Using voxelwise data and principal component analysis to identify patterns of variation across PET measures, we show that striatal D1 and D2/3 receptor availability and a pattern of mesolimbic and anterior cingulate cortex dopamine function are important for adjusting the threshold for leaving a patch to explore, with specific sensitivity to changes in travel time. These findings suggest a key role for dopamine in trading reward benefits against temporal costs to modulate behavioral adaptions to changes in the reward environment critical for foraging.Foraging behavior requires weighing costs of time to decide when to leave one reward patch to search for another. Computational and animal studies suggest that striatal dopamine is key to this process; however, the specific role of dopamine in foraging behavior in humans is not well characterized. We use positron emission tomography (PET) imaging to directly measure dopamine synthesis capacity and D The role of dopamine in foraging behaviour in humans is not well understood. Here, the authors show using PET imaging, that striatal dopamine receptor availability, and dopamine function in the anterior cingulate cortex and mesolimbic areas are related to the decision to explore new environments. In modern humans, foraging decisions typically involve how to spend time and when to switch from one rewarding activity to another, ranging from deciding when to leave one sight to visit another when exploring a new city on vacation to browsing social media4.Foraging is a type of reward-guided behavior that is essential for survival and conserved across species. In contrast to explore-exploit decision-making paradigms where the decision is to repeat (exploit) a familiar action or explore a new one, the important choice when foraging is whether to engage with the current environment or leave and search elsewhere. In foraging behavior, rewards tend to occur in clusters (patches) that deplete over time as the reward is consumed. In a canonical example of an animal searching for food, the reward patch is a tree filled with berries and the foraging decision involves when to leave one tree to search for another5, and experimental evidence supports the notion that this theorem closely describes the behavior of wild animals6 and humans performing a computer-based foraging task2. According to the MVT, animals should leave a patch of rewards in search of a new one when the reward rate in the current patch falls below the average reward rate for the environment5. In other words, when the current setting is less rewarding than the average experienced by the animal, it is optimal to leave and search for something that is likely to be better. In many ecological settings and in our experiment, the average reward rate for the environment depends on two factors: (1) the travel time between patches of rewards and (2) the reward depletion rate within each patch of rewards. For instance, a tourist who is sightseeing in a new city might remain longer at any given attraction if the next location is further away or if the thrill of experiencing the current attraction dissipates more slowly. Therefore, both travel time and reward depletion rate should be accounted for when deciding when to leave a reward patch. Of note, experimental data from humans, nonhuman primates, and other animals has shown a deviation from the MVT such that there is a consistent bias to stay in reward patches longer than optimal9. This could reflect factors not accounted for in the MVT such as preference for immediate over delayed rewards, risk of predation during travel between reward patches, activities that occur simultaneously during foraging , and varied nutritional states (e.g. hungry vs. satiated)9. However, past studies have shown that measuring the relative change in patch-leaving threshold between reward environments controls for individuals’ bias to stay and more closely reflects optimal behavior modeled with the MVT7.Foraging behavior can be quantified using the marginal value theorem (MVT)1. Specifically, the firing rate of neurons in the ACC increases the longer an animal stays in a reward patch until a specific threshold is reached. Longer travel times increased the threshold required for the patch-leaving decision, however, the underlying mechanism for how the threshold is set is not accounted for by ACC neural activity alone. A subsequent fMRI study in humans found that foraging behavior depends on the function of a widespread circuit in the brain including the midbrain, striatum, medial prefrontal cortex, and ACC, with the ACC encoding the average value of the environment and cost of foraging7. While this human study revealed commonalities in the neural basis of foraging behavior across species, it did not use a patch-foraging paradigm and therefore was unable to provide insight into how the patch-leaving threshold is set in human foragers, and was agnostic to putative neurochemical mechanisms proposed in formal computational theory11. One possible mechanism for encoding information about the environment that subsequently modulates the threshold for leaving is through neuromodulation. Dopamine is a neuromodulator that is strikingly conserved across species and known to play a key role in both learning and modulation of circuits in the prefrontal cortex13.Prior work has described optimal foraging behavior and the involved neural circuitry. A seminal study in macaques showed that neural activity in the anterior cingulate cortex (ACC) plays a key role in adjusting the patch-leaving threshold10 and, along with dopamine receptor activation, plays a role in weighing costs and benefits in the decision to exploit known reward sources or explore for new ones15. Tonic dopamine levels are predicted to vary as a function of the average reward rate of the current reward environment, increasing in rich environments when reward patches are abundant and located in proximity compared to poor environments when rewards are sparse and dispersed. Increased tonic dopamine is also thought to drive increased rate and vigor of response seen in animal studies10, as well as modulate how benefits and costs of actions are represented at the time of choice17. Three studies in humans have supported the role of dopamine in foraging behavior. In two studies of Parkinson’s disease, patients off dopaminergic medications tended to wait longer than controls before leaving a reward patch, and dopaminergic medication partially ameliorated this impairment19. A pharmacological study in healthy controls found that administration of a D2 agonist modulated foraging decisions in poor environments only3. However, we lack understanding of the role of D1 receptors, and critically, the spatial localization of dopamine effects on foraging behavior.There is limited empirical evidence on the role of dopamine in foraging behavior in humans. Computational models predict that striatal tonic dopamine encodes the average reward rate of the environment1 receptors in foraging behavior, computational models and work in animals and human genetics suggest that both D1 and D2 receptors are important for decisions that involve weighing costs and benefits and adjusting responses to maximize rewards. Specifically, there is a body of evidence supporting opposing learning effects mediated by D1 and D2 receptors facilitating approach and avoidance learning, respectively17. Tonic dopamine at the time of choice is thought to modulate the D1 and D2-mediated action values to differentially affect representations of benefits and costs. A combined pharmacological and PET study in monkeys revealed that blockade of either D1 or D2 receptors reduced the impact of reward and increased delay discounting through a synergistic effect20. Furthermore, rat and human genetic studies have supported differential roles of D1 and D2 receptor function in learning from positive and negative outcomes in comparative decision making tasks22. In a continuous-space explore-exploit task, genetic variation in D1 and D2 receptor expression was associated with complimentary roles in adjusting response times to maximize rewards, with D1 receptors implicated in speeding up and D2 receptors associated with slowing down responses23. However, a more classic explore-exploit multi-arm bandit task did not find any changes in exploration or exploitation behavior with exogenous administration of a D2 receptor antagonist24. While there have been no prior studies comparing both D1 and D2 receptor function during foraging, there are similarities in the decision-making process that suggest both receptor types may be implicated in foraging behavior as well. For example, both foraging and delay discounting decisions require weighing immediate rewards against the cost of lost time. In addition, like comparative decision making, foraging involves integrating both positive and negative feedback in the form of evaluating whether a reward was received or not at every unit of time. Finally, both explore-exploit and foraging decision-making involve balancing the benefits of exploiting or sticking with a familiar option (e.g. staying in the current reward patch) with exploring alternative options (e.g. leaving for a new reward patch).While there have not been any human studies investigating the role of dopamine D1 and D2/3 receptor availability separately in healthy adults combined with a well-validated19, computer-based task designed to measure foraging decision-making. Based on the non-human primate study showing that leave decisions occurred when ACC neural firing rate reached a set threshold that was modulated by travel time1 as well human studies implicating both exogenous dopamine administration and D2 receptor agonism in patch leaving threshold changes19, we predicted that dopamine function plays a key role in adjusting foraging behavior in response to environmental changes. Specifically, we hypothesized that individuals with higher dopamine synthesis capacity and receptor availability would enact greater adjustments in the patch leaving threshold as the reward environment changed. Preclinical work in rodent models has suggested that presynaptic synthesis capacity may be related to constitutive dopamine neuron population activity (or average number of spontaneously active dopamine neurons), which is thought to influence tonic dopamine efflux27. Furthermore, based on studies of related reward-guided decision-making tasks22, we hypothesized that both D1 and D2/3 receptors would be important for patch-leaving threshold adjustments through synergistic or complimentary effects. Lastly, while there have been no prior studies spatially localizing dopamine effects in foraging humans, we predicted that dopamine function in the ventral striatum would be particularly important for foraging decisions based on computational and animal studies highlighting its role in encoding the average reward rate of the environment28 and evaluating the opportunity cost of time29.To gain a comprehensive understanding of the role of dopamine in foraging behavior in humans, we used positron emission tomography (PET) imaging to directly measure dopamine presynaptic synthesis capacity and dopamine DFifty-seven healthy adults  were recruited from the local community. All studies were completed at the National Institutes of Health Clinical Center and were approved by the Combined Neuroscience Institutional Review Board and the National Institutes of Health Radiation Safety Committee. Participants completed written informed consent and were screened by a clinician-administered history and physical exam, routine laboratory testing, and structural MRI read by a neuroradiologist to rule out confounding medical and psychiatric disorders.18F]-FDOPA (51 subjects) to assess dopamine presynaptic