodynamic characterization of interactions between DNA and small molecules. Small molecules capable of binding to DNA through either intercalation or minor groove binding have long been an intensive focus of research because of their critical roles in therapeutics and biochemical research. Binding affinity, binding site size, sequence selectivity, and enthalpy and entropy changes are critical thermodynamic determinants of the binding behavior and functionality of DNA binders but remain difficult to measure on a single analytical platform. Instrumental methods, such as single-molecule force spectroscopy (SMFS)39. In contrast, we demonstrated that both strong and weak binders could effectively induce strand displacement reactions via the SN2 reaction pathway and could thus be effectively identified using the engineered tandem BIND assay. Our success in finding 8 new binders from a pool of 700 candidate compounds further confirmed the power of BIND as an HTS platform for DNA binders. Moreover, because BIND is practically simple to use with no need for specialized equipment or procedures, we anticipate that BIND will be widely adopted as a one-stop HTS and comprehensive characterization platform to accelerate the discovery of new DNA binders and DNA binding drugs.BIND also provides a high-quality and low-bias HTS platform for discovering new DNA binders. Although DNA binders are an important source of anticancer and anti-infective drugs, there have been very limited HTS approaches for finding new binders. To date, FID is the only HTS assay for DNA binders but is subject to low screening efficiency and significant screening bias because it relies on competitive binding between the binder and the indicator40. Despite the low interferon induction activity, S20 showed relatively high cytotoxicity to MDCK cells (IC50 = 6.6 μM) compared to its derivative. We also found that S20 is also a strong DNA binder, which may contribute to its high cytotoxicity.As BIND is also a powerful characterization platform, we were able to further establish a comprehensive thermodynamic profile of the strongest hit, S20, in terms of its binding affinity, sequence selectivity, and enthalpy/entropy contributions to the binding thermodynamics. S20 was patented in 2012 as an interferon-inducing compound2+  was added to the positive control to ensure complete strand displacement. The solution containing 20 nM CP in 1 × TE buffer was also included as a negative control for fluorescence normalization. Data analyses were performed using Microsoft 365 Excel and GraphPad 8.0.1.All DNA oligonucleotides were purchased from Integrated DNA Technologies  and were purified by IDT using high-performance liquid chromatography. Sequences and modifications were listed in Supplementary Table F is the normalized fluorescence signal of a given sample, FCBC is the normalized fluorescence when the binder concentration equals its CBC, F0 is the normalized fluorescence signal when no binder is added to the strand displacement system, O is the fractional occupancy of the bound binder, Cbinder is the total binder concentration at each sample, CDNA is the concentration of base pairs in CP , Ka is the association constant and n is the binding site size. When taking cooperativity ω into consideration, McGhee and Von Hippel’s binding isotherm algorithm was transformed into:Endpoint fluorescence measurement was used to establish the BIND profile for measuring the binding affinities and binding site sizes of DNA-binder interactions. Briefly, CP was diluted in 1 × TE buffer and then mixed with a given DNA binder at 37 °C for 5 min. To this mixture, invader I was then added to initiate the BIND reaction. The reaction mixture containing 10 nM I, 20 nM CP, and a given concentration of DNA binder was incubated at 37 °C for 2 h before an endpoint fluorescence measurement using a BioTek Cytation 5 Multimode Microplate reader by setting the excitation/emission wavelengths to 640 nm/675 nm. The fluorescence signal was then normalized against the positive and negative controls as outlined above. The association binding constant Ka and binding site size n of each binder were then determined by fitting the fractional occupancy of bound binders using the McGhee and Von Hippel binding isotherm equation:Ka is the association constant, ∆H is the binding enthalpy, R is the universal gas constant, T is the temperature in Kelvin, and ∆S is the binding entropy.Binding enthalpy and binding entropy were determined by measuring the Ka of a given biner using the BIND reaction at 27 °C, 30 °C, 32 °C, 35 °C, and 37 °C and then fit using Van’t Hoff’s equation:CT) that were defined as the binder concentration at 50% displacement yield at the SN1 domain were then plotted against the sink/CP ratios to determine the selectivity factor α, which was defined as the slope of the fitted linear curve.The sequence selectivity of DNA binders was determined using a competitive BIND reaction between CP and stem‒looped sink probes. Each sink probe was designed to contain an 8 bp stem domain of varying ATGC combinations and a 5 nt poly-dA loop domain. For a typical competitive BIND reaction, a given DNA binder was premixed with a sink probe in 1 × TE buffer at 37 °C for 2 h. This reaction mixture was subsequently mixed with CP and I to initiate the BIND reaction using the protocol outlined above. The concentration ratios between the sink and CP probes were set to 2.5, 5, 10, 25, 50, and 100 in the competitive BIND reaction. The threshold concentrations  and incubated at 37 °C for 10 min. To this reaction mixture, a candidate compound at a final concentration of 30 μM was added. After another incubation at 37 °C for 2 h, endpoint fluorescence was measured using a BioTek Cytation 5 Multimode Microplate reader at excitation/emission wavelengths of 640 nm/675 nm. The fluorescence signal in each well was normalized using the positive and negative controls as outlined above. Compounds with numeric values above the threshold (0.25) were considered positive hits and were subjected to subsequent thermodynamic characterization using BIND and thermal melting analysis.Further information on research design is available in the Supplementary InformationReporting SummaryPeer review file


