Datura wrightii, and its hawkmoth pollinator, Manduca sexta, reveals that floral relative humidity acts as a mutually beneficial signal in this system. The distinction between cue- and signal-based functions is illustrated by three experimental findings. First, floral humidity gradients in Datura are nearly ten-fold greater than those reported for other species, and result from active  rather than passive (nectar evaporation) processes. These humidity gradients are sustained in the face of wind and are reconstituted within seconds of moth visitation, implying substantial physiological costs to these desert plants. Second, the water balance costs in Datura are compensated through increased visitation by Manduca moths, with concomitant increases in pollen export. We show that moths are innately attracted to humid flowers, even when floral humidity and nectar rewards are experimentally decoupled. Moreover, moths can track minute changes in humidity via antennal hygrosensory sensilla but fail to do so when these sensilla are experimentally occluded. Third, their preference for humid flowers benefits hawkmoths by reducing the energetic costs of flower handling during nectar foraging. Taken together, these findings suggest that floral humidity may function as a signal mediating the final stages of floral choice by hawkmoths, complementing the attractive functions of visual and olfactory signals beyond the floral threshold in this nocturnal plant-pollinator system.Previous studies have considered floral humidity to be an inadvertent consequence of nectar evaporation, which could be exploited as a cue by nectar-seeking pollinators. By contrast, our interdisciplinary study of a night-blooming flower,  Flowers are well known for attracting pollinators with visual and olfactory displays. Here, the authors show that in a nocturnal, desert pollination system, flower choice by pollinators is also mediated by floral humidity. Although floral scent and color can attract pollinators at a scale of meters9, they cease to be informative once pollinators arrive at a flower’s threshold (mm to cm distance), in the absence of additional information, such as contrasting nectar guides10, scented pollen11 or nectar12. Recently foraged flowers can remain scented, turgid, and pigmented minutes to hours after nectar or pollen has been removed by an earlier visitor, yet it is commonly observed that pollinators reject some flowers upon inspection, without landing14. Pollinators likely make such decisions at a short range from the flowers based on more reliable sources of information as they navigate a patch of flowering plants16. For instance, floral primary metabolism and transpiration produce gradients in carbon dioxide (CO2) concentration and relative humidity (RH) within the headspace of a newly opening flower (mm to cm distance), which more reliably indicate nectar availability before pollinators commit to probing or visiting a flower18.The spatial scale at which pollinators are attracted by floral traits has important consequences for pollinator foraging efficiency19. In the context of communication, when a respiring animal (sender) exhales CO2, it alerts nearby mosquitoes (receivers) to a potential blood meal, with detrimental consequences for the sender of the cue20. In contrast, signals mediate communication between senders and receivers that results, on average, in fitness benefits to both parties21. Despite longstanding debate on signal classification and evolution23, behaviorists distinguish signals from cues using the following criteria: (1) senders provide clear, measurable information, (2) that has evolved for the purpose of communication with receivers, which (3) elicit a distinctive  response from the recipient, resulting in (4) fitness consequences that are favorable, on average, to both parties23. In addition, although exceptions exist, most signals incur significant metabolic, social- or health-related costs that are thought to ensure evolutionary stability against cheating24. In plant–pollinator communication, floral signals may evolve as “indices” , as “icons” (the form is similar to the content but can be decoupled), or as “symbols” .All animals utilize cues—the sensory information available in their environments—to navigate and survive. When cues are produced inadvertently by the movement or metabolism of other organisms, they can be exploited by eavesdroppers2 is a cue by which pollinators can assess nectar presence and profitability. Night-blooming flowers such as Datura wrightii accumulate and release CO2 upon anthesis when nectar is most available, but floral CO2 decays to ambient levels soon after anthesis. Thus, the above-ambient floral CO2 alerts pollinators to the presence of newly opened, profitable flowers25. Flowers with above-ambient CO2 are more attractive to hawkmoths (Manduca sexta) than those with ambient CO226, but in the absence of a sustained difference in profitability, hawkmoth preference for high CO2 diminishes to chance over subsequent floral visits27. Hawkmoths (as receivers) can utilize above-ambient floral CO2 as an ephemeral profitability cue for freshly opened flowers, due to its correlation with unexploited floral rewards. It remains unclear whether plants benefit by furnishing floral CO2 as a cue, as they might derive greater fitness benefits by withholding nectar profitability information28.Current evidence suggests that floral CO2, floral humidity appears to indicate nectar presence to foraging pollinators as a direct physical consequence of nectar evaporation, rather than as a correlated aspect of anthesis. Unlike CO2, floral humidity may therefore alert pollinators to flowers that refill nectar after anthesis. The evening primrose flower (Oenothera cespitosa) presents 4–6% above-ambient RH in its headspace, which decays to ambient levels within 30 min after anthesis29. Floral manipulations revealed that nectar evaporation accounts for half of the floral humidity in O. cespitosa, strongly suggesting a function as a cue. A recent survey30 of floral humidity from 42 plant species in a common garden reported a range of 0.05–3.7% above-ambient RH (henceforth ΔRH) in floral headspace, including species that lack floral nectar31. In the laboratory, Hyles lineata, a common pollinator of O. cespitosa, prefers probing non-rewarding artificial flowers with above-ambient RH over flowers with ambient RH, despite the absence of sugar rewards29. Finally, the generalist bumblebee, Bombus terrestris, has been shown to discriminate ΔRH on artificial flowers when paired with sugar rewards in a lab setting32. Together, these findings indicate that floral humidity influences pollinator foraging decisions, but also suggest that our view of floral RH as an unavoidable consequence of nectar evaporation may be overly simplistic.Unlike CO23. Some of these gaps include the physiological sources of floral RH, the efficacy  of floral RH gradients in the face of environmental noise33, and the mechanisms of pollinator perceptual and behavioral responses to realistic RH gradients in space and time. In contrast to the rapid dissipation (~30 min) of floral RH from the narrow nectar tube and open petals of O. cespitosa29, we hypothesized that larger, trumpet-shaped corollas  might sustain humidity gradients beyond anthesis , exceeding levels previously reported for angiosperm flowers30 by a factor of 10. We find that Datura floral humidity is not a passive consequence of nectar evaporation but, instead, is a persistent floral trait that results from gas exchange through floral stomates. Neurophysiological responses from Manduca antennal hygrosensing neurons confirm that moths perceive minute differences in floral humidity, and experimental occlusion of the hygrosensing sensillum abolishes their innate behavioral preference for humid flowers. We experimentally decouple floral humidity from nectar presence in artificial flowers, measuring moth behavioral responses to three treatments representing alternative functions for floral humidity: an uninformative trait, an informative trait positively associated with nectar, or an informative trait negatively associated with nectar. Our results show that floral humidity benefits Datura plants through increased pollinator visitation and benefits Manduca moths by reducing energetic expenditure related to flower handling time. Moths’ strong innate preference for humid flowers persisted irrespective of the presence or absence of nectar in the artificial flowers. Furthermore, the maintenance of unusually high ΔRH in a desert/grassland plant facing severe challenges in water balance suggests that floral RH in Datura is a costly sexual signal, evocative of an overstated animal courtship display36. In summary, using a neuroethological approach combined with floral physiology, animal behavior, and sensory ecology, we find that the functional role of floral humidity in this nocturnal pollination system is complementary to other attractive signals like floral scent and color, but is more spatially relevant at the threshold of the flower. We discuss the range of possible roles floral humidity can play as an informative trait beyond the (limited) current view that it is a cue for the presence of nectar.We evaluate the role of floral humidity in the well-studied mutualistic relationship between Datura wrightii exhibit an appreciable vertical humidity gradient (ΔRH) within the floral tube, with the greatest ΔRH observed at the base of the flower tube (= 0 mm) , persisting across a broad range of background ambient RH  was recorded when ambient RH was 10–20%, while the lowest ΔRH  was observed when ambient RH was 50–60%. We compared the floral humidity curves across background RH levels using two model parameters: the decay rate (α) of the curve and the intercept y0 (see Methods). Multiple comparisons suggested that the intercept (y0) differs across the range of background humidity but that the decay rate (α) does not , 20–30% , and 30–40% . No other differences were found for floral humidity at any other levels of background RH.At what spatial scale might floral RH be a relevant stimulus, and for how long after flowers open? Flowers of  RH Fig. . At the  RH Fig. . Horizon RH Fig. . At 140 Datura is not an artifact of greenhouse conditions, we sampled from wild D. wrightii plants growing in their natural habitat at multiple locations near Tucson, Pima Co., Arizona, USA . Even under field conditions and at backgrounds of 20–40% ambient RH, we recorded 26.58 ± 6.71% (mean ± SD) ΔRH in the flower tube and 2.64 ± 4.02% ΔRH at the flower opening  and the intercept (y0) of the floral humidity gradient were statistically indistinguishable between the still air and still air + nectar-extracted treatments 37 wing fanning of a hovering moth. To test this prediction, we allowed moths to forage on newly opened Datura flowers while simultaneously recording the humidity in the floral tube  for nectar and only 3.16 ± 0.61% ΔRH for water  corolla limb Fig.  inset inManduca39. The coeloconic type B is a small (2 µm) peg-in-pit sensillum not easily visualized with microscopy. In contrast, the styliform complex is a large (30–40 µm), flexible-peg-type sensillum on the leading edge of each antennal annulus and is easily distinguishable from other sensory pegs  trichoid sensilla on male Manduca antennae 49 or traverse different habitats at the scale of a landscape (m to km).The few studied cases of insect hygrosensory neurons suggest that the response properties of the neurons are a function of both instantaneous humidity and the rate of change in humidity. Typically, the impulse frequencies of the moist sensing neuron increase in proportion to the instantaneous RH and rate of change in RH, whereas the dry sensing neurons respond antagonisticallyManduca with a