synthesis capacity, [11C]-NNC112 (45 subjects) to assess dopamine D1 receptor binding potential, and [18F]-Fallypride (42 subjects) to assess dopamine D2/3 receptor binding potential. Dopamine synthesis capacity was measured over a 90-min period of rest and therefore presumably reflects a basal tonic dopamine synthesis rate27. Thirty-seven individuals completed all three PET scans.These volunteers completed a multi-tracer PET study of dopamine synthesis and receptor availability at a separate time from completing a computer-based patch foraging task. The PET study consisted of three scans, which a subset of the participants completed, including -FDOPA and [18F]-Fallypride scans and 5.5 months between the behavioral task and the [11C]-NNC112 scan. Sensitivity analyses controlling for time between behavioral task and PET scan are included in the To quantify foraging behavior, we used a well-validated computer-based patch-foraging taskand the 1F-FDOPA a19, the optimal leaving rule is to search for a new tree when the expected reward from the next harvest, Εsi+1, is less than the time spent harvesting, h, times the average reward rate, ρ. The state of the tree (si) is equal to the received reward and rewards deplete according to a depletion rate, κ; therefore, the expected reward from the next harvest is equal to the depletion rate, κ, times the received reward, si. Taken together, the MVT predicts that the optimal leave decision occurs when κsi < ρh. Hence, the reward rates at the time of leaving (leaving threshold) should differ between environments with different average rates of reward and should be affected by depletion rate and time costs.Participants aimed to collect as many apples as possible from apple trees. To achieve this goal, they decided whether to stay at a given tree and harvest it for apples or leave and search for a new tree see Fig. . If part3. We tested various dynamics of the patch leaving threshold, which are included in the To assess individual differences in sensitivity to the foraging reward environment, we calculated the difference in leaving thresholds between the reward environments with the highest  and lowest (long travel time and steep depletion rate) average reward rates. This was chosen as our primary behavioral measure of interest based on past studies showing that measuring the relative change in patch leaving threshold between reward environments controls for individuals’ bias to stay and more closely reflects optimal behavior modeled with the MVTp < 0.0373 for decay rate and p < 5.24e−6 for travel time) but not the interaction (p = 0.438). Thresholds for leaving a reward patch reflected both travel time  and depletion rate . Post-hoc t-tests revealed significant differences for all pairs of reward environments except the short steep and short shallow environments , consistent with multiple prior studies30 and likely reflecting factors not included in the MVT and our analyses such as risk of predation when traveling or asymmetrical learning rates30. Notably, behavioral sensitivity to travel time and decay rate parameters were uncorrelated . Some participants were more affected by travel time, others by reward depletion.Participant behavior generally followed the same pattern as predicated by the MVT wherein subjects made foraging decisions on the basis of the average environment value see Fig. . Optimal1 and D2/3 dopamine receptor availability modulates the threshold at which humans leave patches to explore, we first ran a voxelwise GLM for each PET modality including our primary behavioral measure of interest, change in patch-leaving threshold, with age and sex included as covariates of no interest. MNI-space voxelwise data were restricted to the dopamine-rich basal ganglia. We found that the change in exit threshold was positively correlated with dopamine D1 receptor availability in the bilateral ventral putamen and with dopamine D2/3 receptor availability in the bilateral putamen, right ventral caudate nucleus, and bilateral dorsal caudate nucleus , constructing a linear regression that included all PET ROI measures would result in lost information. Principal component analysis (PCA) is ideally suited to capitalize on the unique multimodal design of this study and allow us to identify distinct patterns of variability across the three dopamine PET tracers. We first extracted data from five predetermined ROIs including the ACC, midbrain, ventral striatum, dorsal caudate nucleus, and dorsal putamen  due to low signal reliability in that region, evidenced by poor compartment model fit. The ACC was included as an additional ROI because of prior animal and human fMRI studies showing that neural activity in this region is key for foraging decision-making7. In addition, animal studies have shown that the supragenual ACC is the cortical region with the highest density of dopamine innervation31 and dopamine projections to the ACC have been implicated in effort-based decision making33.Next, we sought to use region of interest (ROI) data to replicate the voxelwise results and further investigate how the different contributors to exit threshold relate to patterns of dopamine function. However, given that the three PET measures are not independent  dopamine presynaptic synthesis capacity and D1 and D2/3 receptor availability along with high presynaptic synthesis capacity and low D2/3 receptor availability in the ACC. We assessed reliability of the PCA solution using an independent sample of 26 individuals and found that components 1 and 2 were stable across samples  whereas components 3 and 4 were not as robustly stable . Second, we identified a more localized pattern of dopamine effects on threshold changes encompassing high mesolimbic PET values in all three dopamine tracers as well as high presynaptic synthesis capacity and low D2/3 receptor availability in the ACC .A linear regression with our primary behavioral measure of interest, total change in leaving threshold, as the dependent variable and the four dopamine PET PCA component scores as the independent variables, revealed two patterns of dopamine variability that correlated with change in patch-leaving threshold  and there were positive trends in several other regions including D1 receptor availability in the putamen and ACC, D2/3 receptor availability in the ventral striatum, caudate nucleus, and putamen, and presynaptic synthesis capacity in the ACC .Next, we sought to examine our secondary behavioral measures of interest, starting with breaking the leaving threshold down into its component parts. In our experiment, reward environments varied on two parameters: travel time between reward patches and decay rate within a patch. To investigate whether the link between the change in patch-leaving threshold and dopamine synthesis and receptor availability was differentially modulated by changes in travel time or decay rate, we measured each individual’s sensitivity to these environmental parameters by calculating the change in exit thresholds due to travel time (average threshold for leaving environments with a short travel time – average threshold for leaving environments with a long travel time) and decay rate  separately. The change due to travel time and change due to decay rate scores were normalized across subjects to account for differences in variation between these two measures. These parameters were uncorrelated  and ventral striatum  with a trend in the putamen (p < 0.1); it was also positively correlated with D2/3 receptor availability in the caudate nucleus  with trends in the putamen and ventral striatum . This dissociation provides evidence that these two patterns of dopamine synthesis capacity and receptor availability are implicated in the valuation of time while foraging.We found that the correlations between patterns of dopamine variability and change in patch-leaving threshold were driven by the travel time and not the depletion rate. To test this, we ran linear regressions for each of the two PCA components that were correlated with total change in patch-leaving threshold (components 1 and 4) including the component score as the dependent variable and the travel time and decay rate change scores as the independent variables. For both components, only patch exit threshold change due to travel time, but not decay rate, was significantly correlated with component score  and presynaptic dopamine synthesis capacity in the midbrain , with trends (p < 0.1) between change in reaction time and D1 receptor availability in the ACC and putamen, D2/3 receptor availability in the ventral striatum, and presynaptic dopamine synthesis capacity in the ACC and ventral striatum. The PCA and ROI results suggest the importance of widespread striatal dopamine receptor availability as well as mesolimbic and ACC presynaptic dopamine synthesis capacity in response invigoration while foraging.Lastly, we analyzed our final secondary behavioral measure, change in response time, to test the computational prediction about dopamine’s role in response invigoration. As predicted by experiment design, we confirmed that the average reward rate was highest in the reward environment with the short travel time and shallow depletion rate and lowest in the reward environment with the long travel time and steep depletion rate see Fig. . We also1 and D2/3 receptors throughout the striatum as well as with a combination of increased dopamine synthesis capacity and receptor availability specifically in mesolimbic regions. Next, we found that dopamine function was linked to behavioral changes related to travel time between reward patches and not to depletion rate of rewards within a patch, suggesting a key role of dopamine in calculating the opportunity cost of time. Lastly, we found that these two patterns of high dopamine receptor availability throughout the striatum and high mesolimbic dopamine synthesis capacity were also related to acceleration of response times in the reward environment with the highest average reward rate.This study provides valuable insight into the role of dopamine in foraging behavior by measuring multiple facets of the dopamine system in the same individuals who completed a patch foraging task. Through our unique study design and utilization of PCA to combine PET measures, we found that the degree to which individuals change their foraging behavior based on the parameters of the experimental reward environment is correlated with two patterns of dopamine synthesis capacity and receptor availability throughout the basal ganglia and ACC. First, we showed that the change in reward patch leaving threshold was positively correlated with the availability of dopamine D1 and D2/3 receptor availability enhances sensitivity to changes in local dopamine release in response to changes in the reward environment. Of note, while we are unable to directly measure tonic dopamine in the synapse, preclinical work has shown that presynaptic synthesis capacity is related to dopamine neuron population activity (or average number of spontaneously active dopamine neurons), which is thought to influence tonic dopamine efflux27. This interpretation builds on computational predictions that tonic striatal dopamine encodes the average reward rate of the environment10 and supports animal models showing ventral striatum dopamine release tracks average reward rate independently of tonic dopamine firing29, suggesting an important role of receptor availability in enacting downstream effects. In addition, this finding builds on prior human studies showing administration of an exogenous dopamine D2 receptor agonist3 alters the reward patch leaving threshold and provides further insight into the role of both D1 and D2 receptors as well as the localization to the mesolimbic dopamine pathway.It is possible