The results show high numbers of Balaenidae ) and grey whale (Eschrichtius robustus) specimens, two taxa no longer present in the eastern North Atlantic. This discovery matches expectations regarding the past utilization of North Atlantic right whales, but was unanticipated for grey whales, which have hitherto rarely been identified in the European zooarchaeological record. Many of these specimens derive from contexts associated with mediaeval cultures frequently linked to whaling: the Basques, northern Spaniards, Normans, Flemish, Frisians, Anglo-Saxons and Scandinavians. This association raises the likelihood that early whaling impacted these taxa, contributing to their extirpation and extinction. Much lower numbers of other large cetacean taxa were identified, suggesting that what are now the most depleted whales were once those most frequently used.Taxonomic identification of whale bones found during archaeological excavations is problematic due to their typically fragmented state. This difficulty limits understanding of both the past spatio-temporal distributions of whale populations and of possible early whaling activities. To overcome this challenge, we performed zooarchaeology by mass spectrometry on an unprecedented 719 archaeological and palaeontological specimens of probable whale bone from Atlantic European contexts, predominantly dating from  This activity, in particular commercial whaling, has completely altered the composition of whale populations and by extension transformed ecosystems . The impe change  on the ohe other .The beginnings of whaling activities in Europe are hard to pinpoint with clarity. Both the use of stranded individuals and active hunting in prehistory have long been recognized , with fiSubfossil whale bone specimens from archaeological and palaeontological contexts hold an important key to revealing the past. However, whale bones are infamously hard to identify to the family or species level. This difficulty can be ascribed to limited osteological morphological differences between the taxa, the often fragmented state of the remains, and the lack of extensive reference collections . As a reZooarchaeology by mass spectrometry (ZooMS) has been shown to be an excellent method for identifying whale bone specimens to family or species . Relevanca 900 BCE to ca 1500 CE), with a few earlier and later specimens providing a broader chronological context. We acknowledge that not all archaeological specimens derive from actively caught individuals, but by studying whale bone remains from sites from cultures frequently associated with whaling, inferences can potentially be made regarding past whaling activities. We address this question by determining the taxa of archaeological finds of cetacean bone using ZooMS. High chronological precision is not a present goal, being a matter of ongoing research given the complexity of radiocarbon dating migratory marine taxa, some of which feed in waters above the northern latitudinal limit of the Marine20 calibration curve .Gadus morhua) in 1992/1993 [Acipenser sturio) in the early twentieth century [Pinguinus impennis) in the mid-nineteenth century [Ectopistes migratorius) in the early twentieth century [In result, using published historical evidence as background information, we infer that now extinct or critically endangered cetaceans—the Atlantic grey whale and the North Atlantic right whale —were especially frequently targeted by human hunters prior to being overexploited. These two taxa were particularly vulnerable to early whaling activities as they were slow swimmers with near-coastal habitats. This interpretation matches expectations regarding North Atlantic right whales, but is an unanticipated discovery for Atlantic grey whales, which are very poorly documented in historical texts and have hitherto rarely been identified in the European zooarchaeological record. In comparative perspective, it is relevant that seemingly superabundant taxa, for reasons of accessibility to potential human harvesters, can be counterintuitively vulnerable to overexploitation or extinction. Classic cases of this phenomenon are known from both the recent and distant past, among both marine and terrestrial/avian fauna. Examples include the collapse of Newfoundland's northern cod  [To assess the past geographical range of whale taxa in eastern North Atlantic waters, and to reconstruct which taxa were most exploited, a total of 719 subfossil probable cetacean specimens were collected from northern and western Europe . The sam500 BCE) . Specimeca 500 mg. Each rotary disc was used once, while the tool was cleaned using ethanol in between sampling. For 474 specimens, collagen was extracted using a modified Longin [et al. [For ZooMS analysis of these 719 specimens, samples were taken using a Dremel® rotary tool removing a small piece of bone weighing up to d Longin  method a [et al. , with thca 8 ml of ultrapure water was repeatedly added to the tube and removed until a pH of 3–4 was reached. Subsequently, 4 ml of 0.5% NaOH was added for 2–4 h at room temperature to dissolve humic acids. The NaOH was removed from the samples and ca 8 ml of ultrapure water was repeatedly added to the tube and removed until pH < 10. Afterwards, 5 ml of 1.22 M HCl was added for 1–2 h to remove atmospheric CO2. The sample was then washed repeatedly with ultrapure water until pH = 3 ± 0.2 and hydrolysed to gelatin at 70°C overnight. Finally, the gelatin was filtered through a prebaked (900°C) quartz filter  and the filtrate freeze dried. The final collagen was taken to the Henry Wellcome Laboratory for Biomolecular Archaeology, University of Cambridge, UK, for ZooMS analysis.Initially, the samples were crushed to small pieces, placed into test-tubes, and cleaned in an ultrasonic bath with 18.2 MΩ cm ultrapure water (type 1) (three times 5 min). The samples were then ultrasonicated for 15 min in dichloromethane and methanol (2 : 1). This step was repeated three or more times, until the solution was clear. Following this, the material was demineralized overnight using 2.44 M HCl (50 ml of solution per 100 mg of bone) in glass tubes within a vacuum desiccator kept at room temperature. The HCl was removed from the samples and Collagen extraction failed for 25 specimens and when sufficient original bone material remained a subsample was taken directly to the Henry Wellcome Laboratory. Additionally, another 237 specimens were analysed by ZooMS without prior collagen extraction. For these 262 (25 + 237) specimens, approximately 30 mg subsamples were taken and processed at the Henry Wellcome Laboratory for Biomolecular Archaeology. The bones were demineralized in 0.6 M hydrochloric acid at 4°C for two weeks. Each sample was then centrifuged, the hydrochloric acid discarded, and the retained material rinsed three times with 200 µl of 50 mMol ammonium bicarbonate (AmBic) pH 8.0 solution, before being gelatinized in 100 µl of AmBic solution at 65°C for 1 h. For the 449 collagen samples prepared at the Norwegian University of Science and Technology (NTNU) , 0.1 mg of collagen was also placed in AmBic solution at 65°C for 1 h.m/z = 904.681, angiotensin I m/z = 1295.685, Glu1-fibrinopeptide B m/z = 1750.677, ACTH (1–17 clip) m/z = 2093.086, ACTH (18–39 clip) m/z = 2465.198 and ACTH (7–38 clip) m/z = 3657.929). Samples were spotted in triplicate, and run on a Bruker ultraflex III MALDI tandem time-of-flight (TOF/TOF) mass spectrometer with a Nd:YAG smart beam laser. A SNAP averaging algorithm was used to obtain monoisotopic masses . Averaged spectra were created from the replicates for each specimen using mMass software [m/z markers for mammals, as presented in Buckley et al. [et al. [et al. [et al. [et al. [For both the 262 and 449 sample groups, the gelatinized collagen, still in AmBic, was incubated with 0.4 µg of trypsin at 37°C overnight, and subsequently acidified with 0.1% trifluoroacetic acid (TFA). The collagen was then purified using a 100 µl C18 resin ZipTip pipette tip (EMD Millipore) with conditioning and eluting solutions composed of 50% acetonitrile and 0.1% TFA and washing solution composed of 0.1% TFA; the samples were eluted in a volume of 50 µl. Equal amounts of the collagen extract and α-cyano-hydroxycinnamic acid matrix solution (1% in conditioning solution) were mixed (1 µl each) and spotted onto a 384 spot MALDI target plate. Each sample was externally calibrated against an adjacent spot containing a mixture of six peptides , Eden's whale , Omura's whale , Rice's whale , Antarctic minke whale , pygmy right whale (Caperea marginata), North Pacific right whale  and southern right whale . Their omission prohibits identification of these taxa and could lead to misidentifications. However, the relevant missing control spectra are of taxa that are unlikely to have been hunted in the North Atlantic within the timeframe this study is concerned with.While ZooMS reference spectra have been created for many large whale species, this has not been undertaken for all. Taxa without existing spectra include the Bryde's whale , the North Pacific right whale (E. japonica) and the bowhead whale . Of these, only the North Atlantic right whale and the bowhead whale are probable in a North Atlantic context. Differentiation between the North Atlantic right whale and the bowhead whale is difficult using ZooMS or osteological morphological identification. Moreover, the distribution of these taxa may have overlapped more substantially in the past than present. This observation is underlined by McLeod et al. [ca 900 BCE to ca 1500 CE, E. glacialis is most likely for Balaenidae identifications. We base this inference not only on the range of the bowhead whale, which is restricted to Arctic and subarctic waters, but  its tendency to stay close to the edge of the sea ice [ca 11.7 ka BP to present), sea ice did not reach most of northern or western Europe [For the taxa of central present concern, d et al. , who usi sea ice . During n Europe .Therefore, we suggest, with some caution, that the vast majority of the identified Balaenidae will probably be North Atlantic right whales, except for the northernmost specimens  and mainland Norway) and a few bones (e.g. from The Netherlands (11 specimens)) that may represent trophies from long-range whaling voyages of the post-mediaeval period. From the early seventeenth century, European whalers ventured towards the Arctic to hunt the bowhead whale and occasionally brought back bones. Often the mandibles were transported, of which 11 have been identified based on morphology after comparison with Turner  and mode. 3Bos taurus/primigenius), elephant/mammoth (Elephantidae), seal (Phocidae), walrus (Odobenus rosmarus) and even human (Homo sapiens). No results were reached for 39 specimens.Using ZooMS, it proved possible to identify 623 specimens to cetacean taxon/ZooMS group level  and the grey whale (110 specimens/15.3%). Specimens of both groups were found along the coast of Atlantic Europe, from Iberia to Norway, although the majority of the grey whale samples originate from The Netherlands figures  and 3. TPhyseter macrocephalus) (43 specimens/6.0%), fin whale  (37 specimens/5.1%) and humpback whale (Megaptera novaeangliae) (34 specimens/4.7%) were identified. Lower numbers were observed for the blue whale , common minke whale  and sei whale .Additionally, some specimens of the sperm whale . These identifications broaden our knowledge of which whales were used in the past. However, they are not as informative as the great whale evidence in terms of the relative frequency of utilization because bones recognizably from small whales were not targeted for sampling. Aside from sperm whale, the identified Odontoceti specimens were highly fragmented pieces of ‘whale bone’ that did not allow . 