Garcinia cambogia, is a natural citrate analog that can inhibit the ATP citrate lyase (ACLY) enzyme with a consequent reduction of inflammatory mediators , reactive oxygen species (ROS), and prostaglandin E2 (PGE2)) levels. Therefore, HCA has been proposed as a novel means to prevent, treat, and ameliorate conditions involving inflammation. However, HCA presents a low membrane permeability, and a large quantity is required to have a biological effect. To overcome this problem, HCA was formulated in liposomes in this work, and the enhancement of HCA cell availability along with the reduction in the amount required to downregulate NO, ROS, and PGE2 in macrophages were assessed. The liposomes were small in size (~60 nm), monodispersed, negatively charged (−50 mV), and stable on storage. The in vitro results showed that the liposomal encapsulation increased by approximately 4 times the intracellular accumulation of HCA in macrophages, and reduced by 10 times the amount of HCA required to abolish LPS-induced NO, ROS, and PGE2 increase. This suggests that liposomal HCA can be exploited to target the citrate pathway involved in inflammatory processes.Hydroxycitrate (HCA), a main organic acid component of the fruit rind of  SB-2049cambogia . Becausecambogia . In thisSalmonella enterica serotype typhimurium (LPS), Roswell Park Memorial Institute 1640 (RPMI 1640), and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich . Soy lecithin was purchased from Galeno .Hydroxycitrate (HCA), L-glutamine, penicillin/streptomycin solution, lipopolysaccharide from For the preparation of HCA-loaded liposomes (Lip-HCA), 120 mg/mL of soy lecithin and 2 mg/mL of HCA were weighed in a glass vial, dispersed in water, and sonicated  with a high-intensity ultrasonic disintegrator .n > 6) were diluted with water (1:100) and analyzed at 25 °C. For comparative purposes, empty liposomes  were also prepared and characterized. The stability of liposomes was evaluated by long-term stability tests, i.e., by analyzing vesicle average diameter, PI, and zeta potential over three months at 25 °C.The average diameter and polydispersity index  of the vesicles were determined by dynamic light scattering using a Zetasizer Nano ZS . Zeta potential was estimated using the Zetasizer Nano ZS by means of the Mixed Mode Measurement Phase Analysis Light Scattering (M3-PALS) technique, which measures the particle electrophoretic mobility. The samples (v/v) fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2 in a water-saturated atmosphere. Pro-monocytic U937 cells were differentiated to macrophages by 10 ng/mL phorbol 12-myristate 13-acetate (PMA). Then, U937/PMA cells were treated with 50 or 500 µM free hydroxycitric acid (f-HCA) or HCA-loaded liposomes (Lip-HCA) and, where indicated, stimulated with 100 ng/mL of lipopolysaccharide from Salmonella enterica serotype typhimurium (LPS).Human monoblastic leukemia U937 cell line (Interlab Cell Line Collection (ICLC) HTL94002) was grown in suspension in RPMI 1640 medium supplemented with 10% (4 cells/well) and treated with f-HCA and Lip-HCA diluted with RPMI medium to achieve the desired concentrations (50 or 500 µM). After 72 h, U937/PMA cells were collected in 1.5 mL microfuge tubes and counted.The effects of f-HCA and Lip-HCA on U937/PMA cell proliferation were determined using a Millipore Scepter™ handheld automated cell counter  72 h after incubation, according to the manufacturer’s instructions. Briefly, cells were cultured into 96-well plates .To evaluate reactive oxygen species (ROS) and nitric oxide (NO) levels, U937/PMA cells were triggered by 100 ng/mL of LPS in the presence or absence of f-HCA and Lip-HCA (50 or 500 µM). After 24 h, ROS and NO concentrations were measured by using 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate  and 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate , respectively. Briefly, cells were collected in tubes and counted. After centrifugation, the cell pellet was resuspended in PBS to have 102 (PGE2) detection, U937/PMA cells were treated with 50 or 500 μM f-HCA or Lip-HCA for 1 h before triggering inflammation with LPS. After 48 h, PGE2 levels were quantified in cell culture medium by using a DetectX® Prostaglandin E2 High Sensitivity Immunoassay Kit  according to the manufacturer’s protocol. Specifically, 100 μL of standards and samples were put into a clear microtiter plate coated with an antibody direct to mouse IgG. A PGE2–peroxidase conjugate was added. The binding reaction, initiated by the addition of a monoclonal antibody to PGE2, lasted overnight. The day after, the plate was washed and the substrate, which reacted with the bound PGE2–peroxidase conjugate, was added. After 20 min of incubation, the reaction was stopped and absorbance at 450 nm was measured using a GloMax plate reader (Promega). Values of each sample were normalized to protein content.For prostaglandin E5 cells/well, treated with 50 or 500 μM of HCA (f-HCA or Lip-HCA) for 24 h, and pelleted at 1200 rpm for 5 min. Supernatants were removed. To extract HCA, 500 μL of 40% MeOH with 0.1% (v/v) formic acid were added to the cells, which were lysed by sonication , mixed, and placed on ice for 15 min. Lysates were then centrifuged at 1500 rpm for 5 min. Supernatants were collected for LC–MS analysis. To ensure that all of the HCA was extracted from cells, another amount of 250 μL of 40% MeOH with 0.1% (v/v) formic acid was added to the cell pellets and suspended again. After 15 min, on ice, the suspensions were centrifuged at 14,000 rpm for 5 min, and all supernatants were pooled together [v/v) formic acid, and 10 μL aliquots were injected in a UPLC–ESI–Qtrap system. The protein concentration in each sample was determined by the Bradford assay [2O (Eluent A) and ammonium acetate 5 mM in MeOH (Eluent B) as mobile phase. The elution conditions were as follows: linear gradient from 2% to 6% of B in 3 min, followed by a faster gradient until 95% of B in 7 min. The flow rate was 0.2 mL/min, and the injection volume was 10 μL. The MS parameters were as follows: precursor ion 207 m/z, product ions 127 and 189 m/z, declustering potential −50 eV, entrance potential −9 eV, collision energy −12 eV, and collision cell exit potential −20 eV [To quantify the cellular uptake of HCA, U937/PMA cells were seeded into 6-well plates at a density of 2.5 × 10together ,17. The rd assay , and celrd assay . After ct-test. Results are shown as means ± SD of, at least, three independent experiments. Differences were considered as significant , very significant , and highly significant .Statistical significance of differences was determined using the Student’s p > 0.05; HCA-loaded liposomes were prepared by a simple method involving the sonication of the phospholipid (soy lecithin) and HCA in water. To evaluate the effect of the encapsulation of HCA on the vesicle arrangement, empty liposomes were also prepared and characterized. As shown by the results summarized in p > 0.05) of the parameters examined, which indicates a good stability of the vesicle formulations.The stability of the prepared liposomes was evaluated by monitoring the average diameter, PI, and zeta potential over three months of storage. The results showed no significant variations  by cell counting. To this end, U937/PMA cells were treated with 50 or 500 µM Lip-HCA or free-HCA (f-HCA), used as control, for 72 h. As illustrated in 2 production [2 secretion. f-HCA was used as control in U937/PMA cells triggered by LPS. As shown in 2 levels were reduced 24% more than with f-HCA (p < 0.01). Surprisingly, 50 µM Lip-HCA completely abolished the LPS effect, and PGE2 secretion was significantly lowered (about 30%) as compared to f-HCA . The conversion of L-arginine to NO and L-citrulline requires NADPH as substrate. Infantino et al.  reportedIn order to evaluate the HCA cell intake after cell incubation with the free compound or with its liposomal formulation, LC–MS analyses were performed. To obtain a good resolution and sensitivity for the HCA detection, different chromatographic conditions, in terms of eluent composition and pH, were tested. LC–MS analyses can provide information on the amount of HCA within cells upon different treatments. U937/PMA cells were incubated with f-HCA and Lip-HCA at two different concentrations (50 and 500 μM) for 24 h. The results confirmed that the use of the liposomal formulation clearly increased the amount of intracellular HCA, at both tested concentrations . The amo2 production in M1 macrophages [HCA has long been known for its anti-obesity activity by inhibiting ACLY, a key enzyme of fatty acid biosynthesis . More rerophages . TherefoNanodelivery systems, among which liposomes are considered one of the most successful, have been shown to boost bioavailability and efficacy of their cargo, as they facilitate the entry through biological barriers, while avoiding the metabolic modifications that can reduce absorption ,24. LipoRecently, HCA has been nanoencapsulated in solid lipid nanoparticles (SLNs). Ezhilarasi et al. reported that HCA-loaded SLNs exhibited a 2-fold higher bioavailability than non-encapsulated HCA and a 1.3-fold higher bioavailability than microparticles, which was ascribed to smaller particle size, longer residence time, and controlled release of SLNs . In that2 production was suppressed by liposomal HCA at a concentration 10 times lower than that of non-encapsulated HCA. Therefore, a small amount of liposomal HCA at the same time inhibits the production of multiple inflammatory mediators, making it very effective in blocking inflammatory responses. Moreover, liposomal HCA, by targeting immunometabolism through ACLY metabolic enzyme, could represent an innovative approach to many pathological conditions, since inflammation underlies countless diseases. Finally, as ACLY is also overexpressed in a wide variety of tumors, such as hepatocellular carcinoma [To the best of our knowledge, this is the first study that reports the nanoencapsulation of HCA in liposomes and the evaluation of the enhancement of its anti-inflammatory activity in macrophages thanks to the delivery by a vesicular carrier system. Our results point to the key role of the nanocarrier in the intracellular uptake process, as liposomes enhanced (by approximately 4 times) the accumulation of HCA within the cells, where the anti-inflammatory activity is exerted. Indeed, NO, ROS, and PGE2. This suggests the use of liposomal HCA to target inflammatory diseases and highlights the need for further research, such as targeted in vivo tests, to validate the in vitro antioxidant/anti-inflammatory potential of HCA. Indeed, the discovery that liposomes potentiate HCA biological activities allows for the development of novel and more effective approaches based on nanotechnologies to treat a variety of conditions that involve excessive inflammation.The results of the present work demonstrate that the prepared liposomes were remarkably effective in delivering the natural molecule HCA, as they promoted its entry in macrophages where the citrate pathway was inhibited, with a consequent suppression of the inflammatory mediators NO, ROS, and PGE