APOE ε4 and ε2 with cognition.This genetic association study analyzes data from 4 Alzheimer disease and cognitive aging cohorts to investigate whether sex and race modify associations of  APOE ε2 and ε4 with cognition, and do these sex effects differ in non-Hispanic Black and non-Hispanic White individuals?Does sex modify associations of APOE ε4 effects on memory, which did not statistically differ by race. Contrastingly, no sex effects were observed with APOE ε2, aside from an intersectional effect of race and sex, whereby the APOE ε2 protective effect on executive function was limited to non-Hispanic White females and non-Hispanic Black males.This genetic association study found that among 32 427 older adults from different racial groups, females showed stronger negative APOE associations with cognition are largely limited to ε4 and do not differ by race.Sex differences in  APOE) ε4 and cognitive impairment in Alzheimer disease (AD). However, it is unclear whether sex-specific cognitive consequences of APOE are consistent across races and extend to the APOE ε2 allele.Sex differences are established in associations between apolipoprotein E . Data were previously collected across multiple US locations from 1994 to 2018. Secondary analyses began December 2021 and ended September 2022.APOE ε4 or APOE ε2 and sex on baseline cognitive scores, while linear mixed-effect models assessed interactions on cognitive trajectories. The intersectional effect of race was modeled using an APOE × sex × race interaction term, assessing whether APOE × sex interactions differed by race. Models were adjusted for age at baseline and corrected for multiple comparisons.Harmonized composite scores for memory, executive function, and language were generated using psychometric approaches. Linear regression assessed interactions between APOE ε2 carriers, and 12 538 (38%) were APOE ε4 carriers. Participants missing APOE status were excluded (n = 9266). For APOE ε4, a robust sex interaction was observed on baseline memory , whereby the APOE ε4 negative effect was stronger in females compared with males and did not significantly differ among races. Contrastingly, despite the large sample size, no APOE ε2 × sex interactions on cognition were observed among all participants. When testing for intersectional effects of sex, APOE ε2, and race, an interaction was revealed on baseline executive function among individuals who were cognitively unimpaired , whereby the APOE ε2 protective effect was female-specific among White individuals but male-specific among Black individuals.Of 32 427 participants who met inclusion criteria, there were 19 007 females (59%), 4453 Black individuals (14%), and 27 974 White individuals (86%); the mean (SD) age at baseline was 74 years (7.9). At baseline, 6048 individuals (19%) had AD, 4398 (14%) were APOE ε4, the APOE ε2 protective effect could vary by race and sex. Although female sex enhanced ε4-associated risk, there was no comparable sex difference in ε2, suggesting biological pathways underlying ε4-associated risk are distinct from ε2 and likely intersect with age-related changes in sex biology.In this study, while race did not modify sex differences in  APOE) is the strongest genetic risk factor for late-onset Alzheimer disease (AD) with the ε2 allele reducing risk and ε4 increasing risk.6 Interestingly, associations between APOE ε4 and AD differ by sex, whereby associations are stronger among females compared with males.14 Likewise, APOE ε4 is associated with greater memory decline in females with both normal cognition and mild cognitive impairment (MCI),13 and although APOE ε2 effects on cognition have not been as thoroughly examined, 1 recent study showed female ε2 carriers had enhanced neuroprotection on memory performance compared with male ε2 carriers.14 Regarding racial differences, several studies have demonstrated that although ε4 frequency is higher in Black participants, associations between APOE ε4 and AD risk are weaker in non-Hispanic Black compared with non-Hispanic White individuals .17Apolipoprotein E  and cognition in racially and ethnically diverse groups remain unclear. The sex- and race-specific effects have important implications for ongoing AD clinical trials, where APOE has been included as a screening criterion in some trials, and both outcomes and adverse events  have been reported to differ by APOE genotype in previous trials.28Nevertheless, studies of APOE effects on cognitive domains,34 with several demonstrating strong APOE effects on memory.33 In contrast, studies of APOE effects on executive function and language are inconsistent with some studies