The area of space immediately around the floral display is likely to have an increased level of humidity relative to the environment around it, due to both nectar evaporation and floral transpiration. This increased level of floral humidity could act as a close-distance cue for pollinators or influence thermoregulation, pollen viability and infection of flowers by fungal pathogens. However, with a few exceptions, not much is known about the patterns of floral humidity in flowering plants or the physiological traits that result in its generation. We conducted a survey of 42 radially symmetrical flower species (representing 21 widely spread families) under controlled conditions. Humidity was measured using a novel robot arm technique that allowed us to take measurements along transects across and above the floral surface. The intensity of floral humidity was found to vary between different flower species. Thirty of the species we surveyed presented levels of humidity exceeding a control comparable to background humidity levels, while twelve species did not. Patterns of floral humidity also differed across species. Nevertheless, floral humidity tended to be highest near the center of the flower, and decreased logarithmically with increasing distance above the flower, normally declining to background levels within 30 mm. It remains unclear how physiological traits influence the diversity of floral humidity discovered in this survey, but floral shape seems to also influence floral humidity. These results demonstrate that floral humidity may occur in a wide range of species and that there might be greater level of diversity and complexity in this floral trait than previously known. Some floral traits are difficult for humans to identify but nevertheless influence floral function, or are detectable to floral visitors . Floral Digitalis, Echium, and Helleborus flowers  was mounted in the center of the sampling zone. A scale floorplan for this room is given in All floral humidity measurements were taken within a 6.11 m × 3.67 m room within the Bristol University Life Sciences Building (51°45′N 2°60′W) that had temperature and airflow controls for maintaining a constant background temperature and humidity. A 3.72 m × 3.67 m section of this room, hereafter referred to as the ‘sampling zone,’ was separated off within a 2 m high wall of 10 mm thick clear polycarbonate. A six-axis articulated The background temperature was kept constant at 23.01 ± 1.38°C , as the amount of water vapor indicated by a relative humidity value varies depending on air temperature . For exaArduino UNO microcontroller . The Arduino transmitted the measurements via a USB port to a PC located outside the sampling zone (behind the polycarbonate screen). Two probes were used in measurements: a ‘background probe,’ which measured background humidity, and a ‘focal probe,’ which measured the humidity in the headspace of the object being sampled. The focal probe was mounted on the robot as detailed in All humidity measurements were taken using DHT-22 humidity probes  attached to an 3 horticultural tubes was fixed along one length of the table (A 75 cm × 90 cm × 74 cm (width × length × height) wooden table was placed in the sampling zone. A rack for 24 cmhe table  where hohe table  in order–1) and paused before starting to record measurements to allow any minute air disturbances to settle. Overall, the robotic arm was also unlikely to change the intensity and gradients of humidity produced by the sampled flowers.The two microcomputers registering the values from the focal and background probes were at a distance of 34.5 and 32.0 cm from their respective probes. They did not heat up significantly or possess any elements that might have generated air turbulence, whilst all elements relating to the control of the robot arm and the data-storing PC were located behind the polycarbonate screen . This me3 plastic horticulture tube. Horticulture tubes were filled to a point 2 cm from the tubes’ lid to provide the flowers unrestricted access to water. Cut flowers were then taken into the lab in these tubes, and measurements started quickly after arrival. If flowers were grown outdoors, care was taken to collect them in dry conditions, so that no standing water from condensation or rain could influence the measurements. Flowers were only used if they were fully open and did not show signs of age, disease or damage.To separate floral humidity from other sources of humidity on the plant, flowers were cut and placed in horticulture tubes prior to the start of the measurements. Furthermore, cutting allowed us to control the orientation of flowers during sampling . Flowersblu tack: Bostik, Paris, France) stuck to the tube lid  was 11:01 h, ranging from 9:10 h to 14:00 h. A full list of the times of the first measurements taken on each individual flower and control are given in All humidity measurements were carried out autonomously by the robot after moving the focal probe to predefined measurement positions in a predefined order, creating a transect-like sequence of humidity measurements. As a first step on a sampling day, the measurement positions of the focal probe had to be adjusted using manual controls, in order to account for different sizes and shapes of flowers. Thus, once flowers were ready for sampling, the focal probe was maneuvered to a ‘transect central point’ above each flower – the point above the center of the flower, 5 mm higher than the flower’s highest point. The transect central point for each flower and a single point less than 2.5 cm of the background probe were stored in the robot’s memory. Once a point is stored in the robot’s memory, the robot can return the focal probe to this position, and positions relative to them, in a predefined sequence.x axis movements are horizontal, moving toward and away from the robot ; z axis movements are up and down . The axes of the robot relative to the room and the table in the sampling zone are indicated in The sequence order in which individual flowers were sampled on each day was selected via a randomization algorithm within the arm’s software. The robot began each sampling sequence with the focal probe to a ‘safe’ position 50 cm above the table . In the x axis, and then along the z axis, relative to the flower  move to the safe position, (2) move to the flower’s or control’s position, start transecting and sampling humidity along the e flower , (3) movx axis from that flower’s transect central point . Measurements were taken in 5 mm intervals along this transect . Humidity measurements occurred regardless of whether the probe was above the flower surface at a sampling point of not (most flowers sampled are less then 60 mm wide). This meant that, even though flowers differed in size, all humidity transects were still the same size, aiding comparisons of floral humidity between different flowers and species. Following completion of measurements of the x axis transect, the robot moved the focal probe to a position that was offset by 5 mm in the z axis from that flower’s transect central point . Again, measurements were taken in 5 mm intervals along this transect . This sequence of movements within the transects, as well as the direction of movements and the orientation of x and z transects relative to the flower and robot, was the same in all transects (as demonstrated in –1). A third (y axis) transect was deemed unnecessary because all flowers and inflorescences that we sampled showed high radial symmetry.In order to start taking measurements whilst transecting the headspace of a flower or probe, the arm moved from the safe position toward the flower or control to a point that was offset by –30 mm in the al point . This poal point . This povia the safe position when moving to and from a probe-control measurement was to avoid disturbances of the headspaces in the flower array.Following completion of both transects on a flower the robot would return to the safe position 50 cm above the table, then move the focal probe to the point near to the background probe (approximately at a distance 2.5 cm from it). Here, it would carry out a probe-control measurement for 230 s, as described below. Following completion of a probe-control measurement, the robot would move the focal probe back to the ‘safe’ position starting the sampling of the next flower or control in the predefined sequence. The movement Only one set of flower arrays was collected and measured per day. The initial randomized order chosen by the robot was maintained across all four replications of the measurement sequence on a sampling day. This meant that each flower was resampled four times within time intervals of 231–308 min. Following completion of the probe control measurement on the final flower in the sequence on the last replicate sample, the robot would return to the ‘safe’ position and power down. All raw data collected are available in At each sampling point along transects , to predict one probe’s measurements from the other when in this position. This was later used to adjust the ‘uncorrected focal relative humidity’ (funcorrected) values for other points usingProbes can vary slightly in their estimations of the same levels of humidity (±5% according to the manufacturer’s specifications). Thus, the probe-control measurements were necessary to calculate how much the probes differed in estimates and reduce this source of inaccuracy. Upon moving the focal probe next to the background probe, the focal probe waited for 30 s before measuring humidity for 200 s, and the background probe made simultaneous measurements. Assuming that both probes are measuring a point of the same humidity during the probe-control measurements we could later compute linear regression parameters, using the MATLAB ‘W and M are, respectively, the slope and intercept parameters obtained from regressing the focal probe measurements against the background probes measurements for the time period of the probe-control measurements. This focal probe correction (Eq. 1) was calculated and applied across each replicate sample of the flowers sampled on the same day . This was done to account for any possibility that the difference between probes may change over time.where To characterize between-species differences in flower size, a flower’s horizontal span was measured on fresh unsampled flowers against a rigid tape measure, or where not possible, estimated based on miscellaneous literature records (cited in the “Results” section). Fresh flowers or literature records, as opposed to measurements from the individual flowers sampled under the robot, were used to measure flower size as such measurement might disturb floral headspace and floral humidity production. Flower or inflorescence structure, where appropriate to each species, was classified based on species descriptions from n = 14) of the species surveyed. Petals were removed from fresh unsampled flowers, and a mold was made of the upper petal surface using dental wax . Again, fresh flowers were used to survey for stomatal presence, as opposed to those sampled under the robot, as such sampling may damage or disturb the floral headspace and floral humidity production. A cast was made from this mold using clear nail polish, and was mounted on a microscope slide using tape . Mounted casts were surveyed from petal base to tip using a light microscope at ×100 magnification, and the presence of stomata was recorded. A species’ presence of stomata was expressed as the percentage of petals surveyed that showed the presence of any stomata.The presence of petal stomata was surveyed in a subset (R 3.4.1 (fcorrected) and the simultaneous background measurements (fbackground) taken throughout the transects can be expressed as a relative humidity whereAll analyses were conducted in R 3.4.1 . The difRH > 0 an increase in relative humidity is detected by the focal probe.Where ΔRH, Eq. 2) was calculated within each measurement period . These averages at each measurement period, were then used for analyses of humidity structure. For each flower species (and control), a series of linear models describing different humidity structures were fitted to this x and z axis data, as described below. How well these different models described humidity structure was compared using AIC to identify the best-fitting model of x and z axis humidity