choice between empty flowers with ambient humidity  vs. above-ambient humidity   Fig. . The humers Fig. . We useders Fig. . Likewisers Fig. . Becauseers Fig. . Neither0) that moths ignore RH when foraging for nectar would be rejected. Instead, we evaluated whether (H1) moths show a fixed preference for floral humidity that cannot be overridden by experience, or (H2) moth preference can be modified in the case of differential profitability  until nectar discovery by moths and used it to calculate net energy gain and expenditure (J) during floral visits as surrogates for moth fitness51.Do moth preferences change in the presence of nectar? We modified the artificial flowers used in our binary-choice assay to selectively include or exclude sugar rewards  increased visitation by moths because, over time, insects can learn trait combinations with rewards, as shown for bumblebees associating dry flowers with nectar presence52 . Accordingly, the increased probing and hovering times translate into more energy (Joules) expended by moths before nectar discovery Fig. . The timDatura  of Datura flowers greatly exceed background noise (plant or leaf headspace) or the calibration errors of most measuring probes (±2% RH)55. The finding that humidity is replenished rapidly after a pollinator’s visit 49. The available evidence supports the conclusion that floral RH is a close-range attractant in the Datura-Manduca system.Regarding proximate mechanisms of floral humidity, little is known about its efficacy: the ability to attract pollinators in the face of background humidity, wind, and temporal dynamicsusly Fig.,30. Suchrs Figs. ,  2f, anManduca sexta possess at least two putative hygrosensing sensilla on each antennal segment: the coeloconic type B (peg-in-pit) and the styliform complex sensillum  can discriminate only 2% ΔRH52. In this context, the ΔRH of Datura flowers constitutes a super-normal sensory stimulus.We used a custom-built stimulus delivery setup Fig.  to reveaons Fig. , a stere RH Fig. . Rates o22. The mechanisms of floral humidity production imply physiological costs with potential fitness consequences to the plant. For example, if floral humidity were an inadvertent nectar evaporation cue in Datura, then the intensity of ΔRH would be merely 3–4%  show that 87.40 ± 1.54% of the fresh floral mass is water, of which nectar mass accounts for only 2.18 ± 0.42%  of floral humidity. In the field of animal communication, there is a widespread expectation that signal evolution involves costs to the sender, that exceed those  associated with cues, especially for icon signalsower Fig.. Moths aower Fig.. Flower 69, including traits associated with primary metabolism. Apart from floral RH, moths also experience floral CO227. In our experiments, moth preference for humid flowers persisted throughout the night. This finding differs from moth preference for floral CO2, which diminishes to chance level after the first choice, in the absence of differential reward27. The variable preference of moths towards floral primary metabolites is consistent with the temporal dynamics of CO2 and humidity in Datura flowers. Floral CO2 in Datura flowers dissipates to background levels within the first two hours after anthesis25, whereas floral humidity remains consistently high throughout the evening, in parallel with floral scent and color . The multimodal interplay between primary and secondary metabolites in shaping pollinator behavior is widespread70 but is rarely addressed. Manipulative experimental studies of the cycad cones of Macrozamia lucida and their thrips pollinators71 revealed that thrips are neutral to CO2 but are repelled strongly by high temperature, humidity, and cone volatile production. Such studies reveal the temporal dynamics of signals and cues in conjunction with pollinator behavior, advancing our knowledge of plant–pollinator communication beyond floral advertisement.Plant–pollinator communication often is multimodal23. In plant–pollinator mutualisms, it is in the plant’s interest to maximize pollination services for minimum cost, whereas pollinators aim to maximize fitness (energy gain) per flower visit72. The evidence presented here confirms that floral humidity is not an uninformative trait for pollinators, but also compels us to examine other possible functions and conditional outcomes. In our study, moths showed a strong sensory bias for humid flowers, whether sugar rewards were absent  preference. Indeed, moths strongly preferred humid flowers whenever they were rewarding  species generally offer copious nectar rewards to hawkmoth pollinators78, there is always a potential for incomplete honesty where flowers could signal high humidity but present low volumes of nectar79. However, our findings predict that (food-deceptive) hawkmoth-pollinated plant species, which always lack nectar , would benefit by adding humidity gradients to their visually conspicuous, fragrant flowers81.These results suggest a potential avenue for plants to use floral RH as a deceptive signal to exploit pollinator preference. For example, rare (nectar-less) mutant ths Fig. . Althoug23, we have characterized the magnitude, spatial-temporal dynamics, efficacy, and physiological sources responsible for the production of a super-normal humidity gradient in the flowers of Datura wrightii as a sender, satisfying the first criterion. Conversely, we have shown that the receiver and primary pollinator, Manduca sexta, responds to changes in floral humidity through highly sensitive antennal sensilla and demonstrates an innate preference for humid flowers, satisfying the third criterion. Moths benefit by reducing their flower handling cost on humid flowers  has been shown to lower seed set and impact plant fitness in related Petunia plants82. The functional role of floral humidity in this interaction fits best with that of a mutually beneficial signal, especially given that Datura nectar is refilled over time25. The only requirement not addressed in our study is the second criterion, which demands evidence that a signal “has evolved for the purpose of conveying information to the receiver”23. This is the most difficult criterion to satisfy without the benefit of trait-independent phylogenetic hypotheses for both sender and receiver lineages19, onto which the kinds of mechanistic data outlined here  can be mapped for sister taxa of Datura plants and Manduca moths85, a task that is beyond the scope of the present study. Such information would allow us to distinguish between alternative evolutionary pathways, such as the “signaler precursor route”, in which signals originate from informative cues provided by the signaler and are honed by selection to convey information more effectively, vs. the “recipient precursor route”, in which signalers evolve to match strong pre-existing receiver biases that arose under contexts independent of communication23. From the receiver end, we know that Manduca sexta moths live longer and imbibe higher sugar concentrations under humid conditions86, prefer to fly towards more humid air in wind tunnel bioassays49 and that a distantly related hawkmoth species (Hyles lineata) also shows an innate preference for modestly humid artificial flowers (ΔRH 5–6%) in the absence of nectar29. Thus, the innate preference of M. sexta moths for humid flowers connects to deeper physiological imperatives independent of flower foraging and may be plesiomorphic among hawkmoths.Returning to the criteria by which signals are defineders Fig. , whereas87. Another possibility is that floral humidity indicates either the presence or the location of nectar. A similar role has been implied for visual nectar guides in bumblebee-pollinated flowers10. Additionally, floral humidity may indicate larger nectar volumes and better nectar quality 88 ensuring that, on average, pollinators benefit from attending to this signal. Maynard-Smith and Harper22 suggest that signals whose information content is similar or related to the associated resource should be called “icons” whereas signals that are mechanistically removed from the resource  would be regarded as “symbols”. Hence, the enormous, sustained ΔRH in Datura flowers could be considered an icon of the plant’s capacity to offer copious floral nectar in a severe desert environment. Our data most closely approximate this distinction because although ΔRH and nectar-standing crops are decoupled, RH may indicate the Datura plant’s capacity to offer rich nectar rewards despite the high cost of water stress. Future studies should test whether variation in floral RH is, in fact, predictive of nectar-standing crops or secretion rates in the wild.Our findings suggest a broader set of roles for the significance of floral humidity to flower-visiting animals. One possibility is that floral humidity serves as an inviting stimulus prompting pollinators to enter a flower. This “microhabitat” hypothesis may also be relevant for the many smaller arthropods that utilize flowers for mating, breeding, and protection without necessarily benefiting the plant89. This notion that floral humidity might be an ancient communication channel in plant–pollinator interaction is in line with the evidence that Ionotropic receptor genes (IR) that mediate hygroreception92 are conserved across arthropods and appear to have preceded the evolution of olfactory receptors93. Collectively, the efficacy of high floral RH in Datura as a trait, the salient behavioral and physiological responses that it evokes from moth pollinators, the non-trivial costs to the plant’s water budget in a desert environment, and the fitness advantages accruing to both sender (Datura) and receiver (Manduca) reveal an unexpected signaling channel in plant–pollinator interactions.Finally, we suggest that floral humidity could have evolved as a communication channel between plants and pollinators in the earliest stages of plant–pollinator diffuse coevolution, in which brood sites or mating sites are proposed to have been more important than energetic rewardsManduca sexta from egg to adult in a laboratory walk-in growth chamber maintained at 24 °C and 50–60% RH with a 16:8 light: dark cycle. Caterpillars were fed a cornmeal-based artificial diet prepared in the lab. Late-stage caterpillars were transferred to individual cavities in wooden pupation blocks. After 7–10 days of pupation in the wooden blocks, pupae were transferred to the greenhouse to a moth breeding cage and left with a tomato plant for egg collection. Pupae used for the experiments were isolated from the lab breeding colony, separated by sex, and placed in 35 × 35 × 60 cm (BioQuip) cages until ready for experiments.We raised D. wrightii seeds from Tucson, Arizona, USA, were requested from the seed bank at Radboud University, Nijmegen, Netherlands (Accession number: 944750169). Seeds were soaked in water for 24 h, followed by a rinse in 50/50 bleach water, and further soaked for 2 days in 0.1% Gibberellic acid. After soaking, seeds were nicked and placed on a wet filter paper in a petri dish until they germinated. Germinated seeds were sowed in 1-gallon plastic pots and placed in the greenhouse facility at Mudd Hall, Cornell University, under a 16:8 light: dark cycle. As necessary, plants were re-potted in a 3-gallon plastic pot, trimmed as needed, and regularly watered with 21-5-20 fertilizer (nitrogen-phosphate-potash). Plants continued flowering throughout the year.Datura plants were brought to a laboratory room from the greenhouse after anthesis. Floral humidity transects were carried out during the first 2 h after anthesis at varying levels of background humidity throughout the year. The room temperature was 23 ± 2 °C and background RH varied from 12 to 68%. Typically, the background humidity was high in the summer months (40–60% RH) and low in the winter