Human interaction recognition technology is a hot topic in the field of computer vision, and its application prospects are very extensive. At present, there are many difficulties in human interaction recognition such as the spatial complexity of human interaction, the differences in action characteristics at different time periods, and the complexity of interactive action features. The existence of these problems restricts the improvement of recognition accuracy. To investigate the differences in the action characteristics at different time periods, we propose an improved fusion time-phase feature of the Gaussian model to obtain video keyframes and remove the influence of a large amount of redundant information. Regarding the complexity of interactive action features, we propose a multi-feature fusion network algorithm based on parallel Inception and ResNet. This multi-feature fusion network not only reduces the network parameter quantity, but also improves the network performance; it alleviates the network degradation caused by the increase in network depth and obtains higher classification accuracy. For the spatial complexity of human interaction, we combined the whole video features with the individual video features, making full use of the feature information of the interactive video. A human interaction recognition algorithm based on whole–individual detection is proposed, where the whole video contains the global features of both sides of action, and the individual video contains the individual detail features of a single person. Making full use of the feature information of the whole video and individual videos is the main contribution of this paper to the field of human interaction recognition and the experimental results in the UT dataset (UT–interaction dataset) showed that the accuracy of this method was 91.7%. Human motion recognition is a key technology for intelligent video surveillance. It is widely used in various scenarios such as human–computer interaction , motion At present, there are many practical algorithms and networks that can be used for human motion video recognition research. Zhao et al.  believe In the field of human interaction recognition, there are many practical algorithms and networks that can be used in human interactive video recognition research. Nilar Phyo et al.  applied This paper focused on human interaction recognition in video. In the existing video research algorithms for human interaction motion, many methods recognize the two sides of the action as a whole and lose the characteristic information that the individual brings. There are also a few algorithms that separate the two sides of the action into two separate individuals for recognition, but they ignore the characteristic information that the whole brings. Therefore, this paper proposed a human interaction recognition framework based on whole–individual detection. This method contains the characteristics of the whole information and the characteristics of the individual information. The whole video includes both sides of the action, and can extract global features such as the relative position and orientation of the action, and the individual detection video contains a single person, where the individual detailed action feature information can be extracted.First, we propose an improved fusion time-phase feature of the Gaussian model to obtain video keyframes and remove the influence of a large amount of redundant information. This method resolves the problem of the differences in action characteristics at different time periods.Then, we propose a multi-feature fusion network algorithm based on parallel Inception and ResNet. This multi-feature fusion network not only reduces the network parameter quantity, but also improves the network performance; it alleviates the network degradation caused by the increase in network depth and obtains higher classification accuracy. This algorithm solves the problem of the complexity of human interaction feature extraction.We have noted that the whole video contains the global features of both sides of action, and that the individual video contains the individual detail features of a single person. Therefore, we combined the whole video features with the individual video features, making full use of the feature information of the interactive video, which will help us solve the problem of the spatial complexity of human interaction.Due to the spatial complexity of human interaction, we propose a two-person interaction recognition algorithm based on whole–individual detection. The video includes two individuals of the action. Information can be extracted such as the relative position and orientation of the interactive action, and the individual detailed action features can be collected from the individual action video. As shown in Due to the spatial complexity of interaction features, the motion characteristics of individual movements and the characteristics of interaction movements, we proposed an interactive motion recognition framework based on whole–individual detection. In the individual detection part of the interactive action video, the moving target is detected for the action video. The video detection is performed according to the detection result, and the algorithm block diagram used is shown in When the HOG feature descriptor and the pedestrian detection algorithm of the SVM classifier are applied to the interactive action video, loss detection and false detection phenomenon may take place. In this paper, a Kalman filter-based auxiliary tracking model was added to the pedestrian detection process to track each individual in the human interaction video. The trajectory and other information detected by consecutive frames provide reasonable prediction of the target’s next frame position. Human body loss detection is greatly reduced by using target tracking. As shown in μ is the expected value of the Gaussian distribution and σ is the standard deviation of the Gaussian distribution, which is related to the magnitude of the Gaussian distribution. It can be seen from the distribution curve that the closer to the expected value, the higher the probability density value. There is a general rule in human interaction video: the closer to the trigger stage of the target action, the higher the distinction between actions. We associated this rule with a Gaussian probability density function that is very similar. In the public dataset taken in this paper, each action video sample could be divided into the action start period, action execution period, and action end period. Based on the analysis, it can be seen that the action execution period had significant feature differences, which can help to classify the movements, and the action frame was concentrated in the center. Analogous to the Gaussian model, we proposed a video downsampling method based on Gaussian probability distribution. In the process of video downsampling, we adopted different sampling methods according to the different action stages of the video. For the video from the beginning to the end of the target action, we sampled at small intervals, and for the redundant video outside the target action, we sampled at large intervals. The selection of the sampling interval needs to be obtained through repeated experiments. The video features will also be more differentiated after Gaussian downsampling processing.When using video images for feature extraction and training, the choice of video sampling methods will directly affect the generalization ability of the classification model. If there is no video downsampling, adjacent video will generate a lot of redundant information, which will increase the burden on the network. Considering the difference in the phase characteristics of the action time, this paper proposed a Gaussian model downsampling method that combines time-phase features. The Gaussian function is also called a normal distribution, and the expression of the probability density function is as shown in Equation (1).In order to improve the accuracy of interactive action video recognition, a convolutional neural network based on parallel multi-feature fusion was proposed for the extraction of interactive feature information. The migration learning method is adopted in the feature extraction process . A blockThe Inception V3  network The Inception structure uses different receptive fields to fuse different scale features, and the obtained image feature information is more complete. At the same time, the series of networks eliminates the full connection layer in the network output layer. This method greatly reduces the parameters of the entire network and improves the efficiency of the training process. With the rapid development of deep learning technology, the Inception network is constantly improving and innovating. For example, Inception V2 network  introducWe continued to explore the feature extraction method based on residual neural network . As the In the residual network structure diagram of lx, ly represent the input and output values of the thl neuron; l)hf(y is the representation activation function. Under the condition of identity mapping: l) = xlhf.There is a residual unit that can be expressed in the form of Equations (2) and (3):lth layer, Equation (5) can be obtained.Similarly, when it is coming to the ε is as Equation (6).It can be seen that when the number of layers is deeper and deeper, the output value of the network is related to the output of the residual in the previous layer and the output of the Lth layer is the sum of the output values of the residuals of the previous layer. In back-propagation, calculation of the partial derivative of the loss function It can be seen from the equation that the process of gradient derivation avoids the possibility that the value in the multiply state is 0, thus avoids the trouble caused by the disappearance of the gradient. After the network is improved in this way, even if the network is deeper, the results of network training will not be too deviated. This improved method avoids the problems of gradient disappearance and gradient explosion, which are caused by the increase in the number of network layers, which leads to maintaining a good network performance.Taking the interactive video in the UT dataset  as an exThe proposed network is a multi-feature fusion convolutional neural network based on Inception and ResNet. At the feature extraction level, feature extraction is performed using the Inception V3 network and ResNet, respectively. Then, the two extracted features are fused at the fully connected layer. Then, training is continued. After multi-feature network convergence, the image