suggesting APOE ε4 carriers perform better on executive function tests,33 whereas others report APOE ε4 carriers have more rapid executive function decline.34 Regarding language function, 1 study suggested APOE ε4 carriers have greater rates of decline in language performance.34 Yet neither APOE ε2 effects nor intersectional effects of race, sex, and APOE on cognition have been thoroughly examined.Progressive cognitive decline in AD is characterized by memory loss and impairments in executive functioning and language. These correlated domains have some unique and shared genetic architecture, which is important to uncover. Prior studies have assessed APOE ε2 and APOE ε4 associations with domain-specific cognitive trajectories over the course of aging and AD. In 4 well-characterized cohorts of Black (n = 4453) and White (n = 27 974) older adults, we leverage 3 harmonized cognitive domains  using robust longitudinal data analysis to characterize the intersectional effects of race, sex, and APOE on cognitive performance along the spectrum of clinical disease. These results provide the most comprehensive picture to date of sex and racial differences in allele-specific associations of APOE with late-life cognitive performance.The goal of this study is to extend previous work by providing a comprehensive picture of the modifying effect of sex on 35; the 3 harmonized cohorts of the Religious Orders Study, Rush Memory and Aging Project, and Minority Aging Research Study (ROS/MAP/MARS)37; the National Alzheimer Coordinating Center (NACC)42; and the Adult Changes in Thought (ACT) study.43 The ADNI and NACC studies enrolled individuals with normal cognition, MCI, or AD, while ROS/MAP/MARS and ACT enrolled participants without known dementia. Participants older than 60 years at baseline were included in this analysis. Sex and race were self-reported. Written informed consent was obtained from all participants in each study, and research was carried out with protocols approved by each site’s institutional review board. These secondary analyses were approved by the Vanderbilt University Medical Center institutional review board. The study followed the Strengthening the Reporting of Genetic Association Studies (STREGA) reporting guidelines.Data were obtained from 4 cohort studies of cognitive aging and AD: the Alzheimer’s Disease Neuroimaging Initiative (ADNI)44 We obtained granular data along with detailed documentation on each item in the neuropsychological protocol from each study, such as test versions, stimuli administered, and response coding. Then, a panel of neuropsychologists and neurologists  assigned test items to a single primary domain: memory, executive function, language, visuospatial ability, or none of these. Differing versions and administration methods were noted at each time point. Next, quality control steps included parsing out missingness codes, recoding, and reverse coding data as needed. We selected the most recent visit for each participant as the reference for co-calibration. These choices enabled us to optimize the spread of cognitive abilities in the data set, which is desirable for ensuring parameters are valid over the entire range of ability levels46 and still including only a single observation per study participant.Harmonization of cognitive data was previously described.47 to co-calibrate separate scores for memory, executive function, and language. We extracted item parameters (loadings and thresholds) for all items, which were used to derive scores in each legacy study separately. These resulted in scores on the same metric.We treated each cognitive item as an ordinal indicator of a domain. Common test items administered and scored the same way across ACT, ADNI, and ROS/MAP/MARS studies served as anchor items to facilitate co-calibration. We used confirmatory factor analysis models with robust maximum likelihood estimation that is robust to missing data in Mplus version 7.4APOE haplotypes for NACC and ACT were determined from the single-nucleotide variants rs7412 and rs42935848 and from pyrosequencing of APOE codons 112 and 158 for ADNI and ROS/MAP/MARS.49APOE ε2 and APOE ε4 were modeled separately using dominant models for APOE ε2  and additive models for APOE ε4. We performed analyses in all participants and restricted to participants who were cognitively unimpaired at their first cognitive visit. We used linear regression for each domain to assess baseline cross-sectional APOE effects. We used linear mixed-effects regression restricted to those with at least 2 cognitive visits, with time (years from baseline) and the intercept as fixed and random effects. All models covaried for mean-centered age at first cognitive visit. Longitudinal models covaried for age at first visit × time. To test the modifying effect of sex, we included 2-way interactions  in cross-sectional models, and 3-way interactions  in longitudinal models. Sex-interaction models were also run stratified by race. The intersectional effects of both race and sex were assessed using a sex × APOE × race interaction in cross-sectional analysis and sex × APOE × race × time interaction in longitudinal models. Significant results were rerun in each cohort separately to assess consistency , covaried for education, or covaried for genetic ancestry via the first 5 principal components.Sensitivity analyses excluded ε2/ε4 individuals (n = 866), modeled APOE status were excluded (n = 9266). Of 32 