Alzheimer's disease (AD) is a neurodegenerative disorder strongly associates with aging. While amyloid plagues and neurofibrillary tangles are pathological hallmarks of AD, recent evidence suggests synaptic dysfunction and physical loss may be the key mechanisms that determine the clinical syndrome and dementia onset. Currently, no effective therapy prevents neuropathological changes and cognitive decline. Neurotrophic factors and their receptors represent novel therapeutic targets to treat AD and dementia. Recent clinical literature revealed that MET receptor tyrosine kinase protein is reduced in AD patient's brain. Activation of MET by its ligand hepatocyte growth factor (HGF) initiates pleiotropic signaling in the developing brain that promotes neurogenesis, survival, synaptogenesis, and plasticity. We hypothesize that if reduced MET signaling plays a role in AD pathogenesis, this might be reflected in the AD mouse models and as such provides opportunities for mechanistic studies on the role of HGF/MET in AD. Examining the 5XFAD mouse model revealed that MET protein exhibits age-dependent progressive reduction prior to overt neuronal pathology, which cannot be explained by indiscriminate loss of total synaptic proteins. In addition, genetic ablation of MET protein in cortical excitatory neurons exacerbates amyloid-related neuropathology in 5XFAD mice. We further found that HGF enhances prefrontal layer 5 neuron synaptic plasticity measured by long-term potentiation (LTP). However, the degree of LTP enhancement is significantly reduced in 5XFAD mice brain slices. Taken together, our study revealed that early reduction of HGF/MET signaling may contribute to the synaptic pathology observed in AD. An estimated 50 million people worldwide are currently living with dementia  receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are expressed in the nervous system from prenatal development to adult life. The human reduced levels of MET protein in the cerebral cortex and hippocampus of patients with AD  and mutant forms of human PS1 gene (M146L and L286V) under the Thy1 promoter  were generated by breeding hemizygote male mice with a floxed Met gene (Metfx/+) and emx1Cre knock-in allele  and their 5XFAD littermates  were used for neuropathological comparisons. All experimental procedures conformed to NIH guidelines and were approved by the Institutional Animal Care and Use Committee of the University of Arizona.The 5XFAD mice used in this study were purchased from The Jackson Laboratory . This line overexpresses both mutant forms of human amyloid precursor protein gene Mice were euthanized with 3–5% isoflurane and transcardially cleared with cold 0.01 M PBS. Four percent paraformaldehyde (PFA) formulated in 0.1 M phosphate buffer (pH 7.4) was then perfused. Brains were dissected and postfixed in cold 4% PFA overnight. After cryoprotection in 30% sucrose for 2 days, brains were embedded in OCT cutting compound and sectioned on a sliding microtome (Leica SR-2000). About 40-μm-thick floating brain sections were washed 3X in 0.01 M PBS and permeabilized in PBS-0.2% Triton. For APP/Aβ+Iba1+Thio-S staining, the free-floating sections were blocked in primary antibody dilution solution  for 2 h. Anti-APP/Aβ primary antibody  mixed with anti-Iba1  was applied for 24 h with slow rotation. Sections were washed 3X in 0.01 M PBS and further incubated with Alexa 555-conjugated goat anti-mouse antibody and Alexa 647-conjugated goat anti-rabbit antibody for 2 h. Sections were extensively washed in 0.01 M PBS and further stained with 0.025% Thio-S (prepared in 50% ethanol−50% PBS) for 10 min. After briefly destaining in 50% ethanol, sections were washed in 0.01 M PBS and mounted on the SuperFrost Plus slides  using DAPI-containing Vectashield mounting medium . Images were acquired on a LSM 710 confocal microscope  with appropriate laser lines and filters. We kept all the acquisition parameters constant through different experiments to allow comparisons, including photomultiplier tube detector gain, pinhole size, laser intensity, and image sizes. To count the number of Iba1+ microglia and coverage of APP/Aβ and Thio-S, we used maximum projection images from a 25-μm-Z stack plane . Pseudo-colors of confocal image channels may be redefined to enhance visualization in figure preparation. To quantify signal area/size and co-localizations between channels, confocal .czi files were imported into Imaris or ImageJ for customized analyses.Western blots was performed using specific antibodies to detect proteins of interest. Total protein content of the micro-dissected PFC and CA1 tissues was quantified using a micro-BCA assay, after being homogenized in NP40 lysis buffer  supplemented with proteinase inhibitor cocktail . The samples are then mixed with equal amount of 2 × Laemmli loading buffer and boiled for 5 min. Samples were separated by electrophoresis on 4–15% SDS-polyacrylamide gels. Proteins were then transferred to PVDF membrane  and blocked with 0.01 M PBS-Tween 20 with 5% non-fat dry milk. Primary antibody was diluted in the same blocking solution and applied to the PVDF membrane in a humidified chamber on a glass plate covered with parafilm. After overnight primary antibody incubation, the PVDF membrane was washed three times in 0.01 M PBS-Tween 20. Secondary antibodies directed to the correct species that are