structure for each species (and control). These best-fitting models were then used to estimate humidity intensity for each species (see below). The most complex model (the ‘full model’) fitted to x axis transect data of each species had a quadratic structure, the most complex model fitted to the z axis data had a logarithmic structure. These full models also allowed humidity to vary between repeated transects. All other models represented simplified versions of the full models. These quadratic and logarithmic models were chosen over more complicated curve fitting methods and functions as, in addition to their relative simplicity, these functions easily condense to other structures as models are simplified . This allows the identity of best-fitting models to indicate differences in floral humidity structure, and changes in humidity over repeat transects. Throughout all floral humidity models, flower identity was included as a random factor influencing the intercept, or amount of humidity produced.The mean difference between the focal and background probe  within rptR 0.9.21  measurements taken during the first 20 s and the last 20 s at each measurement period, denoted The turbulence generated by the movement of the probe may cause humidity to not settle by the end of the 30-s settling time, which could cause a change in humidity measurements at the focal probe between the start of a measurement period (where remaining turbulence would be greatest), and the end (where it would be most stable). It is possible this difference may differ dependent upon the humidity at a sampling point: if little water is present in the air, disruption of that area may affect humidity estimates less than more humid air. To assess the effect of remaining turbulence, the change across a measurement period should therefore be assessed while accounting for how humid the disturbed area is. Furthermore, the directionality (positive or negative) of this change in humidity may differ depending on how the air mixes as a result of turbulence and the humidity of air being mixed in. If turbulence brings in more humid air, humidity may be raised, while the reverse might be expected if less humid air is brought in. To avoid turbulence effects being obscured by conflicting (positive or negative) changes in humidity, the non-directional change in humidity across a measurement period should be considered, rather than just the change in humidity. The presence and extent of remaining turbulence after the settling time was assessed by calculating the means of the focal humidity (c. 100 measurements), x axis transect of a given species or control isThe full model fitted to the X refers to the x axis offset of the sampling point (from –30 mm to +30 mm in steps of 5 mm) relative to the transect central point (where X = 0), t varies from 1 to 4, as the sampling sequence was repeated four times for each flower or control: 1 being the first run of a sampling sequence; 2, 3, and 4 being the second, third, and fourth repetitions, respectively. ΔRHxnt is the mean ΔRH for the measurement period at sampling point X, on transect repeat sample t, on flower n. Ax and Bx are parameters that describe the positioning and slope of the x axis humidity profile in the initial transect. Parameter Ix is the model intercept in the initial transect. Ix is modified by random factor vxn, which represents the change in model intercept, and consequently the intensity of humidity generated, by individual flower (or control tube) n on the x axis transect. Further random factors accounting for individual variation in the shape of humidity structure were not included, in order to simplify models. As flowers may show changes in humidity produced over time across replicate transects, Ax, Bx, and Ix are also modified by parameters ax, bx, and ix, which represent the change in the offset, slope and intercept, respectively  between first and latter replicate transects. The values of ax, bx and ix within each replicate transect are modified as follows:where gtx, ctx, and rtx are the values of ax, bx, and ix during repeat sample transect t, whenwhere gtx, ctx, and rtx are the changes applied to Ax, Bx, and Ix in transect t. Here parameters Ax, Bx, Ix, vxn and all iterations of gtx, ctx, and rtx are parameters to be estimated. The x axis model described by Eq. (4) assumes a degree of symmetry in humidity structure, this is due to the high level of radial symmetry shown by all species sampled. Although some degree of asymmetry, off-center positioning, is included in the model (primarily through the effects of parameters Ax and ax).Consequently z axis transect isThe full model applied to the Z refers to the sampling point’s z axis offset relative to the transect central point  function in the same manner described for respective parameters in the x axis model. Here, parameters Iz, Bz, vzn and all iterations of ctz and rtz are parameters to be estimated.Here, parameter al point . All othx and z transects described in Eqs (4) and (11), as well as simpler versions of these models, were fitted to the x and z transect data of each species and each control group. Simpler models were achieved by removing certain parameters from the full models, done by forcing these parameters to have values of zero. Eleven x axis models and five z axis models were compared for each species. These models are summarized in x and z axis models, respectively. These models were then compared using AIC to select the best models for the x and z transects of each flower species following the guidelines within The full models for the x transect as predicted by the best-fitting humidity model, was calculated for each flower species. This x axis offset (parameter X) value that the best-fitting model predicted to give the largest difference in humidity on transect t, for each species. x axis model of each species. If the best-fitting x axis model was m0 or m4 . In species where the x axis showed a quadratic relationship in the best-fitting model, X value of the vertex of the quadratic curve described by Eq. (4) . Due to this a formula for x axis model can be derived, as detailed in To evaluate the intensity of humidity generated by each flower species and by controls r m4 see , a valuex axis model of each species.xn was set to zero:Once RHx, the In instances where replicate transects had an influence on ΔA phylogenetically controlled analysis was carried out to assess the extent to which floral humidity produced by species . This means that within each measurement period the majority of measurements are about the mean value. This confirms the robot’s consistency of measurements and validates our decision to use mean values of each measurement period for our analyses of humidity structure, as measurements within each period are largely similar.The robot arm showed high repeatability over each set of approximately 100 measurements made in the same measurement period  for each control are given in To detect potential sources of extraneous humidity, we conducted measurements of control samples, such as empty or water filled tubes without flowers placed inside the array . The besblu tack putty gives off a small amount of water as it dries out.When the horticulture tubes had no water in them we saw little change in humidity between focal and background probes, hence the low controls . This suThe addition of water to the controls increased humidity, as seen by the higher controls . This sh control . Humidit control . This isTaken together, it seems that at most humidity extraneous to the flowers from the horticulture tube will be similar to that of the TWL control . Frequently, there was increased variation in humidity about the center of individual flowers, with a positive skew, relative to the rest of the transect  had non-quadratic best-fitting x axis models, as shown in Bx in some way. The x axis humidity structures of six species are given in X = 0) for 22 species, Typically, the humidity structure detected in the al point .z axis were less consistent in structure. The z axis structures of six species are given in z axis models showed a logarithmic relationship with humidity declining with increased distance from the flower (increased z axis offset). In most instances humidity reaching background levels by the end of the 30 mm transect. Although 17 species favored a flat z axis model where humidity remained level across the transect (Humidity profiles in the transect  as seen r), but in 13 species  showed some changes in structure of floral humidity with replicate transects (interacting effects determined by g and c). In most species the humidity decreased in the later replicate transects  explained the data better than the model with BM correlation . The model parameter effects are summarized in p = 0.092).The fitted full phylogenetically controlled model with OU correlation . We note here that we deliberately excluded leaves from our survey, although these were considered with Calystegia silvatica). Varying levels of floral humidity between species may mean that humidity could provide sensory information that pollinators could not only use to determine the presence and location of rewards on a flower, but also potentially facilitate the discrimination between different flower types. The differing floral humidity may reflect different humidity demands on pollen viability, such as the presence of desiccation resistant pollen that may mitigate the need to elevate floral humidity  produced humidity of an intensity that exceeded these  at once. Such procedures would sample the floral headspace more completely and create more three-dimensional measurements of floral humidity structure. Our models assumed a degree symmetry in x axis humidity structure as all species sampled showed a high degree of radial symmetry. For the same reason we did not carry out a y axis transect. Our survey suggests radially symmetrical flowers generally show symmetry in their floral humidity, given x axis humidity structure is quadratic in most species (y axis). Similarly, more complex sampling procedures could be applied to investigate the occurrence of floral humidity in flower species that do not show radial symmetry. It would be expected that such differences in floral shape related to floral symmetry would lead to differences in floral humidity structure compared to radially symmetrical flowers, particularly the symmetry of floral humidity.The transects used in this survey only sampled partial cross-sections of the whole flower headspace  as oppos species . HoweverO. caespitosa were not too dissimilar from those reported by x axis humidity signal of approximately 4% . This would allow floral transpiration and nectar evaporation to occur in a manner more similar to how it would in an open environment, perhaps allowing a more natural humidity equilibrium to be reached. Furthermore, the arm was also capable of more complex motion, allowing us to conduct continuous probe control measurements throughout the experiment, reducing variation as a result of differences between probes.The measurements we made of espitosa , resultiespitosa . It coulespitosa . AlternaOur survey of floral humidity across different plant species has revealed that floral humidity can be detected in many species. Our transects showed that floral humidity intensity and humidity structure can vary between species, although the reasons behind this variation remain unclear. In this way, our transects reveal floral humidity to show a greater level of diversity and complexity than previously known. This wider occurrence of floral humidity could enhance our knowledge of plant-pollinator signaling, as floral humidity is a known cue for at least one pollinating species . SimilarHyles lineata . NH contributed to study conceptualization, funding acquisition, and writing (review and editing). HK carried out robot programming and initial operation. HW contributed to study conceptualization, funding acquisition, and writing (review and editing). SR contributed to study conceptualization, funding acquisition, carried out phylogenetic analyses, and writing .