427 participants who met inclusion criteria, there were 19 007 females (59%), 13 420 (41%) males, 4453 Black individuals (14%), and 27 974 White individuals (86%); the mean (SD) age at baseline was 74 years (7.9). At baseline, 6048 individuals (19%) had AD, 4398 (14%) were APOE ε2 carriers, and 12 538 (38%) were APOE ε4 carriers. Higher proportions of females were cognitively unimpaired at study entry, and APOE allele frequency differed by race.Participant characteristics are presented in APOE ε4 was robustly associated with worse cognitive performance and faster rates of decline across all cognitive domains, and APOE ε2 was associated with better cognitive performance and slower rates of decline. Associations with memory had the largest effects. Results were similar when restricted to participants who were cognitively unimpaired at baseline, although the baseline APOE ε2 associations were attenuated.Results of all models are presented in eTable 2  and eTable 3  in APOE differed by race, we performed 3-way interactions of race, sex, and APOE . APOE ε2 showed a female-specific protective effect among White participants but a male-specific protective effect among Black participants  , whereby the APOE ε4 negative effect was stronger in females compared with males and did not significantly differ among races. Results were similar across sensitivity analyses. There were no significant sex differences in APOE ε4 associations with any of the longitudinal outcomes or when restricting to cognitively normal participants. Among all participants, APOE ε2 did not interact with sex on cognition  . For APOognition .APOE and AD-related cognitive decline. We provide strong evidence of a sex difference in the association between APOE ε4 and cross-sectional memory performance and, for the first time, also extend this sex difference to the language domain. Furthermore, these APOE ε4 effects were stronger in females, and there was no statistical evidence of race specificity. In contrast to APOE ε4, we did not observe evidence of sex differences in APOE ε2 associations with cognition across race. However, we did observe exciting novel evidence of intersectional effects for APOE ε2, whereby sex-specific associations on cognition may differ by racial group, particularly in the executive function domain. Together, our findings solidify current understandings of sex-specific effects of APOE ε4 while highlighting the pressing need to fully characterize intersectional effects of race, sex, and APOE ε2 on late-life cognition.To our knowledge, this study is the largest investigation of the modifying effects of both sex and race on the association between APOE ε4 and cognition among women aligns with and extends past work. APOE ε4 showed stronger effects on baseline memory and language performance among females compared with males, and this effect was similar among Black and White individuals. Interestingly, these female-driven effects of APOE ε4 are only present when evaluating across clinical diagnoses and not among the cognitively unimpaired subset. Indeed, APOE ε4 is a stronger predictor of MCI and clinical AD risk among females compared with males, particularly among those aged 55 to 70 years.16 Likewise, past work has observed female ε4 carriers to have faster memory decline compared with male ε4 carriers.8 That said, there has not been much characterization of sex differences in APOE ε4 effects on language, making our findings of increased ε4 effects among females interesting. The mechanism underlying these female-specific findings, in both our research and that of other groups, remains unclear. Sex hormones may play a role, as studies have demonstrated estrogen levels affect cognition.52 Menopausal loss of estrogen may amplify negative APOE ε4 effects, resulting in greater cognitive impairment compared with males. Additionally, females tend to have steeper cognitive decline in the presence of high levels of AD neuropathology53; therefore, the effects of ε4 certainly could be driven by more advanced neuropathology. Tau pathology has been implicated as a potential pathway previously, as prior studies have highlighted stronger associations between APOE ε4 and biomarkers of tau pathology in females compared with males.55 Regardless of the underlying mechanism, the present results solidify the evidence of robust sex differences in the risk effect of APOE ε4.The strong association between APOE ε2 effects on cognitive decline. Given our large sample size, we were able to provide strong evidence that the sex difference in APOE ε4 risk does not extend to APOE ε2 protection, with strikingly consistent associations between APOE ε2 and cognition in males and females. Fascinatingly, among cognitive normal participants we do provide exciting new evidence of an intersectional effect of sex, race, and APOE ε2 on executive function. More specifically, the APOE ε2 protective effect was female-specific in White individuals but male-specific in Black individuals. Very few studies have evaluated such intersectional effects of APOE ε2 on cognition. In a study of 976 African American and 794 White middle-aged adults, Beydoun and colleagues22 investigated sex and race as potential modifiers of APOE on cognition. While the investigators noted a lack of consistency in associations between APOE ε2 and specific neuropsychological test scores across races, no race- or sex-specific associations were deemed significant.22 