that in the foraging context striatal presynaptic dopamine synthesis capacity enables fine-tuned adjustments in dopamine release and that greater D1 and D2/3 receptor availability throughout the striatum, which could represent a state of increased reactivity to dopamine release26. The positive correlation between dopamine receptor availability and behavioral sensitivity to changes in the reward environment was also reflected in our voxelwise analysis and supports computational and animal work showing that dopamine receptors are important for weighing costs and benefits related to effort costs33. In addition, our results implicating both D1 and D2/3 receptors in foraging behavior adds to prior knowledge about the role of D2 receptors on foraging behavior3 and suggests that both receptor types are important for adjustments in the foraging patch leaving decision threshold. This supports computational models on the dual actions of dopamine receptors for learning and decision making, such that D1 receptors are important for learning the benefits of an action, such as staying in a reward patch, while D2 receptors play a key role in learning about costs, such as the opportunity cost of lost time17. Therefore, both D1 and D2 receptor-mediated functions are essential for accurately weighing both benefits and costs to adjust behavior based on changes in the environment. In contrast to the prior study of the effect of D2 agonist administration on foraging behavior3, our study modulated travel time and not just decay rate and also measured both types of dopamine receptors in the same individuals (D1 and D2/3), allowing us to identify a key role of dopamine in time valuation. However, in our experiment, the travel time and decay rate parameters were held constant within each reward environment leading to an inability to measure differences in sensitivity to reward environment changes within the rich and poor contexts.We demonstrated that the total change in patch exit threshold between the reward environments with the highest and lowest average reward rate is positively correlated with two separate patterns of dopamine variability. The first pattern is characterized by high D1 and D2/3 receptor availability in the ventral striatum, all key regions in the reward network. It was particularly interesting that ACC dopamine synthesis capacity was included in this component, given the prior primate and human fMRI studies showing that ACC neural activity encodes information about the reward patch-leaving threshold and the average value of the environment15, but changes in patch-leaving threshold cannot be accounted for by neural activity alone1. The individual ROI results suggest that ACC D1 receptor availability (included in the first PCA component) may also be important for adjusting the patch-leaving threshold, particularly due to changes in the travel time between patches. It is possible that local dopamine presynaptic release and action at the D1 receptor within the ACC in response to changes in the reward environment may provide a direct mechanism for setting the threshold for leaving a reward patch. In addition, dopamine may modulate the effect of input from other brain regions to the ACC as the striatum and its interactions with the ACC are known to play a role in encoding prediction errors and search costs7. Furthermore, this component also includes a positive contribution of D2/3 receptor availability in the midbrain, a region where D2 receptors play a key role in the autoregulation of dopamine release throughout the brain34. It has been shown that autoreceptors regulate the intrinsic pacemaker activity of dopamine neurons that underlies tonic dopamine levels37, which may be a potential mechanism by which striatal tonic dopamine levels track changes in the average reward rate of the environment. However, the fact that in the individual ROI analyses midbrain D2/3 receptor availability was not correlated with changes in patch-leaving threshold suggest that it is unlikely to have an important role outside the context of dopamine function in other regions. In addition, we suggest caution in the generalizability of conclusions regarding relations with this dopamine component since it did not robustly replicate in our independent sample. However, taken together with our voxelwise results showing that the total change in patch leaving threshold was positively correlated with D1 and D2/3 receptor availability throughout the striatum, these findings demonstrate that dopamine synthesis capacity and receptor availability throughout the reward network predicts greater behavioral sensitivity to parameters in the foraging reward environment. Although our PET measures are not direct assays of dopamine release, we speculate that fluctuations in dopamine function in these two identified localized patterns could reflect one potential mechanism by which information about the reward environment could be signaled to neurons in the ACC and reward network that are key for foraging-based decision-making.The second pattern of dopamine variability that was correlated with the total change in patch exit threshold includes a positive contribution of presynaptic synthesis capacity in the ACC, ventral striatum, and midbrain, as well as dopamine D39. One potential mechanism for dopamine modulating behavioral responsivity to changes in travel time is through negative prediction errors for lapsed time without a reward30. A recent study using a prey selection foraging task showed that individuals learned more slowly from negative prediction errors than from positive ones, contributing to an overestimation of environmental quality when rewards depleted30. While the depleting patch design of the current study does not allow for trial-wise modeling of decision-making needed for fitting a traditional reinforcement learning model and learning rate, we predict that individuals with greater dopamine synthesis capacity and dopamine receptor availability could be better equipped to respond to small negative reward prediction errors that occur over unrewarded travel time.Additionally, we found that dopamine is specifically implicated in behavioral adjustments to changes in travel time between reward patches rather than depletion rate within a patch. This supports prior work showing the selective role of striatal dopamine and neural activity (measured by BOLD activation) in encoding reward timing and the opportunity cost of time18F]-FDOPA tracer quantifies the activity of aromatic l-amino acid decarboxylase (AADC), the enzyme that converts L-dopa to L-dopamine. While in dopamine-rich regions, such as the striatum, it is most likely that variability in this measurement is attributable to dopaminergic terminals, [18F]-FDOPA signal in regions where other AADC-containing cells may be abundant (e.g. the ACC) may not be specific to dopamine41, and interpretations of these data should therefore be cautious.There are limitations to our study that should be kept in mind. Specifically, while PET imaging is the only way to non-invasively directly measure the dopamine synthesis capacity and receptor availability in the living human brain, this methodology has several drawbacks compared to methods used in animal research. These include limited spatial and temporal resolution of neural activity and dopamine system release  as well as the inability to isolate and modulate specific regions or neural types . In addition, we were unable to quantify tonic dopamine or receptor availability during the foraging task itself due to the time course of the PET tracers used in this study, requiring 90–180 min to obtain a single image. Similarly, because PET and behavioral measures were not concurrent, and were in some cases separated by months to years, our findings likely reflect a trait-wise association and cannot address the degree to which dopaminergic variables predict behavior at a more precise point in time. Future studies using a combined MRI-PET scanner and a displaceable tracer could potentially be used to investigate how state-wise regional dopamine release corresponds to local changes in foraging decision-making. Lastly, the -FDOPA PET scan , 45 completed the [11C]-NNC112 scan , and 42 completed the [18F]-Fallypride scan . One participant was excluded due to behavior that suggested they were not following the task instructions (absence of any leave decisions in one of the blocks of the task). All participants provided informed consent and were compensated for their participation. Sex was determined by self-report and was not considered in study design. Study procedures were approved by the NIH Combined Neurosciences Institutional Review Board and Radiation Safety Committee.Fifty-seven healthy volunteers aged 21–57 years  were recruited from the local Washington DC community. Subjects were screened by physician-administered physical and neurological examination, standardized clinical interview (SCID)2. Subjects were shown an apple tree and asked to decide whether to stay at the current tree and harvest it for apples or leave and search for a new tree. If they decided to stay, they would receive a certain number of apples, shown below the tree. The apples earned were summed over the entire experiment and converted to monetary payment that was added to the study compensation. After staying at a tree and harvesting it, the number of apples remaining in the tree would decrease according to a depletion rate, similar to how a tree in the wild would gradually run out of apples the longer that an animal ate from it. Subjects then made the stay or leave decision again. If the participants decided to leave, they had to endure a travel time delay until they reached a new tree. There were an infinite number of new trees available to travel to. Participants completed this game in four different reward environments that varied in travel time delay (short 6 s or long 12 s) and reward depletion rate . Each reward environment, or block, lasted a fixed duration of six and a half minutes. Travel time and depletion rate remained constant throughout the block and the blocks were presented in a random order across participants. If participants did not respond within one second, they received a warning and had to wait two and a half seconds before they were allowed to make another decision. The starting amount of rewards in a tree was drawn from a normal distribution with a mean of 10 and standard deviation of 1, with a maximum value of 15. The reward depletion rates within a patch were drawn from beta distributions such that the steep environments had a mean of 0.88  and the shallow environments had a mean of 0.94 .To assess foraging decision-making, participants played a computer-based game in which they foraged for apples see Fig. . This taTo assess behavioral sensitivity to the variables in this task, we calculated the total change in threshold for leaving a patch between the reward environment with the highest average reward rate  and the one with the lowest average reward rate (long travel time and steep depletion rate). In addition, to assess whether there was a dissociation between changes related to reward timing and magnitude, we determined behavioral sensitivity to travel time and decay rate individually. To measure travel time sensitivity, we took the difference between the average threshold for leaving in the two reward environments with the short travel time and the average threshold for leaving in the two reward environments with the long travel time (short–long). Threshold changes due to depletion rate were calculated as the difference between the average threshold for leaving in the reward environments with a shallow depletion rate and the average threshold for leaving in the reward environments with the steep