conjugated to horseradish peroxidase were applied for 2 h. The protein signal was developed using an enhanced chemiluminescence method  and captured on an X-ray film (Amersham ECL Hyperfilm). The following antibodies were used for this study: From Santa Cruz Biotechnology, mouse anti-MET (Cat# sc-8057); from Millipore/Chemicon, rabbit anti-GluA1 (AB5849), rabbit anti-PSD95 (AB9708); from Cell Signaling Technology, rabbit anti-phospho-MET (Y1234/1235) (#3077), rabbit anti-GAPDH (#5174). The final dilutions of antibodies were between 1:1,000 and 1:2,000. The optical intensity of protein signal band captured on Hyperfilm was digitized by a film scanner (Epson V850) into an 8-bit gray scale image and quantified by densitometry using ImageJ/FIJI.3, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 7 MgSO4, 25-d glucose, 11.6 sodium ascorbate, and 3.1 sodium pyruvate, saturated with 95% O2/5% CO2) to improve brain slice viability. The brains were quickly harvested, and 350-μm-thick parasagittal slices were made on a vibratome  at an angle that preserves intra-cortical L23>L5 connectivity  for 30 min at 35°C and then maintained at 24°C RT until recording.Standard extracellular field excitatory postsynaptic potential (fEPSP) recording was used to investigate long-term potentiation (LTP) changes in the prefrontal cortex (PFC) layer 5 (L5) region. Mice were euthanized using 3–5% isoflurane, followed by intra-cardiac perfusion of ice-cold choline solution  was placed on the L23. The patch electrode had an electrical resistance of 1–2 MΩ at 1 kHz when filled with ACSF. Biphasic electrical stimuli were generated through a Digidata 1440A device  and delivered through an optic isolator . Stimulus (100-μs duration) intensity ranged from 10 to 50 μA and was delivered at 0.05Hz for baseline and LTP recordings. fEPSP signals were amplified using a differential amplifier , low-pass filtered at 2 kHz, and digitized at 10 kHz through the Digidata 1440A board.Brain slices were transferred to an interface chamber (AutoMate Scientific) that is maintained at 32°C and superfused with ACSF saturated with 95% Oabf2load.m function. To test the effects of HGF on LTP in control and 5XFAD slices, 10 nM recombinant human HGF  was added to the ACSF perfusate for 30 min prior to the LTP induction. A subset of slices were collected after LTP studies, and L5 region was micro-dissected for western blot detection of phospho-MET (Tyr1234/1235), which was used as a surrogate of MET activation.For each of the fEPSP recordings in PFC slices, a stimulus-response (input–output) curve was first obtained by measuring the fEPSP slope (first 1-ms response after fiber volley) as a function of the fiber volley amplitude. This curve was then used to quantify the strength of basal synaptic transmission. We adopted a stimulus intensity that produces a ~40–50% maximum monosynaptic fEPSP responses throughout the experiments. A 10-min stable baseline responses of stimulus-evoked fEPSPs were first obtained, and the paired-pulse responses at inter-pulse intervals ranging from 20 to 200 ms were recorded to assess potential changes in presynaptic transmission. Another pre-LTP 10-min baseline was then recorded, and LTP was induced by a theta-burst stimulation protocol, which consists of a 2-s long 5 Hz train (each train consists four pulses at 100 Hz) repeated 5 times at a 10-s interval  that is provided in a 96-well format, according to the manufacturers' instructions. A microplate reader (Tecan) was used to measure absorption at 450 nm.Brains were weighed, sliced, and subjected to sequential Aβ extraction. Prefrontal cortical tissues (L5 region) were dissected and homogenized in 2% SDS-RIPA buffer . The SDS-RIPA buffer also contains protease inhibitor cocktail . Tissue homogenates were incubated on ice for 15 min to extract proteins, followed by centrifugation at 16,000 g for 90 min at 4 C. The supernatant containing RIPA-soluble fraction of AβF test were first employed to test normality and equal variance. All data that passed normality/equal variance tests were reported as mean ± SEM (standard error of the mean). For normal-distributed/equal variance data, Student's t-test or multiple t-tests were used. Statistical analyses and graphing were performed using GraphPad Prism 8.0, Microsoft Excel, and MATLAB. P-Value < 0.05 was considered statistically significant for all tests. Figures were prepared using Adobe Creative Cloud.The experimenters were blinded to grouping/genotype information during data collection and analyses. Sample sizes and number of independent experiments were estimated by power analyses using an R script (“pwr” package on CRAN) that takes pre-specified effect size, type I and II errors as input arguments. Shapiro–Wilk test and p = 0.019, n = 4; HPC-CA1, n = 4, quantification not shown). At P105-120, MET is severely depleted in both brain regions in 5XFAD brain . MET protein reduction in PFC and HPC parallels the abrupt, qualitative increase in Aβ load in the 5XFAD mice at this age . As such, the early reduction of MET protein in PFC and HPC at P45-50 or later cannot be explained by the gross loss of synapse at this stage, as other synaptic proteins markers were not affected.The early reduction of MET proteins in 5XFAD brain can be due to specific reduction of HGF/MET signaling that is related to AD pathology or APP/Aβ overloading, or