Studies are also mixed regarding differential APOE ε2 effects among the sexes, with one study demonstrating stronger effects among cognitively normal males,7 but another highlighting stronger effects among females.14 Additionally, a recent study found that the APOE ε2 association with cognitive decline in cognitively normal White men was stronger than in White women.56 In the present analysis, which included some of the same cohorts, we saw similar evidence of a stronger association between APOE ε2 and longitudinal decline in cognitively normal White men compared with cognitively normal White women, but the APOE ε2 × sex interaction did not reach statistical significance , so this will be an exciting avenue for future work.Because APOE on cognition. However, our results should be interpreted taking weaknesses into consideration. While sex and race effects of amyloid-β and tau pathologies are important to consider in the context of APOE and cognition, harmonized biomarker data were not readily available; therefore, we could not integrate neuropathology and biomarker measures into our models. Additionally, only data for Black and White individuals were included in this study, so our results may not be generalizable to those in other racial and ethnic groups. Moreover, despite the large sample size, the number of ε2 homozygotes was small and warrants further exploration. To maximize statistical power in sex/race stratifications, APOE ε2 was modeled dominantly. Results from dominant and additive APOE ε2 models were, in general, similar (eTables 2 and 3 in APOE ε2/ε3 genotype have a significantly lower risk of AD compared with individuals with the APOE ε2/ε2 genotype.6 Whether this holds true among Black individuals is unclear, again highlighting the need for even larger sample sizes of racially diverse individuals.The present study has several strengths. Longitudinal, harmonized cognitive data across multiple cognitive domains facilitated a large sample size, which allowed for investigation of the intersectional effects of race, sex, and APOE ε4 effects have been shown to vary with genetic ancestry, especially within African American subpopulations.61 While covarying for ancestry principal components did not affect our interpretation of results, evaluation of local ancestry at the APOE locus would allow for better dissociation of population effects.Additionally, though this study leveraged harmonized cognitive scores from multiple cohorts, we observed some heterogeneity of effects across cohorts, as seen in eFigures 1 and 2 in APOE ε4 and memory and provide strong evidence this sex difference does not differ among Black and White individuals. In contrast, we did not see robust sex differences in APOE ε2 effects on cognition, though we provide exciting new evidence of an intersectional effect of sex, race, and APOE ε2 on cognition. Our finding that APOE isoforms were differently modified by sex and race highlights the fact that ε2 and ε4 are not simply opposing sides of the same coin and underscores the need for a comprehensive precision-medicine approach in understanding AD progression.The results of our study confirmed sex differences in the association between


The following corrections were made because of two issues. First, the daylight-saving time in autumn 2017 and spring 2018 in the sensor data of 15 participants was not adjusted, which resulted in a misalignment of plus or minus one hour between the sensor data and the ESM questionnaire data. The problem arose when data collection ran across daylight-saving time, when a sensor was set up before time change and did not automatically adjust for time change but mobile phones automatically did adjust the time. The sensor data of eight and seven participants were adjusted for the time change in autumn 2017 and spring 2018, respectively. Second, some coding mistakes in the time window calculation were found.In the published article, there was an error in Table 1 and Figures 1, 2 as published.In The corrected In the published article, there was an error in The correct Supplementary material appears below.Materials and methods.In the published article, there were errors in Materials and methods, Statistical analysis, paragraph 1. This sentence previously stated:A correction has been made to “The intraclass correlation coefficients of the grand-mean centered variables are 0.45 for positive affect, 0.69 for both negative affect and depressive symptoms, and 0.03–0.15 for the physical activity variables.”The corrected sentence appears below:“The intraclass correlation coefficients of the grand-mean centered variables are 0.45 for positive affect, 0.69 for both negative affect and depressive symptoms, and 0.03–0.13 for the physical activity variables.”Materials and methods, Statistical analysis, paragraph 2. This sentence previously stated:A correction has been made to “Minutes of sedentary behavior, LPA, MVPA, and total physical activity were aggregated by 30-min time windows before the mood ratings…”The corrected sentence appears below:“Minutes of sedentary behavior, LPA, and MVPA were aggregated by 30-min time windows before the mood ratings to create total physical activity…”Results, paragraph 2. This sentence previously stated:A correction has been made to “For positive affect, the effect sizes ranged from 0.05 to 0.10 and the confidence intervals 0.01–0.13. Negative affect and depressive symptoms showed similar effect sizes