4. 4.1Taxa that were the main foci of twentieth-century whaling , and/orca 100 BCE to ca 400 CE) and subsequent Middle Ages (ca 400 to ca 1500 CE). These were centuries of known or hypothesized pre-industrial whaling [By far the highest proportion of specimens has been identified as Balaenidae. While it cannot be excluded that some of these derive from bowhead whales (at least 11 mandibles and one atlas certainly do based on morphology), it is likely, based on the reasoning described above, that many or most represent the North Atlantic right whale. At least a millennium of hunting along the European coastline by various cultures frequently associated with whaling  has led to the complete extirpation of what must once have been an at-least-coastally abundant population ,35,47. T whaling ,34,53.Though ZooMS does not provide data regarding the population size of the different taxa, the high number of Balaenidae, which are assumed, based on previous described reasons, to largely represent North Atlantic right whales, and their spatial distribution, suggests the species was widespread in European coastal waters until at least the Middle Ages. Their representation in the archaeological record implies that they were frequently used. The species has a near-coastal habitat, is a slow swimmer and is naturally buoyant after death .ca 100 BCE to ca 1200 CE. Neolithic specimens from sites of the Vlaardingen Culture demonstrate that grey whales were also used earlier (see below). No closely dated specimens in this dataset are later than the fifteenth century CE, consistent with extinction of the Atlantic grey whale in the middle centuries of the second millennium CE [The high number of grey whale specimens strongly contrasts with the previously known subfossil record  and implnnium CE .et al. [ca 100 BCE and ca 1200 CE  and grey whale, the two species that are currently no longer present in the eastern North Atlantic, are by far the highest represented species, indicates how significantly whale stocks have been altered in European waters after centuries of whaling activities. Alter et al.  previous 1200 CE , insteadet al. [Other whale taxa are notable for their comparative scarcity in the subfossil record, but there is a cluster of sperm whale finds from the southern North Sea area. Unlike the grey whale, these specimens probably do not indicate that the species was especially abundant in the region. The southern North Sea has been described as a sperm whale trap . Young bet al.  also suget al.  providesThe particularly scarce evidence of common minke whales is notable. Based on aerial surveys, minke whales appear to be the most common baleen whale species in the North Sea area . The lowDelphinapterus leucas; an Arctic taxon) from Uddevalla, Sweden, is an outlier, but it is undated and of uncertain significance. Belugas occurred in more southern waters in colder periods in the past [Beyond new evidence regarding the extirpated and extinct right and grey whale, the distribution of the subfossil finds by taxon is broadly consistent with what is known based on modern data. A specimen of beluga , Toranda (probably post 1290 CE), Port of Candás (probably post 1500 CE), San Cibrao (ca 100 BCE to 1800 CE), Oyambre beach (probably post 1612 CE), Porto de Bares (ca 1200–1800 CE) and Luanco (probably post 1622 CE)). Many of these specimens are not precisely dated, but the chronology of site occupation provides a date range or terminus post quem. The Basques are by far the most renowned early whalers based on written evidence [et al. [et al. [Whale bones from seven sites from northern Spain were analysed as part of this study  . To infe. 4.3.4Limited historical evidence suggests the existence of active whaling activities undertaken by Frisians (in what is now The Netherlands and northwest Germany) . There i. 4.3.5Tursiops truncatus), but also common minke whale and sei whale. The diversity of species at Ipswich may suggest opportunistic scavenging, although as observed for Haithabu and Trondheim (see below) this need not be so. The exclusive representation of Balaenidae at Hamwic may suggest active whaling, although the Anglo-Saxon inhabitants of this port might also have relied on trade with Norman whalers [et al. [Little historical evidence exists for active whaling by the Anglo-Saxons in what is now England , but a n whalers . At Flix [et al. . No grey. 4.3.6ca 770–1066 CE) and Trondheim . A variety of historical sources, the first as early as the ninth century CE, suggest that Scandinavians performed active whaling by the Viking Age (ca 800–1050 CE) at the latest. Several forms of whaling are described in mediaeval sources, including spearing (with or without poison), drives and trapping in bays or fjords [ca 550–1050 CE and are also quite frequently found in Sweden, signalling a large-scale distribution of North Atlantic whale products [Whale bones from two relevant centres were analysed: Haithabu  and Hekelingen III (ca 3000–2800 BCE) in particular, plus one whale bone specimen from Pompveld Altena (ca 3500–2500 BCE)) are predominantly of the grey whale. Additionally, the geographically closely positioned Neolithic site of Schipluiden (ca 3490–3380 BCE), which pre-dates the Vlaardingen Culture, has also provided one grey whale specimen. The very high proportion of grey whale findings from the Vlaardingen Culture sites suggests the species was abundant in the Rhine estuarine region and may imply that this particular taxon, which moves slowly in shallow coastal waters based on the behaviour of the Pacific grey whale [To explore the possibility of European whaling even earlier than the mediaeval period, we also considered material from the archaeological Vlaardingen Culture, where marine mammal bones have been recovered from several coastal sites . The whaey whale , was actey whale ,68. Neoley whale . Active Overall, by combining previous historical and new ZooMS evidence it can be suggested that Balaenidae (most likely North Atlantic right) whales were the main target of mediaeval whaling activities, with grey whales targeted as well. The concentration of Balaenidae specimens identified at many of the mediaeval sites considered implies a long and persistent tradition of hunting along the Atlantic coast of Europe starting by or before the eleventh century and continuing up to the early twentieth century . For the. 5This study has highlighted the wealth of historical ecological data hidden in zooarchaeological and palaeontological archives of cetacean bone. Two baleen whale species that are no longer present in the eastern North Atlantic—Balaenidae  and grey whale—were the taxa most identified in the subfossil record of large cetaceans. Early, especially mediaeval, whaling by multiple cultures along potential migration routes is hypothesized to have impacted the populations of both species, which must once have been frequently targeted. Though the eastern North Atlantic grey whale population may already have been declining due to climatic or ecological causes , it is sOdobenus rosmarus rosmarus) of mediaeval Greenland suffered a similar fate [Both the grey whale and the North Atlantic right whale may have fallen victim to a perception of limitless natural abundance, due to ease of hunting and initially high numbers in coastal waters during the inception and early development of European whaling. After the pattern of targeting these once frequently encountered taxa became fixed in whaling practice, they were subsequently hunted to extirpation and extinction regardless of declining numbers. This phenomenon is not without parallels in the annals of human resource exploitation, be it of marine or terrestrial/avian species. The examples of the passenger pigeon , the Eurlar fate ,72. In t