features will be more abundant, which is conducive to improve the accuracy of human interaction video classification and recognition. According to the name of the video, this paper fused two feature files generated by each video file to form a feature file with a size of 4096 × 40 for later training and classification.The decision-level fusion uses different features to obtain the classification results, and then the experimental results are merged. In the classification recognition phase, the whole video, individual video (left), and individual video (right) all produce a preliminary classification result based on probability. In order to make better use of the feature information of video images and improve the action recognition accuracy of interactive video, from the perspective of probability fusion, this paper fused the preliminary classification results at the decision level to obtain the final classification results. As shown in Equation (7), probabilistic weighting is used to fuse the three classification results of each set of action videos to obtain the final classification result:FinalR is the final recognition result, wholeR is the whole video classification result of the double, LeftR represents the classification result after training using the individual video (left), and RightR represents the classification result obtained by using the individual video (right) for model training. wholeP, LeftP, RightP are the weighted probability of the whole video classification result of the two person, the weighted probability of the individual video (left) classification result, and the weighted probability of the individual video (right) classification result, respectively. The weighted probability value was obtained by comparing and analyzing the repeated experiments. A human body interactive video recognition block diagram based on decision level fusion is shown in Among them, This paper conducted the experiments on a computer with a NVIDIA GTX 1080Ti graphics card. The experiment used Tensorflow for the deep learning network framework, Python for programming, MATLAB for other data analysis and preprocessing stages; the software used were Pycharm2018 and MATLAB2016.This paper selected the UT interaction dataset. The UT dataset is a common human interaction video dataset. In order to ensure the effectiveness of the algorithm, the original data are generally divided into a training set and a verification set. This paper used K-fold cross validation (K-CV). This method divides the original data into K groups on average  Each subset of data is used as a verification set, and the remaining K-1 subsets of data are used as training sets. In this way, K models are obtained. Finally, we used the average number of classification accuracy of the final validation set of these K models as the final result. This method is the most widely used as K-CV can effectively avoid the occurrence of over-fitting and under-fitting and the final result has high reliability. The UT-Interaction dataset contains videos of continuous executions of six classes of human–human interactions: shake-hands, point, hug, push, kick, and punch. Ground truth labels for these interactions are provided including time intervals and bounding boxes. There is a total of 20 video sequences whose lengths are around one minute. Each video contains at least one execution per interaction, providing us with eight executions of human activities per video on average. Several participants with more than 15 different clothing conditions appear in the videos. The videos are taken with the resolution of 720 × 480, 30 fps, and the height of a person in the video is about 200 pixels. We divided videos into two sets. Set 1 was composed of 10 video sequences taken on a parking lot. The videos of set 1 were taken with slightly different zoom rate, and their backgrounds were mostly static with little camera jitter. Set 2  were taken on a lawn on a windy day where the background is moving slightly , and they contain more camera jitters. From sequences 1 to 4 and from 11 to 13, only two interacting people appear in the scene. From sequences 5 to 8 and from 14 to 17, both interacting people and pedestrians are present in the scene. In sets 9, 10, 18, 19, and 20, several pairs of interacting people execute the activities simultaneously. Each set has a different background, scale, and illumination. As shown in In addition, in order to verify the applicability of this algorithm, we selected the interactive action video in UCF101  for exte−5, and decay = 10−6. The loss function uses categorical cross entropy. During training, batch size = 32. During the training process, the image in the action video was adjusted to an image of size of 299 × 299. In the feature extraction stage, the image was cropped to a uniform size of 224 × 224. During the experiment, each group of action video clips was 40 frames, so the output size of the network was 2048 × 40.More experimental parameters are as follows. We chose the Adam optimizer in the experiment, the learning rate = 10In order to verify the improved effect of video acquisition based on Gaussian downsampling, we took the interactive video of UT dataset 1 as an example. We adopted equal interval downsampling video processing and Gaussian model based downsampling video acquisition to perform UT whole video preprocessing. In this paper, we selected the Inception network and ResNet for feature extraction and classification recognition processing, and the experimental results are shown in It can be seen from In order to verify the human–interaction recognition algorithm based on the whole–individual detection proposed in this paper, a comparative experiment was carried out on the UT dataset. The UT interactive dataset generates individual video (left) and individual video (right) after the previous interactive video detection process. In this paper, UT individual video (left), UT individual video (right), and whole video were used for the experiments. In order to obtain reliable experimental classification and comparison, the experimental process was based on an Inception and ResNet parallel multi-feature fusion network algorithm. In the video downsampling process stage, we used a Gaussian model with improved fusion time phase features.The experimental results of the UT dataset 1 were analyzed. Among them, the preliminary classification results of the individual actions obtained by the individual (left) video are shown in i, yi), yi is the true value and f(xi) is our predicted value. Use the loss function L(f(xi), yi) to represent the gap between the true value and the predicted value. At the same time, it can be seen from The results of our experiments on the UT human interaction dataset are shown in After the deciding level fusion, the whole accuracy of the video classification results is shown in The comparison experiment results are shown in The experimental results of this paper were compared with the classification results of other experimental methods on the UT dataset in recent years, and the experimental results obtained are shown in In this paper, the human-interaction recognition algorithm based on whole–individual detection was proposed. The experimental verification and analysis work was carried out with the human interaction UT dataset. In the stage of feature information extraction of human interaction, we proposed human-interaction recognition in a parallel multi-feature fusion network. Compared with a single feature information extraction network, the fusion network improved the whole recognition accuracy of interactions. Regarding the complexity of interactive action features, we proposed an improved human body interaction recognition method based on the whole–individual detection. For the differences in action characteristics at different time periods, a Gaussian-based video downsampling method was proposed. This method makes the data acquisition more consistent with the characteristics of each action time. The results show that the whole–individual detection human interaction recognition method based on decision-level fusion proposed in this paper can improve the accuracy of classification recognition. In this paper, the complexity of the algorithm was relatively high. Subsequent work will further improve the complexity of the algorithm to improve the efficiency of recognition.


In this paper, low-density parity-check (LDPC)-coded carrierless amplitude and phase (CAP) modulation with spatial diversity is proposed to mitigate turbulence-induced fading in an underwater visible-light communication (UVLC) channel. Generalized-gamma (GG) distribution was used to model the fading, as this model is valid for weak- and strong-turbulence regimes. On the basis of the characteristic function (CHF) of GG random variables, we derived an approximated bit-error rate (BER) for the CAP modulation scheme with spatial diversity and equal-gain combining (EGC). Furthermore, we simulated the performance of the CAP system with diversity and LDPC for various turbulence conditions and validated the analysis. Obtained results showed that the combination of LDPC and spatial diversity is effective in mitigating turbulence-induced fading, especially when turbulence strength is strong. Recentlribution .To mitigate the effects of turbulence-induced fading, several techniques, such as forward-error correction (FEC) , diversiThis analysis suggests that combining efficient modulation with effective FEC and diversity offers optimal performance for UVLC systems. Hence, in this study, we propose an LDPC-coded CAP modulation scheme with spatial diversity to improve the performance of UVLC systems over a UOT-induced fading channel. In this study, the LDPC outlined in IEEE.802.16 standard, whose fundamental codes can accommodate various code rates and packet sizes , was adoThe remainder of the paper is organised as follows. In N receivers with the same aperture size. The pseudorandom binary streams were parsed into groups of k-bits and encoded using LDPC code, which resulted in corresponding groups of codewords with n-bits, where k and n were the information length and code length of LDPC, respectively. The n-bits in each codeword were then mapped into an M-QAM signal using a Gray mapping rule. The QAM signal was upsampled by a factor of sN followed by separation of inphase (I) and quadrature-phase (Q) sequences, and application of two orthogonal pulse-shaping filters. The impulse responses of the orthogonal filters were given by the multiplication of cosine and sine with a root-raised cosine filter (RRCF) for I and Q signals, respectively, i.e., the impulse responses of pulse shaping filters were given by [cf is the carrier frequency, and T is the symbol duration, and β is the roll-off factor of RRCF.given by ,22.