The relationship between two reflectances of different bands is often encountered in cross calibration and parameter retrievals from remotely-sensed data. The asymmetric-order vegetation isoline is one such relationship, derived previously, where truncation error was reduced from the first-order approximated isoline by including a second-order term. This study introduces a technique for optimizing the magnitude of the second-order term and further improving the isoline equation’s accuracy while maintaining the simplicity of the derived formulation. A single constant factor was introduced into the formulation to adjust the second-order term. This factor was optimized by simulating canopy radiative transfer. Numerical experiments revealed that the errors in the optimized asymmetric isoline were reduced in magnitude to nearly 1/25 of the errors obtained from the first-order vegetation isoline equation, and to nearly one-fifth of the error obtained from the non-optimized asymmetric isoline equation. The errors in the optimized asymmetric isoline were compared with the magnitudes of the signal-to-noise ratio (SNR) estimates reported for four specific sensors aboard four Earth observation satellites. These results indicated that the error in the asymmetric isoline could be reduced to the level of the SNR by adjusting a single factor. Estimation of biophysical parameters from remotely sensed reflectance requires calibration , inter-cA key component of VI model development is the relationship between two reflectances of different bands obtained under fixed biophysical parameter conditions. This relationship produces a reflectance spectrum trajectory in a reflectance subspace attributed to a fixed biophysical parameter value; therefore, this relationship is known as a vegetation isoline. The concept of a vegetation isoline has been used repeatedly to develop optimal VI models ,9,12,16 From the application point of view, understanding of band-to-band relationship would provide information about land cover dependency of calibration coefficients. In retrievals of biophysical parameter, an isoline equation with higher accuracy would lead to better results in retrieved parameters. Moreover, biophysical parameters vary along with the evolution of phenology, which eventually influence on reflectance spectra. Since the derived coefficients of the vegetation isoline depend on the biophysical parameters, the phenology is also related to the variation of the vegetation isoline.Significant efforts have been devoted toward deriving useful analytical formulas based on a model of radiative energy transfer. These derivations used a representation of the top-of-canopy (TOC) reflectance spectrum consisting of photons that were directly reflected by the canopy layer. Because this portion of the reflectance spectrum does not reach the soil surface beneath the canopy, it is called the zero-th order interaction term. Photons that reached the soil surface and were reflected back to the canopy layer by the soil surface only once contributed to the measured reflectance. The ‘one-time reflected’ contributions comprised the first-order interaction term. Analogously, the reflectance spectrum consisting of photons reflected by the soil surface n times was defined as the n-th-order interaction term. The vegetation isoline equations were derived by truncating the second- and higher-order interaction terms. For this reason, the derived isolines are a first-order approximation of the vegetation isoline.The approximation order determines the accuracy of the derived isoline equations. The accuracy of the isoline has been improved by deriving several approximations that considered the second-order terms. The accuracy has been improved by including higher-order terms. The drawback of this inclusion is that the analytical representation is complex. Complex representations hinder the employment of isoline formulations in applications of various types. It would be beneficial to identify ways of improving the isoline approximation accuracy while maintaining the simplicity of the derived formulation.In a previous study, we proposed a derivation technique for satisfying these requirements simultaneously . During This study advanced the investigation one step further. The objective was to introduce a technique for improving the accuracy of the asymmetric isoline equations by optimizing a single factor. The goal of this improvement was to reduce the errors in the vegetation isoline equivalent to a value equal to or smaller than the error induced by the inherent signal-to-noise ratio (SNR) of the existing sensors. The accuracy improvements obtained in this study were validated using a radiative transfer model of a system of vegetation and soil layers, the PROSAIL model ,25. AfteIn this section, two forms of the vegetation isoline equation are introduced. The magnitudes of the errors in the isoline equations differed between the two equations and were characterized numerically.The simplest form of the vegetation isoline equation was derived by truncating the soil–canopy interaction terms at the first-order (single interaction) . The resω, and the variables The fraction of vegetation cover (FVC) is represented by These variables are explained in additional detail elsewhere ,21,23.The second form of the vegetation isoline was derived by only including the interaction terms up to the second-order in the NIR band. The interaction terms in the red band could be expressed using the first-order terms. The asymmetric-order of the approximation in the two bands significantly reduced the error in the isoline relative to the first-order form. The asymmetric form of the vegetation isoline may be written  were computed assuming a fully covered vegetation canopy, where the value of FVC was set to unity. The PROSAIL model was used to simulate the TOC reflectance by varying the leaf area index (LAI) and soil reflectance spectra (from dark to bright soil). We next focused on testing whether the accuracy of the asymmetric approximation was satisfactory from a parameter retrieval point of view. This point was examined by comparing the isoline errors with an error equivalent to the noise level in the reflectance measurements. The comparison was implemented by assuming a simple scenario such that the value of the SNR in the NIR reflectance was 200 and the average value of the NIR reflectance was 0.1 over the entire parameter range. Although this assumption was made for the sake of simplicity, it was a rather conservative assumption because the averaged NIR reflectance is expected to exceed 0.1 in most cases. Under these assumptions, the noise equivalent error could be obtained as 0.0005 in reflectance units over the entire parameter range. With this quick estimate of the noise equivalent error, Improved accuracy was achieved by including the second-order interaction term only in the NIR band. This modification shifted the first-order approximated isoline upward in the reflectance subspace. This gap could be analytically evaluated by clarifying the difference between the first-order and the asymmetric-order isoline equations. The asymmetric-order approximation form of the vegetation isoline was obtained by neglecting the higher-order interaction term Equation . The defEquation , was useAfter rearranging Equation  by notinThe term in the parentheses on the right-hand-side represents the contribution of the asymmetric second-order term, illustrated as the distance between the blue line and the red line in k) introduced into the last term of Equation . The results section focuses on the use of a Spherical model to represent the leaf angle distribution (LAD), with the exception of the simulations presented in The variables used in a series of numerical simulations were computed using the canopy radiative transfer code, PROSAIL , which cξ, was obtained by conducting an additional simulation in which the soil spectrum was even brighter than was assumed in the simulation used to compute The parameters in the isoline equations were computed according to the procedures reported previously ,26. Tvλ2his case . In the is case [ρN≈ωρvN+Tk were analyzed based on numerical experiments in which the k-value was computed in the previous step using Equation (k: (1) FVC was varied using three pairs of fixed LAI and k-values are plotted against FVC for “LAI = 1.0 and k-values were relatively insensitive to the changes in FVC. The differences between the k values for FVC = 0.0 and FVC = 0.9 were less than 1%, and the differences for FVC = 0.0 and FVC = 1.0 were less than 3% for each pair of LAI and k was identified from the large differences between the k-value curves obtained at The dependences of LAI, FVC, and Equation . Three ek-values were relatively insensitive to LAI, as shown in k-values are plotted against LAI for “FVC = 0.3 and k-values for LAI = 0.0 and LAI = 4.0 were less than 5%. The differences between the results obtained for Similarly, the k-values along k-values showed an approximately 50% increase (from 0.9 to 1.35) with increasing k-value to a much smaller degree than did the differences between the maximum and minimum k-values depended heavily on k-values were computed using all possible pairs of the input parameters  as the errors between the true spectra ρ  and the vegetation isolines  × 9261  was obtained. ϵ averaged along the spectral dimensions as a function of the k-values. The error ϵ decreased until the k-value reached 1.25–1.30 and changed to an increasing function upon further increases in the k-value.The Equation ),(17)ϵ, and maximum of ϵ for each k-value were computed and are summarized in ϵ were 8.35 ×k = 1.28. The values of the STD for k = 1.29 were, however, smaller than those obtained for k = 1.28. The same applied to the maximum. Accordingly, the optimum k-value, The optimum values of ϵ over LAI and k  were considered. In ϵ for k = 1.25 and reveals that ϵ was less than 0.00015 for ϵ, however, it increased with increasing k = 1.29, the maximum of ϵ was approximately 0.00025. Overall, ϵ was small across the entire parameter space. The results of ϵ obtained for k = 1.30 were slightly larger than the values obtained for k = 1.29 although ϵ was small for The validity of using ϵ) was smallest for ϵ for k = 1.25 was less than the value obtained for other k-values for ϵ was smallest for k = 1.30 for k-value, ϵ for The error across the entire parameter space  × 9261  were computed for each LAD, and the values of ϵ averaged along the spectral dimensions were computed and plotted as a function of the k-value for each LAD. ϵ versus k-value for the various LADs. The k-values that provided the minimum value of the mean ϵ were approximately 1.2–1.3, except for the erectophile model, indicating that the minimum value of the mean ϵ occurred for The optimum values of k-value and mean, STD, and maximum ϵ, where k-value for each LAD obtained in our simulations. The mean values of the errors were approximately equal to or smaller than 0.0001. The magnitude of the STD of the errors was similar to that of mean. The maximum value of the errors was less than the noise equivalent error (0.0005), except for the erectophile model. The appropriate selection of the optimum k-value thus led to an accurate prediction of the NIR reflectances, but the use of This study sought to decrease the errors associated with predicting the NIR reflectances by using red reflectances in the vegetation isoline equations. The goal was to decrease the errors to the level of the intrinsic errors of the sensor SNR values. Therefore, the errors in the first-order, the asymmetric-order, and the adjusted asymmetric isoline equations with  (MODIS) , the Lan (MODIS) , the Gre (MODIS) , and the (MODIS) , as summr) was computed according to:The ratio of the relative errors in the isoline equations to the sensor SNR  improved the accuracy of the asymmetric-order isoline equations while retaining the simplicity of the equations. The k-value was assumed to be a function of LAI, FVC, and k were much greater than the influences of LAI and FVC such that k could be considered primarily to be a function of k-value to an optimum instead of varying this parameter as a function of The asymmetric-order isoline equations, derived from a previous study, were reformulated as first-order isoline equations plus a correction term multiplied by a parameter The errors in the adjusted asymmetric-order isoline equations were computed using k-value along with each LAD reduced the errors more significantly. In addition, the errors in the adjusted asymmetric-order isoline equations were small over the entire parameter space relative to the noise equivalent errors computed from the SNR of the satellite sensors currently in orbit . The first-order and asymmetric-order isoline equations displayed both superiority and inferiority to the noise equivalent errors by relying on the canopy and soil conditions.The value of k-value, yielding an error that was less than the noise equivalent errors based on the SNRs of some major satellite sensors, without complicating the isoline equations. Validation of the derived equations would require additional numerical experiments involving the application of other radiative transfer models of the vegetation canopy. Improved accuracy in the equations may be necessary if the sensor’s SNR were to increase as a result of technological advancements in the sensor instrument design.This study achieved its goal of reducing the error in the adjusted asymmetric-order isoline equations using a fixed