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Looking for goal-relevant objects in our various environments is one of the most ubiquitous tasks the human visual system has to accomplish Wolfe, . Visual  N = 85 observers]. We have recently referred to this as the “down-side” of spatial context learning  extract and encode repeatedly encountered distractor-target relations in spatial long-term memory, the latter likely involving the medial temporal lobes (MTL). Once established, these memories are retrieved automatically by the search display presented on a given trial, with the activated contextual (distractor-to-target) associations providing pointers to the target location (and thus enhancing its activity) within the search-guiding attentional-priority map. Once consolidated, these representations come to generate memory-related costs in situations requiring contextual adaptation, that is, when the target is consistently repositioned to some other, “new” location within an otherwise unchanged distractor layout: in this case, the distractor context points to the “old” target location, thus cueing attention to the “wrong” location. Given that these memory-based (mis-) guidance signals are triggered automatically, it takes extensive practice to overcome them  signal between the current display input and the expected, that is, initially learned, target position. This mismatch signal would eventually also promote new learning, that is, the adaptation of contextual cueing to the changed distractor-target relations. Recent evidence suggests that the brain indeed works along these lines. For instance, in a recent study  or the rate of change between these displays  “belief” about the general level of regularity prevailing in the current sensory environment, with potential implications for overcoming associative blocking and enabling contextual re-learning.One promising computational mechanism that potentially mediates contextual cueing is spike-timing dependent plasticity  adaptability of contextual cueing. The relevant findings suggest that the relational memory underlying contextual cueing should in principle be flexible and thus be able to incorporate target position changes into previously acquired context memories. For instance, a bulk of neuroscientific data suggest that contextual cueing is supported by the medial temporal lobe (MTL), and especially the hippocampus (HC; e.g., Chun and Phelps, Irrespective of the type of – HC and/or PHC – memory system supporting contextual cueing, a model of the memory-guided search must specify how and when context-based memories interact with basic visual-search processes. This question was addressed in a series of recent electrophysiological and patient studies showing that long-term memory for scenes aids visual processing very rapidly, with the earliest electrophysiological markers of guidance becoming evident only 80 ms post display onset at occipital electrodes contralateral to the hemifield of the target (Zinchenko et al., The phenomenon of contextual cueing illustrates that the human visual system constantly generates predictions from repeatedly encountered events and scenes in our environment that help us to rapidly detect and respond to critical target objects in familiar contexts (Chun and Jiang, We argue that, considered together, these perspectives can provide new impetus for research on the task factors that regulate the interplay of the two types of representation and the effects of more HC-dependent or more PHC-dependent representations on initial contextual learning and subsequent contextual adaptation (for first demonstrations, see, e.g.,: Lleras and Von Mühlenen, The studies involving human participants were reviewed and approved by the Ethics Committee of the LMU Department Psychology. Participants provided their written informed consent to participate in this study.TG, HM, and AZ conceived and designed the perspective article. WS and MC contributed to discussion. TG, HM, and MC wrote the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this work, we report a novel method of label-free detection of small molecules based on direct observation of interferometric signal change in graphene-modified glasses. The interferometric sensor chips are fabricated via a conventional wet transfer method of CVD-grown graphene onto the glass coverslips, lowering the device cost and allowing for upscaling the sensor fabrication. For the first time, we report the use of graphene functionalized by the aptamer as the bioreceptor, in conjunction with Spectral-Phase Interferometry (SPI) for detection of ochratoxin A (OTA). In a direct assay with an OTA-specific aptamer, we demonstrated a quick and significant change of the optical signal in response to the maximum tolerable level of OTA concentration. The sensor regeneration is possible in urea solution. The developed platform enables a direct method of kinetic analysis of small molecules using a low-cost optical chip with a graphene-aptamer sensing layer. Development of label-free, highly sensitive, direct, and real-time-enabled optical biosensors for small molecules (less than 1 kDa in size) is a challenging task of high importance in many areas of health analysis, environmental and food control, agriculture, etc. .In the last decade, the graphene-coated surface plasmon resonance (SPR) interfaces have been actively studied for application in biosensing ,4. Graphwww.realsense.rs), aiming to develop sensors for optimization of a cultivated meat bioprocess, it is important to consider sensing options for mycotoxins as well, including OTA.Ochratoxin A (OTA) is one of the most frequently occurring mycotoxins found in a number of different food products, including meat, meat products, and edible offal . In the The cultivated meat bioprocess starts with the seed train of primary cells, obtained by a biopsy from the animal. Since the animal may have been already fed with mycotoxin-contaminated feed , it is iDetecting the changes in optical properties such as absorption, reflection, and an interferometric pattern in the presence of analytes is one of the prospective methods for mycotoxin detection. Recently, graphene-based refractive index optical sensors were demonstrated for detection of different analytes ,15. The p- polarizations of a single beam [However, interferometric methods are less sensitive to graphene integration and can provide easy functionalization of the sensing area , yieldingle beam . Biorecegle beam . Aptamergle beam . Howevergle beam ,21. It igle beam ,23.Most commonly, interferometric sensors use the competitive immunoassay format or the binding inhibition assay where the target is attached to the surface ,24. VariIn this work, we report, for the first time, the label-free optical detection of ochratoxin A (OTA) in a direct assay using graphene functionalized with the OTA-specific aptamer and implementing the method of spectral phase interference (SPI). The SPI method is based on measuring the phase shift in an interference pattern produced by the reflected light from different surfaces within the sensor chip . This me2 size. The graphene from the backside of the copper foil was etched in solution made of deionized (DI) water, hydrochloric acid (30%), and hydrogen peroxide solution . To remove the copper foil, the sample was etched in aqueous solution of iron (III) chloride for about two hours. Prior to graphene transfer, the surface of the glass cover slips was washed in boiled dimethylformamide (DMF) and activated in oxygen plasma for 5 min. PMMA/graphene stack was rinsed in deionized (DI) water, and transferred onto the glass cover slip. A chip was left under ambient conditions overnight to dry out. To reflow the PMMA and improve the graphene-to-substrate adhesion, we annealed the chip at 150 °C for 5 min [Single-layer graphene chemical vapor deposited (CVD) on 25-µm thick copper foil was provided by Graphenea (Spain). A poly (methyl methacrylate)  was spin-coated with a step-wise increase of the speed from 1500 to 2500 rpm for 1 min on top of the graphene/copper stack and used as a support layer during the transfer. The stack was baked on a hot plate at 150 °C for 5 min. We cut the copper/graphene/PMMA stack to pieces of about 4 × 5 mmor 5 min . Afterwaor 5 min .n-hydroxysuccinimide ester (PBASE) in dimethylformamide (DMF) was spread over the cover glass for 6 h in order to activate the graphene surface. Afterward, the glass was washed thoroughly with DMF, isopropanol (IPA), and DI water for 3 min each. At the last step, the glass was gently blown with the air gun. The OTA aptamer with sequence of GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA [via an n-hydroxysuccinimide cross-linking reaction [Aptamer immobilization on graphene was performed in a way previously described for graphene-based transistor . 100 µL  CGG ACA ,30 with reaction  was carr2 and laser power of 0.5 mW. A Solver Pro atomic force microscope (AFM)  was used to study the morphology of the graphene transferred onto the cover slip. The silicon semi-contact cantilevers NSG03  with resonance frequency of 105 kHz were used in order to avoid any damage to the graphene during characterization.Raman spectra were recorded on the Centaur HR microRaman spectrometer  with a 100× objective at a 532-nm wavelength  with a beam spot of ≈1 μmTo investigate the effect of the small molecules’ conjugations with the modified graphene, we dissolved the OTA solution  in PBS in different concentrations. We used a compact single-channel label-free biosensor without thermal stabilization based on the spectral-phase interferometry, which was powered and controlled via a notebook USB port   is relaWe employed the possibility of the measuring system to follow the kinetics of the binding process to control the aptamers’ immobilization on graphene functionalized by the π-π stacked PBASE. The glass/graphene stack was exposed overnight to a 300-nM OTA aptamer solution in PBS a. The imIn this research, we used the OTA mycotoxin (403.8 Da ) as an eτ was calculated from the time course of the sensogram for the 10-nM concentration of OTA (τ is depending on toxin concentration and rates of association and dissociation of the aptamer and OTA. The results of binding kinetics fitting according to Formula (1) give an approximation of τ ≈ 6 min for 10 nM, which is about 40% lower than previously recorded data for the OTA aptamer by methods of total internal reflection ellipsometry (TIRE) [The response time n of OTA b based oy (TIRE) . In TablBetween measurements, the sensor was regenerated by washing in urea solution. We observed a nonlinear response to OTA concentration with the limit of detection of OTA above 1 nM. The thickness changes occurring upon OTA binding to the aptamer are presumed as the main mechanism of the sensor response. The shift in Fermi level under adsorption or reconfiguration of molecules (aptamer) can be interfered by the thickness change in absorbing molecules as the aptamer transforms from the linear stack to the G-quadruplex  with an However, the response of graphene to small molecules can be even more complex. When the aptamer molecule is attached to the graphene, it will increase electrostatic doping by the negatively charged aromatic bases of the nucleic acid in the aptamer molecule. Furthermore, the conformational change of the aptamer after bonding to the surface leads to decrease in electrostatic doping and, hence, change in the conductivity of graphene . These cIn conclusion, we have developed a proof-of-concept for the robust and scalable methodology based on the interferometric optical sensors for direct, label-free detection of the small molecules, such as mycotoxins by aptamer on a glass surface. We prepared the cover glass/graphene stack as a sensor chip with the OTA-aptamer covalently bonded onto the graphene surface. Implementing a compact interferometric biosensor powered and controlled via a notebook USB port, we demonstrated the sensitivity on the level of 10 nM of OTA with an assay time of 6 min. This technology is very flexible and can provide the basis for a direct assay of various small molecules’ detection based on aptamer-functionalized graphene, potentially in a multiplex format. Given that the interaction of aptamers and small molecules can be registered via changes of the graphene optical properties, it opens the way for utilization of the assay in drug discovery, food, and medical analyses. The described graphene-based measurement system is a potential new tool for cultivated meat contamination assessment as well.