months (10–30% RH). In preparation for measuring vertical or horizontal humidity gradients, flowers were held straight using bamboo sticks and metal wires. The Omega, Inc. hygrosensor probe (model 314 A) was screw-fixed to a syringe pump (kd Scientific model 100) to move the sensor gradually but continuously in either vertical or horizontal transects , a piñon-juniper-boulder habitat at the upper elevational limits of the plant’s distribution at Windy Point  in the Santa Catalina Mountains, and in natural grassland habitat in the Santa Rita Experimental Range . We measured the ambient humidity and temperature adjacent to the flower and noted the weather conditions at each location  flowers in still air. Subsequently, a gentle breeze was applied to the flowers, and another transect was taken for the “breeze” treatment. Next, the nectar was extracted from the flowers, and the fan was turned off to measure the humidity of the nectar-extracted flowers in still air. Lastly, the fan was turned on and another transect was taken for the “breeze + nectar extracted” treatment.Conditions in nature are unequivocally more dynamic than in the laboratory. We expected the wind to reduce the boundary layers of the flower surface. To test that, we generated an artificial breeze of ~0.4 m/s over the flower using a clip-on fan (15 cm diameter) in a laboratory setting. A black air filter pad 50 × 100 × 0.5 cm was placed between the fan and the flower to reduce airspeed and create a laminar flow. The distance between the fan and the flower was roughly 15–20 cm. If the floral RH gradient is an outcome of passive nectar evaporation, we would expect nectar removal during hawkmoth visits to reduce floral RHDatura flower was blocked with petroleum jelly applied locally using a narrow tube connected to a syringe filled with the jelly. An alternative model to produce floral RH gradients is active gas exchange through floral stomata , rather than (or complementary to) diffusion from nectar . To block the stomates of Datura flowers, petroleum jelly was smeared on the inner  surface of the corolla, as shown in Fig. Another way to test the contribution of nectar diffusion to floral RH is to occlude the floral nectar tubes see ref. . AccordiDatura flowers, its corolla was divided into four zones from the base of the flower tube (location 1) to the flower limb  was used for this experiment. The hygrosensor was connected to an Arduino Uno that was connected to a computer and operated through a custom-written Matlab code. The sensor was programmed to collect 10 data points per sec (upper limit) and was left running to collect data for 3 min while a moth was introduced to the insect cage. The background RH was noted at the start of every trial. Light intensity in the room was 0.1 lux, measured using a light meter (Reed LX-1102). Moth visits were filmed using a Canon DSLR camera (EOS Rebel T6i) at 60 frames/sec for the 3-min duration the sensor recorded floral humidity for each trial. The video data were aligned with the floral humidity data using a custom Matlab script94  within a laboratory room. Only male moths were used in this experiment and were isolated from the lab colony on the day of eclosion. Moths were trained to visit and handle Datura flower, based on actual visits of moths in our experiment.To measure the floral humidity experienced by moths as they enter and exit flowers, the hygrosensor was inserted in the flowers for 5–6 s and removed for ~10 s to mimic the behavior of moths when introduced to a cage with a single n = 8 flowers were cut open and flattened for a couple of hours using a wooden herbarium press. Pictures were taken of each flattened flower with a scale reference next to it. The surface area was measured  using Image J software  covered with a white paper towel attached to the plants, to mimic Datura flowers. The spectral reflectance of the paper towel matches closely with the authentic Datura flowers . The ambient flower received air passed through an empty beaker, whereas the humid flower received air pushed through a water-filled beaker resulting in a 0.3–0.4 m/s airflow at both flower openings. A 2 ml syringe plunger was inserted inside the tube of the artificial flower to mimic the grooves of the authentic Datura flowers. A cotton-tipped swab was glued to the center of the plunger to occupy the space taken up by the stamens and style in an authentic flower  in the greenhouse. We used 4–5-day old, starved moths for the experiment. Moths were released in the experimental lab room 3 × 6 m (width × length) at dusk. Two potted non-flowering D. wrightii flowers in greenhouse conditions), only once at the start of the experiment. Depending on the treatment, either one or both flowers were provided with a 22% sucrose reward. The following morning, nectary tubes were checked for consumption of sugar rewards, washed, and dried before using them for another trial.For the rewarded assays, we used only flower-naïve male moths to exclude the oviposition context associated with female moths. The artificial flower was modified by attaching four pipette tips at the edges of the syringe plunger to create four nectary grooves in the artificial flower , we referred to Casey (1976)97, whose data show that a 1.2 ± 0.08 g moth (mean ± SEM) requires 0.237 ± 0.01 W g−1 power to hover. For time values, we plugged in the total flower handling time (sec) for moths until proboscis contact with nectar was established.For power input values 98 with a total sugar content of 44.3 mg per 200 µl solution. Therefore, the energy gain would be 0.0443 g × 4 kcal = 0.1772 kcal/flower , which converts to 741.4 J, because 1 kcal = 4184 J.To calculate net energy gain for moths, we subtracted the energy expended (J) in handling flowers from the energy gained (J) from consuming 200 µl of the Three day old moths were cold-anesthetized in a −20 °C freezer for 10 min. Once anesthetized, moths were viewed under a dissection microscope ventral side up and dorsal side placed over a cold metal block. A UV light-activated glue (Riverruns) was used to occlude the hygrosensors on the moth antennae  with its head attached to a stepper motor (28BYJ-48 ULN2003 5 V Stepper Motor) controlled using Arduino Uno (Arduino Inc.) and a stepper motor driver board (5 V Stepper Motor ULN2003 Driver Board). We used a program written in Arduino IDE (open source) for regulating the airflow rate by controlling the valve opening position, motor speed, and motor acceleration. The valves were regulated such that the air outlets from the two dewpoint generators were antiphase as shown  to continuously push air at a flow rate of 2 L/min. The air was bubbled through an air stone immersed in water at room temperature. The resulting air saturated with water vapor served as input to two dewpoint generators . The dewpoint generators were set to operate in relative humidity controller mode, so that they outputted air at a fixed relative humidity of RH1 = 11% and RH2 = 90% corresponding to the temperature of the area adjacent to the moth antennae, see Fig. . The air99. The sampling rate was set at 20,000 Hz and the individual recording sessions of 3–6 min each, were saved on the computer until further analysis. Selected raw electrophysiology traces were spike-sorted using an open-source Waveclus 3.0 toolbox100. The stimulus and the spikes were aligned, analyzed, and visualized in MATLAB (R2019a) using a custom script94.We immobilized 2–3-day old moths in a 15 ml falcon tube. The falcon tube base was cut off just enough for the moth’s head and antennae to protrude. The head of the moth was prevented from moving by fixing it to the tube base with a collar of dental wax. The moth’s proboscis was extended and fixed to the tube with more dental wax to prevent proboscis movement from interfering with the electrodes. Moths were placed ventral side up under the microscope on a 10 × 10 × 2 cm (l × w × h) plexiglass block. The antenna was adhered to the plexiglass with a hand putty (Blu Tack). The styliform sensillum was viewed under a microscope  at a 40x zoom objective attached with a 1x magnifying lens and 20x eyepiece. For single sensillum electrophysiology, a sharp 2.5 cm wax-coated tungsten microelectrode (MicroProbes) with 2.5 MΩ resistance and a similar reference electrode was attached to a headstage  fixed on a micromanipulator (Narishige). All electrical components of the electrophysiology rig were grounded to a wire in the room away from the rig. The wires attached to the hygrosensor placed next to the moth antenna were wrapped in aluminum foil to reduce electrical noise. The recording electrode was connected to a two-channel high impedance amplifier (A-M systems model 1800), the signal was bandpass filtered for 300 to 1000 Hz, a notch filter was turned on and the signal was transferred to a data acquisition device . The data acquisition device was connected to a computer and the signal was visualized using the open-source software Spike Hound v1.2M. sexta antennae consist of one styliform sensillum on each segment of the antenna; thus, during one recording event, we attempted multiple sensilla on 5–10 segments of the middle portion of the moth antenna. The styliform sensillum is a complex of three to five individual sensing organs (papillae) located at the tip of the peg40. Therefore, a high density of cell bodies is present beneath the sensillum  varies by treatment For the stomatal counts, we performed a Kruskal–Wallis test across the four locations at which we counted stomatal density. For all the behavior data, we performed either one-sample t-tests or Wilcoxon tests, depending on the distribution of the data , with the null hypothesis being that the differences in probing duration and the number of entries between humid and ambient flowers are not different from zero. In other words, the null prediction is that moths cannot distinguish between humid and ambient flowers and visit both flowers equally. For comparisons between the flower handling time and energetics Fig. , we usedWe used MATLAB R2019b to generate the 3D scatterplots of the dry and moist neuron impulse frequency (y-axis) plotted against instantaneous RH (x-axis), and rate of change of RH (z-axis) (Fig. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Reporting Summary