Outdoor air pollutioncauses millions of premature deaths annuallyworldwide. Sulfate is a major component of particulate pollution.Winter sulfate observations in China show both high concentrationsand an accumulation mode with a modal size >1 μm. However,wefind that this observed size distribution cannot be simulated usingclassical gaseous and aqueous phase formation (CSF) or proposed aerosol-processingformation (APF) mechanisms. Specifically, the CSF simulation underestimatessulfate concentrations by 76% over megacities in China and predictsparticle size distributions with a modal size of ∼0.35 μm,significantly smaller than observations. Although incorporating theAPF mechanism in the atmospheric chemical model notably improves sulfateconcentration simulation with reasonable parameters, the simulatedsulfate particle size distribution remains similar to that using theCSF mechanism. We further conduct theoretical analyses and show thatparticles with diameters <0.3 μm grow rapidly (2–3s) to 1 μm through the condensation of sulfuric acid in freshhigh-temperature exhaust plumes, referred to as in-source formation(ISF). An ISF sulfate source equivalent to 15% of sulfur emissionsfrom fossil fuel combustion largely explains both observed size distributionsand mass concentrations of sulfate particles. The findings imply thatISF is a major source of wintertime micron-sized sulfate in Chinaand underscore the importance of considering the size distributionof aerosols for accurately assessing the impacts of inorganic aerosolson radiative forcing and human health. The in-sourceformation mechanism explains not only highsulfate mass concentrations but more importantly the formation oflarge micron-sized sulfate particles observed in winter in China. In comparison to the accumulationmode at a modal size of 0.4–0.8 μm observed in NorthAmerica,28 the dry sulfate in wintertime China appears significantly larger,with a modal size of 0.6–1.4 μm.32 New research noticed the potentialcontribution of plumes in the formation of atmospheric particulatematter,35 whereas the importance of this mechanism to the formationof large sulfate particles under polluted background remains to bedeciphered. Furthermore, observations near coal-fired power plantexhaust in China showed that a large fraction of sulfur emissionsare in the form of SO3,39 which is produced through theoxidation of SO2 during combustion and can also be formedin the exhaust plumes.12 Unlike SO2, SO3 is converted to sulfuric acid virtually instantlyin the troposphere.40 Thus, we hypothesizethat the condensation of sulfuric acid in fresh high-temperature exhaustplumes onto pre-existing atmospheric particles can quickly producemicron-sized particles. We refer to the process of sulfate particleproduction from fresh plumes hereafter as in-source formation (ISF),which may be the missing mechanism of sulfate formation in China.Some studies proposed that aerosol-processing formation (APF),that is, oxidation of SOIn this study, we aim to answer the question of whether ISF canexplain both the observed high mass concentrations and large-sizedistributions of sulfate in the winter in China. Our investigationinvolves simulations of sulfate mass concentrations and size distributionsusing CSF, APF, and ISF, with an emphasis on the formation of largewintertime sulfate particles across China using the Weather Researchand Forecasting model coupled with Chemistry (WRF-Chem) model withdetailed aerosol microphysics. To avoid any confusion, we specificallyuse the term “APF” instead of the commonly used term“heterogeneous sulfate formation” as ISF can also beheterogeneous. We leverage a wide range of diverse observations tothoroughly assess the robustness of our simulations and constrainour results. We pay particular attention to aerosol size measurements,which were not generally considered in previous studies of the wintertimesulfate formation processes. Through the development of a theoreticalmodel and conducting sensitivity experiments, we examine the feasibilityof large sulfate particle formation via ISF. Our study reveals substantialdisparities in particulate size distributions predicted by APF andISF, potentially challenging the current understanding of the effectsof aerosols on radiative forcing and public health implications.2.5 chemical compositionsand gaseous pollutants were monitoredat an urban site in Wuhan, central China  and ultraviolet fluorescence equipment,respectively.41 For MARGA measurements,the sampling flow rate of air was 16.7 L min–1.The recovery rates were controlled to be >95%, and the relativestandarddeviation of the sampling flow was calibrated to <5%. Detailedquality assurance and quality control at the site were performed asdescribed in our previous study.41 Wuhanand its surrounding areas have suffered from severe air pollutionin recent years, which has attracted increasing attention.42 The site in Wuhan is a typical urban site incentral China. Additionally, the time resolution of these data wasparticularly high, and the data fully covered the entire winter season.Currently, hourly measurements of PM2.5 components  are not available at other locationsin China. Thus, we employed these data to evaluate the model performance.Hourly observations of surface PMSupporting Information Table S3); (2) long-term (regular multiple samplings over at least7 days) mass size distribution data of sulfate, nitrate, and ammoniumwere compiled from published articles (denoted by orange circles in Supporting Information Table S4); (3) hourly observationsof SO2 and PM2.5 and monthly meteorologicaldata over China were obtained from the China Ministry of Ecology andEnvironment (http://106.37.208.233:20035/) and the China Meteorological Data Service Centre (http://data.cma.cn), respectively.The inorganic aerosol size-distribution data were selected based onthe following criteria: the sampling site was a nonbackground airsite, sampling time was relatively new (between 2010 and 2017), thenumber of samples was relatively large, and the sample period wasnot affected by special events . The mass size distribution data of inorganic aerosolswere mostly measured by using the following method. First, size-resolvedaerosols were sampled using a multistage cascade impactor (such asthe MOUDI sampler or Andersen sampler shown in Supporting Information Table S4). Previous studies consistentlyshowed that despite potential variations in ambient humidity and aerosolliquid water content between daytime and nighttime, for example, theaerosol liquid water content in PM2.5 during winter nightsin Beijing were more than 2 times higher than daytime levels,43 the size distributions of sulfate sampled atthe same location from day and night were very similar and the modalsize of the size distribution remained relatively consistent.45 Thus, following previous studies, we think the aerosols are basicallyunder dry conditions during sampling.46 Then, compositionanalysis of water-soluble ions in each stage was carried out, andthe mass size distribution data of water-soluble ions  were finally obtained.We also collected severalother types of wintertime observationsacross China to compare with the simulation results: (1) mass concentrationdata of 31 sets of sulfate, nitrate, and ammonium at 25 stations overChina  forthe gas-phase chemical mechanism52 andthe Model for Simulating Aerosol Interactions and Chemistry (MOSAIC)with eight bins (Supporting Information Table S1) for the aerosol simulations.53 MOSAIC includes all major aerosol species and simulates aerosolchemical and physical processes such as nucleation, condensation,coagulation, thermodynamics, and phase equilibrium. More details aboutthe MOSAIC aerosol model are presented in Supporting Information Text S1. Chemical initial and boundary conditionsof WRF-Chem were obtained from simulations of the Community AtmosphereModel with Chemistry (CAM-Chem) model.54 For every simulation and sensitivity experiment, the WRF-Chem modelwas run for 3 months from December 2014 to February 2015, coveringthe entire winter season in China. Observations for the same periodwere used to evaluate the model simulations. The model results forthe first 5 days were excluded as the model spin-up for each monthlysimulation.55 The simulation domain coveredmainland China , with a horizontal resolution of 30 km and 30 vertical levels.Detailed configurations following published literature57 are listed in Supporting Information TableS2.WRF-Chem is a fullyonline coupled meteorology–chemistry model. It has long beenused to simulate regional air quality and evaluate meteorology–chemistryinteractions.58 In this emission inventory,no primary sulfate emissions were included. To enhance the accuracyof emissions data in China, we made some adjustments. Specifically,we replaced the ammonia (NH3) emission inventory with thePKU-NH3 2015 inventory, which was developed based on aprocess-based method to better represent the temporal and spatialdistributions of NH3 emissions in China.56 Additionally, we replaced residential SO2 emissionswith the PKU-FUEL 2014 inventory, compiled based on the latest nationwidesurvey of residential fuel consumption in China.59 The mosaic Asian anthropogenic emission inventory (MIXinventory) was used for the anthropogenic emissions outside China.60 We considered hourly and vertical variationsin anthropogenic emissions based on emission sectors. Biogenic emissionswere calculated online using the Model of Emissions of Gases and Aerosolsfrom Nature (MEGAN) module,61 and hourlyfire emissions were provided by the Fire Inventory from the NCAR (FINN)model.62For anthropogenic emissions, we employed the MultiresolutionEmission Inventory for China (MEIC) 2014–2015 inventory, whichprovides the monthly emissions of major air pollutants from five majoremission sectors. The MEIC inventory uses more data specific to Chinacompared to global inventories and has been widely used in air qualityresearch.2O5) in the model,63 we parametrizedthe process of the heterogeneous uptake of SO2 on aerosolparticles and subsequent oxidation by oxidants to form sulfates inthe MOSAIC module of the WRF-Chem model. The reaction was assumedto be irreversible, and the uptake of SO2 by aerosol particleswas a first-order process, as in a previous work63C2g,SO are the concentrations of gaseous SO2 in the ambientair (mol m–3). β is a hypothetical ratio thatensures that the total amount of sulfate produced by APF and ISF isthe same in China for each month. Here, we introduce a parameter,β, such that the size distribution comparison using differentmechanisms is analyzed under the condition of comparable sulfate massconcentrations. In this manner, the effect of sulfate mass concentrationdifference on size distribution difference was minimized. In thisstudy, the three-month average value of β was 1.10, and thedeviation of the monthly value of β from the average was within0.09. The calculation process of β in the APF simulation ispresented in Supporting Information TextS2. k2,mSO is the first-order mass transfer coefficient for SO2 inbin m (s–1) and is parametrizedasRmp, is the particle radius (cm), D2g,SO is the gas diffusivity of SO2 (cm2 s–1), Nm is the particle number density in bin m (cm–3), and f is the transition regime correction factor  has beenpreviously reported.11 Here, we employed a constantγ2SO of 1 × 10–5, which shows a reasonable agreement with the observations.Two additional sulfate sources(APF and ISF) were considered inthis study. For the APF mechanism, following the existing code ofthe heterogeneous uptake