In current psychiatric practice, antidepressants are widely and with ever-increasing frequency prescribed to patients. However, several scientific biases obfuscate estimates of antidepressants’ efficacy and harm, and these are barely recognized in treatment guidelines. The aim of this mini-review is to critically evaluate the efficacy and harm of antidepressants for acute and maintenance treatment with respect to systematic biases related to industry funding and trial methodology.Narrative review based on a comprehensive search of the literature.It is shown that the pooled efficacy of antidepressants is weak and below the threshold of a minimally clinically important change once publication and reporting biases are considered. Moreover, the small mean difference in symptom reductions relative to placebo is possibly attributable to observer effects in unblinded assessors and patient expectancies. With respect to trial dropout rates, a hard outcome not subjected to observer bias, no difference was observed between antidepressants and placebo. The discontinuation trials on the efficacy of antidepressants in maintenance therapy are systematically flawed, because in these studies, spontaneous remitters are excluded, whereas half of all patients who remitted on antidepressants are abruptly switched to placebo. This can cause a severe withdrawal syndrome that is easily misdiagnosed as a relapse when assessed on subjective symptom rating scales. In accordance, the findings of naturalistic long-term studies suggest that maintenance therapy has no clear benefit, and non-drug users do not show increased recurrence rates. Moreover, a growing body of evidence from hundreds of randomized controlled trials suggests that antidepressants cause suicidality, but this risk is underestimated because data from industry-funded trials are systematically flawed. Unselected, population-wide observational studies indicate that depressive patients who use antidepressants are at an increased risk of suicide and that they have a higher rate of all-cause mortality than matched controls.The strong reliance on industry-funded research results in an uncritical approval of antidepressants. Due to several flaws such as publication and reporting bias, unblinding of outcome assessors, concealment and recoding of serious adverse events, the efficacy of antidepressants is systematically overestimated, and harm is systematically underestimated. Therefore, I conclude that antidepressants are largely ineffective and potentially harmful. Treatment guidelines for major depression (MD) in Europe and the United States typically recommend antidepressant medication with or without psychotherapy . In partResearch funded by the pharmaceutical industry is systematically biased toward their marketed products –15. Thatd or g > 0.5 or a difference >3 points on the Hamilton Depression Scale for Depression (HAMD)  . Based oSome authors deem the small difference between antidepressants and placebo a methodological artifact , 44–46. There are two trial designs to examine effects of long-term antidepressant use. The first is the long-term parallel-arm efficacy trial, where responders to both antidepressants and placebo are followed up. The second, much more common design, is the discontinuation trial, where antidepressant responders are randomly assigned to either continued therapy or rapid discontinuation and switching to placebo. In a recent meta-analysis of long-term parallel-arm RCT of 6–8 months duration, no difference between groups was found with respect to both remission and treatment discontinuation rates . Meta-anSome authors suggest that long-term antidepressant use may increase the vulnerability for (recurrent) depression due to neurochemical sensitization , 59. In placebo groups for adults aged 25–64 years (relative risk: 1.3), and it was even markedly increased in adults aged 65 years and older (relative risk: 2.7). These findings suggest that antidepressants may protect against suicide in middle-aged and older adults, which conflicts with the findings of all other meta-analyses detailed above. However, the work by Stone et al.  by 66% . In anotThe results of this mini-review suggest that the efficacy of antidepressants is systematically overestimated, while potential harms are underreported and neglected. Despite these alarming findings, thought leaders within mainstream psychiatry and official treatment guidelines strongly recommend antidepressant use for acute and long-term therapy. However, mainstream psychiatry is closely tied to the pharmaceutical industry, and most leading psychiatric experts receive substantial amounts of financial support from the industry , 11, 21.MH conducted the literature review and wrote the entire manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Macaca nemestrina, Macaca fascicularis, Macaca mulatta), we build upon this striatal map by systematically mapping inputs from frontal cortex, amygdala, temporal pole, and medial temporal cortex. We find that the VS contains heterogeneous subregions that become apparent when considering both the identities and strengths of inputs. We parcellated the VS into a ventromedial sector receiving motivation and emotion-related information from regions including area TG, ventromedial PFC, ACC, and amygdala; and a more functionally diverse dorsolateral sector that receives this information coupled to cognitive and sensorimotor information from dorsolateral PFC, ventrolateral PFC, premotor cortex, area TAr, and area TEr. Each sector was further parcellated into smaller regions that had different proportions of these inputs. Together, the striatum contains complex, selective input combinations, providing substrates for myriad associations. This VS parcellation provides a map that can guide and interpret functional interactions in healthy individuals and those with psychiatric disorders, and may be useful in targeting treatments for specific psychiatric conditions.The canonical striatal map, based predominantly on frontal corticostriatal projections, divides the striatum into ventromedial-limbic, central-association, and dorsolateral-motor territories. While this has been a useful heuristic, recent studies indicate that the striatum has a more complex topography when considering converging frontal and nonfrontal inputs from distributed cortical networks. The ventral striatum (VS) in particular is often ascribed a “limbic” role, but it receives diverse information, including motivation and emotion from deep brain structures, cognition from frontal cortex, and polysensory and mnemonic signals from temporal cortex. Using anatomical tract-tracing in 17 male adult monkeys ( The striatum is central for learning associations among stimuli, actions, and outcomes. The canonical striatal map is divided into ventromedial-limbic, central-association, and dorsolateral-motor territories. However, the striatum has a more complex topography. We find that the ventral striatum (VS) consists of a ventromedial sector receiving motivation and emotion-related information and a more functionally diverse dorsolateral sector that receives this information coupled to cognitive and sensorimotor information from frontal cortex, temporal cortex, and amygdala. Each sector is further parcellated into smaller regions with different input proportions. These results show the complex, selective combinations of inputs to VS, providing an anatomic substrate for understanding how various functions may be integrated and a guide for interpreting striatal activity associated with different behavioral outcomes.The canonical striatal functional map is composed of a ventromedial-to-dorsolateral gradient of frontal cortical projections . This grThe VS, which includes the nucleus accumbens (NAc) core and shell and adjacent caudate and putamen, is often equated as the part of the striatum that is involved in motivation and reward. However, the VS is a large region, estimated to be 20%–25% of the striatum . It receThe goal of this study was to build on the existing striatal map of PFC and amygdala inputs  by examiG and Figs. 1E and Figs. 1E and r) of the percents of inputs from all examined areas for each pair of injections. Two-tailed p-values were obtained with Fisher’s r-to-t transformation and Student’s t-distribution.Eleven anterograde tracer injections were placed in TP+ and MTC: three in TG, two in area 36, one in TEr, two in EC, one in TH, one in TF, and one in TL G and 3. Macaca nemestrina, eight Macaca fascicularis, seven Macaca mulatta) received tracer injections. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals , wheat germ agglutinin-horseradish peroxidase , or tritiated amino acids , AChE , PV , CB , and NeuN .Eighteen male adult macaque monkeys  of the input percentages for each pair of injections using the corr function in Matlab . Two-tailed p-values testing against the null hypothesis of no correlation were obtained with the same Matlab function, which uses Fisher’s transformation to convert the r value into a t statistic with a Student’s t-distribution.2-D charts of the terminal fields in the striatum from temporal anterograde tracer injection cases were rendered in 3-D and linearly transformed into a standard atlas of the striatum using IMOD (Boulder Laboratories). Temporal projection patterns were assessed both within coronal sections and globally across the striatum. Temporal projections were then overlaid with those from our library of previously reported anterograde tracer injections in the dlPFC (eight injection cases in areas 9 and 46), vlPFC (two injection cases in areas 44/45 and 47), ACC (four injection cases in area 24), OFC , and premotor cortex  consists of the shell of the NAc and the olfactory tubercle. The dorsal VS (VSd) consists of the core of the NAc and the adjacent caudate and putamen . In geneAreas within TP+ were identified based on a parcellation of the human temporal polar cortex . TP refeThe parcellation of the MTC has been previously described in detail . The antBoundaries of frontal cortical areas were identified as previously described  based onAnterograde tracer injections in TG, TEr, and the entorhinal, perirhinal, and parahippocampal cortices of the MTC resulted in distinct corticostriatal projection patterns. TG projections terminated medially, while those from TEr terminated laterally in the VS. Interestingly, projections from the MTC bifurcated into those targeting the VS and those targeting the dorsal edge of the caudate. Overlay of these temporal projections with previously reported frontal inputs  and amygArea TG projects primarily medially to the VS. Specifically, terminals were located mostly along the medial wall of the caudate and in the medial and central VSd, with some scattered patches in the lateral VSd Aii. CaudRetrograde tracer injections in the VSv and the medial, central, and lateral VSd confirmed and extended the input patterns shown by the anterogradely labeled TP+ and MTC projections. Consistent with the TG terminals seen in all sectors of