depletion rate .18F]-FDOPA) and Siemens High-Resolution Research Tomograph (HRRT) scanner . Following an eight-minute transmission scan, dynamically binned emission scans were collected for one and a half hours ([18F]-FDOPA and [11C]-NNC112) and four hours  after tracer injection. The target tracer doses were 16 mCi for [18F]-FDOPA, 5 mCi for [18F]-Fallypride, and 20 mCi for [11C]-NNC112. For all scans, caffeine and nicotine were restricted for four hours preceding the scan, and for [18F]-FDOPA only, food was restricted for six hours preceding the scan to reduce competition for transport of the tracer into the brain. Subjects were pretreated with 200 mg carbidopa one hour prior to injection for the [18F]-FDOPA scan to reduce peripheral degradation of the tracer. Subjects also completed T1-weighted magnetic resonance imaging (MRI) scans used for registration and brain segmentation.The PET scans were all collected on separate days from the behavioral testing using a GE Advance 3D scanner ([http://www.pmod.com/web/). The [18F]-FDOPA specific uptake rate (Ki) was calculated with the graphical linearization Gjedde-Patlak method44, while [11C]-NNC112 and [18F]-Fallypride binding potential  was calculated with the simplified reference tissue model (SRTM) method45.All PET images underwent attenuation correction, reconstruction, and registration to align all timepoints. We then performed both native-space ROI as well as voxelwise standard-space analyses within the basal ganglia and ACC. For both methods, we used non-invasive, reference input kinetic models for PET parameter modeling, implemented with the PMOD software  and manual adjustments to generate standard ROIs of the basal ganglia, where dopamine projections and receptors are most abundant: dorsal putamen, dorsal caudate nucleus, ventral striatum, and dopaminergic midbrain . MRI images were registered to native space mean PET images, and time-activity curves were extracted for each of the ROIs. For voxelwise analyses, each individual’s coregistered anatomical MRI image was warped to a common space (MNI) template using ANTS software (http://stnava.github.io/ANTs/) and the resulting transformation matrix was applied to the PET images. Common (MNI) space PET images were smoothed using a three-dimensional Gaussian kernel of 10 mm3 before undergoing modeling in PMOD.PET images were corrected for inter-scan motion and realigned using FLIRT (https://www.mathworks.com/products/matlab.html). Statistical correction for multiple comparisons was done using a false discovery rate (FDR) level of 0.05 (or 5%) as described by Benjamini and Hochberg46. Specifically, FDR correction was implemented by hand by ranking the p-values of all correlations tested (n) within each specific tracer from lowest to highest . The critical p-value for each correlation was calculated as (1/i)*0.05. The highest p-value that was smaller than the critical value was identified and all correlations with lower p-values were considered significant.For ROI analyses, linear partial correlations with age and sex as covariates of no interest were run in MATLAB (https://www.fmrib.ox.ac.uk/fsl) using randomize  for nonparametric permutation testing and threshold-free cluster enhancement (TFCE)47 with a statistical threshold of p < 0.05, family-wise error corrected for multiple comparisons.Voxelwise analyses, restricted to a MNI-space basal ganglia mask, were run in FSL (n = 37). PET values were corrected for age and sex and z-normalized before being input into the PCA analysis. Normalization was essential prior to PCA analysis because the parameters of interest for the three PET tracers used vary by multiple orders of magnitude. Only components with eignenvalues greater than one were used in the regression analysis with foraging behavioral measures of interest.To take a comprehensive look at dopaminergic function and reduce the dimensionality of our analyses given the five different ROIs for three different PET tracers, we used a PCA to extract patterns of covariance in the dopamine PET data. Only participants who completed all three PET scans were included in this analysis (https://www.mathworks.com/help/stats/fitlm.html) with the behavioral measure as the dependent variable and the dopamine component scores as the independent variables. Lastly, to test for a dissociation between behavioral sensitivity to changes in reward magnitude depletion rate and travel time, the two dopamine PCA components that were correlated with total change in patch-leaving threshold were used as the dependent variables in two separate linear regression models with both behavioral measures included as the independent variables, with a statistical threshold of p < 0.05.Regression analyses were conducted in MATLAB to assess the relationship between foraging behavioral measures of interest and principal components of dopamine variation. For each behavioral measure of interest , we ran a linear regression analysis using the matlab fitlm function (Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


We conclude that the method presented here is a promising research tool to study the kinetics of soluble cytokines over the course of wound healing. More studies are needed to determine the interobserver variation and reproducibility of the NovaSwab method.Cytokines in wound fluid are used as surrogates for wound healing in clinical research. The current methods used to collect and process wound fluid are noninvasive but not optimal. The aim of this prospective study was to evaluate a method (NovaSwab) by which wound fluid is collected by a surface swab and eluted in a physiological buffer for subsequent cytokine analysis. Wound fluid from 12 patients with leg ulcers was assessed by NovaSwab at the start (Day 0) and at the end of a 23‐h collection period of wound fluid retained by foam oblates beneath an occlusive film dressing (Day 1). GM‐CSF, IL‐1α, IL‐1β, IL‐6, IL‐8, PDGF‐AA, TNF‐α and VEGF levels were measured by multiplex and electrochemiluminescence assays. IL‐1α (2.4×), IL‐1β (2.0×) and IL‐8 (1.8×) levels increased from Day 0 to Day 1 as detected by NovaSwab, indicating local production of these polypeptides in the wounds. On Day 1, the NovaSwab method yielded higher levels of IL‐1α (4.0×), IL‐1β (2.7×) and IL‐6 (2.7×), and 35% lower levels of VEGF than those in wound fluid accumulated for 23 h in foam oblates . In vitro experiments showed that the investigated cytokines in cell‐free wound fluid were recovered in a quantitative manner by the NovaSwab method GM‐CSFgranulocyte‐macrophage colony‐stimulating factorILinterleukinPDGFplatelet‐derived growth factorSDstandard deviationTNFtumour necrosis factorVEGFvascular endothelial growth factorWFwound fluid1Wound fluid (WF) is commonly used for studies of the cytokines and growth factors involved in wound healing. WF mirrors the local microenvironment well and is easily accessible.There has been a call for researchers to standardise the methods for WF collection to accurately measure cytokines, but there is a paucity of simple, rapid and reliable methods for WF collection.With the introduction of more sensitive analytic techniques, the demand for WF volumes has gradually decreasing, stimulating the development of simpler sampling methods.Zillmer et al.Our objective was to compare the NovaSwab method to the Zillmer method22.1This prospective, exploratory study involved two sessions (Day 0 and Day 1) for the patients. The study was approved by the Ethics Committee of the Capital Region (H‐19081985) and conducted at the Copenhagen Wound Healing Center, Department of Dermatology, University of Copenhagen, Copenhagen, Denmark in accordance with the Declaration of Helsinki.2.2n = 8) and outpatients (n = 4) with chronic leg ulcers who fulfilled all of the selection criteria were consecutively recruited from our department, a tertiary health care centre. The inclusion criteria were patients aged ≥18 years old, with at least one nonhealing leg ulcer of a duration of ≥2 months, localised between the ankle and the knee ; being informed about the study; being able to comply with the protocol and having given oral and written informed consent. The exclusion criteria were allergies to any of the materials used in the study, the index ulcer judged as having insufficient production of WF, any major changes in antibiotics or immunosuppressive treatment over the previous 7 days, or planned changes during the study. These changes included any initiation, discontinuation or major dosage changes in this treatment. Pregnant women and patients judged unsuitable to participate, for example, patients receiving palliative care, were excluded.Hospitalised patients  from digitised tracings of the epidermal edge on an acetate film.2.3g  for 10 min at 4°C , and the supernatant was stored at −80°C.WF was collected by a sterile swab that was gently pressed to the wound bed in a spiral manner starting at the edge and ending at the wound centre according to the Essen Rotary technique.2.4An overview of the WF samples collected from each patient is shown in Figure On Day 0, WF was obtained with a surface swab before (1st swab) irrigation with sterile 0.9% NaCl  from a 10‐ml prefilled syringe . Excessive saline was dabbed dry with nonwoven compress before swabbing with a 2nd swab.g for 10 min at 4°C). The WF volume was determined by pipetting.WF was then collected according to Zillmer et al.Finally, WF was collected from the wound bed with a 3rd surface swab on Day 1.2.5n = 6) and ∑‐Transwab (n = 6) was estimated using the formula (assuming 100% elution of the proteins from the swabs): 300/(([proteinWF]/[proteinSample]) − 1).Two brands of surface swabs were used: eSwab  with a tip flocked with soft nylon and ∑‐Transwab®  with a polyurethane open‐cell foam tip. The uptake (μl) of WF from the wounds on Day 1 by eSwab (2.6n = 11] and eSwab [n = 1]) and absorbed for 30 s; one swab was processed according to the NovaSwab method  in a 1.5‐ml microcentrifuge tube with a centrifugal filter unit  without the filter membrane. The amputated swab and the filter unit were subsequently discarded, and the WF was transferred to a 1.5‐ml tube .The recovery of cytokines in WF by the NovaSwab and Schmohl methods was studied Figure . Cell‐fr1st extraction]/([protein1st extraction] + [protein2nd extraction]) × 100.The recovery of proteins by the NovaSwab method was estimated. Cell‐free WF (50 μl) from three patients was added to the tip of ∑‐Transwabs and absorbed for 30 s, and the tip was cut off and placed in a 1.5‐ml tube  containing 300 μl of TBS and processed according to the NovaSwab  on a Bio‐Plex 200 system  as described by the manufacturers. Reported sensitivities for each analyte are reported in Table 2.810). Two‐tailed paired and unpaired t‐tests were used in comparative analyses. Bland–Altman plots were created to visualise the agreement in cytokine levels in WF determined by the NovaSwab and Zillmerp < .05. GraphPad Software  was used for statistical analyses.Because it was not possible to estimate the sample size, we decided to include 12 patients. Since the data were not normally distributed for cytokines, logarithmic transformations were performed , six men and six women, were included between May 2020 and October 2021. Six patients had venous leg ulcers, two had pyoderma gangrenosum, one had an arterial ulcer, one patient had calciphylaxis, one was described as idiopathic/inflammatory and one patient had Martorell's ulcer. Ten patients had palpable foot pulses, and ischemia was suspected in one patient.2, range: 5.7–76 cm2). The mean ulcer duration was 15 ± 12 months (range: 2.5–48 months). No ulcers showed signs of clinical infection.Two patients had multiple adjacent ulcers. The sum of these ulcer areas was used for the calculation of the mean ulcer area of the 