The contact barrier potential was tuned using an electric field. Asymmetrical gate behavior was observed owing to the difference between the MoS2 FETs, specifically between the MoS2 FETs with Au/Ti electrodes and those with graphene electrodes. The contact barrier of the MoS2 FETs with Au/Ti electrodes did not change with the electric field. However, the contact barrier at the MoS2–graphene interface could be modulated. The MoS2 FETs with Au/Ti–graphene electrodes exhibited enhanced on/off ratios (~102 times) and electron mobility (~2.5 times) compared to the MoS2 FETs with Au/Ti electrodes. These results could improve the understanding of desirable contact formation for high-performance MoS2 FETs and provide a facile route for viable electronic applications.Two–dimensional materials have the potential to be applied in flexible and transparent electronics. In this study, single-layer MoS Single-layer MoS2 films have a direct bandgap of 1.8 eV, whereas multilayer MoS2 films have an indirect bandgap of 1.2 eV [2 has been intensively studied for electronic and optoelectronic applications. In recent years, it has become possible to synthesize large-area single-layer MoS2 via chemical vapor deposition (CVD) [2 field-effect transistors (FETs) is lower than the theoretically predicted performance [2 [2–metal electrode interfaces is a critical issue. A tunneling barrier that is formed at the interface of a metal contact in an MoS2 device [2. This is one of the main reasons for the poor performance of single-layer MoS2 FETs. Sulfur atoms mediate the hybridization between a contact metal and Mo atoms, resulting in the tuning of the bandgap [2 can be determined by the strength of the Mo–S covalent bonding [2 FETs can help resolve this critical issue and find a reliable method of improving the electrical properties of MoS2 FETs. A charge accumulation region forms at metal–MoS2 interfaces when a metal contact is used. This generally leads to the formation of an interface electric dipole, which modifies the interface band alignment [2. Owing to the challenges associated with metal electrodes, graphene has been considered as a suitable electrode material for MoS2 FETs. Graphene and single-layer MoS2 bond via van der Waals (vdW) forces, thereby creating a pristine interface. Furthermore, the contact barrier between graphene and MoS2 can be controlled by tuning the work function of graphene (4.5 eV), which is quite similar to that of MoS2 [2 can be tuned by applying an electric field.Two–dimensional 2D) materials have attracted significant attention as potential candidates for next-generation electronics ,2. GraphD materiaf 1.2 eV ,16,17. Oon (CVD) ,20,21,22on (CVD) ,24. Moreformance . This dimance [2 ,27,28. D2 device  signific bandgap . Further bonding . Therefolignment . This re of MoS2 . As the  of MoS2 ,34,35,36 of MoS2 . Therefo2 FETs with graphene electrodes that exhibit a considerable enhancement in the on/off ratio (~102 times) and electron mobility (~2.5 times) compared to the MoS2 FETs with Au/Ti electrodes. We show that the contact barrier potential of the MoS2 FETs with graphene electrodes can be effectively tuned by applying an electric field. The work function of graphene becomes higher than that of MoS2 at a negative bias voltage, resulting in the formation of a Schottky barrier. Similarly, the work function of graphene becomes lower than that of MoS2 at a positive bias voltage, resulting in the formation of an ohmic barrier. The contact barrier between MoS2 and graphene can be easily tuned using graphene electrodes. Thus, the on/off ratio and electron mobility of the MoS2 FET can be improved by tuning the contact barrier.Herein, we report high-performance single–layer MoS2 and 15 sccm of CH4 [2 plasma . The Cu foil was completely etched using 0.1 M ammonia persulfate . The PMMA/graphene layer was washed several times with fresh deionized water. Finally, the PMMA/graphene layer floated on the surface of the water, and it was transferred to a SiO2 substrate. The transferred PMMA/graphene layer was patterned using electron beam lithography  as shown in Graphene was synthesized on a copper foil  using CVD at a growth temperature of 1050 °C with 10 sccm of Hm of CH4 . Then, t2 was prepared via mechanical exfoliation from a bulk MoS2 flake . To fabricate MoS2 FET with graphene electrode, a dry transfer process was employed [2 flake after the alignment position using a micromanipulator   a. Au/Ti 2 from bulk MoS2 [2 was transferred onto a silicon wafer with a 300 nm thick SiO2 layer. Raman spectroscopy  was used to determine the number of layers of MoS2 [2 revealed a peak spacing of less than 20 cm−1 between the E2g and A1g modes, indicating that single-layer MoS2 was formed. A 532 nm laser with a power of 1 mW was used as an excitation source. The exposure time was 1 s, and calibration was performed using a reference Si peak position of 520 cm−1. The fabricated MoS2 FETs were loaded into a vacuum chamber (Lake Shore) for electrical measurements. The electrical properties of the MoS2 FETs with graphene–graphene electrodes, Au/Ti–Au/Ti electrodes, and graphene–Au/Ti electrodes were characterized in vacuum (under 10−4 Torr) using a semiconductor parameter analyzer  for comparison.Mechanical exfoliation was employed to extract high-quality single-layer MoSulk MoS2 . Then, s of MoS2 . The Ram2, graphene on single-layer MoS2, and graphene, respectively. The MoS2 and graphene/MoS2 layers exhibit typical single-layer Raman active modes (~18.27 cm−1 of frequency difference between E2g and A1g), and the 2D/G ratio of graphene is about 4.06. Therefore, it can be noted that exfoliated MoS2 flake has a formation of single layer. The Dirac point of intrinsic graphene is at zero gate voltage, the work function of which is approximately 4.5 eV [2 and exposure to oxygen and moisture [2 is shown in 2 were bonded via weak vdW forces. However, MoS2 and the metal interfaces formed covalent interactions, causing a change in the electronic structure [2 FETs were fabricated to investigate the effects of the graphene electrode. The first was a single-layer MoS2 FET with a Au/Ti–graphene heteroelectrode, as shown in 2 layer was used as the substrate. The channel length and width of the mechanically exfoliated MoS2 used in the single-layer MoS2 FET were ~2 μm and ~4 μm, respectively. DS–VDS output characteristics of the single-layer MoS2 FET with the Au/Ti–graphene heteroelectrode without the gate voltage. Different contact barriers were generated according to the contact material. An ohmic contact was formed between single-layer MoS2 and Au/Ti. A Schottky contact was formed between single-layer MoS2 and graphene. DS–Vg transfer characteristics for a positive source–drain voltage (VDS). The on/off ratio and electron mobility (graphene in the heteroelectrode) were >105 and ~3.2 cm2/V∙s, respectively. DS–Vg transfer characteristics for a negative drain voltage. The on/off ratio and electron mobility (Au/Ti in the heteroelectrode) were >102 and ~1.2 cm2/V∙s, respectively. These results indicated that graphene could be used as an ideal electrode in a single-layer MoS2 FET. Mobility was calculated using the following equation: 2. The MoS2 FET with the Au/Ti electrodes exhibited ohmic contact behavior, whereas the MoS2 FET with the graphene electrodes exhibited Schottky contact behavior. Multilayer MoS2 FETs with exfoliated graphene electrodes also showed ohmic contact behavior [2 FET with the graphene electrodes exhibited a Schottky barrier without a gate bias voltage. However, the work function of graphene was electrostatically adjusted to approximately 300 meV for single-layer graphene by tuning the Fermi level (EF) by changing the gate voltage by 50 V [DS–VDS characteristics of the single-layer MoS2 FET as a function of the back-gate voltage. The Schottky barrier between graphene and single-layer MoS2 was enhanced at a negative gate voltage; thus, current could not flow in the negative gate voltage direction  showed almost similar with linear (red dashed line) at a gate voltage of 20 V because the work function of graphene became similar to that of single-layer MoS2 I2 FET with graphene electrodes, the Schottky barrier is decreased dramatically—from 51.5 meV to 0 meV—with the back gate voltage changing from −7.5 to 12.5 V, as shown in 2 FET with graphene electrodes comes from changes in work function of graphene.In the MoS2 FET with graphene electrodes by tuning the contact barrier using an electric field. The MoS2 FET with a Au/Ti–graphene heteroelectrode shows a clear change in the contact barrier between MoS2 and graphene. A Schottky barrier and ohmic barrier exist in the off and on states of the MoS2 FET with the graphene electrodes. The on/off ratio and electron mobility of the MoS2 FET with the graphene electrodes are 102 and 2.5 times higher than those of the MoS2 FET with the Au/Ti electrodes, respectively. The Schottky barrier between MoS2 and graphene is decreased from 51.5 to 0 meV by the back gate voltage. The implication of these results could be of great importance in better understanding the desirable contact formation for high performance MoS2 FETs. This FET may be promising for electronic device applications based on next-generation 2D materials.This work demonstrates the enhancement of the electrical properties of an MoS


Nanofluids made up of propylene glycol, and water and graphene nanopowder dispersed throughout them are the primary focus of our study. Nanofluids were created by mixing propylene glycol and water in quantities of 100:0, 75:25, and 50:50. The essential fluids used in this experiment were propylene glycol and water. Graphene was dispersed in these three different base fluids at percentages of 0.25 and 0.5, respectively. This body of work's fundamental objective is to explore nanofluids' tribological behavior. This behavior was observed with a pin-on-disc device, and the impact for load on wear, coefficient of friction, and frictional force was investigated. The tests were conducted with weights ranging from 1 to 3 kg. It was revealed that as the load ascended, there was a reduction in the amount of wear, the coefficient of friction, and the frictional force for the most of the samples tested. Still, there was an increase in the amount of wear and friction coefficient, including the frictional force for some of the samples. There has been the development of an alternative nanoscale additive for coolants. In the realm of nanotechnology, one of the most recent and innovative developments is the production of nanofluids. Colloids of nanometric materials suspended in liquids such as water, ethylene-propylene glycol, propylene glycol, or oil are referred to as nanofluids. A particle is a little entity that behaves and moves the same way as a complete item and shares its features. On the other hand, ultrafine particles are defined as having a size that ranges from 1 to 100 nm. Nanoparticles could, but it's not a given that they will exhibit size-linked properties that are noticeably different from those of tiny particles or complete materials. Recent years have seen an unprecedented level of interest in nanoparticle research from the fields of mechanical engineering, biological sciences, and electrical engineering5. A nanofluid is a fluid that consists of a base fluid that contains one or more types of nanoparticles scattered throughout the fluid7. Nanofluids are preferable to microfluidics due to their higher thermal conductivity and lower viscosity when it comes to applications that also include heat transfer. This is due to the fact that nanofluids perform their functions more effectively than microfluidic. Since the 1990s, researchers have investigated the possibilities of enhancing heat transfer methods using nanomaterials to get superior outcomes. They devoted most of their efforts to research associated with creating exceptionally effective procedures for heat transfer technology. Nanofluids have been the focus of research in the fields of material science, physics, and chemistry, due to their enormous application potential in various fields, such as chemical and electrical engineering, and vehicle as building construction, microelectronics, and knowledge. Nanoparticles increase cutting fluid thermal conductivity and tribology. Thermally conducting cutting fluids remove cutting zone temperatures better. Tribological features also facilitate oil film formation at tool-chip and tool-work piece contacts. By preventing the cutting temperature from spreading to the tool and workpiece, cutting force, surface quality, tool wear, and tool life may be significantly improved. Nanoparticle additions minimize friction coefficient, energy consumption, and thermal stress to increase tool life and enhance component surface quality.This is because nanofluids can be used in all of these areas. This is because recent years have seen a significant increase in attention placed on researching nanofluids within materials science, physics, and chemistry. In the past, numerous different mixtures of oxide nanoparticle nanoparticles, such as CuO, Al2O3, TiO2, and Fe3O4, have been utilized to make nanofluids and evaluate the transport capabilities of such nanofluids. These mixtures of metallic oxide nanoparticles have proven effective in both endeavors8.Many different kinds of applications