This article presents an overview of twisted bilayer graphene (tBLG) on their fabrication techniques and twisting angle-dependent properties.The properties of tBLG can be controlled by controlling the twisting angle between two graphene sheets. Two-dimensional (2D) materials exhibit enhanced physical, chemical, electronic, and optical properties when compared to those of bulk materials. Graphene demands significant attention due to its superior physical and electronic characteristics among different types of 2D materials. The bilayer graphene is fabricated by the stacking of the two monolayers of graphene. The twisted bilayer graphene (tBLG) superlattice is formed when these layers are twisted at a small angle. The presence of disorders and interlayer interactions in tBLG enhances several characteristics, including the optical and electrical properties. The studies on twisted bilayer graphene have been exciting and challenging thus far, especially after superconductivity was reported in tBLG at the magic angle. This article reviews the current progress in the fabrication techniques of twisted bilayer graphene and its twisting angle-dependent properties. It has attracted abundant interest after its first isolation was achieved through the micromechanical cleavage of graphite in 2004 [Graphene is composed of a one-atom-thick  in 2004 –4. It ha in 2004 –9. Howev in 2004 , their m in 2004 –16. The  in 2004 –19. The  in 2004 , 20, 21. in 2004 –27.θ) [θ = 1.05°) [Tc) ~ 1 K is comparatively high [Twisted bilayer graphene (tBLG) is fabricated by the stacking of two monolayers of graphene with a specific twisting angle (θ) ; in thisθ) , 29. Recθ) –33 and eθ) , which wθ) , 39–41. θ) , 28. Thiθ) . The Fer= 1.05°) ; howeverely high . Meanwhiely high –65. In t2/Si substrates via a simple wet-chemistry method [2/Si in the same way. The prepared sample is baked in the air at 150 °C for 20 min for enhancing the adhesion between the SLG substrate and SLG–SLG [In this section, we reviewed some of the recent advanced synthesis methods used to fabricate twisted graphene sheets. The twisted graphene layers are formed on the surface of crystalline graphite naturally due to the accidental folding of graphene layers onto other graphene flakes or themselves , 67. Sevy method –78, withy method . The twi SLG–SLG . In this2 substrate using a femtosecond laser and was divided into two pieces with a pair of parallel and straight cutting lines, as represented in Fig. θ and were accurately stacked together onto to Si/SiO2 substrate using a suitable transfer method. The control over the twisting angle of tBLG fabricated by this method is better than the other preparation methods.In another report, Kim et al.  reported2/Si substrate to make hydrophilic and hydrophobic sections with a distinct border, (2) the controlled SLG/PMMA delamination from the hydrophilic section in water, and (3) removal of the PMMA layer. The controlled folding in this procedure allows for the fabrication of tBLG structure with distinct stacking orientations; the required twisting angle was achieved by varying the hydrophilic and hydrophobic boundary folding angles. In a recent report, Cao et al. [Wang et al.  preparedo et al.  fabricatθ), as compared to the other [G band resonance and optical absorption and improves the chemical reactivity of tBLG [The variation in the graphene properties is produced due to the mode of stacking orientation among the two graphene layers and the number of stacked graphene layers , 84. Thehe other . The schhe other . The Dirhe other –89. The he other , causinghe other –93, whic of tBLG . The ban of tBLG , confirm of tBLG  by using of tBLG . The moi of tBLG . The sim of tBLG . The tBL of tBLG , 103.FigC) vibration modes and in-plane G and 2D vibration modes. The first-order Raman endorsed peak in graphene is a G band observed at ~ 1584 cm−1, accomplishing momentum conservation, which commands that the scattered phonon must carry no momentum. The D and D′ bands are originated from the intervalley and intravalley double-resonance Raman scattering mechanisms, respectively [R and R′ modes, which are from the TO and LO phonon branches, respectively [θ-dependent q wave vector, which triggers phonons inside of the Brillouin zone, due to which the layer breathing vibrations (ZO′ phonons) can be studied in a first-order light scattering [Raman spectroscopy plays a crucial role in the nondestructive analysis of the lattice structure, as well as the optical, electronic, and phonon properties of graphene , 105. Raectively –110. Twoectively –113, canattering . Lui et attering .−1 Raman shifts and observed two modes in a smaller range of twist angle. Figure G Raman peak intensity is intensely enhanced as a function of twisting angle, which illustrates that these G Raman modes and the lower-energy modes share the equivalent resonance amplification mechanism. The intensity of the enhanced G peak and the 2D band displays a maximum blueshift than the SLG at 12° twisting angle   [−1 are activated by the formation of moiré pattern [L is observed at ~ 94 cm−1. The intensities of (ZO′)L frequency mode, and the background covering on which the lower-energy Raman lines are comprised, demonstrate substantial resonance enrichment near the 12° twisting angle; it is associated with the G Raman peak enhancement. Another Raman mode observed at ~ 52 cm−1 is ascribed to the twisting mode due to the rotation between the two graphene layers with respect to each other. The advancement of the frequency and FWHM of the ZO′ mode as a function of L normalized I2D are shown in Fig. L mode frequency increases with the I2D when the twisting angle is less than the critical angle, and the frequency becomes nearly constant, when the twisting angle is greater than the critical angle. This illustrates that the intense variations in the FWHM and frequency of the (ZO′)L mode occurred when the twisting angle is close to the critical angle (12°). The low-frequency phonon dispersion shown in Fig. −1, and the phonon wave vector kL(ZO′) are in good arrangement with the ZO′ phonon dispersion in tBLG and confirming the obligation of this mode with the layer breathing mode (ZO′)L. The Raman study reveals that the fundamental properties of tBLG are different from the Bernal-stacked bilayer graphene.For investigating the twisting angle effects in tBLG, He et al.  carried le) Fig. a. The phngle (θ) , 117. Th pattern , 117. Th2 substrate which is shown in Fig. G band intensity increases for the ~ 12° twisting angle, attributable to distinctiveness in the joint density of states (JDOS) of tBLG, and its energy is entirely depending on the twisting angle, and the strength of optical transition is directed by the interlayer interactions, which allow direct optical imaging of these parameters, as shown in Fig. G band images of the similar tBLG domain at different excitation energies and the plot of Eex versus θpeak are shown in Fig. The Raman imaging reveals the differences in interlayer interactions, which is useful for the investigation of external physical, chemical, and optical properties of bilayer graphene. The white light dissimilarity enhances the reflection image of single and bilayer graphene on SiOθ = 0.01° to 0.2°) is found in the lower-energy range. The transition developments for the tBLG structures with θ = 10° are shown in the inset of Fig. σ1(ω) spectra of tBLG with different twisting angles show two separate interband transitions  the major absorption peak α originates from the transitions vHs2 → BE1 and BE2 → vHs1 [α-peak toward the higher energy as the twisting angle increases [σLB into two edges with various energies reveals that the Dirac cones of the bottom and top layers are transferred by varying the total energy [α show variation with the gate bias, as the interlayer potential irregularity breakdowns the configuration of the band edge and vHs. The VG-driven variation of σ1(ω) for the hole doping region is shown in Fig. σLB in Fig. α-peak toward lower energy, whereas its intensity is decreased noticeably, which confirms the changing of the band structure of tBLG with the gating. The optical absorption spectra showed notable variations like the shifting of inter-van Hove singularity transition peak and the splitting of interlinear band absorption, as well as the appearance of an extremely strong intra-valence band transition.The decreasing in the twisting angle interference among the two lattice periods creates a moiré pattern with a higher wavelength, where the properties of tBLG such as van Hove singularity and bandgaps seem to be present in the far-infrared region and the band velocity of the Dirac cone is considerably decreased , 119–122ons Fig. b: (1) Th2 → vHs1 , 129. Inl energy . The ints- and p-polarized lights with consequent laser wavelength consistent with the absorption enhancement, as shown in Fig. The PL excitation spectrum recorded after 2-photon excitation, also the twisting angle-associated tunable linear absorption spectrum, is shown in Fig. 2/Si substrate embedded with two terminals are shown in Fig. G band of 13° twisted tBLG domain showed a consistent 20-fold enrichment compared with the 7° twisted tBLG domain, as shown in Fig. Recently, Yin et al.  reportedσ) of the tMLG device in the 90 K to 273 K temperature range is shown in Fig. The perfect and superior characteristics of the bilayer graphene in twisted multilayer graphene (tMLG) than the suspended form of graphene could be attributed to the fact that the tMLG is tens of nanometer in thickness and maintains the graphene layers ultra-clean, as well as free from any substrate influence. These extraordinary properties in tMLG generate from the higher degree of decoupling that occurs from the angular twisting between the layers . RecentlThe interlayer contact conductance among the BLG with different twisting and stacking structures synthesized by the CVD method is recently discussed by Yu et al. . A statiρ) of the tBLG domain (θ = 1.06°) measured near the flat band condition for carrier densities spanning the lower-energy band is shown in Fig. ns/4 and superconducting states at different partial band fillings are revealed in Fig. ρ(T)) increases with the increase in temperature and remains steady with the metallic behavior. The resistivity (ρ(T)) measured in tBLG devices with different twisting angles near − ns/2 is shown in Fig. TH; the resistivity scales linearly with temperature below the temperature TH. At the lowest temperatures, resistivity diverges from a linear dependence on temperature. The tBLG devices clearly show the resistivity saturation, superconducting, or insulating behavior at moderate temperature regimes. The observed three distinctive temperature regimes are noticeable by the different behavior of resistivity (ρ(T)) depending on the carrier density and twisting angle.Polshyn et al.  discusseK or K′ wave vectors for the two graphene monolayers , as a function of temperature (T(K)) for two tBLG devices with 1.16° and 1.05° twisting angles, demonstrated zero resistance at 70 mK revealing the superconducting state  determined using a resistance of 50% of the non-superconducting state value was about 1.7 and 0.5 K for the two tBLG devices. The two-probe conductance versus carrier density at zero magnetic fields and a 0.4 T perpendicular magnetic field of M1 device is shown in Fig. n = 0). It also shows the states of insulating at the superlattice bandgaps n = ± ns. The insulating state at ± 3.2 × 1012 cm−2 is as a result of the presence of single-particle bandgaps in a band structure, as well as the observed conductance