12 patients  was used in seven patients, and a neutral standard dressing was used in five patients. All patients received compression therapy using multilayer or elastic bandages at inclusion and during WF collection with the foam oblates. Two adverse events were registered: skin maceration was noted in one patient, and another patient experienced an allergic reaction to the film dressing.Before inclusion, an active wound dressing .WF was collected with foam oblates  over the course of 23 ± 1.5 h; this process translated into an estimated WF production rate of 0.25 ± 0.14 ml/cmCytokine levels in pg/ml in WF collected by the foam oblates are presented in Table 3.2p = .87) for eSwab (53 ± 24 μl) and ∑‐Transwab (50 ± 31 μl). The results of the cytokine analyses of WFs obtained by eSwab and ∑‐Transwab were aggregated for the statistical analyses.Due to backorders during the COVID‐19 pandemic, ∑‐Transwab replaced eSwab for WF collection from the last six patients. WF uptake from the wounds was similar (p = .016) and IL‐8 by 1.7 times  compared with before irrigation (Table p = .027) after saline irrigation. The total protein concentration of WF processed by NovaSwab was 5.4 ± 3.6 mg/ml before (1st swab) and 3.2 ± 1.6 mg/ml after (2nd swab) saline irrigation (p = .0061).Saline irrigation of the wound increased the levels of IL‐1α by 1.8 times  higher, IL‐1β was 2.0 times  higher, and IL‐8 was 1.8 times  higher on Day 1 versus Day 0 (Table Cytokine levels determined at baseline Day 0 (1st swab) and 23 h later on Day 1 (3rd swab) by the NovaSwab method were compared; IL‐1α was 2.4 times  higher levels of IL‐1α, 2.7 times  higher levels of IL‐1β and 2.7 times  higher levels of IL‐6 than those in WF accumulating for 23 h in foam oblates. VEGF WF levels were 35%  lower with NovaSwab than with foam oblates. Bland–Altman plots showed acceptable agreement between the NovaSwab compared with WF collected by foam oblates. The 95% limits of agreement were broadest for GM‐CSF and narrowest for VEGF , IL‐1α by 1.1 times  and TNF‐α by 1.1 times . IL‐6 levels were 23%  lower with NovaSwab than with the Schmohl method. The total protein concentration of the NovaSwab samples was 6.3 ± 1.6 mg/ml , the WF processed with the Schmohl method was 50 ± 12 mg/ml, and it was 43 ± 10 mg/ml for the controls.WF cytokine levels were studied after being subjected to the NovaSwab and Schmohl methods Figure . In comp4There is a need for convenient and reliable methods to collect WF for cytokine analysis in longitudinal clinical studies of wound healing. Our results support that the NovaSwab method presented here recovers cytokines in collected WFs in a representative manner. Moreover, we confirmed the findings by Zillmer et al.,The cells responsible for the secretion of cytokines have been only sparsely described in the literature on leg ulcers.We had to change the swab supplier in the middle of the study. The two swab types, one with a soft nylon tip and the other with a polyurethane foam tip, were comparable in WF uptake and did not show apparent systematic differences in cytokine levels. Importantly, the same swab type was used for each patient, enabling paired comparisons Table .We chose the Essen Rotary technique for WF collection to account for local variations in cytokine abundances in wounds  with satisfactory recovery of the investigated cytokines in WF, and it is a promising research tool to monitor cytokine fluctuations during wound healing. Future larger studies should assess the interobserver variation and reproducibility of the new method.Kongelig Hofbuntmager Aage Bangs Fond provided funding for the multiplex assay kits.No company or organisation played any role in the initiation or design of the study, collection, analyses, or interpretation of the data, writing of the manuscript, or decision to publish the results. Reponex Pharmaceuticals sponsors Ewa Anna Burian's PhD studies through payments to the department. Ewa Anna Burian is investigating the effect of topical GM‐CSF in venous leg ulcers for Reponex Pharmaceuticals. Tonny Karlsmark is a medical advisor for Reponex Pharmaceuticals. Christian Enevold and Magnus S. Ågren declare no conflicts of interest.Figure S1. Interaction of cytokines in WF by the NovaSwab and Schmohl methods assessed in vitro. (A) Design of the experiment. WF was collected by foam oblates from the 12 patients and centrifuged. Cell‐free WF supernatants (50 μl) were added to the tip of the swab (∑‐Transwab [n = 11] and eSwab [n = 1]) and processed according to the NovaSwab and Schmohl methods or not (controls). (B) Cytokine concentrations were compared between the two methods and to WF alone using a two‐tailed paired t‐test. *p < .05, **p < .01.Figure S2. Effect of saline irrigation on cytokine levels assessed by the NovaSwab method . Open circles represent WF samples obtained by eSwab and closed circles by ∑‐Transwab. *p < .05, **p < .01 (two‐tailed paired t‐test).Table S1. Luminex® Discovery Assay, used for determination of cytokine levels in the study. .Table S2. MSD, S‐plex Platform used for determination of GM‐CSF levels in the study. Kit 151F3S.Click here for additional data file.


Escherichia coli (E. coli) was reported in nine studies. Risk factors for AMU include herd size, vaccine use, and start date of calving season. Conclusions: Overall, a small number of AMR studies were available for synthesis in primarily one population (cows) reporting E. coli AMR. Additional studies targeting reasons for AMU in calves, the impact of management procedures on AMU, potential environmental AMR sources, and AMR in respiratory pathogens and enteric organisms other than E. coli for pre-weaning calves are required to inform AMR risk mitigation strategies.Background: The magnitude and knowledge gaps regarding antimicrobial use (AMU) and antimicrobial resistance (AMR) have not been summarized for the North American cow–calf production sector, although estimates of AMU and AMR are essential to AMR risk analysis. The objectives of this scoping review were to map AMU and AMR in the beef cow–calf sector in Canada and the United States, summarize published AMU/AMR predictors, and identify research gaps. Methods: An electronic search was conducted of four bibliographic databases and Google Scholar, augmented by a hand-search of captured studies. Results: Twenty-three of three-hundred and forty-three publications screened advanced to data extraction. Of these, 10 were conducted in the USA and 13 in Canada. Thirteen studied AMR and twelve studied AMU, with two reporting both. Of twelve captured AMU studies, nine presented counts of herd AMU by antimicrobial class or specific antimicrobial. Antimicrobial resistance in  Limiting the emergence and spread of antimicrobial resistance (AMR) is critical to preserving the world’s ability to treat diseases in humans, animals, and plants, reduce food safety and security risks, protect the environment, and maintain progress toward the Sustainable Development Goals . The AMRThe use of antimicrobials in livestock production has been identified as one driver of AMR occurrence in both human and animal populations . AntimicThe international bodies governing food safety, at both pre-harvest ) and harvest level (Codex Alimentarius) of the farm-to-fork continuum, recommend risk analysis as the framework for the investigation of animal health and food safety problems to inform policy of member states ,4. EstimWhile antimicrobials are important to animal health and welfare in all livestock commodities including the beef industry, information is limited on AMU in the North American cow–calf industry. Antimicrobial use has been reported in pre-weaning calves for treatment of diseases such as calf diarrhea and respiratory disease, and in cows for diseases such as lameness associated with infection, although the proportion of individuals treated within a herd tends to be relatively low, in both the United States of America (USA) and Canada ,7. TheseIn both Canada and the U.S., federal authorities collaborate with national veterinary associations to support veterinarians and ultimately producers in antimicrobial stewardship, by monitoring data on resistance and antimicrobial use ,14. The The magnitude of AMU and AMR have not been specifically summarized for the relatively extensively managed North American cow–calf production sector, although differences in AMU and AMR between the cow–calf and more intensively managed feedlot sectors have been reported ,16. ThisScoping reviews are appropriate for assessing the extent of the knowledge in an emerging field, or to identify, map, report or discuss the characteristics/concepts in that field . In contA scoping review was deemed appropriate as the aim of this review was to describe the published research available to support risk-based policy-making for AMR and antimicrobial stewardship initiatives in the North American beef cow–calf sector. The objectives of this scoping review were to map AMU/AMR research conducted in the North American beef cow–calf sector, summarize published AMU/AMR literature, describe predictors for resistance development in the beef cow–calf sector, and identify research gaps pertaining to the development of AMR risk mitigation strategies. The review specifically targeted animals not identified as being sick to summarize AMR data from the baseline population and to avoid laboratory diagnostic samples from sick animals that had potentially been treated with antimicrobials multiple times.The electronic bibliographic search yielded 343 unique citations, including the additional citations captured in the follow-up search conducted in June 2023. First-level title and abstract screening excluded 294 publications, for reasons including only sampling out-of-scope populations  and sampling locations . Another 32 publications were excluded after full-text review, for reasons including sampling only clinically ill animals, sampling animals not identifiable as beef cow–calf sector, and not reporting primary research. The Google Scholar search additionally identified one relevant thesis , one jouOf the 23 relevant publications, 10 were conducted in the USA and 13 in Canada between 1999 and 2020. Thirteen described investigations of AMR, twelve studied AMU, and two studied both. The most frequently studied population was pre-weaned calves n = 17), followed by cows (n = 14) with breeding bulls the least frequently described population (n = 2). Two AMU studies investigating producer attitudes studied usage at the herd level and did not report usage stratified by class of cattle , followe. The numA map displaying the provinces and states from which samples originated, across the included studies, is presented in While several investigators have identified the value of studying individual animal data in verifying producer reporting of AMU ,41, onlyNine AMU studies described herd-level usage (Yes/No) for classes of antimicrobials  or specific antimicrobials , aggregated as counts of herds that reported use . The panThe objectives for AMU were reported in seven of 12 included studies, ranging from “Prevention, control, or treatment”  to “TreaTetracycline use was reported by most herds in the identified studies, ranging from 36%  to 88%  for injePopulation-averaged individual-level reasons for AMU were reported by U. S. ,24 and CWithin-herd AMU was reported as categorical summaries from herd records in two Canadian studies ,31 for ep < 0. 