over the period of the last few decades have demonstrated that nanofluids provide considerable advantages, and this trend is expected to continue. There is a possibility that the use of certain materials could increase the quantity of heat that is efficiently transported by industrial gear. The fundamental goal of nanofluid research has, from the very beginning, not been to decrease the viscosity of nanofluids but rather to increase their thermal conductivity and their ability to transmit heat3CH(OH)CH2OH. Because it has two different alcoholic functional groups, it is classified as a diol10. It may be dissolved in a wide range of solvents, including water, acetone, and chloroform. In their natural state, glycols do not irritate and have minimal volatility. Propylene glycol has several applications, such as creating polymers, the food and beverage industry, and the medical field. The properties of propylene glycol are shown in Table Propylene glycol, used in these formulations as the fundamental liquid component, was sourced from Naveen Chem. According to the International Union of Pure and Applied Chemistry (IUPAC), propylene glycol is a viscous liquid that is colorless and has a flavor that may be described as slightly pleasing. Another name for propylene glycol is propane-1,2-diol. This substance may be represented by the chemical formula CH12 . This term, derived from the combination of the words "graphite" and the suffix "-ene," describes the tremendous double bonds in graphite. An electron from each atom in a graphene sheet is added to a valence band that spans the whole sheet, and each bit has a strong bond with the three atoms adjacent to it. Carbon nanotubes, PAHs, fullerenes, and even glassy carbon exhibit this kind of bonding to some extent14. Graphene is a semimetal with remarkable electrical characteristics that may be characterized using theories for massless relativistic particles because the conduction band and the valence band are in touch with one another. These theories can explain graphene's properties. The graphene charge carriers have a linear rather than a quadratic dependence of energy on momentum; it is possible for field-effect transistors made from graphene to achieve bipolar conduction. In addition to exhibiting colossal quantum oscillations and enormous nonlinear diamagnetism, the material demonstrates ballistic charge transfer over great distances.Graphene is a kind of carbon nanostructure made up of a single sheet of atoms arranged in a honeycomb lattice over two dimensions15. Graphite has a considerable capacity to absorb visible light, which is why it has a dark appearance17. Still, a single layer of graphene is so thin that it is nearly transparent. This compound is almost one hundred times stronger than the most substantial steel compared to the material's strength. Graphene's extraordinary tensile strength, electrical conductivity, transparency, and the fact that it is the world's most robust material have all contributed to its rise in popularity as a valuable and helpful nanomaterial18. Graphene is the thinnest two-dimensional substance and the world's most robust material. The research and development sectors of the semiconductor, electronics, electric battery, and composites industries accounted for the vast bulk of graphene's demand in 2012, contributing to the global graphene market's value of $9 million20. As a result of the hydrophobic property of graphene, graphene oxide (GO) may be readily suspended in a variety of base fluids.The plane of graphene is a very efficient conductor of both heat and electricityIn comparison to traditional carbon nanotubes, graphene possesses superior heat flow properties, which contribute to the material's increased ability to improve the thermal conductivity of nanofluids. Graphene is idealfor other existing nanoparticles in terms of its thermal conductivity, stability, and resistance to erosion and corrosion. Graphene nanopowder was procured from Ultrananotech Pvt.Ltd. Graphene's description as received is shown in Table Propylene glycol–water samples of three kinds (50:50), (75:25), and 100:0) are prepared in this experimentation. The graphene nanoparticles are dispersed in the prepared solutions with the help of an Ultra probe sonicator 0.25 and 0.5 wt%. Figure 00:0 are 21. Metals wear primarily due to the plastic deformation of the surface, near-surface material, and the breaking of particles that generate wear debris. Other factors that contribute to metal wear include heat and friction. The diameters of particles may vary from millimeters down to nanometers22. For instance, this may occur when one metal comes into contact with another metal, a nonmetallic solid, a flowing liquid, a solid particle, or a droplet of liquid entrained in a flow of gas23. The rate of wear is determined by a variety of different parameters, some of which include the kind of loading , the type of motion , the operating temperature, and the presence or absence of lubrication24. Depending on the tribosystem, it may be possible to identify various kinds and processes of wear. There are a number of tried-and-true methods available for estimating the level of deterioration that has taken place over a certain amount of time and under optimal settings. The ASTM International Committee G-2 is responsible for conducting regular reviews and providing updates to the standards governing wear testing across various sectors and applications. To get a more profound comprehension of tribology, or the study of friction, wear, and lubrication, the Society of Tribologists and Lubrication Engineers has collected data from diverse experiments (STLE). To rate materials in terms of wear resistance, toughness, and other properties, standardized wear tests may be done using the criteria provided in the test's description. Testing in circumstances more indicative of actual use may help improve wear predictions for industrial applications. This is because real-world conditions are more accurate.Wear is the process through which a surface gradually deteriorates over time due to erosion or deformation. In addition to mechanical reasons (such as erosion), chemical variables may also contribute to wear and tear . Tribology studies the processes that lead to wear and the factors that contribute to it. Wear, fatigue and creep are three factors contributing to the progressive deterioration of the functional surfaces of machine components. This gradual degradation might ultimately lead to the breakdown of the material or a loss of functionality. It is predicted that abrasive wear negatively impacts between 1 and 4% of the GDP of affluent countriesf, whereas Fn denotes the normal force. The friction coefficient does not come with any quantifiable units. Because it is scalar, its actual magnitude does not change regardless of the force's direction. The friction coefficient is affected by the many factors that contribute to friction. Although the number is often between 0 and 1, it can be more than 1. The absence of any resistance to motion between two objects is made possible by the superfluidity of the medium. Two things cannot come into contact with one another and not have some reaction to it since friction is required. When the frictional force is equal to one, the normal force is comparable to one. There is a widespread acceptance of the fallacy that the coefficient of friction can only ever be either zero or one. When the coefficient of friction is larger than one, the frictional force, which is measured in comparison to the normal force, is more significant. Silicone rubber is a good illustration of a material with a coefficient of friction much higher than one. The friction force is the resistance to motion produced by a surface whenever an object moves over it or seeks to move over it. This resistance to movement is exerted whenever an item moves over it or attempts to move over it.The friction coefficient quantifies the resistance to relative motion between two surfaces. It's a number used in physics to calculate the average force or frictional force exerted by an item when other methods are not available. The formula denotes the coefficient of friction The frictional force is the name given to the force that is generated when two surfaces come into contact with each other as well as slide against one another. Various sources, including the following, cause the frictional force: the surface roughness, as well as the amount of force that's also driving the particles together, are the two most important parameters that play a role in the determination of these forces. On solid surfaces, friction may take the shape of three distinct phenomena: static, sliding, and rolling. The most effective kind of friction is known as static friction, which is then followed by sliding friction, and finally, rolling friction, which is the least effective form. Because gases and liquids are both regarded to be fluids, the phenomenon of fluid friction may take place in either one.Pin-on-disc instruments assess the wear rate and the frictional force of various materials. The pin-on-disc mechanism consists of a calibrated deadweight-laden pin and a horizontally decaying disc, as shown in Fig. 25. The Table A motor is included to turn the disc.The LVDT sensor is where it shines . Wear on the disc occurs when the LVDT sensor's pin touches the disc as it is spinning. Weight is distributed along the lever arm, which carries the pin. The experiments were conducted by varying the load from 1 to 3 kg. A copper pin was used to observe the wear behavior26. However, propylene glycol dispersed with 0.5% graphene exhibits less wear than others. Propylene glycol dispersed with 0.5% graphene shows much less wear than pure propylene glycol and propylene glycol that has been dispersed with 0.25% graphene. It is possible to see in Fig. The influence of load on the wear rate is seen in Fig. 27, it is demonstrated that the coefficient of friction is minimum for Propylene glycol–water (75:25) dispersion with 0.5% Graphene. Figure 28. From the above results, it can be concluded that the increase in the volume percentage of graphene. There is a decrease in the coefficient of friction.Figure 829. From the above results, it can be concluded that the increase in the volume percentage of graphene decreases the frictional force. Hence graphene was found to be a good alternative for improvement in tribological properties.Figure Water and propylene glycol was chosen as the essential fluids, and nanofluids were created by mixing them in quantities of 100:0, 75:25, and 50:50, respectively. In these two kinds of base fluids, graphene was mixed in at 0.25 and 0.5, respectively. 0.5% graphene-dispersed propylene glycol wears less than pure and 0.25% graphene-dispersed propylene glycol. Propylene glycol–water (75:25) showed a lower wear rate than 0.25% and 0.5% graphene-dispersed versions. Propylene glycol–water (50:50) mixed with 0.5% graphene had a lower wear rate than both 50:50 and 0.25% graphene. 0.5% graphene has a smaller surface area than 0.25%. 0.5% graphene-dispersed propylene glycol offers the lowest coefficient of friction. Graphene is included in both formulations. Propylene glycol–water (75:25) mixed with 0.5% graphene had the lowest coefficient of friction compared to both 75:25 and 0.25% graphene. Analysis proves this. Propylene glycol–water (50:50) with 0.25% graphene had the lowest coefficient of friction. This was the case regardless of whether the graphene concentration was 0.5% or 0.25%. Propylene glycol with 0.5% graphene has the lowest frictional force. Propylene glycol–water (75:25) dispersed with 0.5% graphene has a more downward frictional force than the other two. Propylene glycol–water (50:50) with 0.5% graphene had the lowest coefficient of friction.