(1)fM-QAM symbols.The filter outputs were then summed, i.e.,The signal was then converted to an analogue signal by a digital-to-analogue converter (DAC). Since the IM/DD requires a positive signal, DC bias given by (5)IDC=gη denotes photodetector responsivity, iα, ith branch, respectively. We assumed that path loss σ2. Thus, the electrical signal-to-noise ratio (SNR) of EGC was given byThe modulated beam was expanded and collimated to ease alignment and sufficiently illuminate all receiver apertures. After going through the turbulence channel (details of turbulence channel are given in the following section), the beam was received by spatial-diversity receivers. We assumed that the spatial-correlation width was larger than the aperture diameter of each receiver, and the receiver separations were sufficiently larger than coherence length, whose typical value is on the order of millimeters ,25. Thusymbol is (8)γEGCeAt the receiver, using two matched filters that were the time-reversed versions of transmitter filters, In this paper, GG statistical distribution was adopted to model the UOT, i.e., the fading coefficient given by (10)fα(α10/1) in . Scintillation index fined as 11)δI2=δI2=δI2 iThus, the scintillation index was given byTo ensure that no energy loss or gain occurs during the turbulence-induced fading process, the fading coefficient was normalised, i.e., Optical-intensity fluctuation due to UOT was characterised by multiplicative fading coefficient α .c = , where s represents systematic bits, and p1 and p2 are parity-check bits. Detailed techniques for constructing and encoding LDPC can be found in [M-QAM, followed by M-CAP modulation and transmission through the UVLC channel as described in the section above. At the receiver end, the electrical signal given in Equation (6) was soft-demapped to provide initial bit likelihoods for belief-propagation (BP) algorithm-based LDPC decoder. When the modulated symbols were equiprobable, the log-likelihood ratio (LLR) for the ith bit x(t), which is given by: In this study, we employed a quasicyclic LDPC (QC-LDPC) code based on the IEEE 802.16 standard. Compared to the randomly structured codes, QC-LDPC codes are more advantageous due to lower encoding and decoding complexity, less storage space, more flexibility, and simpler implementation . The outfound in ,29; hencented as (16)L [LLRs obtained by soft-demapping hm (MSA)  based onM-QAM is given by [CAP and QAM have the same BER in theory. An approximated conditional bit-error probability (BEP) of given by (18)p was complicated, as it was very difficult to replace the multidimensional integral resulting from posed in  to deriv1F1M-QAM are given in Using the alternative form of (4.2) in , and acc(4.2) in , the appgiven by (21)Re{φ10/1) in , and parThen, when we derive  is Fox’s H function. Similarly, the imaginary part of Assuming the receiver separations are higher than underwater coherence distance, the fading coefficients are independent; hence, the CHF of Equation (22) is given asailed in . Using Eailed in , the defailed in , we obtaM-QAM with EGC diversity over GG fading channels. The BER of CAP was efficiently calculated on the basis of MATHEMATICA implementation for Fox’s H function presented in [Substituting Equations (21), (24) and (25) in Equation (20), we obtained a closed-form expression in the form of Fox’s H function for the approximated BER of ented in .In this section, the BER performance of the proposed LDPC-coded CAP with spatial-diversity UVLC system was evaluated using a Monte Carlo simulation under different turbulence conditions and diversity orders. Meanwhile, the analytical BER of uncoded CAP with spatial-diversity UVLC system (given by Equation (20)) was verified by comparing analytical and simulation results. The simulation parameters for this study are given in First, the theoretical and simulated PDFs of the GG distribution are presented in N = 1) are also presented. N = 2 needed an additional SNR of 6.5 dB to obtain a BER of 10−3 compared to 4-CAP. Additionally, to achieve a BER of 10−4 at the SNR of ~25 dB, diversity orders were 2 and 4 for 4-CAP and 16-CAP, respectively. Thus, a trade-off between spectral efficiency and reliability should be carefully considered in practical implementation. To obtain further performance improvement, we studied the effect of LDPC to mitigate fading on the basis of MC simulations. The BER performance of LDPC-coded 4- and 16-CAP without diversity in a GG turbulence UVLC channel is shown in N = 2 had better performance than that of the spatial-diversity-only scheme with N = 4, resulting in BER waterfalls at SNRs of ~15 and ~19 dB for 4- and 16-CAP, respectively. In strong turbulence with N = 4 could obtain high coding gain and achieve a BER of 10−4 at SNRs of ~19.5 and ~27.5 dB for 4- and 16-CAP, respectively, while even at the SNR of 30 dB, the BERs of the coded-only systems were larger than 10−2 (shown in N = 4 were larger than 9 −4. So, by comparing shown in , and the−1 at the SNR of 30 dB, while the LDPC-coded CAP with a spatial-diversity system with N = 4 achieved a BER of 10−4 around SNRs of 19.5 and 27.5 dB for 4- and 16-CAP, respectively. Results demonstrated that the effect of turbulence on BER performance could be effectively mitigated by spatial diversity and LDPC codes.In this paper, we studied the performance of LDPC-coded CAP with spatial diversity for UVLC. Expressions for approximated BERs were derived for systems without FEC code. Furthermore, Monte Carlo simulation was used to verify the analytical study of the uncoded system. We showed that the diversity scheme was effective in mitigating the effect of turbulence, and further studied the performance of the LDPC-coded scheme with and without diversity over turbulent channels. Obtained results showed that the LDPC-coded scheme without diversity was not enough to combat strong turbulence, while spatial diversity with LDPC codes offered the best performance. In turbulence with


Malnutrition is a common and significant public health problem, especially for older adults, as the consequences are costly. National guidelines (NICE CG32/QS24) highlight the need to identify and manage malnutrition, the implementation of which was deemed “high impact to produce cost savings”. The ‘Malnutrition Pathway’, endorsed by NICE and other professional bodies, is a practical evidence-based guide to help community healthcare professionals (HCP) to implement guidance on malnutrition management. Published evaluations of its use are needed.This service evaluation in older adults assessed the impact of implementing the ‘Malnutrition Pathway’ on health care use and costs, as well as the acceptability of the management strategies and effect on malnutrition risk.5 GP surgeries in Gloucestershire.163 older adults (80±9 years) with a range of primary diagnoses, living in their own home, were screened using the Malnutrition Universal Screening Tool (‘MUST’) (n50 low risk (LR); n41 medium risk (MR); n72 high risk (HR)). All patients were managed according to risk ; and HR: DA plus two oral nutritional supplements (ONS) .At each review , ‘MUST’ score, compliance and satisfaction to their management plan were recorded. Healthcare use was collected from GP records 6 months before and after implementation of the pathway. A simple cost analysis was completed.Implementing appropriate management of malnutrition led to significant reductions in hospital admissions (p=0.028), length of hospital stay (p=0.05), GP visits (p=0.007) and antibiotic prescriptions (p=0.05). Over 6 months, the costs to manage malnutrition  were more than offset by the savings associated with these reductions in health care use . The proportion of individuals at risk of malnutrition reduced over time, and patients reported being satisfied with the DA (97%) and ONS (96%), consuming 90% of their ONS prescription.Managing malnutrition significantly reduces healthcare use, with a positive budget impact, in older malnourished patients in primary care. This represents an opportunity to improve patient care with benefit on health care spend. Malnutrition is a common and costly problem, with health and social care costs estimated to be around £23.5 billion per year in the UK , around http://www.bapen.org.uk/screening-and-must/must-calculator, which requires measures of height, weight and BMI, and managed according to national guidance .Malnutrition should be identified with a screening tool like the ‘Malnutrition Universal Screening Tool’ (‘MUST’) guidance , 6. Imprguidance . To suppguidance . It was Although there is evidence to support the use of nutrition support strategies including DA and ONS –10 thereThe primary aim of this local service evaluation was to assess the economic impact of implementing the Malnutrition Pathway in older adults (≥65 years) in General Practice, as well as assessing its acceptability and effect on overall malnutrition risk.163 older adults (≥65years), from 5 GP surgeries in Gloucestershire took part in this dietetic led service evaluation and were managed for malnutrition risk according to the Malnutrition Pathway  data was collected at the initial appointment, at 6 weeks (±2 days), 3 months (±2 days), and 6 months (±2 days). Age, gender, primary diagnosis and height were recorded at the initial appointment only. At each review ‘MUST’ score was documented, along with details of compliance and satisfaction with their dietary management and ONS. All pathway group patients were asked the same series of questions regarding the dietary advice they received; were they following it, have they made changes to their diet (both yes/no questions), how easy was it to follow and how satisfied they were with the DA . The HR patients were asked further questions regarding ONS; were they taking their ONS, how easy was it to take, and how satisfied they were with the ONS. For all telephone appointments measures were self-reported, for those attending clinic weight and height were measured to the nearest 0.1kg (Marsden (MS-4202L) calibrated scales) and 1cm (Leicester Height Measure) respectively. If at any of the review points, a patient was no longer at risk of malnutrition they were discharged back into usual care and were not reviewed further.Following the 6 month review a retrospective audit of the patient’s healthcare use , number of GP appointments, total number health care professional visits  and number and type of antibiotic prescriptions) was collected by the dietitian onto standardised forms from the information available in the electronic patient record in each GP surgery. Healthcare use was captured for the 6-month period prior to enrolment onto the Malnutrition Pathway and for the 6-months following its implementation.A simple cost analysis was undertaken on a per patient basis comparing cost of healthcare resource used 6 months before and after implementation of the Malnutrition Pathway. Individual costs Table  includedData was collated and analysed using IBM SPSS Statistics package version 23 . Data was presented as mean values ± standard deviation (SD) unless stated otherwise. A one-way ANOVA was used to compare healthcare use ) from 6 months before and 6 months after implementation of the pathway. Paired t-tests were used to make comparisons of the same outcomes at 2 time points, within the different malnutrition risk groups. As this is a small service evaluation power calculations were not undertaken to determine sample size. A pragmatic approach was taken to recruit as many suitable individuals within the dietetic resource available.2) had a wide range of primary diagnoses including respiratory ); cardiovascular (CVS) (16%); musculoskeletal (14%); endocrine (11%); genitourinary (GU) (10%), gastrointestinal (GI) (7%), neurological (non CVA) (5%), and haematology (2%). Of the total group (n163), 113 (MR+HR) followed the Malnutrition Pathway (pathway group). All 72 patients in the HR group were prescribed ready-made liquid ONS for an average of 14.2±8.6 weeks and received DA. All 41 patients in the MR group were provided with DA and no nutrition support actions were taken with the patients in the LR group (n50).163 patients Table  , and those at HR only, but not in the MR patients Table . In thosThe simple cost analysis, using standard costs Table  was undeOver the period of the evaluation there was an overall improvement in malnutrition risk. Using available risk data at 6 months, of those that were all initially MR, 66.6% had been previously discharged from dietetic care at an earlier visit due to being LR, 13.3% were now LR, 16.6% remained MR, 2.4% had become HR. Similarly, of those that were initially HR, 27.5% had been previously discharged from dietetic care at an earlier visit due to being LR, 7.8% were now LR, 17.6% were now MR, and 47% remained HR.The majority (81%) of all patients on the pathway found the dietary advice they were given easy or very easy to follow and 97% of patients were satisfied or very satisfied with the dietary advice. 92% of the patients also given ONS (the HR group) found that the ONS were easy or very easy to take and 96% were satisfied or very satisfied with the ONS. Patients reported taking a mean of 90% of the ONS they had been prescribed Table .Table 4This service evaluation demonstrates practically the positive impact of following the Malnutrition Pathway for patients at risk of malnutrition in primary care, over a short period of time. It highlights the importance of screening and appropriate management which includes using oral nutrition support and reviewing patients against goals. It highlights the greatest impact for both the patient and healthcare economy is in the management of those at HR, who received DA and high protein ready to drink ONS. Overall patients at HR achieved the greatest reductions in hospital admissions, length of stay, GP contacts, and antibiotic prescriptions, all of which were significantly reduced. Alongside the economic benefits, malnutrition risk was reduced and patients reported being highly satisfied with the interventions (DA and ONS), and had excellent compliance to ONS.Overall the costs associated with managing malnutrition , were more than offset by the savings associated with these reductions in health care use with overall cost savings (per patient over 6 months) estimated between −£278.91 and −£395.64 in the pathway group (MR+HR), and between −£595.95 and −£997.02 for those at HR. When considering a simple budget impact model, extrapolating these savings to a representative population of 100,000 people who are older, live in the community and are at risk of malnutrition, could mean potential cost savings of between +£344,733 and + £641,084 (Table The reason for the greatest benefit in the HR group may be linked to them receiving a combined approach to nutrition support. These patients likely had a poor appetite and dietary intake due to their disease, making improving intake of energy, protein and micronutrients key. Indeed the majority of patients received DA and ONS high in protein, based on clinical benefits and the increased protein needs of the patients , 14 howeMalnutrition screening and management in primary care is a significant clinical issue as recently highlighted –17. It iTo our knowledge this is the first service evaluation to assess in practice the effect of managing disease related malnutrition in older people registered with their GP. It not only provides local data of the potential benefit but also provides evidence to demonstrate that the theoretical high impact cost savings calculated by NICE are achievable in the real world.It should be considered that there are also limitations; it is based on a relatively small number of individuals residing in one local area and has not been undertaken in a randomised manner or with a control group as this is an evaluation of service, real world evidence. The evaluation was undertaken by a registered dietitian, while not a limitation, there are currently insufficient dietitians to support all malnourished community patients, so it maybe that the management of malnutrition in the community be undertaken by other healthcare professionals following the Malnutrition Pathway under the guidance of dietitians. Despite these points the results seen are similar those obtained previously from other service evaluations and randomised trials that produce cost savings , 24, 25.The consequences of untreated malnutrition are costly, implementing the Malnutrition Pathway into primary care including identification and appropriate management is quite simple, has high impact for savings in the short term and follows NICE guidance. With ever-growing cost pressures, an approach to proactively identify and manage malnutrition may be an ideal area to focus especially as it links with so many NHS priorities.


The lateral orbitofrontal cortex (LOFC) is thought to encode information associated with consumption of rewarding substances and is essential for flexible decision-making. Indeed, firing patterns of LOFC neurons are modulated following changes in reward value associated with an action outcome relationship. Damage to the LOFC impairs behavioral flexibility in humans and is associated with suboptimal performance in reward devaluation protocols in rodents. As chronic intermittent ethanol (CIE) exposure also impairs OFC-dependent behaviors, we hypothesized that CIE exposure would alter LOFC neuronal activity during alcohol drinking, especially under conditions when the reward value of ethanol was modulated by aversive or appetitive tastants. To test this hypothesis, we monitored LOFC activity using GCaMP6f fiber photometry in mice receiving acute injections of ethanol and in those trained in operant ethanol self-administration. In naive mice, an acute injection of ethanol caused a dose-dependent decrease in the frequency but not amplitude of GCaMP6f transients. In operant studies, mice were trained on a fixed ratio one schedule of reinforcement and were then separated into CIE or Air groups. Following four cycles of CIE exposure, GCaMP6f activity was recorded during self-administration of alcohol, alcohol+quinine (aversive), or alcohol+sucrose (appetitive) solutions. LOFC neurons showed discrete patterns of activity surrounding lever presses and surrounding drinking bouts. Responding for and consumption of ethanol was greatly enhanced by CIE exposure, was aversion resistant, and was associated with signs of LOFC hyperexcitability. CIE-exposed mice also showed altered patterns of LOFC activity that varied with the ethanol solution consumed. These studies demonstrate that, in intact mice, lateral orbitofrontal cortex (LOFC) neurons are acutely inhibited by alcohol and become hyperexcitable following chronic intermittent ethanol (CIE) exposure. Furthermore, we report that unique patterns of LOFC neuronal activity occur during alcohol seeking and consumption. Interestingly, these patterns of activity are modulated following CIE exposure, particularly when the rewarding properties of the alcohol solution are modulated through adulterations with quinine (aversive) or sucrose (appetitive). Conversely, control animals have considerably more stable patterns of LOFC activity following exposure to air. These unique effects of CIE exposure on LOFC activity likely contribute to the development of excessive alcohol consumption and behavioral inflexibility that are associated with the onset of alcohol dependence.Alcoholism is a chronically relapsing disorder that is characterized by excessive alcohol consumption and a lack of behavioral flexibility surrounding alcohol consumption. Indeed, addicted individuals will continue to consume excessive amounts of alcohol despite the development of a variety of negative social, legal, and health related consequences. Furthermore, this behavioral inflexibility presents a significant barrier to the implementation of efficacious treatment strategies. Accordingly, it is important to obtain a deeper understanding of the brain systems that underly this behavior and to learn how chronic exposure to alcohol impairs neurons in these regions.Behavioral flexibility associated with alcohol drinking can be examined experimentally in rodent models using reward devaluation protocols where stimulus-reward relationships are unexpectedly manipulated . Previouin vivo electrophysiology have shown that LOFC neurons fire in response to reward predictive cues, signaling the predictive value of the reward and also following reward consumption providing information about the actual value of the reward , a subregion of the prefrontal cortex, plays important roles in associative reinforcement learning and modifying behavioral responses when action outcome relationships are changed or devalued . Accordie reward . Additioe reward .Using slice preparations, we have previously shown that the intrinsic excitability of LOFC neurons is reduced by low concentrations of ethanol  and thatDespite these findings, little is known regarding how chronic alcohol exposure impairs the way that neurons in the LOFC modulate their activity when there is a change in the stimulus-outcome relationship during alcohol self-administration. The studies in this manuscript address this issue and use fiber photometry and a mouse model of alcohol dependence to assess LOFC neuronal activity during operant self-administration of ethanol in the absence and presence of aversive or rewarding tastants.Male C57BL/6J mice at four weeks of age were obtained from Jackson labs and allowed one week to acclimate to the Medical University of South Carolina (MUSC) housing facility before surgery and operant ethanol self-administration. Lights in the housing facility were set on a reverse light dark schedule with lights off at 9 A.M. and on at 9 P.M. All drinking and fiber photometry studies were conducted during the dark cycle. All procedures were approved by the MUSC Institutional Animal Care and