the VS, all the retrograde tracer injections labeled cells in TG Ai–Di. ThDetails of frontal and amygdala projection patterns have been previously reported . BrieflyComparison of these frontal and amygdala projections with the present temporal projections suggested two broad convergence patterns in the VS. The first pattern spanned the ventromedial VS, comprised of the medial VSd and the VSv. Here, TG projections converged specifically with the inputs from the vmPFC, including along the medial wall of the caudate A. These The retrograde VS injections confirmed the two broad convergence patterns in the ventromedial and dorsolateral VS and identified the comprehensive set of TP+, MTC, frontal, and amygdala inputs to each VS region. Consistent with the ventromedial VS pattern, in addition to inputs from TG, TI, and MTC, the VSv Aii and mQuantification of the labeled cells within TP+, MTC, and frontal cortical areas from each VS injection and a dorsal caudate injection (Case MF170FS) specified the relative strengths of their inputs to the different VS sectors ; Table 2Furthermore, within these broad convergence patterns, each VS site had a unique combination of inputs . AlthougAs prior studies have noted for the ACC, OFC, and amygdala , certainr = 0.53–0.69, p = 2.0 × 10−5 to 2.1 × 10−3, paired t test), consistent with the high degree of functional diversity in the inputs to the lateral VSd. The distributions of inputs for the medial and central VSd and VSv sites were also correlated with one another, but to a lesser degree , reflecting the more site-specific inputs to these regions. None of the VS sites had distributions that were significantly similar to those of the dorsal caudate injection site.These differences in the distribution of inputs were reflected in the pairwise linear correlations of the areal input percentages for pairs of injection cases . The strThis study examined the patterns of convergent inputs to the striatum from the temporal cortex, specifically TP+ and MTC, in relation to the well-characterized functional topography formed by frontal cortical and amygdala projections. The results demonstrate that the VS contains heterogeneous subregions that become apparent when considering both the identity and strengths of inputs, including those from regions beyond the frontal cortex. Using anterograde tracers, we found that certain temporal areas had novel projection patterns compared with frontal projections: TEr projects to the central and lateral VSd and VSv. In contrast, TG had projections located primarily along the medial wall of the caudate and extending into the central VSd and VSv, which overlapped remarkably specifically with vmPFC inputs. Using retrograde tracers, we comprehensively identified the set of inputs to specific striatal regions. With these expanded criteria, we parcellated the VS into a ventromedial sector receiving motivation and emotion-related information from areas including TG, vmPFC, ACC, and the amygdala; and a dorsolateral sector that receives this information coupled to cognitive and sensorimotor information from dlPFC, vlPFC, premotor cortex, TAr, and TEr. Thus, in general, the ventromedial VS receives the most limited inputs from regions that process information related to reward, emotion, and motivation. In contrast, the dorsolateral VS receives inputs from multiple functional areas that cut across “limbic,” cognitive, and sensorimotor domains.Furthermore, each of these sectors could be parcellated into smaller regions that reflect differences in the proportions of their inputs. Thus, although the medial VSd and VSv both received inputs from largely the same frontal and temporal areas, they differ in the strength of those inputs: the medial VSd received a majority of inputs from the vmPFC and ACC, whereas the VSv received greater MTC inputs. The output of these two regions may therefore be related, but complementary to one another. These results underscore the importance of considering not only the source of afferents, but also their strengths when assessing the nature of the integration occurring in a region. Here, considering input strength identified additional functional heterogeneity than considering input area alone.These results are consistent with human striatal parcellations using neuroimaging methods, which identify both an overall ventromedial-to-dorsolateral gradient  and smalWe previously reported the presence of connectional hubs in the striatum that receive a high degree of diverse functional inputs . With thIn a rare split of projections, all of our anterograde tracer injections placed in the entorhinal, perirhinal, and parahippocampal cortices of the MTC showed terminals in both the VS and the DS. Within the VS, these projections terminated broadly, with the strongest inputs to the VSv, and overlapped with all frontal, TP+, and amygdala afferents. In the DS, these MTC inputs formed a cap at the dorsal edge of the caudate and overlapped with dlPFC, vlPFC, and premotor projections. Interestingly, these dorsally targeting MTC inputs also overlapped with a connectional hub in the rostral dorsal caudate . This huAs a central structure for reinforcement learning and habit formation, the VS has been repeatedly observed to have abnormal activity in psychiatric disorders . By distThis study builds on the well-characterized functional map of the striatum based on frontal cortical and amygdala projections. We present an updated, heterogeneous map of the VS by considering both the identity and strengths of the inputs received, including those from regions beyond the frontal cortex, specifically TP+ and MTC in the temporal cortex. The VS contains a ventromedial sector receiving motivation and emotion-related information from areas including TG, vmPFC, ACC, and the amygdala; and a dorsolateral sector that receives this information coupled to cognitive and sensory information from dlPFC, vlPFC, premotor cortex, TAr, and TEr. Each sector further contains distinct smaller regions that receive different proportional strengths of inputs, suggesting that each region of the striatum has a unique combination and integration of inputs. These results indicate that the striatum contains a complex, selective combination of inputs, providing the anatomic substrates for forming myriad functional associations that add to network interactions in the cortex. This heterogeneous parcellation of the VS provides a map of this combinatorial integration. This map can be used in guiding and interpreting healthy and abnormal striatal activity in normal subjects and those with mental health disorders and for targeting specific functional interactions in different disease conditions.


N,N′‐di‐p‐methoxyphenylamine)‐9,9′‐spirobifluorene in planar perovskite solar cells. The BDTS‐2DPP layer can remarkably passivate the surface defects of perovskite through the formation of Lewis adduct between the under‐coordinated Pb atoms in perovskite and S atoms in BDTS‐2DPP, and also shows efficient hole extraction and transfer properties. The devices with BDTS‐2DPP interlayer show a peak power conversion efficiency of 18.2%, which is higher than that of reference devices without the BDTS‐2DPP interlayer (16.9%). Moreover, the hydrophobic BDTS‐2DPP interlayer effectively protects the perovskite against moisture, leading to enhanced device stability.A high‐mobility p‐type organic semiconductor based on benzodithiophene and diketopyrrolopyrrole with linear alkylthio substituents (BDTS‐2DPP) is used as a dual function interfacial layer to modify the interface of perovskite/2,2′,7,7′‐tetrakis( These additives are hydrophilic and accelerate the degradation of devices.] To improve device stability and efficiency, various interfacial materials, such as metal nanostructures,In most of the high‐efficiency PSCs, 2,2′,7,7′‐tetrakis, as well as compatible energy alignment with CH3NH3PbI3(Cl), and may efficiently extract holes from perovskite to HTL. On the other hand, the electron‐rich sulfur (S) atoms in BDTS‐2DPP as Lewis base can well passivate the under‐coordinated Pb in CH3NH3PbI3(Cl) without annealing. The PSCs with BDTS‐2DPP interlayer at the interface of perovskite/spiro‐OMeTAD show a higher PCE (18.2%) than the control devices without the BDTS‐2DPP interlayer (16.9%). After stored under ambient conditions (humidity of ≈40% and temperature of ≈25 °C) in the dark over 7 d, the PCEs of the unencapsulated PSCs without BDS‐2DPP decay dramatically to 30% of their initial values, while the devices with BDTS‐2DPP interlayer retain about 80% of their initial values, indicating that the BDTS‐2DPP interlayer enhances the device stability.In this work, we demonstrate the first example for p‐type organic semiconductors used as the interlayer at perovskite/HTL interface (BDTS‐2DPP) in conventional PSCs to improve the efficiency and stability. BDTS‐2DPP is based on benzo] Unless stated otherwise, all reagents were obtained commercially and were used without further purification.Device Fabrication: PSCs were fabricated with a structure of ITO/compact TiO2/CH3NH3PbI3(Cl)/HTL/Au. The indium tin oxide (ITO) glass was precleaned in an ultrasonic bath of detergent water, ultrapure water, acetone, and isopropanol. A 40 nm thick layer of a TiO2 nanoparticle in ethanol (5.3 mg mL−1) with the appropriate amount of titanium diisopropoxide bis(acetylacetonate) (TiAcAc) was spin‐coated onto an ITO glass substrate and annealed at 150 °C for 30 min in air. Next, the substrates were transferred into a nitrogen‐filled glove box. A solution of PbI2  was then spin‐coated on ITO/TiO2 substrate at 3000 rpm for 30 s and annealed at 90 °C for 10 min. The mixture of CH3NH3I (50 mg mL−1) and CH3NH3Cl (5 mg mL−1) dissolved in isopropanol was spin‐coated onto the dried PbI2 layer at 3000 rpm for 30 s. Then, the obtained films were annealed at 150 °C for 15 min in air. The dopant‐free BDTS‐2DPP interlayer was spin‐coated onto the perovskite layer at 3000 rpm for 30 s with a concentration of 10 mg mL−1 in chlorobenzene without any annealing treatment. The doped spiro‐OMeTAD was spin‐coated at 3000 rpm for 30 s at a concentration of 80 mg mL−1 with the addition of 35 µL Li‐TFSI/acetonitrile (260 mg mL−1) and 28 µL 4‐tert‐butylpyridine. Finally, an 80 nm gold layer was deposited on the top of HTL through shadow masks via thermal evaporation under high vacuum (≈2 × 10−6 Torr).Measurements: The current density−voltage (J−V) characteristics of photovoltaic devices were obtained using a Keithley 2400 source‐measure unit under simulated sunlight from an Oriel 300 solar simulator, and the light intensity was calibrated using a KG‐5 Si diode. The effective area of each cell was 10.2 mm2 defined by masks for all the photovoltaic devices discussed in this work. The EQE spectrum was measured using a Solar Cell Spectral Response Measurement System QE‐R3011 . The light intensity at each wavelength was calibrated using a standard single crystal Si photovoltaic cell. The morphology was measured using a scanning electron microscope (Hitachi S4800). AFM images were obtained by a Multimode 8 scanning probe microscopy (Bruker). The XRD spectra were obtained from a D/MAX 2400 diffractometer with Cu Kα radiation (Rigaku). FTIR spectra were obtained by a TENSOR27 (Bruker), and Raman spectra were obtained by a LabRAM HR Evolution spectrometer (Horiba). TAS was performed using an IM6ex electrochemical workstation (Zahner), in which the scanning frequency was set between 0.1 and 106 Hz, and the amplitude of the sine perturbation bias was set to 10 mV. XPS was performed on the Thermo Scientific ESCALab 250Xi using 200 W monochromated Al Kα radiation. The 500 µm X‐ray spot was used for XPS analysis. The base pressure in the analysis chamber was about 3 × 10−10 mbar. Typically, the hydrocarbon C1s line at 284.8 eV from adventitious carbon was used as an energy reference. Steady‐state PL and time‐resolved transient‐state PL was measured by FLS980 (Edinburgh Instruments Ltd.) with an excitation at 470 nm. Static contact angles were measured on a Dataphysics OCA20 contact‐angle system at ambient temperature (the test liquid is water). All the measurements of the solar cells were performed under ambient atmosphere at room temperature without encapsulation.SupplementaryClick here for additional data file.