Sarcoidosis can be presented as cardiac sarcoidosis (CS), which is challenging to diagnose due to its clinical silence. Ventricular arrhythmias and atrioventricular blocks can be fatal and cause sudden death in patients with cardiac sarcoidosis. Five percent of sarcoidosis patients have clinically evident cardiac sarcoidosis. However, autopsy reports and imaging studies have shown a higher prevalence of cardiac involvement. Early recognition is important to prevent such detrimental consequences. Cardiac sarcoidosis is increasingly being diagnosed owing to increased awareness among physicians and new diagnostic tools like MRI and positron emission tomography (PET) scan replacing traditional endomyocardial biopsy. A definitive diagnosis of CS remains challenging due to the non-specific clinical findings that can present similar symptoms of common cardiac disease; therefore, the imaging and biopsies are substantial for diagnosis confirmation. Pharmacological and Implantable devices are two main therapeutic approaches in cardiac sarcoidosis, in which steroids and pacemaker therapy have shown better outcomes. This review summarizes the available data related to the prevalence, prognosis, diagnosis, and management of cardiac sarcoidosis. Cardiac sarcoidosis (CS) is diagnosed in almost one-third of patients with systemic sarcoidosis and is mostly diagnosed post-mortem by autopsy, as a very small percentage of patients are symptomatic . The preSince there is no consensus for disease detection, monitoring, and treatment, diagnosis becomes a challenge when the only manifestation is cardiac dysfunction; however, it should be strongly suspected in patients with multi-systemic sarcoidosis . ConfirmA 40-year-old African American female with a history of hypertension presented to the clinic with seven days of dyspnea on exertion, headache, and dizziness. On examination, vitals were normal, chest X-ray showed cardiomegaly, bilateral hilar lymphadenopathy, and multiple lung nodules. Her electrocardiogram (ECG) showed a 2:1 atrioventricular (AV) block and right bundle branch block, followed by echocardiography which revealed right ventricular infiltration and reduction in systolic dysfunction. Based on these findings, differential diagnoses of mediastinal lymphoma, tuberculosis, and sarcoidosis were considered and evaluated.The patient did not have a travel history. Cervical lymph node biopsy showed non-caseating granuloma formation, which confirmed the diagnosis of sarcoidosis. The patient was given corticosteroids 60 mg once daily, and a permanent pacemaker was implanted. At three months follow-ups, the patient's condition improved, and echocardiography findings subsided. Table Sarcoidosis is a granulomatous disease with multi-system involvement ; cardiacEven though the clinical picture of cardiac sarcoidosis is highly variable, conduction abnormalities, arrhythmias, and heart failure are the most common presentation of cardiac sarcoidosis. Sometimes, cardiac sarcoidosis can be the only manifestation of sarcoidosis, and it should be suspected in young patients who are presenting with conduction abnormalities of unknown etiology . CardiacThe diagnosis of cardiac sarcoidosis can be confirmed by direct biopsy. The involvement of the heart is usually patchy, so the endomyocardial biopsy has been positive in only 20% of cases ,20. AlmoMoreover, patients may show ECG abnormalities ranging from nonspecific ECG finding to conduction abnormalities and ventricular arrhythmias, which cannot sometimes be found in 12-lead ECG without Holter monitoring  as in TaPoor outcome is also associated with pulmonary hypertension secondary to extrinsic compression of pulmonary arteries by enlarged lymph nodes or cor pulmonale . In the The two main therapeutic approaches in CS patients are pharmacological management and invasive or device-oriented . SteroidWhen it comes to understanding cardiac sarcoidosis, it looks like we have revealed the tip of the iceberg. Cardiac involvement is often asymptomatic in sarcoidosis cases, so early diagnosis and better screening protocol are essential for early cardiac sarcoidosis diagnosis and management. New imaging techniques have contributed to Global awareness of cardiac sarcoidosis. Capacity-building should be more focused on early detection along with the identification of signs which can be used to identify such cases of sarcoidosis.


In patients, image ratings were similar for all parameters except for “conspicuity of FLLs” which was rated significantly lower on L1-DWI vs. conv-DWI  in group A. In five patients, 11/349 FLLs were not detectable on L1-DWI, but on conv-DWI. L1-regularized iterative reconstruction of single-shot EPI DWI can accelerate image acquisition or improve spatial resolution. However, our finding that FLLs were non-detectable on L1-DWI warrants further research.To investigate whether combining L1-regularized iterative sensitivity encoding (SENSE) reconstruction and single-shot echo planar imaging (EPI) is useful in hepatic DWI. Single-shot EPI-DWI with L1-regularized iterative SENSE reconstruction (L1-DWI) and conventional parallel imaging-based reconstruction (conv-DWI) in liver MRI were compared in volunteers and patients. For the patient cohort, 75 subjects (60 ± 13 years) with 349 focal liver lesions (FLL) were included. Patient groups A and B were used to reduce acquisition time or improve spatial resolution, respectively. Image parameters were rated on a 5-point scale. The number of FLLs was recorded; in case of discrepancy, the reason for non-detectability was analyzed. In volunteers, higher signal-to-noise ratio (24.4 ± 5.6 vs. 12.2 ± 2.3,  First reported by Lustig et al. in 2007, CS uses the sparsity of image data and attempts to avoid undersampling artifacts by using incoherent undersampling with iterative reconstruction7. CS has been shown to yield comparable image quality compared to conventional imaging10. Most research projects focused on the use of CS to accelerate acquisition in dynamic contrast enhanced imaging of the liver and the breast, where fast image acquisition is of key importance13.Numerous MRI techniques have been developed to accelerate acquisition time of MR imaging by skipping data in κ-space such as keyhole techniques, parallel imaging, and, more recently, compressed sensing (CS)6. Similarly, the faster acquisition can be invested to increase spatial resolution at a given acquisition time.Diffusion weighted imaging (DWI) is a pulse sequence that could benefit from the principle of CS; not only for accelerated acquisition as the case with conventional parallel imaging, but also for improved signal-to-noise ratio (SNR) via denoising during iterative reconstruction16. There is even evidence to suggest that DWI is more important for the detection of FLLs than standard structural liver MRI18. Recently wavelet-based denoising as used in the CS framework has been combined with parallel imaging that derives a L1-regularized iterative SENSE reconstruction showing effective in single-shot echo-planar imaging (EPI) based DWI noise reduction20.DWI plays an important role in the detection and characterization of focal liver lesions (FLLs)Therefore, the aim of our study was to investigate CS-based reconstruction using L1-regularized iterative SENSE in EPI DWI (L1-DWI) in healthy volunteers and a cohort of patients undergoing liver MRI in direct comparison to conventional parallel imaging based reconstruction (conv-DWI), for both purposes, i.e. increased acquisition speed and increased spatial resolution.This comparative analysis was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee at the RWTH Aachen Faculty of Medicine (EK 226/20). Written informed consent was waived.For quantitative assessment of the signal-to-noise ratio (SNR) and apparent diffusion coefficient (ADC), L1-DWI was performed in healthy volunteers and compared to conv-DWI. Details are described below in MR Imaging and Image Analysis.For investigation of its potential clinical utility, L1-DWI was applied in therapy-naïve patients, who received standard multi-parametric liver MRI for clinical indications from November 2019 through February 2020 for either search of liver metastases of a newly diagnosed malignancy or diagnosis or staging of primary liver tumors. All patients underwent a standardized multiparametric liver MRI protocol, and conv-DWI plus L1-DWI that were to be acquired in randomized order.To investigate the potential utility of L1-DWI in terms of reduced acquisition time or improved spatial resolution, two different types of L1-DWI protocols were used:During the first half of the study period (November through December 2019), patients underwent conv-DWI plus L1-DWI, where CS-based reconstruction was used to reduce acquisition time of conv-DWI (group A); during the second half of the study period (January through February 2020), CS-based reconstruction was invested to improve spatial resolution compared with conv-DWI (group B). Accordingly, in group A, L1-DWI was acquired with the same spatial resolution as conv-DWI, but faster; in group B, L1-DWI were acquired with similar acquisition time as conv-DWI but had a higher spatial resolution. In both patient groups, the same acceleration factor of 3 was applied as in healthy volunteer exams.If a patient underwent more than one liver MRI study during the study period, only the first examination was included to avoid repetitive observations of the same patient to confound the results.All MR examinations were performed at a 1.5 Tesla system . A torso phased array coil was used in all studies. The standard liver MRI protocol consisted of the following pulse sequences: axial breath-hold dual-echo T1 gradient echo in and opposed phase, axial T2-weighted turbo-spin echo (T2w TSE) sequence with and without spectral-selective fat suppression, coronal T2w TSE-sequence, a dynamic contrast enhanced series consisting of axial T1-weighted turbo field echo (Turbo i.e. gradient echo) sequence before and after contrast media injection (Gadobutrol or Gd-EOB-DTPB Dinatrium), followed by late phase enhanced-T1-weighted High Resolution Isotropic Volume Examination (eTHRIVE) using a fat-suppressed gradient-echo pulse sequence.9. This provided relatively shorter EPI train length, echo time, and repetition time, compared to those in conv-DWI (acceleration factor 2), and resulted in a slightly reduced nominal scan time. For conv-DWI reconstruction, conventional SENSE was applied to the undersampled data. In addition to that, scan parameters of both sequences were matched as closely as possible, with shortest possible repetition times and same number of slices in the interleaved order in each case. For L1-DWI reconstruction, a regularized, iterative L1-norm minimization was applied assuring image sparsity in the wavelet domain and data consistency, as in typical CS reconstruction by taking the SENSE coil information into account28. Standard denoising was applied , anonymized for patient data  and blinded for acquisition parameters .24. In short, identical pulse sequence was performed without radio frequency excitation for the same imaging volume and separate noise-only images in each subject were obtained to account for influence from parallel imaging or CS reconstruction. As it prolonged the examination times, i.e., doubled corresponding L1-DWI or conv-DWI scan times, this evaluation was performed only in the volunteer studies. Regions of interest (ROIs) were carefully placed in the liver parenchyma of L1-DWI and conv-DWI with uniform signal without vessels or artifacts as well as in the corresponding noise map. Two ROIs were placed per volunteer. The mean values and standard deviations from each ROI were extracted to compute SNR and averaged for