05) predictors included herd size, with larger herds more likely to report AMU; and classes of livestock, with greater odds of AMU reported in calves relative to cows for multiple antimicrobials investigated  included in all but two of these studies and other enteric bacteria  less frequently reported , and all E. coli studies used breakpoints endorsed by the Clinical and Laboratory Standards Institute (CLSI) or its predecessor, the National Committee for Clinical Laboratory Standards (NCCLS). Other breakpoints  were reported in three studies investigating Campylobacter spp. Only three studies reported Salmonella, and in one of the two studies, there was only one isolate.Broth microdilution was the most frequently used method of antimicrobial susceptibility testing  associated with AMR in one or more studies included herd attributes such as calf mortality  [Predictors significantly (ith AMR)  and calfith AMR) , with a ith AMR) . An incrith AMR) . One stuith AMR) . The magp < 0.05) significant. Ionophore feeding was reported to have a significant (p < 0.05) association with decreased odds of cefotaxime-resistant bacteria [E. coli resistance to ≥2 antimicrobials [Several herd-level management practices, such as regularly disinfecting water troughs, and quarantining new arrivals to the herd, were associated with significantly reduced odds of AMR . While mbacteria . Herds icrobials .Individual-level factors significantly associated with AMR included calf age, with calves greater than 10 days of age having increased odds of AMR relative to those less than three days old , and preE. coli bacteria was resistance to one or more antimicrobials, reported in five publications representing fifteen studies across various populations and hierarchical levels of aggregation. A summary of the most frequently reported quantitative AMR outcomes in enteric bacteria originating from North American beef cow–calf herds is presented in The most frequently reported AMR outcome in E. coli resistance in cows to one or more of the following antimicrobials: cephalosporins, tetracyclines, sulphonamides, or aminoglycosides. Resistance varied across antimicrobials, populations sampled, and level of aggregation. Resistance in E. coli, aggregated at the isolate level, was relatively low, with AMR occurring more frequently in samples originating from calves relative to cows  or diamino-pyrimidines  . FindingOverall, a relatively small number (n = 23) of relevant publicly available studies of AMU or AMR in North American beef cow–calf production were captured. These largely originated from several research or surveillance groups: the Western College of Veterinary Medicine (n = 10 studies), the United States Department of Agriculture ) (n = 3), Ontario Veterinary College (n = 2), and the College of Veterinary Medicine of the University of Tennessee (n = 2). However, this coverage emphasizes the major beef-producing regions of the U.S. and Western Canada ,7.The most recent sampling captured is from 2019–2020, reported in only one Canadian study . SignifiThe value of using multiple information sources to validate AMU data, especially including individual treatment records, has been previously reported ,42. HoweMost (n = 11) of the AMU studies in this review reported count-based AMU metrics only. This is especially noteworthy as one study that presented multiple usage metrics found comparative usage across groups varied based on the outcome metric selected . This obStudies reporting herd-level AMU alone do not fully capture herd exposure to AMU. Two studies capturing AMU choices for herds, as well as the proportion of individual animals treated within herds, reported a relatively small proportion of individuals being treated within herds reporting usage of a given antimicrobial ,31. PopuInvestigation and reporting of beef cow–calf sector AMU are contingent on producers securing this information in a retrievable form, preferably with individual animal records. One study included in this review  reportedInvestigation of whether antimicrobials are being used prudently  similarly would require knowing the true diagnosis, the producer’s diagnosis, what the veterinarian prescribed for treatment, and whether/how accurately this was administered by the beef producer. Confirmation of producer diagnoses by a veterinarian is not practical or even possible in many instances due to the distance of cattle from the nearest clinic, as well as limited opportunities for observation, restraint, and timely treatment in extensively managed animals. Telemedicine opportunities are increasing, but cell phone coverage is inconsistent in many areas used for grazing. These questions also necessitate accurate individual treatment records, as well as substantial time and resources to correlate records to other information sources including herd protocols, drug invoices, and garbage can audits. Studies assessing prudent use are rarely reported, even in more intensively managed and therefore less logistically demanding sectors such as dairy and swine production, and no such investigation of prudent use in the beef cow–calf sector was captured in our search. When executed, these studies can be informative: a survey of Danish dairy farms validating treatment records with other information sources reported that of 12,930 beta-lactam drug use records, 1669 were under the manufacturer’s recommended daily dose and 6814 were over . HoweverUnderstanding the herd manager’s process for making treatment decisions is fundamental to assessing the prudent use of antimicrobials in beef cow–calf operations. This intrinsically involves documenting the use of treatment protocols drafted by the attending veterinarian. One Tennessee study of producer attitudes towards antimicrobial use reported that of 147 cow–calf producers responding to this question, 15% (22/147 (respondents) had written treatment protocols for the treatment of sick animals . An earlWhile studies aiming to characterize antimicrobial exposure should ideally include both the level  and duration of exposure, this information was reported in only one included study . This stIf the AMU data are to be used for benchmarking, comprehensiveness of the data and representativeness of study target populations are crucial. Antimicrobial use is intertwined with herd health and influenced by a range of management practices such as calving heifers and cows together, or failure to sort cow–calf pairs out of the calving area . ReportiThe chronological time of sampling is a potential predictor of interest to industry and policy-makers. However, given the relatively small number of studies included in this review, the range of outcomes and other potential predictors  , expecteComparability is an important consideration across study populations, sub-populations , or herds. The population at risk can vary by population size and structure, average weight at treatment, as well as the risk period for being treated . Within E. coli AMR studies tended to test for a group of antimicrobials similar to the Canadian Integrated Program Antimicrobial Resistance Surveillance (CIPARS) panel [E. coli AMR studies used NARMS breakpoints. The 2008 USA NAHMS studies [Several differences were noted between Canadian and USA AMR studies. The Canadian S) panel , using C studies  represenEscherichia coli was the most frequently reported enteric bacterial target, possibly due to its value as an indicator organism with potential public health impact [E. coli precludes the effective assessment of the samples for resistance to many macrolides important in veterinary medicine due to intrinsic resistance [h impact . Selectisistance , leavingCows were sampled more frequently than calves, likely reflecting the relative ease of handling for both groups. The outcomes measured were reported at levels ranging from isolate to industry sector , and this contributed to a relatively small group of surveys per outcome which could be compared or potentially pooled for summary estimates.Mannheimia haemolytica (n = 2) and those studying enteric organisms (n = 10). The two studies of respiratory pathogens did not report risk factors occurring pre-weaning for AMR, given the studies’ focus on AMR trends throughout the production cycle. In combination with the relatively small number of herds sampled , research gaps remain in improving our understanding of AMR prevalence and risk factors for important pathogens. The finding of “unhealthy” calves having increased odds of AMR suggests the possible importance of some herd health and herd-level management procedures in mitigating AMU [The twelve captured studies of AMR fell into two broad groups: those studying respiratory pathogens such as ting AMU . A recenting AMU .Multiple potential sources of bias exist within and across the included studies, including selection bias at both the herd and individual levels of sampling. Recruitment of herds to observational studies requires owner consent, and this inherently introduces selection bias for owners having the interest and willingness to contribute time and resources to allow physical sample or data collection . IndividFor some time, technological advances have been suggested as potential opportunities to reduce AMU or mitigate AMR . Within Given the complexity of the dataset, with a myriad of potential herd- and individual-level predictors, and the identified data gaps, the studies captured in this review collectively provide incomplete evidence required by policy-makers to support benchmarking or to inform risk models in support of public health. For example, when reporting AMU by drug class, an accompanying listing of the specific drugs captured by the study survey would be helpful in supporting the assessment of the comparability of findings across studies. More detailed reporting of drugs captured by each survey would also support differentiating the use of “long-acting” vs. repeatedly dosed products, which is of interest pertaining to the logistics underlying drug choices, and also the potential for longer exposure to a lower level of the active drug. However, an issue in extensively managed operations is that short-acting drugs might only be used once due to difficulty in capturing the animal a second or third time—leading to incomplete treatment and increased risk of AMR. Single-dose drugs could logistically be preferable at times, especially in animals on pasture, in that single-use drugs could be more effective in clearing the original infection if used in the right animals at the right time.Producers and veterinarians additionally need to continue to improve documentation and tools to promote responsible management of AMU in the beef cow–calf sector, to inform the social license to operate, and to prevent potential treatment failure associated with AMR. This requires identification of the major purposes for AMU, and those diseases most frequently associated with the use of drug classes important in human medicine. In Canada, MIAs are categorized as Very High Importance, High Importance, or Medium Importance based on indications for usage in human medicine and available alternatives . In the E. coli as the microbial target of interest, additional studies of AMR with other enteric organisms as well as for respiratory pathogens in pre-weaning calves would be useful in prioritizing disease syndromes for AMU mitigation strategies. Pre-weaning calves are of particular interest to the industry as they represent the risk of AMR in calves entering the feedlot, potentially limiting options for treating bovine respiratory disease.Current findings across this dataset suggest a relatively small proportion of cattle receive antimicrobials in the cow–calf sector. There is, however, evidence to suggest areas for potential improvement. Beef calves in spring are the most likely group on-farm to receive treatment  includinE. coli isolates from cow–calf operations suggest the importance of further investigating other potential drivers of AMR such as the environmental burden of AMR in cow–calf production [One included study describes the isolation of AMR bacteria from the environment of cow–calf