minima connected with many body gaps [12 to − 1.9 × 1012 cm−2 electrons per unit cell, the conductance was significantly high for nil magnetic fields than in the vertical 0.4 T magnetic field (B⊥), which reveals the presence of superconductivity at the magic angle. The density-dependent resistance of the tBLG device with a 1.14° twisting angle plotted almost over the complete flat band density range is shown in Fig. δn < 2 × 1010 cm−2), and at the magic angle (θ = ~ 1.1), the resultant hybridization and moiré superlattice among the graphene layers caused the development of a remote flat band at the charge neutrality point (CNP) [ns/2 and +3 ns/4. The superconductivity regions emerge in both electron- and hole-doped regions at ~ 10 mK base temperature with the dropping of resistance to zero. However, for ± ns/2 densities, below the base temperature, no sign of superconductivity is observed. The superconductivity appears considerably absent or weak on the lower-density side and extra stronger in the higher-density side of the insulator in both bands.The unusual superconducting behavior of different materials has been studied broadly for the last decades. The weak interlayer interaction creates the interlayer coupling in tBLG, and the strength of interlayer coupling as well as twisting angles equally affects the Fermi velocity and the VHSs of tBLG, which makes the novel electronic state of tBLG, which is different from those in SLG –140. Theers Fig. b. The inate Fig. c. The crody gaps . At 70 mnt (CNP) , 143. Inθ = 1.1 ± 0.1°) [B) and longitudinal resistance (Rxx) versus an elongated gate voltage (Vg) range at a 1.7 K temperature (Landau fan pattern) are shown in Fig. Vg = ± 0.85 V, as the lower-energy moiré bands are filling at densities nm = ± 4 (where nm is the number of charges per moiré unit cell) [Vg = 0.43 V from which an alongside set of Landau levels emanate. The peak on the half-filling and the twofold degeneracy of Landau levels illustrate the breaking of the symmetry of spin valley [nm = 0, ± 4, and +2, the resistance peaks are clearly observed in Rxx(Vg) at B = 0 and for different temperatures (0.28 to 5.2 K)  decreases, ρ decreases to zero with the two consecutive steep successions at T ~ 0.3 K and T ~ 1.5 K  curves at two descriptive densities (Vg = 0.58 V and Vg = 0.50 V) are shown in Fig. Ic) is observed at Vg = 0.58 V and observed the supercurrent for an elongated range of density with Ic (~ 1 to 15 nA). The temperature dependence of the resistance peaks is nearly invisible, as the temperature is enhanced above ~ 5 K  , 140. Thit cell) , 141. Thn valley  and the  K) Fig. b. The Rx5 K Fig. c; it cou5 K Fig. d. The Ar and hBN , 148; thL mode illustrates the influence of the twisting angle-dependent electronic band overlaps, onto the Raman spectrum. The continuous variation in optical and electrical properties of tBLG is strongly dependent on the twisting angle (θ) among the two graphene layers. We believe that the development and variation in the optical properties of tBLG would be extensively used in the future in the field of optoelectronics. The tBLG devices displayed non-monotonous conductivity variation, which reveals a semiconductor to metallic transition. The superconducting properties observed in tBLG are due to the electron interactions, which can distinctly influence the properties of moiré superlattices at higher densities and smaller twisting angles. There are quite a few challenges that are related to achieving a control over the twisting of two graphene layers for the development in the fabrication and characterization of twisted bilayer graphene (tBLG). It is expected that theoretical studies will be published in the future to search for novel superconducting and insulating phases of tBLG at a lower temperature.The objective of this review paper is to provide detailed information regarding the fabrication of twisted bilayer graphene (tBLG) via different methods, its properties, as well as its technological applicability. The tBLG-related research field has developed at an enormous speed. The prominent tBLG fabrication methods such as micromechanical exfoliation, CVD, graphene flake pickup, CRS, stacking methods, and their unique properties are summarized in the initial sections. The control over the twisting of two graphene layers is the major challenge in the fabrication of tBLG. The twisted bilayer graphene (tBLG) is a novel arrangement, which shows the basic properties are different from those of the stacked bilayer graphene. The variation in line width and position of the (ZO′)


Pituitary pars intermedia dysfunction (PPID) is the most prevalent endocrine disorder of older equids. To date, key research areas likely to have the greatest impact on equine health have not been identified. In human medicine, public and patient involvement is widely used to inform research agendas. This study aimed to engage with veterinary surgeons and horse owners to identify evidence gaps (‘uncertainties’) and prioritise these into a list of the 10 most important PPID research questions. The James Lind Alliance (JLA) Priority Setting Partnership (PSP) Framework was adapted. Questions about the diagnosis, treatment and prognosis of PPID were gathered via an online survey targeting veterinary surgeons and horse owners with experience of PPID. Thematic analysis was used to form a longlist of collated indicative research questions (CIRQs), defined by the JLA as true ‘evidence uncertainties’ when not answered by a published, clinically relevant, up-to-date systematic review. In an interim prioritisation survey, questions were ranked by weighted scores creating a shortlist of 25 that were taken forward to the PSP workshop, where participants reached a consensus on the top 10. Useable responses containing ≥1 question were received from 524 respondents . After screening for relevance, 1,260 individual questions were included in thematic analysis, resulting in 47 CIRQs. Interim prioritisation votes for the CIRQs were received from 360 respondents. The top 10 questions prioritised at the PSP workshop focused on long-term prognosis, diagnostic accuracy, efficacy of pergolide treatment, alternative treatment/management strategies and potential treatment options for poor responders to pergolide. The quantity of questions generated indicates an extensive number of uncertainties regarding the diagnosis, treatment and prognosis of PPID. The top 10 research questions will help to inform key areas for evidence synthesis and knowledge translation, and to direct future research into areas most important to end users involved in caring for and treating animals with PPID. Pituitary pars intermedia dysfunction (PPID) is a common age-associated equine neurodegenerative disorder ,2. WhileEnsuring that research is relevant and applicable to those who can improve patient care is essential. However, researchers do not always investigate areas or answer questions important to the end users ,21. InclVeterinary surgeons and horse owners with experience of PPID are best placed to identify questions about the disease most in need of answering. Therefore, the aim of this study was to engage these end users in order to identify their top 10 research priorities for the diagnosis, treatment and prognosis of PPID.This study did not include animal participants, therefore international, national, or institutional guidelines for humane animal treatment are not applicable. This project received institutional ethical approval from the University of Liverpool and Animal Health Trust. Following institutional ethical approval, the six steps of the JLA framework  were adaIdentification of, and contact with, collaborators to form a steering group.Development and dissemination of surveys to the target audience to gather questions they have regarding PPID.Collation, categorisation and refining of the questions submitted by participants into a longlist, and formatting these collated questions in PICO  format where possible.Searching the literature to identify if the questions are ‘uncertainties’. An uncertainty is defined by the JLA as “a question which cannot be answered by a relevant, up-to-date systematic review” .If >30 uncertainties are identified, interim prioritisation is undertaken to form a short list of 25 questions taken forward to the PSP workshop.The organisation of a PSP workshop.A steering group was established to run and oversee the Priority Setting Partnership (PSP). The steering group consisted of veterinary surgeons, including specialists in evidence-based medicine and equine internal medicine, as well as horse owners with experience of PPID. A protocol detailing the specific objectives of the PSP was developed in accordance with the JLA guidelines ,33 S1 A and adapth April 2017 to 9th June 2017.A survey was developed to collect questions from respondents based on the JLA guidelines using the freely available online survey tool, Kwiksurveys  S2 Appe. The surResponses were downloaded into a Microsoft Excel spreadsheet and anonymised. Each individual question was screened for relevance and to ensure the inclusion and exclusion detailed in the JLA PSP protocol  were metThe literature was searched for relevant, up-to-date systematic reviews using appropriate databases including MEDLINE, CABI, SCOPUS, Web of Science (WOS) and the online database of citations for systematic reviews of relevance to veterinary medicine and science VetSRev . Broad srd October 2017 to 20th November 2017. Questions were ranked via weighted scores, depending on the number of votes received, to ensure equal weighting of veterinary surgeon and horse owner responses. Separate rankings were then combined to form a final ranked list of all questions. The top 25 questions were taken forward to final PSP workshop.From the initial survey, more than 30 uncertainties were identified; therefore an interim survey was undertaken to prioritise which uncertainties should be included in the PSP workshop . A link Participants from both surveys who expressed an interest in participating further, and had provided contact details for this purpose, were invited to attend the final PSP workshop. The final priority setting was conducted in a face-to-face workshop using both small and whole group discussions, utilising an adapted nominal group technique ,37. ThisA total of 524 usable responses, which contained at least one question about diagnosis, treatment and/or prognosis, were received from veterinary surgeons and/or horse owners with experience of PPID in the initial online survey. The majority of responses were from owners , of which 438 currently owned a horse with PPID and 47 had previously owned a horse with PPID. The remaining 39 responses were from veterinary surgeons, of which 35 were currently in practice treating horses with PPID, and four were also current owners of a horse with PPID. The majority of respondents were from GB , and 7.3% (n = 38) were from other regions including North America (n = 12), Europe (n = 10), Ireland, the British Channel Islands and Isle of Man (n = 9), Australasia (n = 6) and Asia (n = 1). A further two respondents did not provide information regarding their country of residence.The process of rationalising the list of questions is summarised in th December 2017 . Median participant ratings for their overall experience, usefulness, purpose and organisation of the PSP workshop out of 10, were 9.5, 9, 10 and 10, respectively.This PSP achieved engagement with horse owners and veterinary surgeons across two phases of prioritisation to identify research priorities for PPID. The