comparison. The same criteria of ROI selection also applied to quantification of the apparent diffusion coefficient (ADC).For signal-to-noise ratio (SNR) evaluation, additional noise maps were acquired for both L1-DWI and conv-DWI in volunteer measurements. The method was previously describedFor qualitative image analysis, two radiologists with 10 years (AB) and 4 years (MB) of experience in interpretation of abdominal MRI reviewed the CS- and conv-DWI data sets side-by-side, yet independently and blinded to the clinical information as described above. Image quality was visually scored according to a 5-point scale regarding the following features: (1) sharpness of the liver contours, (2) delineation of intrahepatic vessels, (3) signal homogeneity of the liver parenchyma and (4) conspicuity of FLLs, from 1 = poor to 5 = excellent; (5) image noise and (6) motion artefacts were evaluated using a 5-point scale ranging from 1 = severe to 5 = none.During a separate reading session done more than 16 weeks after the first session on image quality assessment, the two readers, again blinded to the type of DWI acquisition, reviewed the images in randomized order to compare the detection of FLLs. The number of FLLs for each DWI study of each patient was recorded.When a FLL was missed on one of the pulse sequences (L1-DWI or conv-DWI), we investigated the respective cause: Whether the FLL was missed because of a reading failure, or whether, also in retrospect, i.e. with knowledge about the location of the FLL, the FLL was not visible; in this latter case, we recorded possible reasons for non-detectability such as local image degradation due to motion or pulsation artefacts.In case of a discrepancy of the number of visible FLLs between L1-DWI and conv-DWI, we used all pulse sequences of the multiparametric hepatic MRI protocol, plus follow-up information of further MRI studies to clarify whether or not the DWI finding corresponded to a true FLL or to an artefact.We also assessed the respective size and clinical relevance of the missed FLLs in terms of their implications on further patient management.t-test. For each image quality parameter, the mean and SD were calculated based on the ratings of the two readers and the resulting scores for L1-DWI and conv-DWI were compared by using a Wilcoxon rank sum test. The level of statistical significance was set at α = 0.05. The interrater variability was assessed by using weighted Cohens Kappa for each rated criterion at a 95% confidence interval. A coefficient value < 0.00 indicated poor agreement, 0.00–0.20 slight agreement, 0.21–0.4 fair agreement, 0.41–0.6 moderate agreement, 0.61–0.8 substantial agreement and 0.81–1.00 almost perfect agreement25. Statistical analyses were performed using IBM SPSS Statistics for Windows, version 25.0.Data were reported as mean and SD. Differences in continuous variables were evaluated with a paired Twelve healthy subjects  with different body sizes (1.57 to 1.93 m) and no known illness were included in the volunteer group. Figure Accordingly, the final patient cohort consisted of 75 patients. Patient demographics and details on their primary tumors are given in Table Of the 75 patients, 57 patients had at least one and up to 30 FLLs. In group A, 101 FLLs were observed in 20/28 patients. In group B, 248 FLLs were observed in 37/47 patients.2  and 800 s/mm2 . Liver ADC values were deemed significantly different between L1-DWI and conv-DWI , whereas SD of ADC value in L1-DWI was found significantly lower than that in conv-DWI .In general, reduced background noise and better structural delineation could be visualized in L1-DWI compared to conv-DWI, as shown in Fig. p = 0.006).Results are given in Table In group B, image quality ratings did not differ significantly; although also here, a tendency for somewhat higher FLL conspicuity in conv-DWI was observed, this feature did not reach statistical significance. For representative images of group A and group B see Fig. The two readers showed slight to substantial agreement for the rated image quality parameters  were not detected on L1-DWI, but on the patient’s conv-DWI. Even in retrospect, these two FLLs were not visible on L1-DWI. An analysis of why these two FLLs were occult on L1-DWI showed that one FLL happened to be obscured by pulsation artefacts at the site of the FLL on L1-DWI. For the other missed FLL, no apparent reason could be identified , 9/248 FLLs (4%), between 3 and 7 mm in diameter, were not prospectively detected on L1-DWI, but prospectively called on the respective patient’s conv-DWI. Even in retrospect, these lesions were not visible on L1-DWI  to note that non-detectability of lesions in L1-DWI was not explainable by or attributable to e.g. artefact degradation, but was observed also in L1-DWI images that appeared to offer perfect image quality.CS has been introduced to accelerate data sampling in MRI. Most studies on the clinical application of CS focused on its use to shorten acquisition time, and analyzed the resulting image quality. Most authors found that CS allows a reduction of image acquisition without loss of image quality—which is in good agreement with our findings. However, much less information is available on the diagnostic accuracy of such heavily accelerated pulse sequences. One study, by Nam et al. investigated the use of CS for dynamic contrast enhanced liver MRI including detection of FLLs in a multi-reader study and found a similar diagnostic accuracy of CS Dixon-gradient recall echo over standard non-CS Dixon-gradient recall echo. However, in that study, no head-to-head comparison of individual detected lesions was performed.26. Higher ADC values in L1-DWI compared to conv-DWI has also been reported previously in different anatomies including brain and prostate26. A possible cause may be a shorter repetition time ) used in L1-DWI27 or higher noise in conventional SENSE images using parallel imaging. However, in the current study a quantitative SNR improvement in volunteer scans was not reflected by qualitative scores in patient exams, where no improvement was shown for L1-DWI in image noise and artifacts rating. While there may be various factors contributing to this discrepancy, including local ROI selection vs. global visual inspection, body size and composition variabilities in the volunteer vs. patient cohorts, etc., detailed investigations are needed to study its impact in image quality and, particularly, delineation of normal appearing tissues and lesions28.The observed SNR improvement in L1-DWI in volunteer scans most likely benefits from the iterative denoising process in the wavelet domain, in which the noise patterns are sparsely represented, as well as a shorted echo time due to higher acceleration factor used in L1-DWI. Lower standard deviations in ADC of L1-DWI have shown less variability and may indicate a higher precision and reproducibility in ADC assessment, which is in accordance to other studiesThe positive impact of CS on image quality could be expected to improve detectability of FLLs, e.g. by reducing motion artefacts in protocols that exploit fast data acquisition with CS, or by shortening the echo train length and thus improving SNR in DWI with CS. On the other hand, with the undersampling that is employed for CS, it is conceivable that small lesions are “levelled out” and thus not displayed with the true signal intensity that they should have on DWI or structural MRI. Accordingly, there is a need for research that compares the detectability of such small lesions in heavily accelerated pulse sequences.In our study, 2 and 4% of FLLs observed in 4 and 6% of patients of group A and B, respectively, were detected by conv-DWI, but were not visible on L1-DWI. The majority of patients in our cohort, including the five patients who had FLLs missed on L1-DWI, had multiple FLLs. Therefore, the missed FLLs would not have led to a change of patient management. However, it is possible that in future patients, e.g. with colorectal cancer in preparation of major liver surgery, detectability of even small additional metastases may do impact the treatment plan.A further analysis of the type of FLLs missed on L1-DWI revealed that all missed FLLs were small, 7 mm or less in diameter, i.e. had a size similar to that of the section thickness of our DWI protocol. Small FLLs can always be missed on DWI in case there happens to be image degradation due to artefacts at the site of a lesion, or in case partial volume averaging leads to reduced contrast. In these latter situations, however, there should at least be a subtle signal intensity difference on a post-hoc analysis of the respective images—which was not the cases where FLLs were missed in L1-DWI in our study cohort. Also, a post-hoc analysis of image quality at the site of the missed FLLs revealed no artefact degradation as a possible explanation for such a misdetection.28, the same equidistant k-space acquisition pattern as in conventional single-shot EPI was used here for CS-based reconstruction, instead of a specifically tailored incoherent sampling pattern7. This might also limit the denoising performance. On the other hand, different imaging parameters between L1-DWI and conv-DWI such as echo times, and therefore scan time, may also contribute to lesion conspicuity, for example, due to induced image contrast change. Consequently, a systematic evaluation of the scan set ups in clinical settings is needed. The setting of the parameters should not only be chosen based on the image quality, but also based on the visualization of small FLLs.One explanation of missed FLLs in L1-DWI in group B is because of their small size and reduced SNR in the image associated with higher spatial resolution. Moreover, the contrast to noise may be further decreased due to CS denoising that already occurred in every DWI measurement in the resource images. In this case, although the background tissue, i.e. liver parenchyma, appears smoother and less noisy via CS denoising, the visibility of small FLLs may become less apparent, which can no longer be retrieved by the sparsifying constraint in the CS-based reconstruction with L1-regularized SENSE including wavelet-based denoising, even after adding multiple measurements or acquisitions (see Figure, Supplemental Digital Content 29, in which case the minimum repetition time should be determined by T1 of the background tissue to reach thermal equilibrium30.Our study has several limitations. First, the study cohort was relatively small. In addition, the subgroups differed in the number of patients, which is due to the fact that the enrollment of patients was fixed at two months for each group in our study design. Second, the study cohort consisted of a heterogeneous patient population. On the other hand, this may also be considered advantageous because we assessed the feasibility of L1-DWI in various clinical settings. Third, most FLLs were not confirmed by pathology. Accordingly, although we did our best to provide a sound composite ground truth, it is conceivable that detected and non-detected small FLL did not correspond to a relevant pathologic condition. Finally, our study design did not include having fewer number of signal averages as another way to reduce the scan timeIn conclusion, although L1-DWI can be used to reduce acquisition time or increase spatial resolution, and although measured SNR and stability of ADC values are improved, its image quality in clinical patients appears to be mostly comparable, while detection of FLLs is affected. Further investigations on optimal scan setups are needed before implementation of L1-DWI in clinical practice and should be done not only based on image quality but also visualization of small FLLs.Supplementary Information.