The limits of detection and quantification in the urine are 0.5 ng·mL−1 and 1 ng·mL−1, respectively, and the recovery clenbuterol rates are 82.8–92.4% with coefficients of variation <9.4%. The day-to-day variation of the detection is less than 6.41%, and the shelving life of the SERS substrates is no less than 4 weeks. All these indicate that this proposed SERS detection protocol for clenbuterol is reproducible, reliable, and can be easily developed for the routine monitoring of the illicit use of clenbuterol in animal farming.Combining surface-enhanced Raman spectroscopy (SERS) of aggregated graphene oxide/gold nanoparticle hybrids with immunomagnetic bead sample preparation method, a highly sensitive strategy to determine the clenbuterol content in animal urine was developed. Based on a linear calibration curve of the SERS characteristic peak intensity of clenbuterol at Δ Zhu et al. reported a SERS detection of clenbuterol based on a competitive SERS immunoassay with a LOD of 0.1 pg·mL−1et al. prepared a modified AuMBA@Ag - Antibody probe ) and demonstrated a LOD of 0.24 pg·mL−1 for quantitative detection of clenbuterol from urineRecently, many rapid screening methods have been proposed for clenbuterol detection, such as enzyme-linked immunosorbent assay technique101416−1 and 1 ng·mL−1, respectively. A sufficient linearity (R2 = 0.976) was found in the concentration range of 1–20 ng·mL−1 in urine, and the recoveries of clenbuterol from spiked urine samples were in the range of 82.8–92.4% with relative standard deviation (RSD) values in range of 0.81–4.32% (n=6). The total detection time was approximately 15 min/sample.In this study, we designed a unique hybrid graphene oxide/Au nanoparticle(GO/AuNPs) SERS probe for clenbuterol detection from urine sample.The hybrid probe was stable within four weeks under 4 °C storage temperature, and the LOD and the limit of quantitation (LOQ) based on SERS for real urine sample were 0.5 ng·mLMaterials and Methods” section. Below we discuss the most important results obtained.The general clenbuterol detection strategy can be divided into the following three steps as shown in λ0 = 530 nm, which is a typical localized surface plasmon resonance (LSPR) wavelength of AuNPsλ0 = 580 nm. Such a significant red shift could be due to the clustering of AuNPs or due to the plasmonic coupling between AuNPs and GO sheetsA photograph of the suspensions of AuNPs, untreated GO/AuNPs, and reoxizided GO/AuNPs is shown in −1 clenbuterol standard solution was used to evaluate the SERS performance of different solutions to detect clenbuterol, see v = 1265, 1474, and 1602 cm−1, respectively. The Δv = 1474 cm−1 peak corresponds to the in-plane stretching vibrations coupled with the anilinic C-N stretching vibration; the Δv = 1602 cm−1 peak is assigned to the C=C stretching band of the aromatic ring, and the Δv = 1265 cm−1 peak results from the anilinic C-N stretching coupled with in-plane ring deformationsI1474 at Δv = 1474 cm−1 is 2986 counts for the reoxidized GO/AuNPs, as compared to 88 counts for the GO/AuNPs. Thus, the second oxidization treatment is indeed very necessary to improve the SERS detection sensitivity. Therefore, we choose the reoxidized GO/AuNPs for the clenbuterol detection.In fact reoxidized GO/AuNPs show the best SERS performance for detecting clenbuterol. 20 ng·mLC = 1, 2, 5, 10, and 20 ng·mL−1 (n = 6) and clenbuterol inoculated urine samples. v = 1236, 1325, 1532, and 1593 cm−1. Those peaks may correspond to the vibrations of the solvent molecules or the background in the control samples. However, for all the spectra of clenbuterol, the three characteristic peaks at Δv = 1265, 1474, and 1602 cm−1 appear, and the intensities of these peaks increase as the C increases. To quantify the SERS peak intensity and clenbuterol concentration, we plot the peak intensity at Δv = 1474 cm−1, I1474, as a function of C in I1474 versus C follows a linear relationship, and a liner fit shown as the solid line in −1/intensity count. The limit of detection (LOD) is the lowest concentration at which I1474 is significantly larger than that from a control sample. We use 3-sigma rule of the SERS spectra from a control sample to set the cut-off I1474: the average SERS intensity value at Δv = 1474 cm−1 plus three times the standard deviation of SERS intensity of the control samplev = 1474 cm−1 plus ten times the standard deviation of SERS intensity of a control sample. From our measurements for the purified clenbuterol solution, LOD is approximately 0.5 ng·mL−1 and the LOQ is1 ng·mL−1.The LOD of SERS on clenbuterol detection has been evaluated through the SERS measurements of different concentrations of clenbuterol standard solutions t and the elution frequency play the most important roles. The incubation is required to allow the clenbuterol antibodies on the magnetic beads to have enough time to capture the clenbuterol molecules in urine as much as possible. To investigate the effect of the incubation time on SERS detection of clenbuterol from urine, the urine sample inoculated with 1 ng·mL−1 of clenbuterol was used with a fixed washing time N = 6 and the Ab-MBs incubation time in urine samples was varied as t = 1, 2, 5, 10, 15, and 20 min, respectively. The SERS intensity (I1474) of the extracted clenbuterol as a function of t is plotted in I1474 increases almost linearly with t. When t = 10 min, I1474 reaches a maximum. Further increase of t does not increase the SERS intensity. Therefore, t = 10 min is the optimum incubation time. The elution is required to further remove contaminants in the Ab-MBs extracted from urine. Such a process not only can reduce the contaminants, but also could remove captured clenbuterol on Ab-MBs. Therefore, there must be an optimal elusion frequency to retain most of the clenbuterol while to remove most of the contaminants. By keeping the same inoculated urine sample and fixing t = 10 min, the relationship between the SERS intensity I1474 and the elusion frequency N  was investigated, and is plotted in N= 6 is the optimal elution frequency.The LOD from urine sample is determined by three primary factors, the clenbuterol extraction efficiency from urine, the potential interference from contaminants in the extracted sample, and the intrinsic SERS detection LOD of pure clenbuterol. Urine samples are complex matrices containing multiple contents, such as urea, creatinine, uric acid, and small proteins. Many of them could generate SERS signals if not removed, and will introduce interference for clenbuterol detection. In order to remove the interference in the urine and obtain clenbuterol SERS spectra with a high signal-to-noise ratio, we use the Ab-MBs to extract the clenbuterol molecules from urine. For such an extraction process, the incubation time t =10 min and N = 6, the quantitative relationship between the SERS spectra of the extracted clenbuterol and the inoculated clenbuterol concentration C = 0.5, 1, 2, 5, 10, and 20 ng·mL−1, respectively, in urine was investigated. I1474 as a function of C, as shown in −1/intensity count, which is comparable to that of the standard solution. Based on the previously stated criteria, we determine that the LOD is approximately 0.5 ng·mL−1 and the LOQ is 1 ng·mL−1 for the inoculated urine samples.Under the optimal sample treatment condition, I1474, applied the LOD criteria, and used CSERS in urine is very close to those determined by LC-MS/MS, CLC-MS. CSERS versus CLC-MS. Clearly the data follows a linear relationship with a slope of ~1.1. In all the cases, SERS gave a slightly higher concentration than that determined by LC-MS/MS. However, a slope of 1.1 demonstrates that SERS results are highly correlated to LC-MS/MS results.The practical application of the proposed SERS method for clenbuterol detection is validated using the real urine samples. 40 different urine samples from sheep, pig, and cow obtained from the Chinese National Feed Supervision from 2012–2014, previously tested by LC-MS/MS−1 of clenbuterol were prepared. We adapted the optimal Ab-MBs treatment methods to remove the interferences of the urine samples, and did the SERS measurement as mentioned above. The analysis results was expressed as “detected values” (ng·mL−1). The recoveries values were the ratio between the detected values and the spiked values. The recoveries were found to be 92.4, 83.4, 91.5, 88.4, and 82.8%, respectively , as summarized in −1 for each individual agonist respectively, were used in the specificity test. After the Ab-MBs treatment, the SERS spectra were shown in v = 1474 cm−1 peak versus the clenbuterol concentration, and the sensitivity is 0.020 ng·mL−1/intensity count, which is almost the same as those shown in The specificity of the present method was explored against two other kinds of β4·4H2O, analytical grade, >99.9%) and sodium citrate  were acquired from the Sinopharm Chemical Reagent Co., Ltd.Graphene Oxide (GO) solution , carboxylic acid-magnetic beads , analytical grade bovine serum albumin (BSA), 2-(N-Morpholino) ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and all other chemicals from Sigma Aldrich Co., Ltd. were used as received. Ultra-pure water  was used in all experiments. All of the glass vials were cleaned using a concentrated piranha solution , then rinsed twice using ultra-pure water, and dried for 2 h under airflow in an oven at 100 °C.The non-contaminated urine samples used in the study were obtained from Small Tailed Han Sheep and were tested by LC-MS/MS. Clenbuterol hydrochloride and clenbuterol monoclonal antibody were purchased from Dr. Ehrenstorfer GmbH and Kwinbon Biotechnology, respectively. Chloroauric acid tetrahydrate  were added. The solution was incubated with vortex mixer at ambient temperature for 30 min. After incubation, magnetic beads were separated from the solution, and were washed twice. Then, a 10 μL aliquot of the clenbuterol monoclonal antibody was added to the magnetic beads, and 25 mM MES buffer (pH = 5) was added, to a final volume of 100 μL. The solution was incubated for no less than 30 min at ambient temperature with a vortex mixer. Finally, the antibody functionalized magnetic beads (Ab-MBs) were collected from the solution.To activate the immunomagnetic beads, 100 μL carboxylic acid-magnetic beads thoroughly resuspended were washed by adding 100 μL of a 25 mM MES buffer solution at pH 5 and incubated for 10 min, and a 50 μL aliquot of 50 mg·mLTo block any unreacted carboxylic acid groups, the Ab-MBs were incubated in a 100 μL 50 mM Tris buffer solution at pH = 7.4 for 15 min and washed four times in Tris buffer. The resulting beads were resuspended in 200 μL phosphate buffer solution (PBS) solution and were stored in the dark at 2–8 °C for three months.