Use Committee and are consistent with NIH guidelines concerning the use of animals in research. We chose to focus these initial studies on male mice as they show robust and reproducible increases in alcohol drinking and self-administration following exposure to repeated cycles of CIE. In contrast, there is significant variability in CIE-induced drinking in female mice with many studies including our own  showing n = 3 Air, n = 2 CIE).Mice were deeply anesthetized with isoflurane vapor  and 300 nl of AAV1-CaMKII-GCaMP6f (Addgene) was injected into the LOFC . A custom-made optical fiber and ferrule  were implanted at these coordinates and secured in place using Herculite. Mice recovered in their home cage for 7 d before beginning lever-press training. All mice were inspected postmortem to ensure proper viral expression and implant location. Any mice with inaccurate placements or lacking viral expression were removed from the study. Mice with damaged fiber optic implants but accurate expression were excluded from photometry analysis because of poor signal quality but were included in drinking microstructure analysis  and visual (house light off) cues for 1.5 s while 20 μl of 10% ethanol (v/v) solution were delivered to a drinking well using calibrated Med Associates pumps. The active lever had a 1.5-s time out during the presentation of the cue and reward delivery. A second inactive lever was included in the box that when pressed produced no cues or delivery of the ethanol solution. Drinking at the alcohol well was monitored using Med Associates Lickomoters and licking microstructure was analyzed by grouping bursts of licks into drinking bouts. A drinking bout was determined to be at least three licks occurring with <1 s in between, similar to F/F time series. Video of the test sessions were recorded using a C930e webcam (Logitech) affixed to the top of the operant chamber.Data were acquired using custom-built imaging equipment based on that described by the Deisseroth laboratory , with mom quinine, and on the last day they drank 10% ethanol + 5% sucrose . Average values were obtained from all drinking bouts or lever presses in each session and used for comparison across group and drink type. Outliers were identified using Grubb’s test. When appropriate, repeated measures (RM) one-way ANOVAs with the Geisser–Greenhouse correction were used to compare effects of different drinking solutions on calcium signals within groups. In other instances, RM two-way ANOVAs with the Geisser–Greenhouse correction were used to compare data between groups with the repeated measure being within subject values. ex vivo slice preparations showed that low concentrations of ethanol reduce current-evoked spiking of LOFC neurons  = 13.73, p = 0.015, Dunnett’s multiple comparisons test was significant at 10 min: q = 4.91, p = 0.014 and 20 min: q = 3.69, p = 0.036, n = 5; F = 19.74, p < 0.001, Dunnett’s multiple comparisons test was significant at 10 min: q = 4.68, p = 0.016, and 20 min: q = 5.82, p = 0.008, n = 5;F = 2.52, p = 0.18, n = 5;F = 2.50, p = 0.18, n = 5;Results from our previous studies using  neurons . In the n 10 min A, 2D, con 10 min . A dose n 10 min  reduced m) while on day 5, mice self-administered 10% ethanol sweetened with 5% sucrose.Mice were trained to lever press for 10% ethanol under an FR1 schedule of reinforcement. After eight weeks of baseline drinking in daily 30-min sessions, they were separated into CIE or Air groups counterbalanced by baseline ethanol consumption. Mice were then exposed to four consecutive weeks of CIE or air exposure, as described in previous studies . CIE-expF = 38.7, p < 0.0001, Dunnett’s multiple comparisons test significant for post-CIE drinking: q = 4.38, p = 0.0029 n = 12;m  = 4.57, p = 0.04 n = 9, Dunnett’s multiple comparisons test was significant for quinine: q = 3.7, p = 0.015;F = 30.82, p < 0.0001, and drink: F = 24.46, p < 0.0001, significant interaction: F = 12.05, p < 0.0001. Sidak’s multiple comparisons test was significant for post-CIE/Air: t = 2.98, df = 14.85, p = 0.037; quinine: t = 5.29, df = 18.93, p = 0.0002; and sucrose: t = 4.76, df = 10.9, p = 0.0024).Consistent with previous literature , mice exF = 12.35, p = 0.0014, Dunnett’s multiple comparisons test was significant for sucrose+ethanol: q = 3.12, p = 0.035;F = 24.4, p < 0.0001, Dunnett’s multiple comparisons test was significant during sucrose+ethanol drinking, q = 5.86, p = 0.0003, and trended toward significance with quinine+ethanol drinking  = 32.41, p < 0.0001; but not CIE: F = 1.768, p = 0.20; or interaction: F = 2.45, p = 0.073, Sidak’s multiple comparisons test was insignificant across all solutions, p > 0.05;The microstructure of alcohol drinking in Air-exposed and CIE-exposed mice was assessed by analyzing the number of licks per bout, the number of bouts per session and the number of licks per second. Bout size in air-exposed mice varied significantly across the different drinking solutions  = 3.34, p = 0.058;F = 23.34, p < 0.0001, Dunnett’s multiple comparisons test was significant at post-CIE: q = 5.34, p = 0.0007, quinine: q = 6.58, p = 0.0001, and sucrose: q = 11.04, p < 0.0001;F = 17.7, p = 0.0005, and drinking solution: F = 17.71, p < 0.0001, significant interaction: F = 7.75, p = 0.0002, Sidaks multiple comparisons test was significant for quinine: t = 4.46, df = 18.94, p = 0.0011, and sucrose: t = 5.45, df = 16.68, p = 0.0002;There were no significant differences in the number of bouts per session in air-exposed mice for the different drinking solutions  = 0.34, p = 0.74). CIE treatment produced a small but significant effect on lick rate  = 4.89, p = 0.014;p = 0.013). However, this effect did not reach statistical significance when lick rates between Air-treated and CIE-treated mice were compared with one another  = 0.63, p = 0.4369, and drink: F = 0.28, p = 0.74, interaction F = 2.41, p = 0.076;Finally, there were no differences in lick rate across drinking solutions for air-exposed animals  = 7.06, p = 0.021, and drink: F = 8.64, p = 0.004, and a significant interaction, F = 3.28, p = 0.038, Dunnett’s multiple comparisons test revealed significant differences during sucrose drinking at 2 s: q = 4.26, p = 0.0055, 3 s: q = 4.01, p = 0.0079, and 4 s: q = 2.90, p = 0.044 before the bout and during quinine drinking at 2 s: q = 4.16, p = 0.0063 before the bout;F = 1.41, p = 0.29, and drink: F = 0.49, p = 0.61, and no interaction: F = 1.32, p = 0.31;Differences in the magnitude of these three features from the preexposure baseline drinking sessions were calculated for each of the post-CIE/Air drinking sessions. CIE-exposed mice had a significant increase in the size of the ramp-up in GCaMP6f signal, which occurred earlier in CIE-exposed mice during drinking of either of the novel ethanol solutions  = 11.02, p = 0.0009, Dunnett’s multiple comparisons test was significant during sucrose drinking, q = 4.05, p = 0.0075, and was trending toward significance during quinine drinking, q = 2.59, p = 0.07;F = 1.44, p = 0.29;F = 4.29, p = 0.017, CIE: F = 1.65, p = 0.22, interaction: F = 6.65, p = 0.0009, Sidak’s multiple comparisons test revealed significant no significant differences across drinking sessions;CIE-exposed mice also showed a significantly larger dip in activity during the drinking bout that was most pronounced during ethanol+sucrose drinking  = 3.54, p = 0.07  = 1.98, p = 0.16  = 1.39, p = 0.26, CIE: F = 0.6, p = 0.45, interaction: F = 4.8, p = 0.0058, Sidak’s multiple comparisons test was not significant for any drinking solution;In both Air-exposed and CIE-exposed mice, there were no within group differences in the size of the postbout spike in GCaMP6f signal . HowevF = 15.34, p = 0.002, drink: F = 1.16, p = 0.32, interaction: F = 1.38, p = 0.26, Sidak’s multiple comparisons test revealed significant differences during quinine: t = 3.64, p = 0.014, and sucrose drinking: t = 4.32, p = 0.0029;F = 1.13, p = 0.31, drink: F = 55.35, p < 0.0001, interaction: F = 0.60, p = 0.62; Sidak’s multiple comparisons test > 0.05;F = 12.06, p = 0.0011, Dunnett’s multiple comparisons test was significant across all drinks, post-CIE: q = 3.55, p = 0.019, quinine: q = 3.35, p = 0.022, and sucrose: q = 4.43, p = 0.0043;F = 1.94, p = 0.21;F = 0.72, p = 0.52, drink: F = 0.51, p = 0.68, interaction: F = 0.41, p = 0.97;F = 8.47, p < 0.001, drink: F = 0.36, p = 0.77, interaction: F = 0.59 p = 0.87; Dunnett’s multiple comparisons test > 0.05;F = 3.51, p = 0.029, drink: F = 2.94, p = 0.046, interaction: F = 2.62, p = 0.001, Dunnett’s multiple comparisons test revealed significant differences during post-CIE drinking at the earliest two time points 3–2.5 s: q = 2.95, p = 0.0028 and 2.5–2 s: q = 2.63, p = 0.0094, and quinine drinking at the earliest two time points 3–2.5 s: q = 2.40, p = 0.046 and 2.5–2 s: q = 2.52, p = 0.033;F = 3.13, p = 0.045, drink: F = 0.54, p = 0.66, interaction: F = 0.33, p = 0.99; Dunnett’s multiple comparisons test > 0.05;Upon pressing the active lever, mice were presented with both auditory (1.5-s tone) and visual cues (house light off for 1.5 s) followed by delivery of 20 μl of solution in the drinking well. Mice quickly learned to press the active lever to gain access to ethanol containing solutions. Consistent with the changes in drinking noted above, CIE-exposed mice had more active lever presses compared with air-exposed mice during ethanol+quinine and ethanol+sucrose drinking  = 6.98, p = 0.014, Dunnett’s multiple comparisons test was significant for post-CIE: q = 2.84, p = 0.048, quinine: q = 2.91, p = 0.043, and sucrose: q = 2.84, p = 0.048;F = 1.20, p = 0.34;F = 3.13, p = 0.13  = 0.50, p = 0.57  = 0.50, p = 0.63  = 0.62, p = 0.54 . Previous studies from our lab, using electrophysiological recordings from ex vivo slice preparations, have shown that LOFC neurons are acutely inhibited by bath application of ethanol at concentrations associated with voluntary consumption  and sedating (3.5 g/kg) doses of ethanol significantly reduced the frequency of GCaMP6f peaks with the higher dose inhibiting ∼80% of activity. In further agreement with previous in vitro studies  compared with other studies  alter the structural, molecular and physiological framework of the LOFC  as well The LOFC is thought to maintain a pliable representation of the anticipated value of a reward . In thisIn conclusion, the results of this study show that repeated cycles of CIE exposure increase alcohol consumption, decrease the devaluing effect of quinine and produce time-locked alterations in the activity of LOFC neurons during operant ethanol self-administration. While these studies did not distinguish between projection specific subpopulations of LOFC neurons, future studies should examine how CIE alters the signaling characteristics of LOFC neurons in neural circuits involved in anticipation of reward delivery, evaluation of reward value, and implementation of flexible behavior.