and confidence intervals; negative affect:−0.06 to−0.09 (standardized beta) and−0.02 to−0.13  and depressive symptoms:−0.07 to−0.11 (standardized beta) and−0.03 to−0.15 .”The corrected sentence appears below:“For positive affect, the effect sizes ranged from 0.03 to 0.08 and the confidence intervals 0.00–0.12. Negative affect and depressive symptoms showed similar effect sizes and confidence intervals; negative affect:−0.05 to−0.08 (standardized beta) and −0.01 to −0.12  and depressive symptoms: −0.06 to −0.11 (standardized beta) and −0.02 to −0.15 .”Results, paragraph 3. These sentences previously stated:A correction has been made to “The effects of physical activity on positive affect increased during the 180 min before the mood rating, they were the lowest in the 180–150 min before the mood rating  and the largest in the 30 min before the mood rating .”“Significant interactions were observed for physical activity measured 30–0 min before the mood rating predicted positive affect  with stronger associations in participants with higher baseline levels of depressive symptoms .”The corrected sentence appears below:“The effects of physical activity on positive affect increased during the 180 min before the mood rating, they were the lowest in the 180–150 min before the mood rating  and the largest in the 30 min before the mood rating .”“Significant interactions were observed for physical activity measured 30–0 min before the mood rating predicted positive affect  with stronger associations in participants with higher baseline levels of depressive symptoms .”Results, paragraph 4. This sentence previously stated:A correction has been made to p < 0.001), negative affect , and depressive symptoms  with physical activity measured 0–30 min after the mood rating were significant in the group with higher baseline levels of depressive symptoms but they were not significant in the group with lower levels of depressive symptoms .”“The associations of positive affect , negative affect , and depressive symptoms  with physical activity measured 0–30 min after the mood rating were significant in the group with higher baseline levels of depressive symptoms but they were not significant in the group with lower levels of depressive symptoms , negative affect  and depressive symptoms . MVPA measured 30–0 min before the mood rating significantly predicted positive affect  and depressive symptoms .”“Applying the isotemporal substitution model , LPA measured 90–60 min before the mood ratings significantly predicted positive affect  and depressive symptoms .”“Applying the isotemporal substitution model , 6, LPA Discussion, paragraph 1. These sentences previously stated:A correction has been made to “The size of the association appeared to be relatively constant for physical activity across the 180 min before the mood rating .”“Moreover, exploratory analyses using isotemporal substitution approach showed that there were associations of both MVPA and LPA with subsequent levels of positive affect, negative affect and depressive symptoms, but we did not detect stronger effects of MVPA compared to LPA on average nor a meaningful temporal pattern of the effects over the 180 min before the mood rating, which suggests that both MVPA and LPA might both have positive effects on mood.”The corrected sentences appear below:“The size of the association appeared to be relatively constant for physical activity across the 180 min before the mood rating .”“Moreover, exploratory analyses using isotemporal substitution approach showed that there were associations of LPA with subsequent levels of negative affect and depressive symptoms, but we did not detect a meaningful temporal pattern of the effects over the 180 min before the mood rating, which suggests that LPA might have a positive effect on mood.”The authors apologize for these errors and state that these do not change the scientific conclusions of the article in a substantial manner. However, the significant associations between light physical activity (LPA) measured 90–60 min before the mood ratings and positive affect and between moderate and vigorous physical activity (MVPA) measured 30–0 min before the mood ratings and positive affect and depressive symptoms were no longer significant. The original article has been updated.


Many of these applications rely on the assumption that the surface Earth Si isotope budget is in mass balance with bulk silicate Earth, such that trends in δ30Si over time can be attributed to changes in flux to or from key silica reservoirs. We compiled δ30Si data from modern reservoirs representing the major sources and sinks of surface Earth reactive silica, to which we applied an inverse model to test assumptions about mass balance. We found that δ30Si values of reverse weathering products must closely match those of diatoms, conflicting with previous assumptions. Model results also revealed that of the 19 to 21 teramoles per year Si released during silicate mineral weathering, ~10 to 18 teramoles per year is stored in terrestrial silica sinks, consistent with assumptions of incongruent weathering reactions. Our results demonstrated that the modern silica cycle summary is in isotopic mass balance.Geological reservoir δ 30Si values of surface Earth reactive silica informs weathering stoichiometry and utility as a proxy.Inverse modeling of δ We can now measure nuances in the silica cycle that we were unable to measure before, such as