JLA framework was successfully applied to an equine disease of complex pathophysiology, indicating this methodology could be effectively applied to other equine diseases. The research priorities, within the pre-defined topics of diagnosis, treatment and prognosis, included: [i] long-term prognosis, [ii] diagnostic accuracy, [iii] efficacy of pergolide, [iv] alternative treatment/management strategies and [v] potential treatment options for poor responders to treatment with pergolide. As the study was conducted in GB and respondents were primarily from GB, differences in horse populations, management and treatment practices around the world means the questions raised here may not be globally representative.The PSP followed a rigorous and transparent predefined process to create a top 10 list of shared research priorities. The process gathered a large number of responses, comparable with other JLA PSPs ,40. The Various methods were used to distribute the survey with some being more successful than others. Advertising via social media was effective for recruiting horse owners but not veterinary surgeons. A large number of veterinary surgeons were contacted via BI’s mailing list. However, the number of veterinary responses compared to horse owners was disappointing. This may have been for a number of reasons, such as they were not interested in engaging in this type of research, the open question design of the questionnaire discouraged them from participating or they felt there were no gaps in the evidence and therefore did not have a question to pose. A more targeted approach or a prior, formal collaboration with veterinary practices may have improved veterinary responses. However, a low response rate from veterinary surgeons and veterinary practices has been noted in other studies ,42. Explet al (2014) [Despite the low number of responses, questions posed by participating veterinary surgeons did not markedly differ from those posed by owners. Around a quarter of questions raised by participants were not specific to diagnosis, treatment or prognosis and instead covered other PPID-related subjects. This is comparable the previous veterinary PSP conducted by Dean l (2014) . The majl (2014) . Many ofl (2014) . This alThe ‘Choose Ten’ interim prioritisation approach was chosen because it is an uncomplicated way of allowing participants to consider the whole list then make choices that involve genuine shortlisting . DespiteDuring the adaptation of the JLA protocol, it was decided that the JLA definition of an ‘uncertainty’ would be applied for the equine veterinary setting. There are few systematic reviews in equine medicine; at the time of writing only 23 were available on the VetSRev database. This meant it was likely all questions posed by participants would be defined as uncertainties. However, it was agreed by the steering group that the level of certainty could not be lowered and the quality of evidence should remain the same across fields. Systematic reviews collate data from numerous studies and offer the highest level of evidence , and if The final PSP workshop allowed an open and thoughtful exchange of views between horse owners and veterinary surgeons. This enabled consensus to be developed and facilitated the identification of the top 10 research questions. Both groups were represented by an appropriate number of participants ,33. All Two of the top 10 questions related to the accuracy and interpretation of diagnostic tests. In several studies, utilising a variety of different clinical reference standards, the commonly used basal ACTH test has been reported to have good sensitivity and specificity ,50,53–55The majority of questions centred around treatment , with quPriority questions identified in this PSP are potentially methodologically complex to answer in terms of study design, implementation, ethical concerns and financial limitations. The JLA process is not concerned with how the questions raised will be answered: its function is to provide a platform for the involvement of end users –27. HoweThis study shows that horse owners and veterinary surgeons can be involved in identifying and prioritising uncertainties. The involvement of veterinary surgeons and owners at this stage of the research process has the ability to improve available evidence, ensure research is relevant to end users and aid decision making. During the PSP process horse owners acted as a proxy for the patient. The JLA has previously been utilised in this way for feline medicine . This adThe JLA methodology can be successfully adapted into an equine veterinary setting and applied to the diagnosis, treatment and prognosis of PPID in horses. The response and quantity of questions generated indicates an extensive number of uncertainties about the disease. However, as the research was undertaken in GB, it is possible that the research questions prioritised may differ from unanswered questions of veterinary surgeons and horse owners in other countries. Identifying the top 10 research questions for a disease or condition, especially those that require long-term management, will help to direct evidence synthesis, knowledge translation and future research into areas most important to the end users.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.S4 Appendix(XLSX)Click here for additional data file.S5 Appendix(XLSX)Click here for additional data file.S6 Appendix(XLSX)Click here for additional data file.


It is assumed that THK5317 PET/MRI may also depict tau protein in the skeletal muscles of patients with sIBM. Here we introduced a novel application of tau PET in diagnosis of sIBM in a rare case.Sporadic inclusion body myositis (sIBM) is a chronic progressive idiopathic inflammatory myopathy, with characteristic rimmed vacuoles and sarcoplasmic abnormal tau protein deposits. THK5317, an We presented a 46-year-old woman who suffered from progressive lower limb weakness for one and a half year.Needle electromyography showed myogenic damage. Characteristic myopathological changes of sIBM were discovered, and abnormal tau protein deposits were identified by tau immunostaining. Genetic testing ruled out the GNE myopathy, a hereditary distal myopathy with rimmed vacuoles. The patient was finally diagnosed as sIBM.18F] THK5317 PET/MRI on the patient.We performed  THK5317 PET can reveal muscular tau deposition in vivo, which provides a new and noninvasive diagnostic method for sIBM and offers the opportunity to monitor the progression of tau pathology along with muscle impairment.[ Further research investigated that a variety of abnormal protein deposits similar to those in the brain of patients with Alzheimer's disease (AD) have been discovered in sIBM muscle fibers, such as phosphorylated tau protein. This suggests that sIBM and AD have similar pathogenesis and pathological characteristics. In vivo study on patients with AD using [18F] THK5317 has shown considerable cortical uptake and temporal lobe retention, in comparison to healthy control individual. Based on the similarities of pathologic features shared by sIBM and AD, we tentatively display tau protein deposits in vivo by using [18F] THK5317 PET/MRI in a female patient with typical pathological features of sIBM. This is the first reported sIBM case who was performed by [18F] THK5317 PET/MRI and muscle biopsy.Sporadic inclusion body myositis (sIBM) is described as an uncommon, progressive, inflammatory myopathy, mostly involving patients older than 50.This study passed the ethical review by the Medical Ethics Committee of Chinese PLA General Hospital.2A 46-year-old woman was presented to our hospital with progressive lower limb weakness for one and a half year with no family history of neuromuscular diseases. Her previous medical history was normal. Neurological examination showed normal speech and mental status, and the cranial nerve examination was normal. Manual muscle strength testing was entirely normal in the upper extremities, and lower in bilateral proximal (grade 3) and distal (grade 4) of pelvic limbs. Neck flexor and extensor strength were normal. Deep tendon reflexes were normal, without Hoffman signs or clonus, and plantar responses were flexor. The sensory examination was normal. No truncal or appendicular ataxia was noted. Serum creatine kinase (CK) level was 689.6 U/L , and lactic dehydrogenase (LDH) level was 243.7 U/L .The patient's nerve conduction studies were normal. Her needle electromyography demonstrated fibrillation potentials in quadriceps and anterior tibialis muscle, with small motor unit potentials and rapid recruitment in these and upper and lower extremities muscles, including first interosseous muscle, quadriceps and anterior tibialis muscle. Muscle biopsy of the left quadriceps muscle revealed some necrotic fibers, rimmed vacuoles in muscle fibers with basophilic deposits Fig. A, B, and18F] THK5317 PET/MRI was performed on the patient after she signed the informed consent. Forty minutes after intravenous injection of [18F] THK5317, whole body distribution of radioactivity was measured with a Siemens PET/MRI scanner (MAGNETOM Biograph mMR). PET image reconstruction based on MRI was performed to analyze the difference of radioactive uptake in muscles. Reading of [18F] THK5317 PET/MRI image was performed by at least two nuclear medicine physicians, looking for focally enhanced uptake within skeletal muscles. The case presented with increased [18F] THK5317-SUV levels in the biopsy site of the left quadriceps with corresponding tau positive immunostaining. Tau uptakes of bilateral quadriceps were increased to SUV level of 0.73/0.67 (right/left) (Fig. Subsequent  THK5317-PET can depict muscular tau deposits in vivo. Although muscle biopsy can reveal abnormal deposition of tau protein in sIBM, but it cannot provide the condition of tau deposition within all skeletal muscles. Our case further confirms that the muscles of patients with sIBM can show tau-tracer binding in vivo. The application of tau PET has allowed identification of tau deposition in vivo and can help to improve for the ability and efficiency of sIBM diagnosis.In our study, we introduced a novel application of tau PET in diagnosis of sporadic inclusion body myositis in a case which is diagnosed with pathological definite sIBM. To our knowledge, this is the first report on using of the tau PET/MRI within sIBM patient, which presented a significantly increased uptake of tau protein within involved muscles. This suggests that  THK5317 could be an excellent imaging agent for tau protein in sIBM patients and offer the opportunity to monitor the progression of tau pathology along with muscle impairment.Maetzler et al previously reported that 18F] THK5317-PET can reveal muscular tau deposition in vivo, which provides a new and noninvasive diagnostic method for sIBM and offers the opportunity to monitor the progression of tau pathology along with muscle impairment. While due to only one patient included, our findings must be considered as preliminary and further study in a larger number of participant patients is needed to confirm these findings.In conclusion, [We thank the patient and her family for their kind cooperation.Conceptualization: Ke Li, Qiang Shi.Data curation: Yutong Zhang, Ke Li.Formal analysis: Yutong Zhang.Funding acquisition: Ke Li, Chuanqiang Pu, Qiang Shi.Investigation: Yutong Zhang.Methodology: Haodan Dang, Jiajin Liu.Project administration: Qiang Shi.Resources: Ke Li.Supervision: Chuanqiang Pu.Visualization: Haodan Dang, Jiajin Liu.Writing – original draft: Yutong Zhang, Ke Li.Writing – review & editing: Qiang Shi.