−1 GO solution was added to 20 mL 30% H2O2 solution at room temperature, followed by stirring for 4 h under ultraviolet light excitation . The resulting 3.5 mL GO solution were mixed with 350 μL 1 wt % aqueous sodium citrate and 40 mL distilled water in a 100 mL Erlenmeyer flask. With 800 rpm vigorous stirring the solution was heated to reflux, and 500 μL 1 wt % HAuCl4 was added within 2 s. The solution remained under reflux for another 10 min, then the heating source was removed while the solution was kept continuously stirring and cooled down to ambient temperature within 10 min. The resultant GO/AuNPs hybrids were washed twice by distilled water after centrifugation at 2800 rpm for 5 min (DAIHAN WiseSpin CF-10), and were stored at 4 °C for no longer than four weeks (the storage time on SERS response is shown in 20 mL 0.2 g·L3 (1 wt %) was added, and the Ab-MBs were extracted by a permanent magnet. The remained solution was used for SERS measurements and detection.A 50 μL Ab-MB stock solution was washed twice with 1.5 mL deionized (DI) water, then mixed with a 2 mL urine sample. After shaking the tube for 60 s, the mixture was incubated at ambient temperature for 10 min and washed six times with 1.5 mL DI water. Finally, 1 mL HNO−1. Each spectrum was an average of two scans.60 μL of above solution was gently mixed with 600 μL GO/AuNPs for 10 s , then th−1 were analyzed using the RamanAnalyzer software . The spectra baseline was offset using the Savitzky-Golay second derivative transformationThe SERS spectra ranging from 500–2500 cmThe study was approved by the Animal Research Ethics Committee of Chinese Ministry of Agriculture and supported by a special fund for Chinese Agro-scientific Research in the Public Interest (No. 201203088-1). The whole experiment was carried out in accordance with the guidelines of the animal experiment and some relevant bulletins published by the ministry of agriculture of the People’s Republic of China. The urine samples were obtained from the Chinese National Feed Supervision Mission from 2012–2014.v = 1474 cm−1versus the spiked clenbuterol concentration, the quantity of clenbuterol in real animal urine samples can be determined. These results matched well with those determined by LC-MS/MS. The limits of detection and quantification for detecting clenbuterol from the urine samples were 0.5 ng·mL−1 and 1 ng·mL−1, respectively, and the recovery clenbuterol rates were 82.8–92.4% with coefficients of variation <9.4%. Furthermore, the day-to-day variation of the detection was less than 6.41%, and the shelving life of the SERS substrates is no less than 4 weeks.SERS is an emerging analytical technique with satisfactory performance in the detection of trace amount of targeted analyte. Herein, based on a unique GO/AuNPs hybrids, a rapid, selective, and sensitive SERS method combined with immunomagnetic was developed to detect the clenbuterol content in animal urine. We demonstrated that based on a linear calibration curve of the SERS characteristic peak intensity of clenbuterol at ΔCurrently, the Chinese government surveys the illicit use of clenbuterol in animal farming using the GC-MS or LC-MS/MS methods. These standard methods not only require time-consuming sample pretreatment, with approximately 30 min/sample, but also need expensive chromatography instruments operated by experienced, well-trained personal. The SERS method we developed here only require a portable Raman instrument and the specific designed Ab-MBs & GO/AuNP assays with similar sensitivity, while the total detection time is only approximately 15 min/sample. Thus, the proposed SERS method holds a great promise for field-based clenbuterol detection in animal farming.This protocol could be developed for routine on-site monitoring of urine for the purposes of government and market surveillance.How to cite this article: Cheng, J. et al. Highly Sensitive Detection of Clenbuterol in Animal Urine Using Immunomagnetic Bead Treatment and Surface-Enhanced Raman Spectroscopy. Sci. Rep.6, 32637; doi: 10.1038/srep32637 (2016).


This observational study was undertaken to verify whether breath-hold F-18-FDG-PET/CT (bhPET) detects additional foci compared to free-breathing PET/CT (fbPET) in cases of malignant melanoma, and to assess the impact of breath-holding on standard uptake values (SUV) and metabolic isocontoured volume  were assessed for both methods and evaluated by linear regression and Spearman correlation. The impact of lesion size and time interval between investigations was analyzed.Thirty-four patients with melanoma were examined. BhPET and fbPET findings of 117 lesions were compared and correlated with standard contrast-enhanced (ce) CT and MRI for lesion verification. Quantitative parameters (SUVmax, and SUVmean proved significantly higher and mVic40 significantly lower than at fbPET. The positive effect on SUVmax and SUVmean was more pronounced in smaller lesions, whereas the time interval between bhPET and fbPET did not influence SUV or mVic40.In 1 patient, a CT-confirmed liver metastasis was seen only on bhPET but not on fbPET. At bhPET, SUVIn our patient cohort, bhPET yielded significantly higher SUV and provided improved volumetric lesion definition, particularly of smaller lesions. Also one additional liver lesion was identified. Breath-hold PET/CT is technically feasible, and may become clinically useful when fine quantitative evaluations are needed. However, performing the scans in free-breathing generates artifacts in the form of breath-related blurring.The F-18-fluorodeoxyglucose positron emission tomography (F-18-FDG-PET) is an established method for the imaging of malignant melanoma.,5Two methods have been tested to limit the impact of breath-related blurring in the application of F-18-FDG-PET/CT: respiratory gating (rgPET) and breath-hold PET (bhPET).,7 In contrast, bhPET has no additional costs and requires only limited effort, but image quality largely depends on the ability of the patient to hold his breath. For chest findings, in particular, investigations on the effects of bhPET/CT with F-18-FDG have shown that the standard uptake value (SUV) is typically higher and the isocontour volume smaller than with free-breathing (fb) PET/CT.,10 The ability to detect additional tumoral foci, normally missed by the free-breathing technique, has been demonstrated in a case of carcinomatous lymphangitis and in a liver metastasis of a colorectal carcinoma.Disadvantages of rgPET are the need of dedicated hardware and software, the technical effort and costs, the long scanning time, a high radiation exposure, and prolonged reconstruction times. These problems have resulted in limited application of rgPET in clinical routine.The goals of this study were to evaluate whether F-18-FDG bhPET/CT allows the detection of additional tumoral foci in patients with melanoma compared with fbPET/CT, and to verify the impact of breath-holding and free-breathing on quantitative parameters.22.1The observational study sample comprised 34 consecutive adults , with clinically and histologically confirmed metastatic melanoma . Diabetes mellitus type II was present in 5/34 (14.7%) patients (both tablet- and insulin dependent). 264 ± 18 MBq F-18-FDG (7.13 ± 0.49 mCi) was administered in 10 mL of 0.9% saline as a bolus, followed by flushing with the same amount and concentration of saline according to current guidelines. Scanning procedures were performed 101 ± 24 minutes (range 56–180 minutes) after tracer injection.The preadministration blood glucose levels were 5.6 ± 1.2 mmol L2.3Each patient was positioned in the PET/CT system  with arms beside the body and in supine position. Automatic voice announcements instructed the patient to breath in a regular and shallow fashion. A noncontrast low-dose CT scan for attenuation correction and anatomical reference was obtained . Whole-body fbPET scans were then performed, extending from the vertex to the feet. In total 12 to 14 bed positions were acquired .2.4A bhPET/CT including one bed position was performed 31 ± 7 minutes (range 17–60 minutes) after the fbPET/CT. In each case the localization of the breath-hold bed position was selected based on the clinical indication and query of the referring physician. In total 86 thoracic and 31 extrathoracic lesions were examined Table .First, a bhCT was performed in deep end-inspiration. CT parameters were the same as for the fbPET/CT scan. For the acquisition of bhPET, the patient was instructed to repeat the breathing exercise in the same way as long as possible. This phase was supervised by a radiographer and the scans were manually stopped when the patient resumed respiration.2.5Morphological sectional images used for method correlations were contrast-enhanced CT (ceCT) in 39/46 investigations (84.8%) and contrast-enhanced magnetic resonance imaging (ceMRI) in 1/46 investigations (2.2%). In 6/46 investigations (13%) ceCT could not be performed for the following reasons: renal insufficiency, allergy to contrast media, and lack of recent laboratory tests (TSH and/or glomerular filtration rate). For these patients only a low-dose CT (ldCT) was available.A ceCT was performed in parallel to the PET/CT in 32/39 investigations (82.0%), whereas in 7/39 investigations (18.0%) recent ceCT examinations were considered for comparisons. The mean time interval between ceCT and PET was 10.9 ± 27.6 days. Follow-up examinations were available for 21/34 patients (61.8%).2.6max and SUVmean) and metabolic isocontoured volumes (mVic40) were assessed in both investigations.Comparisons were performed by an experienced nuclear medicine specialist assisted by a doctoral candidate on a consensus basis. Data were presented on a dedicated multimodal evaluation console  with the aid of software for PET/CT analysis, and were visually assessed for increased tracer accumulation. First the attention was aimed at detecting focal findings seen at bhPET but not at fbPET. Then a targeted comparison was performed with the available CT and MRI in order to identify subtle morphological correlates. A 3-dimensional volume of interest (VOI) was drawn over the entire lesion of interest. Maximum and mean standardized uptake values /fbPET × 100.The percentage differences between bhPET and fbPET for SUV2.7max, SUVmean, and mVic40) were tested using 2-tailed and paired t tests. Associations were tested by linear regression and Spearman correlation (rho). The corresponding P values were additionally reported to avoid false correlations due to outliers. P values <0.05 were considered significant. Data were illustrated by Tukey box and whisker blots. Boxes represented the first, second, and third quartile, while the whiskers represented the lowest/highest value still within 1.5 interquartile ranges of the lower/upper quartile. Outliers were not shown.Differences between bhPET and fbPET measurements .33.1In the patient population analyzed in the present study, the bhPET identified 1 additional lesion not visible at fbPET but corresponding to a lesion identified at ceCT  and spine (15%). In 21% no primary could be identified (CUP) whereby diagnosis was confirmed by histological examination of metastases . The same was true for the SUVmean . In contrast, the mVIC40 was significantly smaller at bhPET than at fbPET . In terms of %bh-index, the SUVmax was increased by 40.4 ± 81.6% and the SUVmean by 35.4 ± 90.7%. The metabolic volume was reduced by 22.2 ± 38.8%. The impact of bhPET on the SUVmax was larger than the impact on the SUVmean . The tumors imaged with bhPET showed a significantly higher SUV3.4max  and SUVmean  than in larger lesions. The size of the lesions did not significantly affect the mVic40  . In smaller lesions, the bhPET had significantly larger effects on SUV3.5max , SUVmean , or mVic40  . The time interval had no significant influence on SUV4 In this study, the bhPET technique was evaluated exclusively in patients with histologically proved malignant melanoma. The examinations were performed for staging, restaging, and surveillance, therefore only metastatic tumors were taken into account.Breath-hold and free-breathing methods are available to limit the influence of respiratory motion during PET examinations. A bhPET would be preferable in terms of reduced time efforts and required equipment, but its feasibility largely depends on compliance and general health status of the patient. and colorectal carcinoma.Previous studies have not elaborated on the detection of additional lesions in bhPET, reports being limited to lymphangitis carcinomatosaSide-by-side interpretation of fbPET and bhPET studies was preferred instead of a blinded approach. The intention of bhPET in our setting was not to replace fbPET but to use it as a supplementary option to improve general diagnostic performance. Moreover, the side-by-side appraisal corresponds to the current use in clinical routine. In our patients 11% of the metastases were in the liver and 26% in the lungs  displayed a 40.4% higher SUVmax.In fbPET the respiratory motion in the 2-minute scanning time per bed position induces a blurring of focal lesions, which in turns leads to changes of SUVmax is certainly a simple and robust marker of the area with the highest uptake of tracer, but sometimes it corresponds only to 1 voxel. Instead, SUVmean reflects the tracer accumulation during the whole process, hence more closely reflecting the investigator's visual impression. The influence of bhPET on SUVmean was investigated in only 1 study, also revealing a significantly higher SUVmean compared to fbPET. Data on the metabolic volume are also scarce, for example at F-18-FDG bhPET this proved to be 20% smaller,,18 given that the spatial fixation during the breath-hold phase reduces the blurring and better delineates the lesion. The relative difference found in the present study (−22.2%) is therefore well compatible with the published data. Thus, the presented data on the use of bhPET in melanoma confirm the results obtained with other tumors, that is, the more commonly used fbPET underestimates the SUVmax and SUVmean and overestimates the mVic40.The SUVmax and SUVmean at bhPET, that is, in particular smaller lesions were more sharply delineated and easier to identify. This result confirms similar observations in lung carcinoma, that SUVmax differences between standard and bhPET are significantly more pronounced in small lesions.,19 There was no statistical significance of the lesion size regarding to mVIC40. The lack of impact on the mVic40 remains to be verified in other studies, as no comparable data are available in the literature.In this melanoma patient population, the size of the lesions had a significant influence on SUV,20A potential confounder in this study is the time interval between fbPET and bhPET (mean 31 minutes). As radiotracers have specific kinetics, the quantitative parameters can change over time. This confounder is mentioned in other F-18-FDG studies, but statistical analyses of the data have not been published. Investigations on when the plateau is achieved in a given tumor have provided different results. An in vitro F-18-FDG study with different subtypes of cell lines of malignant melanoma has shown that the SUVmax steeply increases within the first 60 minutes, and then progressively less until 120 minutes postradiotracer administration. A clinical study on breast cancer has shown a rapid increase of uptake until 90 minutes post-F-18-FDG injection, followed by a less rapid uptake until 180 minutes. A dynamic study with nonsmall-cell lung carcinoma (NSCLC) has shown that a plateau was not reached before 2.5 hours after F-18-FDG administration. The data of the present fbPET/bhPET study were acquired at a mean of 101 and 132 minutes, respectively, with a mean time difference of 31 minutes. This implies that the investigations took place in a phase of slower tracer accumulation. Although a certain influence of the FDG kinetic cannot be excluded, the results showed that the time interval between fbPET and bhPET did not significantly influence SUV and mVic40. This result is analogous to a study with PET acquisitions at 50 and 90 minutes (time difference of 40 minutes), showing that the later acquisitions had a significantly higher SUVmax, but the time difference between the acquisitions did not play a significant role.In general, the SUV is higher in malignant and granulomatous diseases compared to benign diseases, whereas over time a rapid uptake is typically followed by a plateau. Another phantom study postulated that the breath-hold should be greater than 90 seconds, but consisting of 8 intervals of 12 seconds each. Since breath-hold periods with the same respiratory depth are difficult to achieve without additional intervention, other authors opted for a single episode of sufficient breath-hold, lasting for 30 seconds for investigation of large tumors, or 20 seconds in a phantom study.The present study has some limitations. Routine PET scans are usually acquired with 2 to 3 minutes per bed position, but patients cannot hold their breath for the entire time, especially in view of their impaired health status. The shorter scan time for bhPET  leads to reduced signal statistics and higher background-noise-ratio. Some authors have attempted to estimate the optimal scanning time for bhPET, given that PET scanners of different companies vary regarding technical specifications. A phantom study with PET acquisitions stopping during simulated respiratory pauses, but with addition of short breath-hold phases, showed that acquisition times of 45, 60, and 120 seconds had a significantly higher diagnostic precision than a fbPET at 120 seconds, suggesting that a breath-hold of at least 45 seconds is necessary.–30The present study was based on a single bhPET. Therefore, it must be considered that the respiratory depth during the PET and the CT acquisition may have differed, leading to a mismatch between the 2 examinations and also to a suboptimal attenuation correction.A further drawback of this breath-hold study was that the scanning area was limited . The lungs, and possibly also enlarged livers, were not completely included. In addition, the upper and lower edges of the scanning area showed some imaging artifacts.Finally, the retrospective nature of the study implied that not all patients had ceCT or ceMRI available for morphological comparison. Some of the patients, in addition, did not undergo ceCT or ceMRI because of contraindications to the use of contrast media, and only 60% had follow-up investigations for further verification.5ic40 than fbPET. On bhPET, SUV significantly depended on lesion size, but not on the time difference between fbPET and bhPET acquisitions.In summary, the use of bhPET with F-18-FDG yielded higher SUV and lower mVThe better definition obtained with the bhPET technique may become a useful diagnostic option when fine quantitative evaluations for staging, recurrence, prognosis, and therapeutic response are needed, particularly for small lesions. bhPET enabled the identification of 1 additional liver metastasis, which was not clinical relevant for the further management of this particular patient with multiple metastases, but could have been decisive for a patient without known metastasis. In this respect, bhPET may be recommendable in selected cases. Future developments of more sensitive technologies with reduced acquisition time, or use of larger detectors with larger field-of-view, may enable a wider use of bhPET as a feasible alternative to the complex rgPET method.