of dinitrogen pentoxide  was calculated using the followingequationdp is thediameter of the particle (m), Mi is the molar mass of sulfuric acid vapor (98 × 10–3 kg mol–1), Di is the diffusion coefficient of sulfuricacid vapor in the air (m2 s–1), and Pi is the vapor pressure ofsulfuric acid in the gas phase (Pa), calculated from the concentrationof SO3 in the flue gas and the temperature of the fluegas. Based on the observations of SO3 and temperature,38 these two variables were set to 5 mg m–3 and 50°C, respectively. Pieq, is the equilibrium vapor pressure on the particle surface.As sulfuric acid is nonvolatile, Pieq, was set to zero during the calculation. R is the gas constant (8.314 J K–1 mol–1), and T is the temperature of thesurface of the particle (K), which was set equalto the temperature of ambient air (0 °C). fn is the transition regime correction factor expressed as66Kn is theKnudsen number, defined as Kn = (2 ×λ)/dp (where λ is the meanfree path),53 and a isthe mass accommodation coefficient with a value of 0.65.67 Finally, according to the law of conservationof mass, the growth rate of a particle can be expressed as follows–3).To examine the particle growthprocess during ISF inside or nearsmokestacks, we employed MATLAB R2020b to develop a theoretical modelto calculate the particle size growth over time. There are many pre-existingparticles in the polluted atmosphere that provide abundant surfaceareas for the condensation of sulfuric acid vapor in the exhaust.Following a previous study,68 For the directradiative effects, we calculated the all-sky direct radiative effectsfor different size distributions. At every time step, we kept allof the meteorological parameters, cloud properties, surface properties,and aerosol mass loadings the same and used the APF or ISF size distributionsof sulfate, nitrate, and ammonium in the model. We then calculatedthe aerosol radiative properties  with two different size distributionsand obtained two sets of radiation flux data at the surface. In additionto direct radiative forcing, the size distribution of aerosols caninfluence the formation of CCN and aerosol-cloud indirect effects.69 However, in the WRF-Chem model, aerosols, clouds,and meteorologies are coupled and affect each other, making the indirecteffects challenging to quantify. Here, we calculated the CCN concentrationand total radiative forcing in the APF and ISF simulations with the same total sulfate mass emissionin China but different particle size distributions of secondary inorganicaerosols. Finally, we compared the results of the two simulationsand computed the impact of different aerosol size distributions onthe total radiative forcing at the surface.We calculated theradiative forcing of aerosols using the WRF-Chem model Rapid RadiativeTransfer Model for General Circulation Models (RRTMG) longwave andshortwave radiation scheme.70 which provides size-resolved particle depositionfractions in different regions  in the human respiratory system. We appliedfitted equations from the ICRP model , which was calculated by averaging thedata from different exercise levels for men and women.71 Because the original model did not account forthe moisture absorption and growth of particles in the lungs, we usedthe approach of Kodros et al. to calculate the hygroscopic growthof particles in the lungs using the hygroscopicity parameter computedwith 1072Dp,equilibrium_lung is the particle size afterwater uptakeand equilibrium in the lungs, Dp,dry isthe diameter of the dry particle (m),RH is the relative humidity in the lungs, and κ is the hygroscopicityparameter.73 RH and the temperature inthe lungs were set to be 99.5% and 37 °C, respectively, followingthe published literature.74 The total particleκ value was calculated as the volume-weighted average of allindividual species.72 The time requiredfor the particles to reach equilibrium τ (s) in the lungs wascalculated using Dp,ambient is theinitial particle diameter inthe ambient environment (m), Dg is thediffusion coefficient of water vapor(m2 s–1), β is the correction factor, M is the molar mass of water vapor (18 g mol–1), ρ is the density of particle (g m–3),and C∞ and Cs are the water vapor concentration in the lung and at theparticle surface (mol m–3), respectively. 72Dp,lungisthe diameter of the particle in the lung (m) and τlung is the residence time in the lung (2.5 s).72 Finally, the particle diameter associated with deposition in thehuman body Dp,deposition, which is theaerodynamic diameter after considering the water uptake in the lung,was expressed as72particle and ρwater are the density of theparticle (g cm–3) and water (1 g cm–3), respectively.The InternationalCommission on Radiological Protection (ICRP) particle deposition modelwas employed to evaluate the size-resolved health impacts on humans,–3; range: 0.3–22.9 μg m–3) were much lower than the observations  , with an accumulationmode at a median modal size of 0.91 μm . At a few sites, we also observed a small peak in thecoarse mode, which is likely attributable to the sulfate formationon dust particles.77 In contrast, the simulationresults using CSF revealed particle size distributions characterizedby an accumulation mode with a median modal size of 0.35 μm(ranging from 0.34 to 0.46 μm), which was more than two timessmaller than the observed values. Under CSF, in-cloud and gas-phaseoxidation are the primary pathways of sulfate production,4 suggesting that the sulfate mechanism overlookedin the model may also contribute to the formation of large sulfateparticles.We found that neither CSF nor APF couldexplain the observed mass concentrations and size distributions ofsulfate in China. The WRF-Chem simulation results show that the standardCSF mechanism substantially underestimates sulfate concentrationsin winter. For instance, the simulated sulfate concentrations fromDecember 2014 to February 2015 at the Wuhan station (mean: 5.4 μgmμg m–3) 1a. From μg m–3) 1e. These2 using the APFmechanism were also ameliorated , and this result was similarto that of CSF.When compared to that of CSF, the utilization ofAPF demonstrates a substantial enhancement in predicting sulfate concentrations.This is evidenced by a notable reduction in the normalized biasesof sulfate concentrations from −76% to −23% at the Wuhansite 1b. Theseliorated 1b,e. How78 In this study, aerosol surface area distributionsof aerosols in APF simulation showed a unimodal distribution and peakedat ∼0.2 μm , which was similar to the typical urban aerosol surfacedistributions presented in the previous study.4 Under this mechanism, high aerosol surface area concentrations weremainly located in the range of 0.1–0.4 μm diameter, andaerosol surface area concentration limited the APF reactions;78 thus, we posit that this mechanism might beunable to capture the observed sulfate size distribution.The APF mechanism occurs at the surface of thesuspended aerosolparticles, while the sulfate production rate of the APF reaction isproportional to the aerosol surface area concentration.11 and sensitivity analysis, we adopt a constant γ, which mayintroduce uncertainties on the quantity of sulfate particle productionbut not on the sulfate size distribution. It is unlikely that γin the APF reactions varies significantly with the aerosol particlesize because many meteorological variables affecting γ  of different particle sizes inthe same location are the same. At present, observations regardingthis issue are scant, and future research is needed. Further, ourresult of the APF simulation was consistent with the analysis by Mengand Seinfeld,79 who suggested that largeparticles produced from fog evaporation may be a major source of aerosolswith a size of 0.7 μm in California. While the modal sizes ofsulfate particles are mostly >0.7 μm in the observationsacrossChina .40 Finally, as a high-temperatureexhaust plume mixes with the ambient atmosphere, the cooling processrapidly decreases the saturation vapor pressure of sulfuric acid andwater vapor, leading to condensation onto existing atmospheric aerosols,the number concentration of which is usually high in wintertime China.81We found thatISF largely explained both the observed mass concentrationsand size distributions of sulfate in China. There are many potentialpathways for the ISF. Here, we provide detailed analysis of the Ht is SO2 3b.Previ4 and the exhaust plume was assumed to have an SO3 concentrationof 5 mg m–3.38 The theoreticalmodel simulation result showed that the pre-existing particles quicklygrow to a size of 0.9 μm in 2.0–3.3 s . The aerosolnumber concentration comparison is also important for determiningthe mechanism; however, there is a lack of relevant observations.For more details, please refer to the discussion in Supporting Information Text S3.To analyze the impact of ISF onthe formation of large sulfateparticles, we utilized a theoretical model that focuses on the rapidgrowth of these particles through the condensation of sulfuric acidonto small pre-existing particles. According to previous observations,the initial diameters of the pre-existing particles in the accumulationmode were set from 0.1 to 0.5 μm,.0–3.3 s 3c, sugge79 The rapid coolingof water vapor can result in the formation of water droplets, providinga surface for the adsorption of sulfuric acid. As more ambient airmixes into the exhaust, the relative humidity decreases, and the waterdroplets evaporate, leaving behind large sulfate particles.82 In winter, lower ambient air temperature canresult in more serious white plumes and likely enhance the formationof large sulfate particles.83 We hypothesizethat the third potential pathway involves the catalytic oxidationof SO2 to sulfuric acid by transition metals.13 The high temperature, acidity, and water contentof the exhaust can greatly accelerate catalytic SO2 oxidationcompared to that in the ambient atmosphere.84 It is possible that similar heterogeneous processes occurring onthe hot surface of a smokestack coated with transition metals couldexplain the significant portion of SO3 observed in coal-firedpower plant exhaust. In the future, additional high-precision andmultivariate simultaneous measurements are needed in the plumes and surrounding areas in China to enhanceour understanding of the processes in the ISF mechanism.We conjecture that anotherpotential pathway for ISF is the formationand evaporation of large