Moreover, the minimum TWR obtained for graphene-based dielectric, i.e., 0.17 mg/min is approximately 1.5 times less than that achieved with kerosene.Titanium alloys, especially Ti-6Al-4V, which is considered a difficult-to-cut material, bears numerous applications in aerospace and biomedical sectors. The criticality of the accurate formation of the machined cavity for the said applications and properties of Ti-6Al-4V accentuated the use of electric discharge machining (EDM). However, the issues of lower material removal rate (MRR) and tool wear (TWR) discouraged the use of EDM. These inherent issues hold a pivotal role regarding the sustainable machining of Ti-alloy. Therefore, in this research the potentiality of kerosene-based dielectric, having graphene nanoparticles, is comprehensively examined for the sustainable EDM of Ti-6Al-4V, which was not focused upon yet. Experimentation was performed under Taguchi’s design (L18) with three types of electrodes, namely Aluminum, Brass and Copper. In total, 36 experiments were conducted, of which 18 were with graphene-mixed dielectric and the remaining were with kerosene. Experimental results reveal that the brass electrode with negative tool polarity yields higher MRR for both types of dielectrics. The maximum MRR (7.602 mm Titanium (Ti) and its alloys are extensively employed in the manufacturing of different parts/products in many industrial and commercial applications. Especially, Ti-6Al-4V bears a range of industrial applications, owing to its fabulous characteristics, such as high-temperature stability, remarkable strength-to-weight ratio and excellent corrosion resistance. These attributes make Ti-6Al-4V useful in the chemical industry, medical science, aerospace, automotive industry, energy sector, food industry, and defence industry etc. ,2,3. DesEDM is considered as a valuable machining technique for the cutting of hard-to-machine materials, such as Ti-6Al-4V ,11,12. HMRR is described as the volume of the material dislodged from the specimen during the machining time. The productivity of EDM typically depends on the MRR, which reflects the operative speed of production . Another2O3 nanoparticles in deionized water lowers the micro-cracks on the machined cavity in the EDM of super alloy Inconel 825 with a copper electrode [Many researchers have revealed that the dielectric with particles is generally preferred for improving MRR and TWR in EDM. Erden and Bilgin  investiglectrode . Anotherlectrode . The outlectrode .Although significant efforts have been made in the past for achieving high MRR and low TWR in the EDM of Ti6Al4V, considering different means, i.e., by adding certain additives or by adjusting the parameters or by some modification in the cutting mechanism. However, the potential of graphene nanoparticles mixed with kerosene dielectric has not been explicitly studied so far in the EDM of Ti6Al4V. It is pertinent to mention that graphene has attracted the focus of the researchers, owing to its remarkable characteristics. Therefore, this research aims to comprehensively inspect the influence/impact of graphene-based dielectric on the EDM of Ti6Al4V in terms of MRR and TWR. The selection of these two response attributes is based on their well-proven contribution towards ensuring the sustainability of the machining process. The achievement of higher MRR by the addition of graphene in the kerosene at same electrical input is a depiction that lesser resources are consumed at the power generation station. So, the low level of pollution contributed to the environment if graphene-based dielectric is engaged in EDM. In other words, it helps to maintain a clean environment. The aspect of TWR is linked with sustainability as it accounts for the wastage of material. Hence, its lower value is deemed necessary for warranting the sustainability of the EDM. Considering the importance of the set responses, experimentation has been planned under Taguchi’s design (L18). In total, 36 experiments were performed: 18 with kerosene and the remaining 18 with graphene-mixed kerosene. The experimental results were analysed with the statistical tools. An optical microscopic analysis has also been done for providing the insight of the parametric effects in relation to the selected responses. Optimal settings have also been developed, based on the parametric trends.Ti-6Al-4V was taken as a workpiece herein, which has numerous applications in aerospace, automobile, chemical, and biomedical applications . The worIn this setup, a separate container with a stirrer inside was used for adding graphene nanoparticles in the kerosene. The stirrer was used to ensure the homogeneity of the mixture during experiments. In each of the experiments, a cavity with a diameter of 9 mm and depth of 0.3 mm was produced, using an electrode of 9 mm. The three different electrode materials, namely copper, brass and aluminium were engaged in this research to select the best tool material for the sustainable cutting of the selected work part. The cross sections of all the electrodes are shown in Pulse time ratio (PTR) is the ratio of on-time to off-time. The selection of these control variables was made because their impact was rated significant with regard to MRR and TWR in the EDM of Ti6Al4V ,42,43,44Experimentation was planned according to Taguchi’s experimental design, which was proved as a promising design technique in a similar study conducted on the same material . ConsideInitial and final masses of workpiece and tool have been measured. MRR was calculated by volumetric method, whereas TWR was assessed in terms of mass reduction. MRR and TWR were found using the relations mentioned as Equation (1) and Equation (2), respectively.x, y, d and t represent lateral dimension, axial dimension, depth and cutting time, whereas, iM and fM denote the initial and final masses of the electrode, respectively.Here, After calculating the MRR and TWR for all the experiments, the results have been comprehensively analysed using statistical techniques, such as parametric line plots and bar charts, etc. These plots have been drawn considering the mean values of the responses. Against each of the parametric levels, the values of MRR have been noted for all the experiments conducted on the specified level value of the parameter. As polarity has two levels, this means that nine experiments were performed at each tool polarity. However, in a case when a parameter has three levels, as in the case of current against each of the levels, six experiments were performed. For analysis purposes, the mean value of the response against the defined parametric level was computed. Since current has three levels, this means three data points are available that are linked with a line to depict a parametric trend for the set response, i.e., MRR. The similar procedure is opted for in the case of TWR. Moreover, optical microscopic analysis was performed to have physical evidence of the findings. A detailed comparison has been made for the results achieved with the graphene-mixed dielectric and simple kerosene. Finally, an optimal combination of input variables has been developed based on parametric analysis.Experimental results are provided in It is observed that discharge current is the main governing factor that affects the MRR in the EDM of Ti6Al4V using a graphene-based dielectric, as illustrated in As the value of current increases from 6 to 8 amps, the MRR decreases because of the presence of graphene particles. Their availability in the discharge gap creates more hindrance in front of sparks, which results in a lesser MRR. However, the magnitude of MRR sharply improved as the discharge current value raised from 8 to 10 amps. The rise in MRR is associated with the generation of intense heat energy in the gap between the tool and workpiece. At high energy, a larger quantity of material was removed in the form of melted debris from the workpiece surface, resulting in higher MRR. In any case, given the large amount of heat input, the higher the magnitude of current, which also yields powerful discharges. These discrete penetrating discharges intensively hit the liquid material in the liquid puddle and caused the ejection of more material, leaving deeper craters in the machined region . The avaThe experiment results declared that the kerosene-based dielectric showed a somehow similar trend for MRR, as that was portrayed by the graphene mixed dielectric. The MRR is decreased with the rise in current up to 8 amps due to the deposition of the carbon layer over the surface of the tool, which causes improper discharging between the workpiece and the tool . SubsequBy examining the results (one with a graphene-based dielectric and other with a kerosene-based dielectric), it has been confirmed that the material removal rate is higher for a graphene-based dielectric. This is attributed to the fact that the presence of graphene nanoparticles influences the spark erosion phenomenon in EDM. Their existence in the dielectric fluid provides a powerful explosion, which induces a strong penetrating heat flux in the workpiece. Thus, a higher MRR is achieved in the EDM of Ti6Al4V with the application of graphene nanoparticles in the dielectric in contrast to simple kerosene, as demonstrated in Three electrodes—aluminium, brass and copper—were used for experimentation. Their effect is shown in The cutting performance of all the electrodes was also compared in terms of MRR in the EDM of the Ti-alloy using a graphene-mixed dielectric, as shown in In case of kerosene dielectric, the electrode of Al provides the highest MRR amongst the available alternatives. The decreasing trend of MRR is observed for the kerosene-based dielectric from aluminium to copper, as presented in Polarity is another important control variable for getting higher MRR. Two types of polarities are engaged in the EDM process, i.e., positive and negative polarity. Usually, polarity refers to the “polarity of the tool”. Positive polarity means positive polarity of the tool and, similarly, negative polarity designates the negative polarity of the tool. In the EDM process, if the tool is positively charged then obviously the workpiece is negative and vice versa. The effect of polarity on MRR in the EDM of Ti6Al4V using both kerosene and graphene-based dielectrics is demonstrated in However, the MRR found at negative polarity with the graphene–kerosene mixture is notably higher in contest to that achieved with kerosene. Hence, negative polarity is more suited when graphene particles are mixed in the dielectric for getting the high value of MRR. It has been confirmed from the results that MRR is higher when machining has been performed at negative polarity because the spark formation is greater, which forced the material to wear out.Pulse–time ratio (PTR) is another important factor which greatly affects the MRR in EDM, as highlighted in In the case of the kerosene dielectric, a decreasing trend of MRR is observed for a pulse–time ratio from 0.5 to 1.5, as presented in Flushing is a phenomenon of removing debris from the gap between tool and workpiece. It is considered as an important function in Electric Discharge Machining. There are various types of flushing techniques, such as pressure flushing, suction flushing, and jet flushing, etc. Each technique has its own application, as per the need and requirement. M.M. Makenzi et al.  reportedThe larger value of flushing pressure provides better MRR when the graphene-based dielectric is employed, as depicted in the The