Human T-lymphotropic virus (HTLV) infection has been under enhanced surveillance in England and Wales since 2002, however, little is known about testing patterns. Using data from two surveillance systems held at Public Health England, we described HTLV antibody testing patterns between 2008 and 2013 and the demographic and clinical characteristics of persons diagnosed with HTLV in England and Wales between 2004 and 2013. An increase in HTLV testing was observed in England between 2008 and 2013 . Most tests  occurred within secondary care, 0.5%  of persons were reactive for HTLV antibodies and 0.3%  were confirmed positive. Increasing age and female sex were predictors of a reactive HTLV screen and confirmed diagnosis. Testing in primary care including sexual health and antenatal services was infrequent. Between 2004 and 2013, 858 people were diagnosed with HTLV, most of whom were female , of black Caribbean ethnicity (60%), not born in the United Kingdom  and asymptomatic at diagnosis . Despite increased testing, the epidemiology and clinical features of those diagnosed with HTLV have remained consistent. Apart from donor screening, testing for HTLV infection remains uncommon, except to diagnose associated disease. The human T-lymphotropic viruses (HTLV), discovered in the early 1980s , have noStrongyloides stercoralis. There is no vaccine or effective treatment to reduce or eliminate HTLV viral load and treatments available for those who have malignant or inflammatory manifestations of HTLV infection are limited.Although the majority (ca 90%) of HTLV-1-infected individuals remain asymptomatic carriers, the other 10% will develop one or more of several diseases : 2–6% wiIn endemic areas, HTLV is predominantly acquired through mother-to-child transmission (MTCT), primarily through breastfeeding , and thrHTLV-1 testing was introduced in the UK in 1986 and routine testing of all blood donations was introduced in 2002 . SurveilTwo data sources were used for our analysis: (i) SSBBV, to look at HTLV antibody testing in 2013 and changes in testing between 2008 and 2013, and (ii) the enhanced surveillance of new diagnoses of HTLV to describe the epidemiological and clinical characteristics of persons diagnosed with HTLV between 2004 and 2013 .SSBBV collects information on tests for hepatitis A–E, HIV and HTLV, regardless of result, from 21 participating sentinel laboratories in England. Individuals are de-duplicated and linked to all their test results, using a combination of coded surname (soundex), first initial, date of birth, National Health Service (NHS) number and clinic number . AlongsiEnhanced surveillance of new diagnoses of HTLV in England and Wales has been conducted since 2002 by PHE ,16. In bDemographic and testing data for all individuals tested for HTLV antibodies in 2013 from 10 participating centres in England were extracted from SSBBV to give a snapshot of HTLV testing, as 2013 had the highest number of laboratories providing HTLV antibody tests for a full year. Where individuals had been tested more than once for HTLV antibodies, a reactive test was recorded over any negative or equivocal tests. Where all tests within 2013 were negative or equivocal, information from the most recent test was used.To investigate trends, testing data for the period 2008–13 from the five sentinel laboratories with complete testing data for the 6-year period was used. Demographic and testing data for all individuals tested for HTLV antibodies between January 2008 and December 2013 were extracted from SSBBV. A cohort approach was used, with each individual recorded only once in each year tested. Where individuals had been tested more than once for HTLV antibodies in a year, a reactive test was recorded over any negative or equivocal tests. Where all tests in a year were negative or equivocal, information from the most recent test was used. Individuals who were reactive for HTLV antibodies were excluded from appearing in subsequent testing year cohorts.In both datasets, tests were linked to information on the location of the service requesting the test. Age at test was calculated using date of birth. Individuals younger than 1 year were excluded from the analysis, as a reactive result at that age may reflect maternal HTLV antibody status. Individuals with a reactive HTLV antibody test were considered to have confirmed HTLV-1 or HTLV-2 infection if a Western blot or PCR performed at the VRD at PHE, which provides the confirmatory HTLV diagnostics, was positive. Requesting services were grouped into primary care , genitourinary medicine (GUM), accident and emergency (A and E), occupational health, prisons and drug dependency units) and secondary care services, along with an additional category called ‘other’, which included tests requested by blood and stem cell banks, research studies and where requester information was not available.Data for the years 2004 to 2013 were used for these analyses, based on reports received by end of October 2015. Numbers may rise as further reports are received, particularly for recent years.Unless otherwise stated, HTLV in this paper refers to both HTLV-1 and HTLV-2. Stata SE (Version 13.1) was used, with Wilcoxen rank-sum tests to compare continuous variables and chi-squared and Fisher’s exact tests for categorical variables. A multivariate logistic regression, adjusted for age, sex, service type and year of test, was used to look at predictors of a reactive HTLV antibody test and confirmed HTLV diagnoses in those tested between 2008 and 2013. This dataset was used as more people were tested between 2008 and 2013 compared with the 2013 only dataset.Joinpoint regression analysis was used to analyse changes in testing and positivity rates between 2008 and 2013, using Joinpoint Software (Version 4.3.1.0). Joinpoint identifies significant increases and decreases in testing and positivity rates over time, and whether there was a point at which a statistically significant change in the slope occurred during the period. The annual average percent change (APC) in testing numbers, with 95% confident intervals (95% CI), are reported. All proportions reported are excluding unknowns, and tests were considered statistically significant if the p value was lower than 0.05.During 2013, 10 laboratories participating in sentinel surveillance reported HTLV testing in England, with 11,213 HTLV antibody tests conducted on 9,302 persons. Median age at testing was 45 years (interquartile range (IQR): 31–58 years), and men were older than women  .The majority  were tested within secondary care. Overall, the most common settings were haematology services , specialist renal services  and general medical or surgical departments . Among primary care settings, the most common for HTLV testing was GP with 265 tests , followed by GUM services . In 2013, women’s services requested just 62 tests (0.7%), comprising 22 HTLV tests within antenatal care, 12 in gynaecological services, 18 in birthing and obstetric services and 10 in postnatal services. During 2013, there were 664,517 live births in England [Overall, 0.5% (n = 48) of persons tested were reactive for HTLV antibodies in 2013. HTLV antibody-reactive women were older than men , and the proportion of reactive tests was highest in persons aged 45–59 years. More than half (n = 27) of those reactive for HTLV were confirmed HTLV-positive by the VRD at PHE. The age and sex distribution of those with a confirmed HTLV-positive result was similar to those reactive to HTLV antibodies.Five sentinel laboratories consistently reported HTLV testing each year between 2008 and 2013, with 29,519 HTLV antibody tests performed on 27,206 persons . Overall, the number of persons tested increased by an average of 9.5% (95% CI: 7.4–11.7) annually between 2008 and 2011, and by 22.2% (95% CI: 17.5–27.2) annually between 2011 and 2013 .The number tested each year between 2008 and 2013 increased by 17.8% (95% CI: 12.3–23.5) in women and by 10.7% (95% CI: 6.8–14.7) in men . OverallIn the five sentinel laboratories, 0.6% of persons tested (n = 176) had a reactive HTLV antibody result between 2008 and 2013, with no significant difference in the proportion positive by year (p = 0.2). All other variables remained stable. The specialities that found the most number of persons reactive for HTLV were haematology (n = 53) and general medical and surgical wards (n = 20). Among those with an initially reactive HTLV antibody result, 50.6% (n = 89) were subsequently confirmed to have HTLV, a prevalence of 0.3%.Using a multiple logistic regression , the oddBetween 2004 and 2013, 858 new HTLV diagnoses were reported to PHE in England and Wales, averaging 86 cases per year (range: 76–98) .Where reported, more women were diagnosed with HTLV than men . Median age at diagnosis was 52 years (IQR: 42–66), with no difference between men and women . The majority of diagnoses were among persons of black Caribbean ethnicity , followed by white , black African , other/mixed , south Asian  and black other ethnicities . When compared with white individuals, persons of other/mixed and South Asian ethnicity were younger , whereas persons of black Caribbean ethnicity were older . Where country of birth was reported , the majority of HTLV-positive persons of black African (51/56), black Caribbean (231/284), South Asian (14/17) and other/mixed ethnicity (17/25) were born abroad. Overall, 5.0% of persons diagnosed with HTLV (n = 43) were co-infected with HIV.The majority of HTLV infections diagnosed, where type was reported, were HTLV-1  and only 4.9% (n = 40) of persons were infected with HTLV-2. Most persons diagnosed with HTLV-1 were of black Caribbean origin , whereas most persons with HTLV-2 were of white ethnicity (n = 18).Where reported , the most common reason for testing for HTLV was clinical symptoms , followed by blood donation . Persons diagnosed following blood donation were younger than those tested because of symptoms  and a higher proportion of women tested positive in the context of blood donation than men . There was no difference by sex in the proportion of persons presenting with symptoms . Persons of black ethnicity  and those diagnosed with HTLV-1  were most commonly diagnosed following the presentation of symptoms, whereas persons of white (46/98), other/mixed (13/23) and South Asian ethnicity (14/19) and those with HTLV-2 (10/19) were most commonly diagnosed when tested in the context of blood donation.Probable route of infection was reported for 46.7% (n = 401) of persons between 2004 and 2013. Among those, just over a quarter were probably infected through heterosexual sex , another quarter  via MTCT, and 38.2% (n = 153) reported both MTCT and heterosexual contact. MTCT and heterosexual contact were the most common routes of probable infection regardless of ethnicity or sex, but men were more likely to report other transmission routes than women  and almost all (9/10) persons with a probable infection following injecting drug use were of white ethnicity. A smaller proportion of persons infected with HTLV-2 reported a probable infection route of heterosexual contact and/or MTCT than those with HTLV-1 , with injecting drug use accounted for the remaining infection in HTLV-2 (n = 8).The majority of persons with symptoms reported were asymptomatic , 20.5% (n = 122) had ATLL, 15.0% (n = 89) had other or non-HTLV related symptoms, 11.9% (n = 71) had TSP/HAM and 7.7 (n = 46) had other malignancies/neurology .Persons who were asymptomatic at diagnosis were younger than persons with ATLL and TSP/HAM . Regardless of ethnicity, most persons were asymptomatic at diagnosis, but almost all persons with ATLL were of black ethnicity (90/94). Where reported, a higher proportion of persons with HTLV-2 were asymptomatic than with HTLV-1 .Of all persons diagnosed between 2004 and 2013 in England and Wales, 96 (11.2%) are known to have died from HTLV-related illness. Deaths most commonly occurred in women (n = 57), persons of black Caribbean ethnicity (n = 47), persons with HTLV-1 (n = 89) and persons with ATLL (n = 53). Median time between HTLV diagnosis and death was 156 days (IQR: 28.5–324.5).An estimated 22,000 persons are infected with HTLV in England and Wales, yet each year an average of only 86 HTLV diagnoses are made. To explore why there is this discrepancy, we investigated HTLV testing patterns from sentinel laboratories in England. The numbers of persons tested for HTLV increased between 2008 and 2013, and the majority of HTLV tests occurred in secondary care. The biggest proportional increases in testing were seen among women and in primary care services but from a very small baseline, and testing in these settings remains uncommon. Although more testing occurred in men, women are more likely to have a reactive HTLV test and confirmed HTLV diagnosis. This paper also updates the epidemiological picture of HTLV provided through enhanced surveillance since it was last described in 2002. The number of persons being diagnosed with HTLV remained constant over the 10-year period. The majority of persons diagnosed were female, of black Caribbean ethnicity and were born outside the UK. HTLV-1 remains the most common form of HTLV