nonconservative behavior in estuaries . As2 concentrations over long time scales is thought to be chemical weathering of terrestrial silicate rocks ; dissolved inorganic carbon, mainly in the form of HCO3−; cations; and a host of trace elements to solution. The feedback of silicate weathering on atmospheric CO2 concentrations is often modeled by applying a stoichiometric relationship between Si and C (3) The stoichiometry of dissolved Si to CO2 consumption; in other words, some of the dSi released by weathering is incorporated back into a mineral phase. The stoichiometry of Si to C in incongruent silicate weathering reactions can be represented by the incomplete dissolution of the aluminosilicate K-spar (KAlSi3O8)2 consumed. Another example of an incongruent weathering reaction where Na-rich plagioclase (“An20”) is altered to halloysite that has been used recently is as follows , and approached the evolution of Earth’s climate by modeling C cycle summaries , enumerated as following: marine silica sinks: diatoms: n = 240 from water column samples and n = 50 from sediments; siliceous sponge spicules: n = 72; marine silica sources: aeolian dust: n = 14, assuming that this source is well-represented by loess; hydrothermal fluids: n = 2; river dSi: n = 372, including a subset of n = 23 samples from river mouths; river aSi: n = 75; submarine groundwater discharge (SGD): n = 31, including subsets of n = 11 marine SGD  and n = 5 freshwater SGD ; and (sub)polar glacial meltwaters: n = 40; and terrestrial silica sinks: secondary clay minerals formed in soils : n = 104; plant phytoliths: n = 216; and freshwater diatoms: n = 33. Because of lack of data, low temperature silicate weathering (a marine silica source) was assumed to have a similar δ30Si distribution as river aSi . Compiled δ30Si data are summarized in tableS1, and δ30Si distributions are shown in 30Si values are not used in the inverse model as they are not a source or sink of dSi to the global ocean (seawater or marine porewater) or are an unconstrained but likely very small sink (radiolarians) but have been compiled here and are available in the Supplementary Materials.To provide δ30Si value of a source/sink or the size of a source/sink in terms of a flux) based on the distributions of δ30Si values of each of source and sink drawn from our data compilation and the size of each source and sink (F), including uncertainty. For most of our simulations, we used flux estimates from the most recent marine silica cycle summary , we inverted the data to estimate δ30SiRW assuming isotopic mass balance between marine Si sources and sinks. By evaluating whether these δ30SiRW estimates are reasonable, we assess whether the underlying assumption of Si isotopic mass balance is correct.Following the logic in a recent Precambrian silica cycling study   sinks  is weighted toward lower δ, 1.12‰) . Further30SiRW would be offset to lower δ30Si values from the materials being altered  by 1 to 2‰  does not differ strongly from global average δ30SiDiatom values  , (iv) the representativeness and parameterization of river dSi δ30Si data, and (v) the representativeness of the sponge δ30Si data  .Although it has been commonly assumed that δ± 0.74‰) . This fi30Si values of both marine silica sources and sinks are not in isotopic mass balance with BSE (Fpedogenic clay), plant bSi , and freshwater diatoms (Ffreshwater diatoms) . Rather than imposing assumptions on the absolute or relative sizes of each of the individual terrestrial sinks, we explored the parameter space of possible three-component mixtures.We also used our model to examine another poorly constrained sink—silica burial in terrestrial settings—by assuming that the combination of marine and terrestrial sinks must be in Si isotopic mass balance with BSE. Flux-normalized δwith BSE , althougSi sinks , we esti30Si distributions for each terrestrial Si sink are shown in 30Si distributions (both with and without reverse weathering products) are almost all greater than δ30SiBSE, the flux-weighted terrestrial Si sink δ30Si distribution must be less than δ30SiBSE to fulfill the presumed condition that the combined sink is in Si isotopic mass balance with BSE. However, the distributions of δ30Si values of both freshwater diatoms and phytoliths are biased to values similar to or greater than δ30SiBSE  (Compiled δδ30SiBSE . Therefool/year) .Fterr-sinks estimates for each of the possible three-component mixtures to yield estimates for the sizes of pedogenic clay , phytolith , and freshwater diatom  sinks (fig. S3).We unmixed the Fpedogenic clay to solve for the average stoichiometry of weathering reactions that produced those pedogenic clay minerals for each of the mixtures. Our model demonstrated that the Si stoichiometry of silicate mineral weathering reactions required for the global silica sinks to be in isotopic mass balance with BSE is 0.54 to 0.74 moles of Si stored in pedogenic clays per mole of silica released as dSi   is freed as dSi via terrestrial silicate mineral weathering , much higher than what has been used in the past to model the modern silica cycle [e.g.,  to ocean is 6.2 Tmol/year, then gross dSi produced during silicate mineral weathering before formation of secondary minerals should be ~21 Tmol/year. These values for dSi release via silicate mineral weathering, 19.1 Tmol/year and 21 Tmol/year, are in close agreement.We used the estimates of e, 0.72) , consiste, 0.72)  or the