effect of spark voltage on the defined response by applying the two types of dielectrics in EDM of Ti-alloy is provided in The effect of discharge current on TWR is shown in The micrograph shown in The comparison of the TWR achieved against the two different dielectrics revealed that the rate of tool wear is significantly lower with a graphene-based dielectric if an 8 A current is used in contest to that found with kerosene.The influence of the electrode material for the set response is shown in the plot presented in The one-to-one comparison of the electrode materials in terms of TWR was also performed and is highlighted in It is noticed that the magnitude of TWR is different at positive and negative tool polarities in the EDM of the selected workpiece material, as demonstrated in In a nut shell, the positive polarity of the tool is appreciated in minimizing the wear of the tool. This polarity of the tool ensures the excessive heat dissipation in the surface of the workpiece at the end of the discharge duration and helps in the stabilisation the spark. Ultimately, TWR is decreased.on from 25 to 50 µsec while keeping Toff constant at 50 µsec. This carbon layer acts as a wear-resistant layer for the electrode and assists in reducing its wear. Another reason that contributes to the lowering of TWR is the expansion of the plasma channel at larger values of on-time. Such a kind of expansion of the plasma channel hinders the effective transfer of heat energy to the workpiece and the tool as well. Since a meagre heat input is experienced by the tool, thereof its wear in notably reduced. It is pertinent to mention that the trend of said control variable is similar for graphene-mixed and kerosene dielectrics in the EDM of Ti6Al4V, as depicted in The effect of the pulse–time ratio (ratio between pulse on-time to pulse off-time) on tool wear rate is illustrated in the Flushing time is the time which is required to remove the debris between the workpiece and electrode gap. The impact of flushing time on tool wear rate is displayed in The behaviour of flush time (under kerosene as dielectric) for TWR is dissimilar to that observed in the case of nano particle slurry. Initially, TWR decreases with the rise in said input parameter up to 6 µsec but, afterwards, the opposite trend is noticed. The lowering of TWR until 6 µsec is credited to the solidification of the melted droplets on the electrode surface because of the phenomenon of quenching . HoweverThe influence of spark voltage on TWR using the graphene- and kerosene-based dielectric is provided in Considering the effects of all the parameters accumulatively, it is concluded that the optimal combination of parameters for achieving the maximum MRR at negative tool polarity using brass under the graphene-mixed dielectric is current = 10 A, pulse–time ratio 1, flushing time = 6 µsec and spark voltage = 4 V. On the other hand, to get a lower value of TWR, the most preferable levels of parameters are  under graphene nanoparticle slurry as the dielectric. The optimum conditions in the case of the kerosene dielectric are current (6 amps), electrode material (copper), polarity (positive), pulse–time ratio (1.0), flushing time (6 µsec), and spark voltage (3 V) as present in 4 S/m. Therefore, the presence of graphene nanoparticles influences the discharge characteristics. Their availability is a source of plasma channel’s expansion between tool and workpiece. Ultimately, the strength of the discharge breakdown got affected. These particles also played a pivotal role in the flushing of etched chips, thus, reducing the probability of abnormal sparking. Subsequently, higher MRR is the outcome. Furthermore, in the initial discharge phase of EDM the current magnitude gradually improved which reduced the impact of electron-to-electrode attack. Hence the material loss from the electrode surface notably reduces.It is inferred that, when graphene nanoparticles are used in the kerosene dielectric, then the material removal rate is enhanced, and tool wear rate is reduced significantly. Basically, the conductivity of these particles is quite appreciable, i.e., 8 × 103/min, which is 64.5% higher in comparison to the maximum MRR obtained using the kerosene dielectric, i.e., 4.62 mm3/min.The graphene-based dielectric outperforms MRR when compared with kerosene. The maximum value of MRR achieved with the graphene-based dielectric is 7.60 mmWith respect to the tool material, the electrode of brass provides the highest value of MRR when graphene mixed dielectric was used, whereas the maximum MRR in the case of kerosene is obtained with the aluminium electrode.The tool wear of the Cu electrode is found to be minimal (0.17 mg/min) when graphene nanoparticles are added in the kerosene. This value of is approximately 1.5 times lower than that achieved with kerosene only.The magnitudes of MRR and TWR are especially sensitive to the variation in the tool polarity. The MRR and TWR are both at maximum at negative polarity. Hence, negative polarity is good for achieving a high material removal rate. However, tool wear rate for all electrode materials is lower at positive polarity, thus positive tool polarity is suitable for TWR.The optimal combination of parameters for achieving the maximum MRR at negative tool polarity using brass under the graphene-mixed dielectric is current = 10 A, pulse–time ratio 1, flushing time = 6 µsec and spark voltage = 4 V.The minimal magnitude of TWR was attained with the positive polarity using a copper electrode in graphene-based slurry while keeping the values of current, pulse–time ratio, flushing time and spark voltage at 8 A, 1, 4 µsec and 3 V, respectively.This research focuses on evaluating the cutting performance of the graphene-based kerosene dielectric for the two core contributing aspects (MRR and TWR) of the sustainable electric discharge machining of titanium alloy (Ti6Al4V). Experiments were performed using three different electrode materials, i.e., aluminium, brass and copper, at both positive and negative tool polarities. In total, 36 experiments were performed; 18 were with kerosene and the rest of the experiments were with the graphene-based dielectric under L18 Taguchi design. The dielectric is used with the powder-mixed graphene-based nanoparticles. The MRR and TWR have been investigated using the graphene dielectric to achieve the optimum parameters for the machining of titanium alloy. The selection of the most preferable tool material with suitable polarity was carried out in order to have maximum material removal rate and minimum tool wear rate. Based on the experimentation findings and their detailed dissuasion, along with the physical evidence, the following conclusions are drawn:Comparing the results obtained for graphene mixed dielectric for the kerosene, it has been revealed that the addition of the graphene nanoparticles is appreciable in achieving the sustainable EDM of Ti-alloy.In the future, a detailed investigation will be carried out seek the impact of particles’ morphology on the cutting characteristics. Moreover, the surface integrity of the machined cavity will also be comprehensively examined.


Tachycineta bicolor). We address two questions: (1) do the cloacal bacterial communities differ between female and male tree swallows within a population? and (2) do pair-bonded social partners exhibit more similar cloacal bacterial communities than expected by chance? To answer these questions, we sampled the cloacal microbiome of adults during the breeding season and then used culture-independent, 16S rRNA gene amplicon sequencing to assess bacterial communities. Overall, we found that the cloacal bacterial communities of females and males were similar, and that the communities of pair-bonded social partners were not more similar than expected by chance. Our results suggest that social monogamy does not correlate with an increased similarity in cloacal bacterial community diversity or structure. As social partners were not assessed at the same time, it is possible that breeding stage differences masked social effects on bacterial community diversity and structure. Further, given that tree swallows exhibit high variation in rates of extra-pair activity, considering extra-pair activity when assessing cloacal microbial communities may be important for understanding how these bacterial communities are shaped. Further insight into how bacterial communities are shaped will ultimately shed light on potential tradeoffs associated with alternative behavioral strategies and socially-transmitted microbes.Host-associated microbial communities can influence the overall health of their animal hosts, and many factors, including behavior and physiology, can impact the formation of these complex communities. Bacteria within these communities can be transmitted socially between individuals via indirect  or direct  pathways. Limited research has been done to investigate how social interactions that occur in the context of mating shape host-associated microbial communities. To gain a better understanding of these interactions and, more specifically, to assess how mating behavior shapes an animal’s microbiome, we studied the cloacal bacterial communities of a socially monogamous yet genetically polygynous songbird, the North American tree swallow ( Host-associated microbial communities can contribute to the overall health of their animal hosts –4, and mTachycineta bicolor) and house sparrows (Passer domesticus). More recent work using culture-independent methods has revealed that pair-bonded social partners experimentally blocked from making cloacal contact have significantly less similar cloacal bacterial communities compared to control pairs in black-legged kittiwakes (Rissa tridactyla) )munities . Given tStaphylococcus spp., Campylobacter spp., Salmonella spp., and Shigella spp. In our study, we used culture-independent, next-generation sequencing. We did not find that any of the cloacal bacteria they cultured were members of the core bacterial communities of birds within our study population. While Staphylococcus spp. and Lactobacilli were detected, their relative abundances were <0.001%. This suggests that culturing of cloacal bacteria may not consistently result in isolation of the most dominant bacteria in the system. It is also possible that the bacteria cultured by Lombardo et al. [Previous work examining the similarity of the cloacal bacteria of social partners in tree swallows used culture-dependent methods . The isoo et al.  are rareIn conclusion, our results suggest that the cloacal bacterial communities of female and male tree swallows are similar within our study population and that pair-bonded social partners do not share more similar cloacal bacterial communities than expected by chance. Given that tree swallows exhibit high variation in rates of extra-pair activity, we argue that considering rates of extra-pair activity or the number of sexual partners per bird when assessing cloacal bacterial communities may be important for understanding how cloacal bacterial communities are structured. Also, since cloacal bacterial communities comprise bacteria derived from both the reproductive and digestive tract, diet should also be considered when assessing cloacal bacterial communities. Lastly, we suggest that pair-bonded social partners should be sampled simultaneously to control for any temporal shifts in individual behavior and physiology that may influence shifts in cloacal bacterial community structure across breeding stages.