water droplets, which can promote the generationof large particles.Supporting Information Text S4). This improved agreement can be attributed, in part, tothe spatial distribution changes resulting from the different formationmechanisms (Supporting Information TextS5), while APF and ISF simulate similar SO2 concentrations. Mostimportantly, the inclusion of ISF in the model resulted in a remarkableagreement with the observations, demonstrating a simulated accumulationmode with a modal size of 0.92 μm . This outcomesignifies that ISF not only enhanced the accuracy of predicted sulfatemass concentrations but also provided a comprehensive explanationfor the observed aerosol particle size distributions. The main differencebetween the APF and ISF mechanisms lies in the total processing volume.In the ISF mechanism, the processing volume is limited to fresh emissionplumes, while the APF mechanism encompasses the entire boundary layer.Therefore, the APF processing volume is orders of magnitude largerthan that of ISF. While the total supply of sulfate production ison the same order for the two mechanisms, the much smaller ISF processingvolume means that the number of pre-existing particles onto whichH2SO4 condenses is much smaller than those ofAPF. The much smaller number of ISF particles can therefore grow tomuch larger sizes than APF particles.Byintroducing the ISF into the atmospheric model in light of theobservations and conducting simulations, we found that, compared withCSF, ISF greatly ameliorated the simulation of sulfate concentrations were 0.62 ± 0.09 μm, significantly lower thanthe observations . In the second simulation (S2), we increased the modalsize of the ISF source from 0.9 to 2 μm while keeping its sourceat 50% of sulfate. The large difference in the size distributionsof the APF and ISF sources resulted in two modal distributions peakingat 0.35 and 2 μm, respectively, whereas the observations haveonly one peak in the accumulation mode in most cases . Although thesulfate mass concentrations were close to the ISF simulations in bothS1 and S2 simulations, the simulations cannot reproduce the observedsize distributions. The observations also showed that the wintertimesize distributions of nitrate and ammonium were generally similarto those of sulfate . Taken together, the observed size distributions of sulfate,nitrate, and ammonium and the sensitivity experiments indicated thatthe observed distributions provide an important constraint on thesources of sulfate, which is the observation basis for the ISF sourcebeing the integral source of sulfate in the winter in eastern China.Two sensitivity experimentswere conducted to examine ISF sourceparameters using the WRF-Chem model. In the first simulation (S1),we assumed that 50% of the sulfate was from the ISF source, and theother 50% was from the APF and CSF sources. The average modal sizesat the 12 observation sites . The ISF high-temperaturecan greatly increase the Mn(II)-catalyzed oxidation of SO2 to sulfuric acid since a 10 K increase from 278 K can speed up theaerosol-surface reaction by up to a factor of 10.13 The observations of similar size distributions betweensulfate and Mn aerosols with a modal size of ∼1 μm13 are also consistent with this process. Dependingon the efficiency of the ISF metal-catalyzed oxidation of SO2 on the aerosol surface or in aerosols, ISF sulfate formation mayrequire a much smaller fraction of SO3 emissions than weassumed in this study. The sulfate production rate at high temperaturesof exhaust plumes is currently unknown. Future studies are neededto account for this ISF oxidation pathway of SO2 basedon experimental data as well as the variations in metal and SO3 emission rates from different combustion sources in orderto quantify the contributions by SO3 conversion and heterogeneousSO2 oxidation to the ISF sulfate source.A recent study found that the sulfate formation rate of dissolved-manganese(Mn) catalyzed oxidation of SO–2 over eastern China due in part to a decreasein the total aerosol scattering cross section and an increase in theasymmetry factor compared to the APF simulation.85 The largest ISF effects were found in Central China (1.7W m–2) and Southwest China (1.9 W m–2), primarily due to high anthropogenic emissions and the abundanceof solar radiation.Previousmodel simulations using standard regional models were unable to accuratelysimulate micron-sized particles. Consequently, previous evaluationsof aerosol radiative and health impacts may not accurately reflectthe actual influence of micrometer-sized particles observed in China.To emphasize the significance of considering aerosol size distributionswhen assessing the impacts of inorganic aerosols on radiative forcingand human health, we conducted an examination of the effects of APFand ISF sulfate sources on the evaluations of aerosol climate andpublic health impacts across various particle size distributions.We found that APF presumably underestimates the impact of aerosolson surface radiative heating, overestimates the deposition of particlesin the tracheobronchial and alveolar regions, and underestimates thedeposition of particles in the entire respiratory system. The totalsulfate source was the same in both the APF and the ISF simulations.Although the APF and ISF simulated mass concentrations were more similarto each other than these two simulations were to the CSF simulations,the size distributions of the aerosols were substantially different2. We use69 we found that ISF induced a 64% reduction in the CCN . For example, because of the change from APFto ISF, the simulated increase in total deposition over the NorthChina Plain, Sichuan Basin, Yangtze River Delta, and Pearl River Deltareached 12% to 22%, while a minor decrease occurred over sparselypopulated western China. Thus, we argue that inorganic aerosols mayhave a greater health impact on the entire respiratory system anda smaller health effect on the bronchus and alveoli than previouslythought.The change inthe size of inorganic aerosols from the APF to ISFsimulation also significantly alters the assessment results for publichealth. The respiratory deposition model showed that the depositionof aerosols into the respiratory system peaks for small (<0.01μm) and coarse (>5 μm) particles 4f. The i11 because sulfate aerosols are strongly related toclimate change and human health impacts.8 We show in this work that the observations of aerosolsize distributions with a modal size of 0.6–1.4 μm inChina32 are much higher than that of 0.4–0.8 μm in North America.28 We find that APF and ISF mechanisms can simulate similar sulfatemass concentrations that are consistent with observations. However,their impact on the sulfate particle size distribution differs. Specifically,only the ISF mechanism can explain both the mass concentrations andthe size distributions of sulfate. Our multiple lines of evidencedemonstrate that ISF is most likely the dominant source of micrometer-sizedsulfate particles in winter in China. There are several pathways ofISF through the condensation of existing ambient small aerosols ofsulfuric acid produced by SO3 in fresh exhausts39 or rapid high-temperature catalytic SO2 oxidation bytransition metals13 on acidic aerosolsfrom the conversion of SO3 to sulfuric acid or the acidsurface of the smokestack from the deposition of sulfuric acid. ISFmay also explain the observed large sulfate particles with a modalsize of 0.8–1.5 μm in other countries such as India,Vietnam, and Singapore.93 An increasing number of studies have shown that SO3 is important for air quality.94 Our findingsfurther reinforce the critical role of sulfuric acid produced fromthe combustion emissions of SO3 in the formation of largesulfate particles. We suggest that policymakers pay more attentionto controlling the direct emissions of SO3 and sulfateformation in the plumes. In the future, considering expected changesin emission sources due to the air pollution control policies, changesin the size distribution of particles may also occur due to modificationsin emission sources and their associated particle size characteristics.Accurately assessing the effects of inorganic aerosols on radiativeforcing and human health relies heavily on understanding the sizedistribution of aerosols. Thus, continuous monitoring and assessmentsare essential to evaluate the long-term impact of air pollution controlmeasures on ISF.A much greater focus has been placed on the formation of wintertimesulfate masses in China96 In conclusion, our findings suggestthat future models should prioritize refining the processes involvedin sulfate formation and incorporating the influence of the particlesize. By doing so, we can effectively reduce uncertainties in climatechange simulations and improve the accuracy of human health assessments.On a broader scale, our research reveals thatthe ISF source of large sulfate aerosols has substantial implications.These aerosols contribute to increased surface heating and alter aerosoldeposition in the human respiratory system, thereby influencing ourunderstanding of the overall impacts of aerosols. That is, we showthat an underestimation of inorganic aerosol sizes could lead to anoverestimation of the aerosol radiative cooling feedback to increasethe surface concentrations. Thus, our study is helpful in improvingsulfate simulations in China by using atmospheric and climate models.Particle size plays a crucial role in the deposition of particulatematter in the human body. However, it is often overlooked in manyhealth risk assessments, where the effects of different particle sizeson human health are implicitly disregarded. Keeping the mass concentrationconstant, ISF can lead to a higher total mass of larger particlesdeposited throughout the entire respiratory system. However, largerparticles have a weaker ability to penetrate deep into the respiratorysystem, resulting in a smaller total mass deposition in the tracheobronchialand alveolar regions. Consequently, the health impact of larger particleson the tracheobronchial and alveolar regions is smaller. Another implicationof ISF is that it may change our understanding of the biogeochemicalcycle of elements such as sulfur and nitrogen because the particlesize is a key factor that impacts atmospheric sulfate and nitratedeposition, which greatly affects soil pH, water quality, and greenhousegas emissions.