operations , as do ooduction .This scoping review had several potential limitations. We did not chart data specifically pertaining to risk of bias (ROB) per se given the current lack of widely endorsed guidelines for assessing individual observational studies for ROB or incorporating ROB in furthermore focused syntheses such as systematic review and meta-analysis of observational studies ,58. We dThis scoping review was conducted following a protocol informed by Joanna Briggs Institute (JBI) Evidence Synthesis  and the Given the aim of the review, the condition, context, and population (CoCoPop acronym) was used to characterize eligibility criteria . The criCondition: Antimicrobial use or antimicrobial resistance, or risk factors potentially associated with either of these, reported in any metric.Context: Environmental factors can have an impact on the frequency of occurrence of the conditions of interest [interest , and thePopulation: Studies with healthy/asymptomatic animals produced in beef cow–calf herds in Canada and the USA were eligible. Studies of all classes of livestock within the cow–calf sector  were eligible. For studies reporting data from cow–calf operations as well as other enterprises such as stockers, backgrounding or feedlots, or studies sampling operations which combined one or more of these enterprises with cow–calf production, sampling from the populations of interest was deemed relevant, and sampling originating from populations outside of scope  was not included in data extraction.Inclusion: Primary research investigating beef cow–calf populations in Canada and the USA, of any size, was eligible with no restrictions on year or language of publication. If the research included AMR outcomes, only data originating from sampling clinically healthy as opposed to ill animals were deemed relevant. Journal articles, government or industry publications, and theses were all deemed relevant.Exclusion: Studies conducted in countries other than Canada and the USA were excluded to restrict data to regionally relevant herd management practices, licensed antimicrobials, and regulations limiting access. Additionally, AMR studies were excluded if they only sampled clinically ill or symptomatic animals, or they did not report outcomes identifiable as originating from clinically healthy animals or collected as part of routine surveillance from privately owned herds. Calf rearing operations, in which dairy calves not destined for the breeding herd are fed to slaughter, were outside scope, even if reporting on calves of beef breeds. Studies investigating cattle in feedlots or only, or only sampling cattle raised within organic or antibiotic-free production systems, or herds purposively selected for known minimal antimicrobial use, were also deemed outside of scope.An initial search was conducted on 22 January 2022 in three electronic bibliographic databases: Web of Science, Scopus, and AGRICOLA, using a three-term search. One term described the populations of interest . The second term described the outcome of interest . The third term described location . Examples of the specific search strategies employed in each database are presented in the study protocol in A commercial spreadsheet program was used to manage the data . First-level relevance screening was performed independently by two reviewers on abstracts captured by the search. The first level screening form asked four questions: “Is the literature in the form of a journal article, thesis, or government report?”; “Does the research take place in Canada or the United States?”; “Does the study include data from cow-calf herds?”; and “Does the article contain information on AMU or AMR?”. Those citations deemed relevant using this form were promoted to second-level relevance screening, and the full paper was obtained.The objective of second-level relevance screening was a full-text review by two independent reviewers to confirm eligibility. Relevant publications contained information on AMU or AMR in the cow–calf sector of the beef industry, specifically the Canadian or American cow–calf industries. First- and second-level relevance screening conflicts were resolved through discussion and confirmation by the senior author.Third-level data extraction was performed by a single reviewer using a pre-tested data extraction form capturing major characteristics of each relevant study, such as year of sampling, populations studied, broad outcome(s) reported  and metrics used, as well as level of data aggregation . Copies of the three relevance screening forms used are available online in For purposes of this review, a study was defined as an investigation of AMU or AMR in a defined population, identifiable by beef cow–calf sub-populations . A publication (synonymous with “paper”) was defined as a publicly available description of one or more studies. The same study population could be sampled for different studies . Multiple studies could originate from the same overall study population, within one publication . For purposes of mapping the literature, all relevant publications were included in data extraction. If multiple publications sampled the same population for different studies and reported different outcomes, these were indicated with a superscript in tables.Descriptive statistics were computed in a commercial spreadsheet program .E. coli for pre-weaning calves are required to inform the development of AMR risk mitigation strategies. Studies specifically describing the decision-making processes of producers in AMU, which report AMU or AMR as an outcome of interest, are currently research gaps.The dataset captured focused predominately on one AMU metric (count), studied at one hierarchical level (herd), and one population (cows) was investigated for AMR, leaving several major information gaps in most of the published literature. A small number of risk factors significantly associated with AMU  and AMR (biosecurity procedures) were reported. Additional studies targeting major reasons for AMU in calves, the impact of management procedures on usage, potential environmental sources of AMR, and AMR in respiratory pathogens and enteric organisms in addition to This research is especially important given the importance of AMU in cattle relative to overall livestock use in Canada and the USA.


Escherichia coli BL21 and further application in releasing FA. After screening the candidate signal peptides, the optimal one for each enzyme, which were selected as SP1 and SP4, respectively, was integrated into the vectors pET22b(+) and pETDuet-1. Among the generated E. coli strains SP1-F, SP4-X, and SP1-F-SP4-X that could express extracellular enzymes either separately or simultaneously, the latter one performed the best in relation to degrading the biomass and releasing FA. Under the optimized culture and induction conditions, the strain SP1-F-SP4-X released 90% of FA from 10% of de-starched wheat bran and produced 314.1 mg/L FA, which was deemed to be the highest obtained value to the best of our knowledge. This result could pave a way for the re-utilization of agricultural residues and enhancing their add-value.There is still a large amount of ferulic acid (FA), an outstanding antioxidant, present in agricultural residues. Enzymatic hydrolysis has been regarded as the most effective way to release FA. This present study therefore selected feruloyl esterase (FAE) and xylanase (XYN) from the metagenomes of a cow rumen and a camel rumen, respectively, for their recombinant expression in  Ferulic acid (FA) is widely distributed in grains, fruits, vegetables, and agroindustry-derived byproducts, accounting for the highest dry weight of cells of up to 3% . The preIn the plant cell wall, FA mainly exists in its bound form and acts as a cross linker between arabinoxylan and a lignose. It could be obtained using the chemical extraction approach and enzyme hydrolysis strategy; the latter one is comparatively more environmentally friendly and specific . Feruloyfae (Genbank accession no. HQ338149.1) obtained from the China Holstein cow ruminal metagenome has been verified to produce FAE with a high thermal and pH stability, as well as a broad substrate specificity, making it a good candidate to produce FA by degrading biomass [xylCM5 gene (Genbank accession no. KX644152.1) obtained from the camel ruminal metagenome was found to encode a β-1,4-endoxylanase XylC with a xylanase domain Xyn, a polysaccharide-binding domain CBM, and a carbohydrate esterase domain CE. Both the wild XylC enzyme and the truncated Xyn-CMB form exhibited a great activity towards the oat xylan within a broad pH range, and the truncated Xyn-CMB form showed a greater thermal stability [The ruminal metagenomes of the cow and camel have been considered as treasure troves of glycoside hydrolases. The  biomass . The xyltability .fae and the partial sequence xyn-cbm of the xylCM5 gene were fused with their optimal signal peptides (SPs) for their extracellular expression in Escherichia coli [E. coli strains were used as the cell factory for producing FA from wheat bran, one of the current agricultural residues that are still used as the low-value feeding stuff. This work could enhance the value of the use of wheat bran and pave the way for a greener and more effective production of FA.In the present study, the full-length hia coli ,14,15,16E. coli strains and plasmids used in this study are listed in E. coli JM109 and BL21 (DE3) was used for DNA manipulation and protein expression, respectively. The E. coli strains were routinely cultured with Luria–Bertani (LB) medium at 37 °C, with ampicillin supplemented at a final concentration of 100 μg/mL when required. The Ezup bacterial genomic DNA extraction kit, Rapid Taq Master Mix, restriction enzymes, and T4 DNA ligase were purchased from TaKaRa Biotech Co., Ltd. , Vazyme Biotech Co., Ltd. , and Sango Biotech Co., Ltd. . FA, arabinoxylan, and methyl ferulate (MFA) standards were purchased from Sigma-Aldrich . The wheat bran sample was provided by Jiangsu Agribusiness group Corp Ltd. All other chemicals were purchased from Sinapharm .All the fae and xyn (xyn-cbm of the xylCM5 gene) were synthesized according to the E. coli preference of codon usage and ligated with pET22b(+) to fuse the signal peptide pelB with these two enzymes. The SP1~SP5 were then substituted with pelB through fusion PCR, using pET22b-fae and pET22b-xyn as their templates, respectively , verified using colony PCR, and stored in 20% glycerol at −80 °C. All the primers used in this study are listed in In order to clone the two enzymes of interest, the genes ectively . The PCRplasmids  were furE. coli strains were inoculated, cultivated, and induced with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 30 °C, 150 rpm, when the OD600 reached 0.6. The samples were then taken and the subcellular fraction’s enzymatic activity of each recombinant strain were monitored after 12 h of cultivation. BL21, containing the blank pETDuet-1 vector, was used as the negative control. Subcellular fractionation of recombinant FAE and XYN was achieved using the osmotic shock method to isolate the cytoplasmic, periplasmic, and cytoplasmic cell fractions [To select the optimal SP, the recombinant ractions .fae and xyn genes, with their optimal SPs, were integrated into the same pETDuet-1 vector, which was then transformed into the BL21 strain.The 2HPO4 (pH 6.0) to react at 40 °C for 10 min [To determine the FAE activity, 100 μL of purified FAE was mixed with 900 μL 1 mM methyl ferulate in 0.05 M citric-Nar 10 min . The rear 10 min . The sta2HPO4 (pH 6.0) to react at 40 °C for 5 min. The reaction was terminated by adding 200 μL of 3,5-dinitro-2-hydroxybenzoic acid (DNS) [For XYN activity measurement, 100 μL of purified XYN was mixed with 100 μL of 10 