Waf/CIP1 is a small unstructured protein that binds and inactivates cyclin-dependent kinases (CDKs). To this end, p21 levels increase following the activation of the p53 tumor suppressor. CDK inhibition by p21 triggers cell-cycle arrest in the G1 and G2 phases of the cell cycle. In the absence of exogenous insults causing replication stress, only residual p21 levels are prevalent that are insufficient to inhibit CDKs. However, research from different laboratories has demonstrated that these residual p21 levels in the S phase control DNA replication speed and origin firing to preserve genomic stability. Such an S-phase function of p21 depends fully on its ability to displace partners from chromatin-bound proliferating cell nuclear antigen (PCNA). Vice versa, PCNA also regulates p21 by preventing its upregulation in the S phase, even in the context of robust p21 induction by γ irradiation. Such a tight regulation of p21 in the S phase unveils the potential that CDK-independent functions of p21 may have for the improvement of cancer treatments.p21 Waf/CIP1/CDKN1A) we mention that, even though the cytoplasmic functions of p21 were described [Before starting to discuss nuclear functions of cyclin kinase inhibitor p21 , a protein that was discovered in 1978 and was initially described as an essential factor for proliferation . Many yeCdt2. PIP degrons are formed by a threonine and an aspartic acid (TD) within the PIP box, and a basic residue (R) in the +4 position from the last residue of the PIP box [PIP boxes reside in intrinsically disordered regions of proteins. The PIP box consensus sequence was defined as Qxxϕxxψψ, where, by definition, Q is glutamine, x is any amino acid, ϕ is a hydrophobic residue, and ψ an aromatic residue (commonly Phe or Tyr) . The p21 PIP box . A very  PIP box . In conc PIP box . Experim PIP box . A schem PIP box , the int PIP box ,34. HencPCNA is a highly abundant protein, especially in cancer cells ,36 ThereSkp2, CRL4Cdt2, MDM2/X (or HDM2 in humans), CRL2LRR1, and APC/CCdc20. For details on such degradation mechanisms, please refer to [Skp2, HDM2, and CRL4Cdt2 peaks at the beginning of and during the S phase, and could counteract the p53-mediated accumulation of p21 [Skp2 did not contribute to such γ-irradiation-associated p21 proteolysis in the S phase [Skp2 degrades cyclin–CDK-bound p21 [Skp2, while the contribution of CRL4Cdt2 to p21 degradation was central and required to promote both irreversible S-phase entry and to prevent premature S-phase exit upon DNA damage [Cdt2-dependent degradation of p21 was triggered by ATM (ataxia telangiectasia mutatated) activation [It is an acknowledged fact within the research community that the accumulation of p21 triggers cell-cycle arrest outside the S phase. In this section, however, we focus on a not-so-familiar yet remarkable fact about p21. In the S phase, p21 is never upregulated, even when treating cells with DNA-damaging agents that cause p21 accumulation to an extent that is easily detectable through a Western blot. It is well known that p21 levels in cells are controlled by different mechanisms that include but are not exclusively restricted to the activity of the following E3 ligases: SCFrefer to ,60. Morerefer to . p21 levn of p21 ,63,64. In of p21 . Moreoven of p21 . Live-cen of p21 . StrikinA damage . After γtivation .cdt2 ligase, which degrades PCNA-bound p21 in the S phase [Skp2 and Mdm2, the other two ligases that mediate p21 degradation during the late G1 and early S phases, contributed only marginally to the prevention of p21 accumulation in the S phase of γ-irradiated cells [cdt2-mediated proteolysis. Alternatively, liberating p21 from PCNA interaction may cause dysregulated interaction between p21 and CDKs that favors genomic instability with an unknown mechanism. In this regard, it should be mentioned that, in a manner that depends on CDK inactivation, p21 was reported to regulate the homologous recombination-mediated repair of double-strand-breaks (DSBs), a DNA lesion that is induced by γ irradiation [Would p21–PCNA interaction then be needed for S-phase onset and shut-down? A recent report that monitored single cells for 20 hours, combining noninvasive live-cell imaging of CRISPR/Cas9-endogenous fluorescent p53 and p21 reporters and computational-based analysis of multiple single-cell events, addressed such questions . In agre S phase . In conted cells ,67. Intred cells ) caused ed cells ,44. Moreadiation . A modelIn the previous section, we discussed different reports demonstrating that, regardless of p53-dependent p21 mRNA induction, p21 levels in the S phase are strongly downregulated. Moreover, γ-irradiation cannot trigger p21 accumulation in cells arrested in the S phase using ribonucleotide reductase inhibition by hydroxyurea . Such obCdt2 ligase saturation. The dependency on PCNA was confirmed using a p21 mutant with a disrupted PIP box , which did not cause CDT1 and CDC6 upregulation [Caenorhabditis elegans, p21 accumulation triggered by CRL4Cdt2 and SCFSkp2 inactivation causes nuclear retention of CDC6 and rereplication [(a) p21 levels influence the turnover of licensed replication origins. By exploring markers of active replication (KI67) and p21 levels in human tumors, unexpected colocalization of p21 and ki67 was observed in approximately 5% to 7% of cells within p53-deficient tumors . Since sgulation . p21-ovegulation , DSBs wegulation , and posgulation . Becausegulation ,75. Reinlication . We conc(b) p21 levels influence replication-fork speed. Endogenous p21 levels in unperturbed p53-expressing cells are lower than in the scenario discussed in (a). Such p21 is considered residual as it does not modulate unperturbed cell-cycle progression ,78. HoweThe S-phase-specific impairment of p21 upregulation after γ-irradiation, NCS, and nutlin-3 was discussed in previous sections. We also debated evidence indicating that such low p21 levels accumulated in unperturbed or γ-irradiated S phase cells are insufficient to attempt CDK inhibition, but enough to modulate the occupancy of PCNA trimmers at replisomes. p21 is capable of modulating the recruitment of many partners to the PCNA IDCL pocket without interfering with the recruitment of replicative DNA polymerase δ to DNA . Such a Cdt2 and a noncanonical type of ubiquitination at its N-terminus [Since the levels of p21 in an unperturbed S phase do not suffice to inhibit the DNA synthesis by replicative DNA polymerases, they may not suffice after γ irradiation, NCS, or nutlin-3 treatments as well ,67,68. Iterminus ,94,96,97Cdt2-dependent proteolysis of p21 to allow the utilization of damaged DNA as templates for DNA synthesis by specialized DNA polymerases [The results in the previous section indicated that p21–PCNA interaction may be actively prevented after different types of genotoxic stress. A hint on the relevance of p21 downregulation in S-phase post-UV irradiation came from the correlation between the timing of p21 degradation and the recruitment of specialized DNA pol η to replication factories or chromatin-bound PCNA . The recymerases . A modelBecause TLS events may protect tumor cells by favoring DNA replication, TLS is considered a potential target in cancer therapy ,108,109.While the concept of p21 as a cyclin kinase inhibitor is massively accepted by the research community, the possibility that such a renowned function of p21 exerts a “secondary” role is far from being acknowledged. For multiple rounds of cell division, cells that cycle freely may not accumulate enough p21 to inhibit CDKs. However, in every cell cycle, cells express p21 in the S phase in such amounts that are enough to fulfill PCNA-mediated functions. Despite the average Western blot showing a faint, almost undetectable p21 band, such an amount of p21 is enough to regulate key aspects of DNA replication events in every cell cycle. This notion is supported by several reports ,79,83,86


There has been considerable interest in empirical research on epistemic emotions, i.e., emotions related to knowledge-generating qualities of cognitive tasks and activities such as curiosity, interest, and surprise. One big challenge when studying epistemic emotions is systematically inducting these emotions in restricted experimental settings. The current study created a novel stimulus set called Magic Curiosity Arousing Tricks (MagicCATs): a collection of 166 short magic trick video clips that aim to induce a variety of epistemic emotions. MagicCATs are freely available for research and can be used in a variety of ways to examine epistemic emotions. Rating data also support that the magic tricks elicit a variety of epistemic emotions with sufficient inter-stimulus variability, demonstrating good psychometric properties for their use in psychological experiments. Recent years have seen a surge of interest in the topic of so-called epistemic emotions. Epistemic emotions refer to a group of emotions which are related to the knowledge-generating qualities of cognitive tasks and activities , researchers need a number of short repeated trials to ensure the reliability of the task. However, triggering epistemic emotions in such a short time frame is challenging because epistemic emotions, by definition, require people to cognitively process the task; epistemic emotions are not something that can be immediately triggered upon presenting a stimulus. In addition, even if it is possible to induce epistemic emotions in a short time frame, the magnitude of the emotion is likely to be insufficient to cause a psychological response and/or behavioral change. Thus, there are limited experimental materials that claim to induce epistemic emotions. In fact, the vast majority of studies in experimental psychology use trivia questions or similar knowledge questions to induce epistemic emotions, particularly curiosity : a collection of 166 novel short magic trick video clips that trigger people's epistemic emotions in experimental settings. The videos of MagicCATs are available for researchers (see "Stimulus Availability" section in Methods) and the current article reports basic characteristics and the norms of these magic trick video clips. There are already several studies that have used magic tricks as stimuli to trigger epistemic emotions, including neuroimaging studies , which is thus likely to induce relatively strong feelings of epistemic emotions. An advantage of magic tricks is that they mainly consist of nonverbal information such as vanishing or appearing materials. This nonverbal nature of the stimuli