Thrombin is a key enzyme involved in blood clotting, and its dysregulation can lead to thrombotic diseases such as stroke, myocardial infarction, and deep vein thrombosis. Thrombin aptamers have the potential to be used as therapeutic agents to prevent or treat thrombotic diseases. Thrombin DNA aptamers developed in our laboratory exhibit high affinity and specificity to thrombin. In vitro assays have demonstrated their efficacy by significantly decreasing Factor II activity and increasing PT and APTT times in both plasma and whole blood. Aptamers AYA1809002 and AYA1809004, the two most potent aptamers, exhibit high affinity for their target, with affinity constants (Kd) of 10 nM and 13 nM, respectively. Furthermore, the in vitro activity of these aptamers displays dose-dependent behavior, highlighting their efficacy in a concentration-dependent manner. In vitro stability assessments reveal that the aptamers remain stable in plasma and whole blood for up to 24 h. This finding is crucial for their potential application in clinical settings. Importantly, the thrombin inhibitory activity of the aptamers can be reversed by employing reverse complement sequences, providing a mechanism to counteract their anticoagulant effects when necessary to avoid excessive bleeding. These thrombin aptamers have been determined to be safe, with no observed mutagenic or immunogenic effects. Overall, these findings highlight the promising characteristics of these newly developed thrombin DNA aptamers, emphasizing their potential for therapeutic applications in the field of anticoagulation therapy. Moreover, the inclusion of an antidote in the coagulation therapy regimen can improve patient safety, ensure greater therapeutic efficacy, and minimize risk during emergency situations. Thrombosis is a serious condition in which a blood clot forms inside a blood vessel (artery or vein). If thrombosis is untreated, the clot can slow, block, or travel in the blood flow, causing life-threatening emergencies such as thromboembolic stroke, heart attack, venous thromboembolism (VTE), or pulmonary embolism (PE). It is estimated that  from PE .Among the activated coagulation proteinases, thrombin is a unique coagulation protein that plays a central orchestrating role in procoagulation, anticoagulation, and platelet activation . In procDabigatran etexilate (Pradaxa) is an FDA-approved direct and reversible thrombin inhibitor. It is an orally administered anticoagulant specifically targeting thrombin, and has been approved for various indications, including prevention of systemic embolism in non-valvular atrial fibrillation, prevention of recurrent venous thromboembolism (VTE), and prevention of stroke. Dabigatran is an active form of dabigatran etexilate that competitively and selectively binds thrombin, preventing the conversion of soluble fibrinogen to insoluble fibrin and inhibiting thrombin-induced platelet aggregation . It is aAptamers are RNA and DNA oligonucleotides that bind to specific target molecules with high affinity and specificity, in which they are similar to antibodies; however, they have many advantages over antibodies, such as long shelf life, ease of modification, cost-effectiveness, and lower immunogenicity. In the coagulation cascade, aptamers have been developed to interact with multiple coagulation factors and cofactors, offering antidote-mediated controllability that safeguards the intrinsic coagulation capacity from exhaustion ,14. As aThe first anti-thrombin DNA aptamer  to select aptamers that bind to the active site of thrombin. Several anti-thrombin aptamers with binding high affinity were successfully generated. The two most potent aptamers, AYA1809002 and AYA1809004, exhibit high affinity for their target, with affinity constants (Kd) of 10 nM and 13 nM, respectively. Furthermore, they demonstrate in vitro assay activity by effectively decreasing Factor II activity and increasing both prothrombin time (PT) and activated partial thromboplastin time (APTT) in plasma and whole blood in a dose-dependent manner. Crucially, the thrombin inhibitory activity of these aptamers can be effectively reversed through the use of reverse complement sequences. This feature provides a valuable mechanism to counteract their anticoagulant effects, enabling timely and precise intervention in emergency scenarios. Our results show that these two aptamers are stable in whole blood at room temperature and 37 °C for at least 24 h. To enhance their stability in the bloodstream, both aptamers were chemically modified through crosslinking with TAG, PEG, or cholesterol moieties. These modified aptamers successfully demonstrated in vitro activity, indicating that they retain their functionality. Additionally, both of these thrombin aptamers were determined to be safe, without any observed immunogenic or mutagenic effects. Our findings suggest that AYA1809002 and AYA1809004 are effective and safe anti-thrombin candidate aptamers that can potentially be used to treat thrombosis in the future.The immobilization of human alpha-thrombin native protein  on PierceTM NHS-Activated Magnetic Beads  was performed as described by standard protocol, as follows. One mg of thrombin (MW 36.7 kDa) was dissolved in coupling buffer  procedure was performed as previously described in . A synthThe single-stranded DNA (ssDNA) obtained after each round of SELEX underwent library preparation for Illumina sequencing using the TruSeq ChIP sample preparation protocol. A total of nine DNA libraries comprised of paired-end indexed sequences were created by pooling the samples. The Illumina TruSeq ChIP sample preparation kit reagents were utilized for this process to facilitate cluster generation and subsequent DNA sequencing. The initial DNA input (50 µL of 200 pg/µL) underwent processes to blunt-end and phosphorylate the molecules. Additionally, a single “A” nucleotide was attached to the 3Native human alpha-thrombin protein  was added to each well of a 96-well plate  at a concentration of 250 nM in Tris-buffered saline with a volume of 100 µL per well. The plate was then incubated at 4 °C for 16 h to allow the wells to become coated with thrombin. After removing the thrombin solution, the wells were washed and blocked using blocking buffer  for a 1 h duration. Next, biotinylated aptamers at the indicated concentrations in a volume of 100 µL were added to the wells in an incubation buffer  and incubated for 1 h at room temperature. In the competition binding assay, unlabeled aptamers were added along with the biotinylated aptamers, with the unlabeled aptamers being present in a 100-fold excess compared to the biotinylated aptamers. Following three washing steps with wash buffer , streptavidin-HRP  was added at a dilution of 1:5000 in the incubation buffer. The plate was washed again three times to remove any unbound reagents. The bound biotinylated aptamers were then detected using 3,3The RNAfold webserver was used to predict the secondary structure of the aptamers. This server is available online and can be used for RNA and ssDNA prediction. Minimum Free Energy (MFE) secondary structure prediction aims to find the structure with the lowest free energy. It predicts the structure by minimizing the thermodynamic free energy of the nucleic acid molecule.g. In vitro activity assays were performed on ACLTOP analyzer manufactured by Instrumentation Laboratory, which is a type of coagulation analyzer commonly used in clinical laboratories to measure clotting times and assess coagulation parameters. The ACLTOP analyzer is designed to perform a wide range of coagulation tests, including Factor II activity, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, and other related assays. ACLTOP analyzer uses HemosIL reagents developed by Instrumentation Laboratory company  to perform coagulation tests.Sterile human blood in sodium citrate was purchased from the Rockland Immunochemicals, Inc . Plasma was prepared by centrifugation for 10 min at 2000× g for 10 min at 4 °C. For the dose-dependent study, concentrations of 1 µM, 2 µM, 3 µM, and 4 µM of AYA1809002 and AYA1809004 were utilized.For screening in vitro activity of the aptamers, human citrated plasma (0.5 mL) was incubated in the absence or presence of 2 µM thrombin aptamers or dabigatran for 2 h. Factor II activity was measured using human plasma immunodepleted of Factor II for the quantitative determination of Factor II activity in citrated plasma based on the prothrombin time (PT) assay, for which an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory  was used. To evaluate the dose-dependent activity of the aptamers in whole blood, human blood samples were collected in citrate-treated tubes. Subsequently, the blood samples were incubated at room temperature for 2 h in both the absence and presence of various concentrations of thrombin aptamers. To measure the Factor II activity using the ACLTOP coagulation analyzer, plasma was separated from the cells by centrifugation at 1500× g for 10 min at 4 °C. For the dose dependence study, concentrations of 1 µM, 2 µM, 3 µM, and 4 µM of AYA1809002 and AYA1809004 were utilized.Human citrated plasma (0.5 mL) was incubated in the absence or presence of 2 µM thrombin aptamers or dabigatran for 2 h. PT time was measured using RecombiPlasTin 2G for quantitative determination in human citrated plasma of PT and Fibrinogen on an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory . To assess the activity of the aptamers in a dose-dependent manner in whole blood, human blood samples were collected in citrate-treated tubes. The blood samples were then incubated at room temperature for 2 h in the absence or presence of varying concentrations of the thrombin aptamers. To measure the Prothrombin Time (PT) on the ACLTOP coagulation analyzer, plasma was obtained by centrifuging the samples at 1500× Human citrated plasma (0.5 mL) was incubated in the absence or presence of 2 µM thrombin aptamers or dabigatran for 2 h. APTT time was measured using the synthetic phospholipid reagent SynthASIil for quantitative determination in human citrated plasma on an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory .A Thrombin Inhibitor Screening Assay Kit (fluorometric)  was employed to test the direct binding ability and inhibitory potential of the AYA1809002 and AYA1809004 aptamers against thrombin. The procedure was carried out following the standard protocol provided with the kit. In brief, aptamers at different concentrations, dabigatran at 2 µM, and controls were added to their respective wells on the plate . Subsequently, a thrombin enzyme mixture was added to each sample and control well. Following an incubation period of 20 min, a substrate mixture was introduced into each sample and control well. Fluorescence measurements were taken using a SYNERGY/HTX multi-mode reader  with the excitation/emission wavelengths set at 360/460 nm. The measurements were recorded for 45 min at a temperature of 37 °C while ensuring protection from light.To evaluate the stability of the selected aptamers  and compare them to dabigatran, citrated blood collected from a donor was incubated at room temperature in the absence or presence of 1 µM of AYA1809002, AYA1809004, or dabigatran for 0.5, 2, 4, 6, and 24 h. At each time point, blood samples from both the treated and untreated groups were collected and plasma was obtained by centrifugation. The collected plasma samples were then frozen for further analysis. Factor II activity was measured using human plasma