mg/mL wheat xylan in 0.05 M citric-Na600 = 0.5, 0.8, 1.0, 1.5, and 2.0, respectively), five levels of IPTG concentration , five levels of induction temperature , as well as three levels of glycine concentration  were all evaluated while maintaining the other parameters at a constant level. Experiments were performed in a 250 mL flask containing 50 mL of the medium. After induction for 12 h, the cell-free supernatants were collected for FAE and XYN activity measurement.In order to optimize the secretory expression of FAE and XYN, five levels of initial cell density  was prepared as previously reported [600 reached 2.0, and then incubating at 30 °C for 48 h. SP4-X was induced with the optimal secretion parameters for XYN, i.e., supplementing 0.5 mM of IPTG and 10 g/L of glycine when the OD600 reached 1.0, and then culturing at 30 °C for 48 h. SP1-F-SP4-X was cultured in two batches: one induced with the optimal parameters for FAE (SP1-F-SP4-X1) and the other with those for XYN (SP1-F-SP4-X2), respectively. The SP1-F (OD600 = 0.2) and SP4-X (OD600 = 1.0) were mixed (SP1-F+SP4-X) and induced with 0.2 mM of IPTG plus 10 g/L of glycine at 30 °C for 48 h. The total amount of FA in the DSWB was determined through alkaline hydrolysis (2.0 M NaOH containing 1% Na2SO3 for 10 h at 25 °C in dark condition). After acidification with HCl, the samples were extracted using ethyl acetate and then analyzed with HPLC, as described above [To assess the FA-release capability of the constructed recombinant reported , and theed above .The results were presented as the mean ± standard deviation and all experiments were performed in triplicate. The date was analyzed using ANOVA analysis using SPSS version 13.0.fae gene was ligated into the pET-22b(+) vector to be fused with the intrinsic signal peptide pelB. Thereafter, the pelB SP was substituted with six different SPs using fusion PCR, including one Bacillus alkaline cellulase SP, four Lactobacillus FAE SPs, and the osmotically inducible protein Y (OsmY), respectively. After 12 h of induction, these signal peptides were able to assist FAE secretion in most recombinant strains, which were then verified by monitoring the FAE activity in each subcellular location and conducting SDS-PAGE analysis of these extracellular proteins. The SP1-F exhibited the highest activity (0.087 U/mL) among all the strains, and the extracellular activity of other recombinant strains were less than half of this. Subsequently, the more cytoplasmic and periplasmic FAE could be detected and its highest level was detected as 1.17 U/mL. In contrast, the OsmY-F and pelB-F exhibited a relatively low enzymatic activity in all three components, suggesting that OsmY and pelB may cause FAE misfolding, and are not suitable for its expression and secretion , which was in consistence with its greatest abundance detected during SDS-PAGE analysis. Since the SP4-X showed a high XYN expression level, and the proportion of extracellular enzyme activity was up to 23.6%, SP4 was subsequently selected as the best signal peptide for XYN secretory expression .fae and xyn genes, as well as their best signal peptides SP1 and SP4, were then integrated into the pETDuet-1 vector, generating the expression plasmid pETDuet-SP1-fae-SP4-xyn, which was then verified with PCR using the specific primer pairs F1-Nde I/R-Xho I-xyn. The strain SP1-F-SP4-X, harboring this plasmid, was consequently constructed for the simultaneous expression of FAE and XYN with the synergic degradation of biomass.The E. coli. Although FAE and XYN were able to be expressed and secreted by SP1-F-SP4-X, their activities were too low to cause the release of all the FA from the agricultural residues. Hence, a single-factor design of experiments, including initial cell density (OD600), concentration of IPTG, induction temperature, and concentration of glycine in 250mL flasks was applied to further improve their expression. As shown in A large number of factors are known to affect the expression of recombinant proteins in For IPTG induced fast synthesis of recombinant enzymes, the lower temperature could prevent the misfolding and consequently improve the solubility of the enzyme. The optimized induction temperature was 30 °C for FAE and 37 °C for XYN C, respecE. coli [Glycine is known to interfere with peptidoglycan synthesis and therefore affect the integrity and permeability of the outer membrane permeability of E. coli . In the 600 reached 2.0 and supplemented with 10 g/L of glycine. For XYN, the parameters were optimized with the initial OD600 of 1.0, 0.5 mM IPTG, 37 °C, and 10 g/L of glycine. Under their optimal conditions, the extracellular activity of FAE and XYN reached 0.94 U/mL and 0.87 U/mL, which were 10.9 and 2.5 times higher than that before their optimization, respectively. These findings could indicate that a large amount of FAE was secreted into the medium, with the extracellular activity accounting for 30.6% of the total FAE activity after optimization, whereas it was only 4.1% before optimization , containing 3.49 mg/g of FA, was supplemented into the LB medium as a carbon source for E. coli cell growth, and also acted as the substrate for producing FA. SP4-X expression only supported the functions of recombinant XYN, and the control BL21 strain harboring the blank pETDuet1 vector was not able to produce any FA, indicating that FAE was essential for releasing FA from the biomass. With FAE alone, 1.64 mg/g DSWB of FA was obtained with the SP1-F strain in 48 h, with the release rate of 47.0%. By the joint actions of FAE and XYN using the strain SP1-F-SP4-X under the FAE optimal secretion condition for 48 h, a 91.1% (3.18 mg/g DSWB) release rate of FA was achieved, producing 127.2 mg/L of FA.The direct use of the recombinant It was noted that a relatively lower yield (release rate) and titer was obtained when the strain was induced under the XYN optimal secretion condition, suggesting that the FAE-catalyzed reaction was the limiting step during FA release. The yield of FA using the mixed strains of SP1-F and SP4-X was almost the same as that with the single strain SP1-F-SP4-X1 .Lactobacillus alone achieved the highest FA release rate of 1.48 mg/g of DSWB [Furthermore, 8% and 10% of DSWB was supplemented in the releasing system, which increased the FA titer to 256.8 mg/L and 314.1 mg/L at 48 h, respectively, without causing a decrease in the yield of FA. In previous studies, actions of the FAE enzyme from  of DSWB ,19, and  of DSWB ; while iFA has also been reported to be the precursor of vanillin. With the metabolic pathway from FA to vanillin integrated into the SP1-F-SP4-X1 strain, the tentative plan of vanillin production from agriculture residues, as exemplified by wheat bran, could be established. At the same time, the final productivity of the reducing sugar reached 4023.4 mg/L, indicating that SP1-F-SP4-X also demonstrated a great application potential in the production of reducing sugars (xyloses/xylo-oligosaccharides) .FA has been widely used as a functional ingredient across the cosmetics, medicine, and food industries. As a natural source of FA and an agricultural residue with a low price, wheat bran has become an excellent raw material for natural ferulic acid production. The present study attempted to recombinantly express the ferulic acid esterase and xylanase with a greater activity and superior tolerance from the metagenomes of the cow and camel rumen, which are treasure troves of glycoside hydrolases, to enzymatically produce FA from agricultural residues. By screening seven candidate signal peptides, the optimal ones, SP1 and SP4, were selected and fused with ferulic acid esterase and xylanase, respectively. After optimizing the inducing conditions of the constructed strain SP1-F-SP4-X, these two enzymes were expressed and secreted successfully, with extracellular activities of 0.84 U/mL and 0.62 U/mL, respectively. When cultured with 10% DSWB, a titer of 314.1 mg/L FA with a release rate of 92.0% was obtained after 48 h of induction. A reasonable quantity of reducing sugar consisting of xylose and xylo-oligosaccharide were also obtained at the same time, indicating the great potential of this constructed strain.


Endometrial diseases are the most common gynecological pathologies in Western Countries. Among these, endometrial cancer (EC) is the most common gynecological malignancy , the fifEndometrial hyperplasia is considered a precursor of endometrial carcinoma and according to the World Health Organization (WHO) classification of 2014, and it is classified in atypical hyperplasia or non-atypical hyperplasia ,6,7. At Thus, in recent years, numerous studies have been developed to refine the clinical management and personalization of patient therapy with EC, considering not only traditional prognostic factors but also an innovative molecular analysis with the aim of defining different classes of risk and developing therapies targeted to the molecules involved in carcinogenesis.In 2013, the Cancer Genome Atlas (TCGA) Research Network changed the approach to EC classification with the integration of molecular characterization, resolving the numerous limitations in risk stratification that, for decades, were based only on tumor grade and histotype, depth of myometrial invasion and cervical and adnexal involvement. Based on mutations and somatic copy-number variations, genome and exome sequencing and microsatellite instability (MSI) assay, it is possible to divide EC into four groups, each with a different prognosis in term of specific progression-free survival and recurrence risk: Polymerase epsilon (POLE) ultramutated, MSI hypermutated, copy-number (CN) low and CN high . The firConsidering the strong scientific evidence and the results we will obtain in ongoing studies, in recent years, EC therapy is increasingly becoming tailored for the various subclasses even if no level A evidence has supported the use of mutational and genomic profiling in the selection of adjuvant treatments in patients with early-stage disease. To date, only advanced or metastatic stages could benefit from targeted adjuvant therapies based on molecular alterations, particularly considering advanced MSI-H/MMR-deficient (dMMR), numerous studies have evaluated the efficacy of monoclonal antibody therapy directed against immune checkpoint-associated proteins, expressed at high levels within the tumor microenvironment and making tumor cells susceptible to immune system response .However, molecular analysis could guide the therapeutic strategy for the treatment of precancerous EC lesions involving premenopausal women, nulliparous or with pregnancy plans, who would prefer conservative treatment. There are few data available in the literature that analyze how molecular classification might predict which subclasses have highest risk of evolution in EC. Results confirmed that, also in endometrial atypical hyperplasia POLEmut and CNL groups have a good prognosis compared to CNH and MSI-H groups  and PhosUnfortunately, few data are available for the use of molecular analysis for such diseases and improving risk classification for patients who may undergo fertility-sparing is one of the future targets. Endometrial diseases include a variety of pathologies that even within the same class may differ in histopathological or molecular features. Therefore, detecting and validating the use of molecular classification in precancerous lesions or analyzing different molecular markers could change therapeutic strategy, increasing the follow up of fertility-sparing patients or tailoring surgical radicality, reserving demolition surgery only for patients at high risk of cancer progression. More studies are needed to validate this scientific evidence that could revolutionize the clinical management of endometrial disease.