diagnosed in England and Wales and most patients were asymptomatic at diagnosis and infected through either heterosexual contact or MTCT. In contrast, the smaller fraction diagnosed with HTLV-2 were more likely to be white, asymptomatic and have a history of injecting drug use.Our results collected through the enhanced surveillance are consistent with previous descriptions of HTLV-diagnosed persons in England and Wales ,16, withThe vast majority of studies worldwide present data through blood donor testing, antenatal screening and enhanced surveillance, with limited information on general testing patterns. Sentinel surveillance of blood-borne virus testing presents a unique opportunity to gain an insight into where HTLV tests are being requested in England and reviewing changes in testing and prevalence estimates.Like HIV and hepatitis, HTLV has a long latency period, and a person can be infected for many years before receiving a diagnosis or presenting symptoms. Therefore, it is likely that the vast majority of infections in England and Wales, estimated at 22,000 persons, remain undiagnosed . The intOur testing data suggests potential missed opportunities for the prevention of maternal transmission of HTLV. While we observed some testing in antenatal, birthing and post-natal units, it is likely that much of this testing is being driven by the screening of tissue for storage and donation, rather than prevention of HTLV transmission, and primary care testing remains infrequent, implying that testing for HTLV infection is uncommon within sexual health services in England.Prevalence of HTLV in some groups, particularly older individuals and women, was high. Higher positivity rates in older persons are likely to be due to low disease awareness and the long latency period where a person will remain asymptomatic before developing symptoms and being tested. Higher positivity rates in women are probably due to a higher prevalence in women overall, which may result from differences in the efficiency of HTLV transmission between male and female sexual partners, with transmission rates higher from HTLV-positive male partners ,19. WorkThe high rate of confirmation of HTLV infection following a reactive result in the initial test supports the high specificity of the currently available assays. This is important as there is concern, particularly in low prevalence regions, that even minimal lack of specificity places a large burden on diagnostic laboratories to investigate many false-negative results. Our finding that more than half of reactive antibody tests were confirmed positive is thus reassuring.Although 72% of HTLV infections occurred in persons of black Caribbean or black African ethnicity, 94% of diagnoses of ATLL occurred within these ethnic groups. The Caribbean as well as western and southern Africa are endemic regions for HTLV infection and thus there is greater potential for mother-to-child HTLV transmission. HTLV-1 infection acquired perinatally or in infancy through breast-feeding has been associated with risk of ATLL, whereas HTLV-1 infection acquired in England and Wales is more likely to have been due to behaviour during adulthood. These data therefore support the assumption of ATLL risk and also draw attention to the need to consider HTLV-1 infection in any patient with a T-cell malignancy, but especially if they belong to a high-risk ethnic group.There are two key limitations in our study. The first is the small number of persons with a positive HTLV screen and confirmed HTLV diagnosis. Despite our testing data indicating an increase in testing, with similar proportions testing positive for HTLV, we have not seen the number of persons reported to the enhanced surveillance of HTLV increase over the same time period. In addition, the small number of people diagnosed with HTLV prevented us from looking at demographic and clinical trends. Secondly, we were unable to look at trends in ethnicity in HTLV testing through the SSBBV testing database. It would have been particularly interesting to see whether particular groups are being targeted for testing in line with demographics, with persons of black Caribbean ethnicity representing the majority of those diagnosed with HTLV in England and Wales. Despite these limitations, enhanced surveillance of HTLV has high case ascertainment of persons diagnosed with HTLV in England and Wales, as all confirmatory tests are directed to PHEs VRD, ensuring that our sample is representative.Since 2013, there have been significant changes to the surveillance of new diagnoses of HTLV, which has resulted in key clinical information being under-reported (< 30% completion rates), making comparison with more recent data problematic. Work is ongoing to improve the completeness of the dataset. However, we do not expect the characteristics of persons newly diagnosed with HTLV between 2014 and 2015 to differ significantly from those diagnosed between 2004 and 2013.Although characteristics of those with a confirmed diagnosis of HTLV reported to the enhanced surveillance in England and Wales do not appear to have changed over the past 10 years, HTLV testing has increased in England. This increase occurred predominately through increased awareness of HTLV, associated with symptom presentation. Although there have been increases in testing, testing frequency in primary care services, including sexual health services, is still suboptimal and continued work is needed to ensure that HTLV is considered among those at greater risk, tackling the undiagnosed fraction and reducing transmission.


Combinatorial external and internal triggering events enable hydrogel to control protein release by mimicking signal transduction of the cell in response to metabolism. A variety of hydrogels have been synthesized for controlling the release of signaling molecules in applications such as drug delivery and regenerative medicine. However, it remains challenging to synthesize hydrogels with the ability to control the release of signaling molecules sequentially or periodically under physiological conditions as living cells do in response to the variation of metabolism. The purpose of this work was to study a novel biomimetic hydrogel system with the ability of recapitulating the procedure of cellular signal transduction and controlling the sequential release of signaling molecules under physiological conditions. In the presence of a small chemical, the signaling molecule is regulated to change from a DNA-bound state to a free state and the freed signaling molecule is able to regulate intracellular signal transduction and cell migration. Moreover, periodic exposure of the hydrogel system to the small chemical leads to sequential protein release. Since signaling molecules are important for every activity of the cell, this hydrogel system holds potential as a metabolism-responsive platform for controlled release of signaling molecules and cell regulation in various applications. For instance, thyroid hormone and insulin needs to be delivered periodically for treatment of skeletal development or diabetes in response to the progress of tissue growth or the variation of glucose concentration;6 and vascular endothelial growth factor and platelet-derived growth factor BB (PDGF-BB) need to be delivered sequentially and periodically for treatment of cardiovascular ischemia.7 Thus, there is a great need to develop novel hydrogels that can mimick the releasing function of the cell.Hydrogels have been widely studied for controlled release of various cargoes due to their biocompatibility and functional similarities to human tissues.Cells respond to and release signaling molecules during the variation of metabolism through a series of stepwise signal transduction. Moreover, the cells do not sacrifice their integrity during the procedure of signal transduction or significantly leak signaling molecules under a nontriggering condition. Thus, when needed, the release of signaling molecules from the cells can be repeated over multiple cycles. The ability to mimic this mechanism observed in living organisms would lead to broad applications such as drug delivery, regenerative medicine, and molecular biosensing. The purpose of this work was to explore a hydrogel system that can recapitulate the procedure of cellular signal transduction to control the release of signaling molecules in response to a small chemical. In particular, we applied the principles of DNA–DNA and DNA–protein interactions to develop the biomimetic hydrogel for controlled protein release.via Watson–Crick base-pairing hybridization reactions;8 moreover, the duplexes can undergo dissociation via strand-displacement  reactions under physiological conditions.9 These hybridization and displacement reactions are fundamental molecular mechanisms observed in the biological systems and have been well studied from the view-points of thermodynamics and kinetics.10 The basic understandings of these reactions have allowed the use of DNAs as basic reactants to synthesize versatile materials such as molecular motors,11 DNA computers,12 catalysis machines13 and delivery systems.14 In addition to DNA–DNA interactions, basic studies have shown that DNA–protein interactions can be regulated by DNA. For instance, the states of transcription factors in binding to specific DNA sequences for transcription are regulated by nucleosomes in nuclei.15 Inspired by these biological mechanisms, we explored the hydrogel system to control the release of signaling molecules in response to a small chemical. The overall concept is shown in DNA strands form duplexes 16 The core compartment contained the AA–TS duplex; and the shell compartment contained the molecular complex made of AP and protein  and the signaling molecule binding to AP, respectively. Their aptamers were previously selected and well studied.18The hydrogel system was made of two compartments responsible for the displacement and hybridization reactions, respectively . The hyd protein . These tTo develop the functional core compartment, it was important to ensure that AA and TS could form a stable duplex and meanwhile this duplex can be displaced in the presence of adenosine. A truncated adenosine aptamer was used to design AA with three functional regions . The fir19 By measuring the variation of fluorescence intensity, we were able to determine whether AA was in the form of single strand or helical duplex. As shown in We first evaluated the stability of the AA–TS duplex. AA was purposely modified with an internal 2-aminopurine since the fluorescence intensity of 2-aminopurine in a base pair can be significantly quenched in comparison to an unpaired form .19 By meAfter the examination of the stability, we investigated the capability of adenosine in displacing AA from TS. The concentration of adenosine was fixed at 5 mM. A parabolic relationship between the number of base pairs and the enhancement of fluorescence intensity was observed  and S3†.We further studied the effect of the concentration of adenosine on TS displacement. versus the adenosine concentration was similar to that observed in the aqueous solution , the AA-conjugated hydrogel displayed strong fluorescence whereas the native hydrogel exhibited very weak fluorescence . It demosolution  and S4†.i.e., ACCTGGGGG) at the 3′ end  could only sequester ∼1%  into a protein signal output . The core–shell hydrogel was treated with a series of adenosine solutions. It showed that the PDGF-BB release increased with the concentration of adenosine  exhibited a strong calcium response. Moreover, the pattern of this calcium response  increases.22We further studied whether the output PDGF-BB can regulate signal transduction of smooth muscle cells (SMCs) at the levels of ions and proteins. Calcium ions serve as a second messenger in cells, playing important roles in cellular signaling.response  was consresponse . We furtresponse . It demo23 As cell migration is essential for the development and maintenance of tissues and organs,24 a cell migration study was carried out to illustrate not only the outcome of signal transduction at the cell level but also the potential biomedical application of this hydrogel system. The results showed that adenosine-triggered PDGF-BB signal output from the hydrogel induced SMC migration, demonstrating that periodic exposure of the hydrogel to adenosine led to periodic PDGF-BB output for regulatory cell migration  and their corresponding aptamers. Similarly, PDGF-BB and its aptamer can be replaced with other signaling proteins  and aptamers. Meanwhile, it is worthy of noting that the aptamer sequences after molecular design need to be carefully evaluated to ensure that they do not lose their ability of sequestering target proteins in the hydrogel. With rational design, this biomimetic hydrogel system would constitute a general platform of controlling the output of signaling proteins for versatile potential applications such as drug delivery, cell regulation, molecular sensing and regenerative medicine.In summary, we have demonstrated a biomimetic hydrogel system that has the ability of transforming a small chemical signal into a protein signal output There are no conflicts to declare.