immunodepleted of Factor II for quantitative determination of Factor II activity in citrated plasma based on the prothrombin time (PT) assay, for which an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory was used. PT time was measured using RecombiPlasTin 2G for quantitative determination in human citrated plasma of PT and Fibrinogen on an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory. The stability experiments at 37 °C were conducted as follows: citrated blood was collected from a donor and incubated with or without 1 µM AYA1809002 or AYA1809004 at 37 °C. At each time point, blood samples from both the treated and untreated groups were collected and plasma was obtained by centrifugation. Factor II activity, PT time, and APTT time were measured for each sample. To assess the relative activity, the measured activity of the treated whole blood sample was divided by the activity of the corresponding untreated sample collected at the same time point.To provide additional evidence of the aptamers’ stability in an in vitro setting, we conducted the same stability assay using whole blood at 37 °C. At various time intervals, we utilized biotinylated reverse complement sequences along with streptavidin-coated magnetic beads to selectively isolate the aptamers from the blood samples. Following elution from the magnetic beads, the aptamers underwent analysis using a Fragment Analyzer system  to assess their integrity.Citrated blood collected from a donor was incubated in the absence or presence of 1 µM AYA1809002 at room temperature. After 2 h incubation, the indicated concentration of the reverse complement strand was added to the citrated blood sample. After an additional incubation time of 2 h, plasma was obtained through centrifugation. Factor II activity was measured using human plasma immunodepleted of Factor II for the quantitative determination of Factor II activity in citrated plasma based on the prothrombin time (PT) assay, for which an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory  was used. The PT time was measured using RecombiPlasTin 2G for quantitative determination in human citrated plasma of PT and Fibrinogen on an ACLTOP coagulation analyzer manufactured by Instrumentation Laboratory . As a control, 1 µM of dabigatran was incubated with the citrated blood.g for 15 min at room temperature. The resulting supernatant consisting of platelet-rich plasma (PRP) was collected and aliquots of 500 µL were transferred to microfuge tubes containing 50 µL of 300 mM CaClFor the inhibition study of clot-bound thrombin, we utilized the method previously described in ,33 with Modified aptamers chemically crosslinked with TAG, PEG, and cholesterol entities at the 3Human PBMCs were isolated from buffy coats provided by Carter BloodCare. Isolation was carried out through density-gradient centrifugation utilizing Ficoll-Paque Plus from GE Healthcare. The isolated human PBMCs suspended in cRPMI media were subjected to stimulation with or without the AYA1809002 and AYA1809004 aptamers and a control aptamer. Various concentrations  of these aptamers were used, along with positive control stimuli such as LPS (200 ng/mL), ODN 1826 , or a combination of LPS (100 ng/mL) and ODN 1826 (10 µM). Following incubation periods of 24 and 72 h, the cell supernatant was collected via centrifugation at 200 g for 3 min. Cytokine levels were evaluated using the LEGENDplex Human Inflammation Panel 1 from BioLegend  following the manufacturer’s protocol, soluble analytes were acquired using a Navios EX flow cytometer , and subsequent analysis was performed using BioLegend’s LEGENDplexThe mutagenic potential of AYA1809002 and AYA1809004 was evaluated using the Ames test with the Xenometrix Ames MPF PENTA I kit, including S9 and Positive Controls . The assay procedure followed the protocol outlined in our previous publication .t-test for comparisons between two groups. Data are presented as mean ± standard deviation (SD). A p-value of ≤0.05 was deemed statistically significant, and is indicated by an asterisk (*), while a p-value of ≤0.01 is denoted by a double asterisk (**).Statistical significance was assessed using GraphPad Prism version 10.0.0 (131), 2023  employing nonparametric methods such as the Mann–Whitney test or unpaired Student’s TM NHS-Activated Magnetic beads following established protocols. It is worth noting that the immobilization process of thrombin on NHS beads involves a random attachment in which lysine (Lys) residues can potentially bind to NHS groups on the beads. In light of the steric challenges posed by the structure of activated thrombin, specifically the deep groove of its active site, utilizing lysine residues located within or near the active site for binding to the beads might be intricate. However, even if such binding occurs, it is important to note that other molecules of thrombin with accessible binding sites remain available for subsequent binding of aptamers  and wp2 uvrA [To assess the mutagenicity of the aptamers, we performed Ames test, a widely used assay for evaluating the mutagenicity of chemical compounds. The Ames test is a bacterial reverse mutation assay that detects the ability of a substance to induce mutations in the DNA of certain strains of bacteria. It utilizes various bacterial strains, including wp2 uvrA ,41,42,43Thrombin plays a crucial role as a central orchestrator in the blood coagulation cascade, encompassing various essential functions such as pro-coagulation, anticoagulation, platelet aggregation, and inflammatory activities. From hirudin, to parenteral direct thrombin inhibitors , to oral direct thrombin inhibitors (ximelagatran and dabigatran), scientists have been working hard to investigate and develop a range of thrombin inhibitors in recent decades. However, the use of thrombin inhibitors is limited or contradicted in clinics due to drug interactions, side effects, risk of bleeding, and cost. The biggest challenge in developing thrombin inhibitors is the need to reduce bleeding-related risks while maintaining high anti-thrombin efficacy. Therefore, developing a thrombin inhibitor that is efficient, safe, affordable, and has minimal side effects remains highly necessary. In this study, we employed theSystematic Evolution of Ligands by Exponential Enrichment (SELEX) method to generate a diverse range of aptamers targeting the active site of thrombin. Notably, during the SELEX procedure we introduced dabigatran as a competitor to enhance the selection specificity.Employing this approach, we successfully obtained a pool of aptamers that displayed high affinity and selectivity toward thrombin. Within this enriched pool, aptamers AYA1809002 and AYA1809004 demonstrated the highest binding affinity for thrombin among all of the identified candidates. The effectiveness of these aptamers was evaluated in vitro using an FDA-approved ACLTOP instrument in both plasma and whole blood samples. The assessment focused on measuring Factor II activity, PT time (Prothrombin Time), and APTT time . The results of the study demonstrated a clear dose-dependent effect of both aptamers on Factor II activity. As the concentration of the aptamers increased, there was a corresponding reduction in Factor II activity. This indicates that the aptamers effectively inhibited the functionality of Factor II, a key component of the coagulation cascade. Furthermore, the study revealed an increase in both PT time and APTT time with increasing concentrations of the aptamers. PT time is a measure of the extrinsic pathway of the coagulation cascade, while APTT time assesses the intrinsic pathway ,46,47,48Extensive research has been conducted over the years to find reversal agents for anticoagulation therapy. Numerous studies have focused on identifying and evaluating effective strategies to reverse the anticoagulant effects of various anticoagulants, including thrombin inhibitors ,50,51,52Chemical modifications such as crosslinking with TEG, PEG, or cholesterol entities were employed to enhance the stability of the aptamers in the bloodstream. Remarkably, these modifications did not compromise the effectiveness of the aptamers in terms of inhibiting Factor II activity and prolonging PT and APTT times. The modified aptamers retained their inhibitory effects on the measured coagulation parameters, demonstrating their potential as stable and functional candidates for anticoagulation therapy. Aptamers AYA1809002 and AYA1809004 were shown to be safe, as they demonstrated a non-immunogenic nature. This indicates a favorable safety profile for these molecules, making them potentially well-tolerated in therapeutic applications. Additionally, a mutagenicity assessment, specifically the Ames test, confirmed that these aptamers do not possess mutagenic properties. These findings provide strong evidence supporting the safety and biocompatibility of AYA1809002 and AYA1809004, reinforcing their potential as promising candidates for therapeutic interventions. It is worth noting that the selected aptamers are rich in G. This observation suggests the potential formation of G quadruplex structures, a prevalent motif found in numerous DNA aptamers. This structural characteristic could potentially enhance the stability of these aptamers. When Bock and Toole discovered the first single-strand DNA aptamer against human protease thrombin, they discovered that the sequence of GGTTGG was the most conserved sequenced in almost all of their selected clones . Greatlyormation . Among tThe selected aptamers were subjected to testing to evaluate their ability to inhibit both fluid-phase thrombin and clot-bound thrombin. This comprehensive assessment allowed us to determine their efficacy in targeting thrombin in different contexts. By inhibiting fluid-phase thrombin, the selected aptamers can directly prevent excessive thrombin activity in the blood, while their ability to inhibit clot-bound thrombin offers the potential to prevent further thrombin-mediated clotting events and inhibit the propagation of the clot. This dual inhibitory action further highlights their versatility and therapeutic value in managing thrombotic conditions.In this study, we have demonstrated the properties and anticoagulant activity of two anti-thrombin aptamers. Our findings suggest that AYA1809002 and AYA1809004 can be effective, safe, and affordable potential therapies for thrombosis.Our primary goal in developing these new anti-thrombin DNA aptamers is to broaden the selection of aptamer candidates that could eventually be considered for clinical applications. Our comprehensive investigation has revealed compelling evidence supporting the efficacy and safety of AYA1809002 and AYA1809004 as potent anti-thrombin candidates. These aptamers demonstrate remarkable performance in vitro in inhibiting thrombosis, effectively countering excessive clot formation, and their activities are easily reversible by administration of the antidote. Furthermore, a comprehensive assessment of their safety profile through in vitro studies demonstrated minimal negative impacts, reinforcing their potential in clinical applications. It is important to note that in vitro evaluations serve as only an initial step in assessing the effectiveness of aptamers and their potential clinical applications. Further studies, including in vivo investigations and clinical trials, remain necessary to validate these findings and determine the safety and efficacy of the aptamers in more realistic and dynamic physiological environments.