makes it easier for people to intuitively understand the content. As a result, magic tricks can trigger epistemic emotions regardless of the participant’s language, educational and cultural backgrounds. Of course, we are not claiming that magic tricks are superior to existing stimuli to trigger epistemic emotions. There are some obvious limitations such as difficulties controlling stimulus length. However, given the unique advantages of magic tricks, we believe that the current stimulus set provides complementary benefits to the researchers studying epistemic emotions.There are different ways to utilize MagicCATs in research, but one common context is an experiment in which researchers intend to elicit different levels of epistemic emotions on a trial-by-trial basis to examine the within-person correlates of epistemic emotions. We have already conducted one neuroimaging experiment using MagicCATs and validated the effectiveness of the stimuli. In Lau, Ozono, Kuratomi, Komiya, & Murayama , we pres“science of magic”, which investigates human cognitive mechanisms using magic tricks  with sufficient inter-stimulus variability.Four male magicians, including a champion of an international magic competition, performed 145 magic tricks in total. These performances were filmed in two studios (one studio for one magician and another studio for the other three magicians) by professional photographers using high resolution video cameras. The magicians selected magic tricks that would maximize the variety of materials  and the types of the tricks . Magicians also ensured that the magic tricks were heterogeneous enough to induce different levels of epistemic emotions . As most previous research on curiosity and interest were comparing the responses to the stimuli that induce either a low or high-level of curiosity and interest  software to have a similar monotonic (dark) background, size (720 x 404 pixels) and viewing focus. The videos were muted, and English subtitles were added in a few videos, when necessary. The face of the magician was obscured as much as possible to avoid potential distraction due to their appearance and facial expressions. This editing also helps minimize potential responses to the gender of the magicians reported by Gygax, Thomas, Didierjean, & Kuhn . Twenty-A total of 495 participants took part in the rating task through Amazon Mechanical Turk. A total of 470 US participants were paid $3.5 for approximately 35 min of study participation, whist the first 25 participants were paid $2.5 before our re-calculation of the more realistic study completion time. Prior to the main data analysis, we excluded 44 participants who either (a) took more than +2 SD longer than average to complete the experiment (there is no participant who took shorter than 2SD below the average duration); (b) gave identical ratings on more than three questions for all trials; (c) answered "no" to the "clarity of the trick" question for all the presented video clips (see below); (d) indicated some issues in video presentation and internet connection in post-questions; or (e) indicated that they were distracted during the experiment or had already taken part in a similar experiment previously. This exclusion led to the final sample of 451 participants; 259 males and 192 females .It was impractical for any single participant to view and rate all the 166 video clips in MagicCATs, particularly as there were some duplicated magic tricks due to the short- and long-version as described above. To address this, we split the 166 video clips into nine lists and only presented each participant video clips from two of the lists. Our design is an adopted version of a Balanced Incomplete Block (BIB) spiraling procedure in the literature of test theory Fleiss, .The nine lists (Lists 1–9) were created in the following manner. Lists 1 and 2 consisted of 21 tricks from the short and long versions of the video clips mentioned earlier. Short and long versions of the video clips were assigned evenly to these lists . Video clips from the same magic trick were not assigned to the same list, thus when List 1 included a short version of a magic trick, List 2 included a long version of the same magic trick. The other 124 video clips were assigned to the remaining 7 lists (List 3 to List 9), resulting in 17 or 18 video clips for each of the lists. When creating these lists, we attempted to minimize the difference in the total duration of the video clips between lists. Consequently, the total duration of the video clips in Lists 1 and 2 were 16–17 min and 9–10 min in Lists 3 to 9.clarity of the trick); (b) how surprised they were at the magic trick (surprise in response to the trick); (c) how interesting the magic tricks were (interest in the trick); (d) how confident they were that they had figured out the solution to the trick (confidence in the solution); and (e) how curious they were about how the magic trick was done (curiosity of the solution). Clarity of the trick, which assessed whether participants understand what happened after seeing the video clip, was rated using a binary response scale (Yes/No); we included this rating to be used as an exclusion criterion. The other four questions were rated on 10-point Likert scales ranging from 1  to 10 (very much).Participants were presented with video clips from two of the nine lists in a randomized order. The assignment of the lists was randomly determined with the constraint that participants were not presented both Lists 1 and 2 because they included video clips from the same magic tricks but of different lengths. For each video clip participants gave five different ratings: (a) whether they understood the intention of the magic trick or not  and whether they perform magic tricks themselves . Second, they reported any video presentation or internet connection problems that had occurred during the experiment; whether they were doing anything else during the experiment and whether they had already participated in another experiment with the same videos. We emphasized that their compensation would not be affected by their responses. As indicated above, these questions were used to exclude participants from the main data analysis who seemed to have been disengaged during the study.Analysis on the post hoc questions showed that participants were generally interested in the magic tricks M = 7.13, SD = 2.05. Only six participants indicated that they frequently performed magic tricks; 379 participants indicated that they had never performed a magic trick. In the following, we did not use the data for Trick 9 because it mistakenly contained sounds . These average rating values were consistent with the fact that we intended to include both surprising and less surprising magic tricks to ensure the heterogeneity of the stimulus set. The average confidence in the solution is relatively low (M = 4.14). Note that this is a subjective rating of confidence and we do not have objective data to demonstrate that participants indeed correctly guessed the solution behind some of the magic tricks.To further examine whether our new stimuli can appropriately evaluate within-person variability of epistemic emotions, we applied a mixed-effects model to the data to decompose the three distinct variance components: participant variance, video variance, and participant x video variance. Participant variance represents overall individual differences between participants , whereas video variance represents differences in ratings between video clips . Participant x video variance represents individual differences in participants' responses to different video clips . Note that participant x video variance also includes variance from measurement errors, which we cannot statistically separate. In most of the previous literature on epistemic emotions . On the other hand, confidence was negatively associated with age . There were no statistically significant gender differences . General interest in magic tricks is significantly and positively associated with epistemic emotions and confidence . Experience with magic tricks has a strong positive association with confidence , and a weak positive relationship with interest . Finally, number of trials was negatively associated with epistemic emotions and confidence, indicating that there was a general declining trend for these ratings over trials .We also examined the extent to which these ratings can be explained by the individual difference variables we assessed  by including these variable as additional fixed-effect predictors in the model. We also included a fixed-effect of trial number and its random slopes as an additional within-person predictor to explore the potential role of familiarization in epistemic emotions. Table To further examine the differentiation of these epistemic emotions, we also computed the correlations between surprise in response to the trick, interest in the trick, confidence in the solution, and curiosity in the solution at the within-person level. More specifically, we calculated within-person correlations for each participant  and then computed the mean and SD of the correlations across participants (please see Table https://osf.io/ad6uc/) along with the stimulus list .The current article introduces 166 magic trick video clips (MagicCATs) as a novel stimulus set to induce epistemic emotions. MagicCATs include a variety of magic tricks with various lengths (8–155 s) and diverse materials , making it easy for researchers to select and use the subset of stimuli best suited to their own research purposes. Furthermore, rating results showed sufficient within-person variance with moderate mean levels of epistemic emotions, meaning that these video clips are suitable to examine these emotions on a trial-by-trial basis. MagicCATs video clips are available for research purpose, and stimulus list and raw rating data are also available online , whereas interest ratings asked whether they were interested in the magic tricks themselves . Curiosity in the solution focused on the subjective motivation to close the knowledge gap (Loewenstein, Some additional points are worth discussing. First, our findings from the rating analysis may have a limitation in that we relied on a single-item measure to assess epistemic emotions, which are expected to be less reliable than multiple-item measures. However, we believe that simple subjective emotional feelings such as surprise can be assessed reliably and validly with a single-item measure (see also Diamantopoulos et al.,