Graphene-copper nanolayered composites have received research interest as promising packaging materials in developing next-generation electronic and optoelectronic devices. The weak van der Waal (vdW) contact between graphene and metal matrix significantly reduces the mechanical performance of such composites. The current study describes a new Cu-nanoporous graphene-Cu based bonding method with a low bonding temperature and good dependability. The deposition of copper atoms onto nanoporous graphene can help to generate nanoislands on the graphene surface, facilitating atomic diffusion bonding to bulk copper bonding surfaces at low temperatures, according to our extensive molecular dynamics (MD) simulations on the bonding process and pull-out verification using the canonical ensemble (NVT). Furthermore, the interfacial mechanical characteristics of graphene/Cu nanocomposites can be greatly improved by the resistance of nanostructure in nanoporous graphene. These findings are useful in designing advanced metallic surface bonding processes and graphene-based composites with tenable performance. Surface bonding is an essential step in three-dimensional electronic packaging, as it provides mechanical support, heat transfer, and electrical integration . A low-tSince its isolation as a two-dimensional (2D) system, graphene-copper nanolayered composites have been widely acclaimed and proved to be the effective alternatives to pure metal materials owing to their outstanding mechanical and heat transfer properties ,13,14,15Here, we report a simple new Cu-nanoporous graphene-Cu based bonding technology that is of low bonding temperature and high reliability. We transfer nanoporous graphene to the copper surface and form copper nanoparticles on nanopores by depositing copper atoms before thermocompression bonding. Since the copper nanoparticles deposited in the nanopores form a metal bond connection with the copper bonding surface, they effectively enhance the interfacial connection and, consequently, lead to higher interfacial shear strength between graphene and Cu surface. At the same time, the metal nanoparticles contribute to solid-state diffusion and intermixing of surface atoms between the interfaces at low temperatures. The present study aims to quantitatively investigate the above-mentioned process. Comprehensive molecular dynamic simulations are carried out, and the results reveal that the copper-nanoporous graphene-copper sandwich structure can be bonded at low temperature by the thermocompression method, and the interfacial shear strength of the interface can be significantly increased by nanoporous-nanometal particles hybrid structure.x and y directions while the nonperiodic boundary condition is applied along the z-direction. The equilibrium structures are achieved using the canonical ensemble (NVT) [For molecular dynamics simulation of Cu-nanoporous graphene-Cu bonding process and the reinforcing effect evaluation, the setup of the simulations performed in the present work is shown in le (NVT)  of the cNext, the bottommost two layers are fixed. The other atoms above the fixed layers are defined as temperature control layers that control the system temperature using the canonical ensemble (NVT), and the time step is set to 0.5 fs. In the condition of copper sputtering deposition simulation, the deposition rate of incident Cu atoms is 0.25 atoms/ps. The coordinates of incident Cu atoms were distributed randomly within the defined insertion volume, which is 50 nm above the graphene surface, as shown in Then the bonding model is constructed by introducing a single crystal copper cell of the same size as the previous composite model as shown in x-direction until it is completely pulled out from the bonding interface as shown in −5 Å/fs is adopted to 245 atoms treated as a rigid body on the rightmost side of the graphene. A temperature of 300 K is employed for all simulations, and a time step of 0.5 fs is adopted throughout the whole simulation process. The boundary condition for the simulation box is periodic in the y directions while the nonperiodic boundary condition is applied along the x and z directions.Finally, the pull-out simulation is used to investigate the interfacial shear strength of Cu nanocomposite. During the pull-out simulations, the upmost two layers and the bottommost two layers of Cu atoms are fully fixed. The pull-out simulations using the canonical ensemble (NVT) are performed by applying a constant velocity to the graphene along the The embedded atomic method (EAM)  models sFirst, the Cu atoms deposition onto the nanoporous graphene surface is investigated. As shown in Then thermocompression bonding simulations are performed to exploit the bonding mechanisms of nanoporous graphene-based hybrid architecture. A constant temperature of 300 K and pressure of 0.5 MPa are applied to the model. In the initial state, two contact interfaces form between nanoislands and the bulk copper surface as shown in x-axis, a constant velocity is delivered to the graphene. Pull-out simulations of the nanoporous graphene from the metal matrix are used to investigate the reinforcing mechanisms of Cu-nanoporous graphene-Cu nanocomposites. Along the When the sliding distance is larger than 4 Å, the force decreases to about 18 nN when the first column of nanoporous is fully pulled out. As mentioned above, the deformation resistance of the nanostructures obtained by depositing copper atoms in nanoporous is the key to enhancing the interfacial mechanical properties of composites. For this purpose, the effect of the number of deposited copper atoms on the pull-out force is investigated in the present work. The influence of nanoporous diameters on the reinforcing effect of graphene/Cu nanocomposites is also further studied. We have considered three different nanoporous diameters: small (3.45 Å), medium (8.07 Å), and large (11.93 Å). The number of deposited atoms is 500. As shown in In this study, we report a novel Cu-nanoporous graphene-Cu based bonding technology that is of low bonding temperature and high reliability. The process of depositing copper atoms and thermocompression bonding has been numerically investigated by employing MD simulations. Numerical results show that deposition of copper atoms onto nanoporous graphene can help to generate nanoislands on the graphene surface, facilitating atomic diffusion bonding to bulk copper bonding surfaces at low temperatures. Moreover, the resistance of nanostructure in the nanoporous can dramatically improve the interfacial mechanical properties of graphene/Cu nanocomposites. It is worth mentioning that an increase in the number of deposited atoms enhances the maximum pull-out force, but too many atoms deposited will reduce the interfacial strength. While larger nanoporous helps to form bigger and stronger nanostructures in the nanoporous, which are not deformable and require greater pull-out forces to destroy the nanostructures. Decidedly, the research findings of this study provide a feasible and facile route for low-temperature metal surface bonding with a high-performance metal nanocomposites interface layer reinforced by nanoporous graphene.


Underlying the regulation of mammalian gene expression at the level of transcription is the structure and modifications of chromatin. Understanding the twisting structures of DNA wrapped around histones and their higher-level ordering allows us to peek into a vast regulatory landscape. Looking closer, the physical accessibility of the genome provides information on the positions of nucleosomes and biologically-active regions — promoters, enhancers, insulators, and other regulatory elements. Currently, Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a widely used technology for detecting genome-wide chromatin accessibility These advantages of ATAC-seq — simplicity, speed, and low input material requirements — have made it highly popular in recent years. Publications and datasets using ATAC-seq have increased exponentially e.g., reads under peak ratio, background, promoter enrichment, and subsampling enrichment) in conjunction with other traditional QC metrics, Liu et al. conducts QC checks at different steps of data processing and provide a user-friendly visualization of quality reports. Even further, Liu et al. incorporate a PE-asSE strategy to better analyze the Tn5 transposase insertion event within ATAC-seq data. Briefly, Liu et al. shift each end of the non-redundant uniquely mapped read pair +4 bp/−5 bp to define the Tn5 insertion position and then further extend 75 bp in both directions around the Tn5 insertion position. By applying this strategy, one non-redundant uniquely mapped read pair is divided into two single-end fragments. Applying the PE-asSE approach to ATAC-seq data of GM12878 cells leads to the identification of ∼ 99.9% peaks that can be identified by traditional strategies and an additional ∼ 23%  peaks that cannot be discovered by traditional methods. Further analysis shows that most of these PE-asSE-specific peaks overlap with known GM12878 DNase I hypersensitive sites (DHSs) and are highly enriched in all active histone modifications. Taken together, the additional open chromatin regions (OCRs) identified by PE-asSE are likely to be true functional regulatory elements rather than false positives, highlighting dramatic improvements in ATAC-seq OCR discovery. Given these improvements, Liu et al. further extend the usage of AIAP to differentially accessible region (DAR) analysis, applying AIAP to ATAC-seq data of mouse liver at embryonic day 11.5 (E11.5) and postnatal day 0 (P0). Peak differential analysis of data collected at these two time points results in an ∼ 35% increase in the number of DARs identified. Overall, Liu et al. present an ATAC-seq QC and analysis pipeline that dramatically improves the sensitivity of both peak calling and differential analysis.In this issue, Liu et al. In another paper of this issue, Qiu et al. present the Containerized Bioinformatics workflow for Reproducible ChIP/ATAC-seq Analysis (CoBRA), which provides a comprehensive and customizable ChIP and ATAC-seq analysis pipeline In short, AIAP and CoBRA, the two useful ATAC-seq analysis pipelines developed by Liu et al. and Qiu et al., will provide a convenient entry and general guideline for scientists with limited computational experience to explore ATAC-seq data. Both pipelines use docker to allow compatibility on different operating systems and are able to generate high-quality figures automatically. These two studies represent significant progress in ATAC-seq analysis. Despite their benefits, more efforts are needed to extend the power of these ATAC-seq pipelines in the future. For example, more alternative tools may be included for each analytical step. Currently, both AIAP and CoBRA pipelines only include a popular software MACS2 Ya Cui: Conceptualization, Writing – original draft, Writing - reviewing & editing, Supervision. Jason Sheng Li: Writing – original draft, Writing - reviewing & editing. Wei Li: Conceptualization, Writing - reviewing & editing, Funding acquisition, Supervision. All authors have read and approved the final manuscript.2021 The Authors. Published by Elsevier B.V. and Science Press on behalf of Beijing Institute of Genomics, Chinese Academy of Sciences / China National Center for Bioinformation and Genetics Society of China.This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Competing interestsThe authors declare no competing financial interests.Ya Cui: Conceptualization, Writing – original draft, Writing – review & editing, Supervision. Jason Sheng Li: Writing – original draft, Writing – review & editing. Wei Li: Conceptualization, Writing – review & editing, Funding acquisition, Supervision.