Diisocyanates are a group of chemicals widely used in different industrial applications. The critical health effects related to diisocyanate exposure are isocyanate sensitisation, occupational asthma and bronchial hyperresponsiveness (BHR). Industrial air measurements and human biomonitoring (HBM) samples were gathered in specific occupational sectors to examine MDI, TDI, HDI and IPDI and the respective metabolites from Finnish screening studies. HBM data can give a more accurate picture of diisocyanate exposure, especially if workers have been exposed dermally or used respiratory protection. The HBM data were used for conducting a health impact assessment (HIA) in specific Finnish occupational sectors. For this purpose, exposure reconstruction was performed on the basis of HBM measurements of TDI and MDI exposures using a PBPK model, and a correlation equation was made for HDI exposure. Subsequently, the exposure estimates were compared to a previously published dose–response curve for excess BHR risk. The results showed that the mean and median diisocyanate exposure levels and HBM concentrations were low for all diisocyanates. In HIA, the excess risk of BHR from MDI exposure over a working life period was highest in the construction and motor and vehicle industries and repair sectors, resulting in estimated excess risks of BHR of 2.0% and 2.6%, and 113 and 244 extra BHR cases in Finland, respectively. Occupational exposure to diisocyanates must be monitored because a clear threshold for DI sensitisation cannot be established. Diisocyanates (DIs) are chemicals that contain two isocyanate functional groups . DIs are widely used in different industrial applications, most notably in the manufacturing of polyurethanes and as hardeners in industrial paints, glues, varnishes and resins ,2. In thThe most commonly used DIs include 4,4′-methylene diphenyl diisocyanate , toluene diisocyanate , hexamethylene diisocyanate  and isophorone diisocyanate . MDI, TDI and HDI account for more than 95% of the total volume of DIs used in the EU. DIs share common toxicological properties with the functional NCO groups. The harmonised EU Classification, Labelling and Packaging (CLP) system (EU regulation 1272/2008) classifies these widely used DIs as dermal and respiratory sensitisers, and as eye, skin and respiratory irritants [The use of DIs is not expected to decline in the near future because there are no suitable alternatives for most DI applications. To minimise the health risks caused by DIs, the European Commission recently adopted a restriction under the European regulation concerning the Registration, Evaluation, Authorization and Restriction of Chemicals  for the use of DIs. According to this restriction, DIs cannot be placed on the market or used in concentrations above 0.1% in weight unless the industrial and professional users have completed adequate professional training in the safe use of DIs [Occupational exposure to DIs primarily occurs as inhalation and dermal exposure but may also occur through the gastro-intestinal tract after hand-to-mouth exposure , althougThe monitoring of inhalation exposure to DIs is technically challenging due to their reactivity . Air meaIn this paper, we summarise data gathered from the Finnish Institute of Occupational Health (FIOH) databases of industrial hygiene air measurements and HBM samples, covering DI exposure in several industrial sectors in Finland during 2008–2021. These data were used for a health impact assessment (HIA) together with ECHA’s RAC dose–response curve  and a phIndustrial hygiene air measurements of MDI, TDI, HDI and IPDI and urinary measurements of DI-derived diamines 4,4′-methylenedianiline (MDA), toluene diamine (TDA), hexamethylene diamine (HDA) and isophorone diamine (IPDA) were gathered for specific sectors from the Finnish registers of industrial hygiene and biomonitoring measurements between 2008 and 2021. These registers are upheld by FIOH, according to the law on the activities and funding of FIOH (STM 159/1978) and contain measurements taken by FIOH as part of its expert services to Finnish workplaces. The law on the activities and funding of FIOH (STM 159/1978) defines the information gathered in the FIOH databases and gives FIOH permission to use the collected data for research purposes. The database, which is not publicly available, conforms to the European General Data Protection Regulation . It consists of the original analytical results and is not a biobank, as no samples are stored. Informed consent, including consent to store the measurement results in the FIOH database, was obtained from all the workers who provided samples for analysis. The contextual information stored in the biomonitoring database concerns sample timing, sex, smoking information, job titles and company information.www.ttl.fi/biomonitorointi accessed on 29 December 2022). Information on this worker population is given in the The air monitoring samples included in the current data analysis were collected from the personal breathing zones of the workers. The workers’ post-shift urine samples were collected at the end of the week or after the working period that involve the use of DIs, in accordance with the FIOH sampling protocol (3 using molecular weight (MW) to make the DI levels comparable. Conversion was performed using the equation:The air samples were analysed at FIOH using either liquid chromatography mass spectrometry (LC-MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS). The limit of quantitation (LOQ) for the air measurements was 0.004 µg DI/sample for MDI, 0.002 µg DI/sample for TDI, 0.002 µg DI/sample for HDI, and 0.004 µg DI/sample for IPDI. Urinary diamines were analysed either by FIOH  or by hired subcontractors in the United Kingdom  or Germany . The LOQs for the urinary diamines were 0.1–1.0 µg/L for MDA, 0.1–0.6 µg/L for TDAs, 0.1–0.6 µg/L for HDA and 0.1–0.9 µg/L for IPDA. In statistical modelling, air and urinary values below LOQ were replaced with values of LOQ/2. Air concentrations for different DIs were converted to µg NCO/mMDI and TDI exposure reconstruction was performed using a PBPK model . This moThe distribution of urinary biomarker levels for each industry was estimated from the quantiles of the empirical distribution of the HBM samples using linear interpolation. The average air exposures were then estimated by Monte Carlo (MC) integration  using a total of 10,000 iterations.Reverse dosimetry was performed using a Bayesian Metropolis Hastings Monte Carlo (MHMC) algorithm  and a we3) and urinary HDA concentrations (1.36–27.7 µg HDA/g creatinine). The HDI and HDA concentrations were calculated using the equation:As there was no PBPK model for HDI, the exposure reconstruction for HDA data was performed using the equation reported by Maître et al. , which iTo reduce overestimation caused by possible non-occupational background exposure, we subtracted an estimated non-occupational background level of 0.2 µg/L from the measured exposure levels used for exposure reconstruction. The background level of 0.2 µg/L was based on the available literature data on the diamine levels in the general population ,19,20,21The estimated external DI levels were compared to the exposure–excess risk relation for BHR and air NCO concentration estimated by the RAC . The excThe number of workers exposed to DIs in Finland  was estiThe polyurethane, plastic products or furniture manufacturing sector included workers from both the plastic industry (manufacturing of polyurethane and rigid foam products) and the furniture manufacture sectors . These s3) were for MDI n = 2 (0.5%), TDI n = 0 (0%), HDI n = 2 (1.4%) and IPDI n = 0 (0%).The highest measured MDI and HDI levels were in the motor vehicle manufacturing and repair sector, whereas TDI exposure was highest in the manufacture of polyurethane, plastic products or furniture, and among assemblers of industrial products. Due to the low number of measurements, the IPDI data were not further stratified by industrial sectors. The number of air monitoring samples exceeding Finnish short-term (15 min) OEL for DIs .We summarised the data gathered from the FIOH databases of industrial hygienic air measurements and HBM samples, covering DI exposure in several industrial sectors in Finland in 2008–2021. After exposure reconstruction, HBM data were utilised for the human HIA of excess risk of BHR according to ECHA’s RAC dose–response curve .3. No 8 h time-weighted average (TWA) OELs have been set for DIs in Finland. The European Commission’s Advisory Committee on Safety and Health at Work (ACSH) has recently proposed a binding occupational exposure limit of 10 µg/m3 , reducing it to 6 µg/m3 from 1 January 2029 [3 and 34 measurements (4.9%) exceeded 6 µg NCO/m3. Although the Finnish mean/median air monitoring data levels were well below these levels in the analysed period, even these levels may not be without a risk, as it is currently not possible to identify a threshold for the sensitising properties of DIs [In the Finnish data, the mean and median air exposure levels and HBM concentrations were low for all DIs in all the occupational sectors . Howeverary 2029 . Twenty s of DIs .3 for TDI and HDI, respectively) when sampled at the end of the shift. Similarly, the Deutsche Forschungsgemeinschaft (DFG) has given a biological tolerance (BAT) value of 15 µg/g creatinine, measured as urinary HDA, for HDI and a BAT value of 5 µg/g creatinine, measured as the sum of urinary 2,4- and 2,6-TDA, for TDI [However, it should be noted that in most of the known high exposure-level work tasks, such as spray painting and polyurethane blasting, the workers used RPE, and the air samples were collected outside the respirator. When RPE is used, biomonitoring can give a more realistic overview of the real exposure. Biomonitoring can also give information on exposure via other routes, including via dermal absorption or ingestion (due to hands-to-mouth exposure). Dermal exposure has shown to contribute to the sensitisation risk of DIs ,10. Ther for TDI ,30.3. However, in spraying applications, the highest HDI air concentrations have been measured during spray painting and coating (>400 µg NCO/m3) [3 [3 [In addition to the RAC restriction proposal on DIs , occupat NCO/m3) ,33,34,35O/m3) [3 ,36,37,383) [3 [3 ,42,43.In the construction sector, the Finnish HBM data  for MDI The HDA levels reported in the motor vehicle manufacturing and repair sector in the UK have been lower . The levels in the range of <0.1–0.87 µg/L (range) for urinary MDA have been reported also by Robert et al. [There are also significant uncertainties related to the highly skewed nature of exposure data on the risks and on human HIA. As can be seen in t et al. . The Natt et al.  reportedThe arithmetic mean values in each sector were affected by a few high values, which were above the levels for which ECHA’s RAC  has a deOne important uncertainty in the human HIA was related to the number of exposed workers. In this HIA, we used estimated figures of exposed workers for each sector. However, these estimates are generally difficult to extract. In this case, we used the average numbers of exposed workers estimated on the basis of the FIOH FINJEM database, the FIOH biomonitoring register, and the number of workers in Statistics Finland records, as well as estimates made as part of REACH diisocyanate restriction . BecauseIn The advantages of performing risk assessments or HIA by using HBM data instead of air monitoring data are related to RPE use and skin exposure. The only way to ensure RPE effectiveness when working with DIs is to take HBM samples, as air concentration measurements would overestimate personal exposure. In addition, skin exposure to DIs contributes to the risk of BHR and can be monitored by HBM. For example, in the motor and vehicle manufacture and repair sector, many paints and coatings contain HDI, which cannot be applied without protective clothing and RPE. However, it remains challenging to relate reconstructed exposure levels based on HBM to a risk relationship based on inhalation exposure.Although the observed exposure levels were generally low in comparison to the available OELs for DIs, high exposures may occur in some tasks, resulting in an elevated risk of BHR. The highest numbers of excess BHR cases were estimated to occur in the construction sector and motor vehicle manufacturing and repair, which also showed the highest numbers of exposed workers. In the future, the sensitivity of the analysis methods in DI screening should enable the detection of even lower HBM levels of these compounds to efficiently monitor exposure. This requires also better characterisation of general population background levels from environmental sources and consumer products, as the current information on background levels in adult population is rather limited. The exposure levels of DIs may also be lower in the future, as the new EU regulation/restriction (EC 2020/1149)  on worki