ie, 0.72)  (17). Bo30Si value lower relative to the flux-weighted silica source δ30Si values , wh30SiRW value  needed to balance the budget closely matches the median δ30SiDiatom value , although the distribution of δ30SiRW estimates  encompasses a greater range than δ30Sidiatom . Model solutions at the lower end of the distributions in the δ30SiRW values would be consistent with some dSi released via sponge or aSi (tableS1) dissolution being sequestered into reverse weathering products. Model solutions at the higher end of the distributions could result from an imbalance in the most recent 2021 summary (30SiRW is similar to median δ30SiDiatom) is the same if the fluxes are forced to be in mass balance (fig. S1B). Some findings of reverse weathering along the shelf , consistent with direct observations of authigenic clay formation rates  the fraction of δ30SiRivers that is sourced from proglacial rivers varies and the relative contribution from δ30SiISM increases or decreases through time; (ii) the δ30SiDiatom distribution changes over glacial-interglacial time scales . The stoichiometry of dSi:CO2  to global alkalinity budgets and the portion of alkalinity that is estimated to originate from silicate mineral weathering. The global alkalinity river flux from weathering of carbonate rocks is ~17.3 Tmol/year of 30.1 Tmol/year total  supports the interpretation that the global silica cycle is in isotopic mass balance with BSE, because our estimates are conditioned on that assumption.Our findings have substantial implications for applications of δ30Si distributions from those precursors. On the one hand, this suggests that, in lieu of direct measurement methods, δ30Si values of authigenic clays formed via reverse weathering could be estimated from δ30Si distributions of diatomaceous sediments. However, on the other hand, the sizes of two sinks with indistinguishable δ30Si distributions cannot be differentiated—the inverse modeling approach could only be used to reconstruct the sum of the two sinks but not the relative contributions of each to that sum. Would the same process dominate before the evolution of diatoms? Diatom frustules are more reactive than radiolarian tests or sponge spicules  might not differ significantly in their δ30Si compositions should be applied and interpreted for the Precambrian rock record. Assuming that, along with cherts and iron formations, reverse weathering products were a major silica sink in Precambrian oceans  of these authigenic clays formed via alteration of an abiotic aSi precursor , then we must question the assumption that reverse weathering products are isotopically distinct from chert and/or BIF. A better understanding of the mechanisms of marine clay authigenesis in Precambrian seawater and methods to uniquely identify and measure the δ30Si compositions of reverse weathering products are both needed to advance our ability to interpret the Precambrian δ30Si record.Our findings also inform how inverse modeling of the marine silica cycle via δFor the marine silica cycle, Si isotope mass balance can be expressed mathematically as in The left-hand side terms are the silica sources: aeolian dust, hydrothermal fluids (HT), dSi in rivers, aSi in rivers, fresh and marine submarine groundwater discharge (FGW and MGW), dissolution of lithogenic silica (W), and silica from glacial sources (ISM). The right-hand side terms are the silica sinks: bSi in the form of diatoms and sponge spicules and authigenic clays formed through reverse weathering reactions (RW).30SiRW, we compared two approaches. In the first approach, we solved 30SiRW as in To invert the data to estimate δFRW was constrained using the most recent marine silica summary estimate  the size of the diatom sink, (ii) the representativeness of the diatom δ30Si data, (iii) the representativeness of river dSi δ30Si data, and (iv) the representativeness of the sponge δ30Si data. (i) With respect to the size of the diatom sink, we compared model δ30SiRW estimates for smaller and larger FDiatom (7 and 14 Tmol/year) ; under both scenarios, we used FRW assuming flux mass balance. (ii) With respect to addressing the representativeness of the diatom data, which is primarily from Southern Ocean samples, we explored whether surface seawater dSi δ30Si, for which there is better geographic data coverage, could be used to estimate diatom δ30Si values via the well-characterized diatom Si isotope fractionation factor. We explored the relationship between surface seawater dSi δ30Si and diatom δ30Si values and determined that, even in the Southern Ocean, using seawater dSi δ30Si to estimate diatom δ30Si values introduces more uncertainty due to Rayleigh-type fractionation (fig. S4). (iii) With respect to addressing possible bias in river dSi δ30Si due to changes in δ30Si along the length of each river  (fig. S1E) and developing a flux-weighted dSi δ30Si estimate by matching each river in our database to catchments  following a coastal segmentation study  rather than pedogenic clay minerals, particularly given the reported ~−2‰ fractionation  to the total flux of dissolved Si produced by silicate weathering, for which we used the sum of the fluxes of fluvial dSi + aSi, submarine groundwater discharge, ice sheet meltwaters, freshwater diatoms, and phytoliths.To estimate the global stoichiometry of silicate weathering reactions, we calculated the ratio of the flux of Si into storage as secondary clay minerals (