it could be simply a non-specific effects of early synapse loss. We therefore probed levels of PSD95, a postsynaptic protein, and GluA1, an AMPA receptor subunit of the excitatory synapse, in the PFC-L5 tissues. After normalizing to the GAPDH loading controls, quantification of MET, PSD95, and GluA1 is shown in Met cKO mice (Metfx/fx:emx1cre) to the 5XFAD mice (refer to Methods). Emx1cre drives inactivation of the Met gene from mid-gestation stage. We compared the 5XFAD mice  with their littermate mice harboring the inactivated Met gene (5XFAD:MetcKO) for their neuropathological markers. We immunostained PFC sections for Iba1, a marker for reactive microglia; APP/Aβ, and Thio-S, a marker for non-soluble fibrillary β-sheet forms of amyloid plaques  = 2.43, p = 0.006]. Next, we quantified the APP/Aβ+ plaque coverage as a percentage of the imaged L5 area. 5XFAD:MetcKO mice show significantly increased percentage areal coverage of APP/Aβ . To estimate the density of non-soluble fibrillary forms of amyloid plaques, we quantified the Thio-S+ areas. 5XFAD:MetcKO mice also show significantly increased percentage areal coverage of Thio-S staining . Taken together, these data show that genetic ablation of MET signaling developmentally aggravates or accelerates neuropathology in the 5XFAD mouse model.We first counted the number and density of Iba1+ microglia from L5 mPFC regions in 12-week-old  5XFAD and the 5XFAD:Met cKO may exacerbate production of Aβ, which is an essential component of the phenotypic signature of AD. We again used the ~12-week-old mPFC tissues and micro-dissected the L5 regions (1−42 species (refer to Methods). We found that Met cKO significantly increased the levels of soluble Aβ1−42 . In addition, the amyloid plaque associated insoluble fraction of Aβ1−42 was even more dramatically elevated . These data are consistent with the neuropathological findings and suggest that MET signaling could be a neuroprotective factor during AD pathogenesis.Based on the observed changes in neuropathology, we asked whether  regions . We bioct(10) = 27.2, p < 0.0001]. We also quantified HGF-induced LTP enhancement. HGF leads to significantly lower LTP enhancement in 5XFAD slices .It has been previously shown that HGF, when acutely applied to juvenile rat hippocampus slices, enhanced synaptic LTP in the HPC-CA1 region. This enhancement is most likely due to augmenting NMDA receptor-mediated currents in slices by HGF . These data suggest that baseline activation of MET is much lower in 5XFAD mouse brain at a pre-symptomatic age, yet the endogenous level of MET is still capable of responding to HGF-induced signaling.The reduced level of theta-burst-induced LTP and HGF enhancement of LTP magnitude in 5XFAD PFC slices may reflect the overall lower levels of MET at this age  or reducMet results in exacerbated pathological changes in the 5XFAD mice. These are reflected by the increased amyloid plaque deposition, dense core fibrillar forms of plaque, increased soluble and insoluble forms of Aβ, and increased microglia activation. All of these observations suggest that HGF/MET reduction may contribute to AD pathogenesis in this mouse model featuring aggressive amyloid deposition.In this study, we found early reduction of MET receptor tyrosine kinase in the 5XFAD mouse model for AD. This is consistent with clinical literature reporting a reduction of MET protein in the AD brain, including the hippocampus and cortical regions  mice, there was a disruption in hippocampus LTP and an early cognitive decline  to control actin dynamics . The signaling is capable of promoting dendritic spines/synapses morphogenesis de novo in response to neuronal activity .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Channel state information (CSI) provides a fine-grained description of the signal propagation process, which has attracted extensive attention in the field of indoor positioning. The CSI signals collected by different fingerprint points have a high degree of discrimination due to the influence of multi-path effects. This multi-path effect is reflected in the correlation between subcarriers and antennas. However, in mining such correlations, previous methods are difficult to aggregate non-adjacent features, resulting in insufficient multi-path information extraction. In addition, the existence of the multi-path effect makes the relationship between the original CSI signal and the distance not obvious, and it is easy to cause mismatching of long-distance points. Therefore, this paper proposes an indoor localization algorithm that combines the multi-head self-attention mechanism and effective CSI (MHSA-EC). This algorithm is used to solve the problem where it is difficult for traditional algorithms to effectively aggregate long-distance CSI features and mismatches of long-distance points. This paper verifies the stability and accuracy of MHSA-EC positioning through a large number of experiments. The average positioning error of MHSA-EC is 0.71 m in the comprehensive office and 0.64 m in the laboratory. The positioning algorithm is the basis of Location-Based Service (LBS). People need location information in many scenes such as production and life, entertainment, and public services. Traditionally, LBS is limited to outdoor activities, but the expansion of building scale and the increase of people’s activities in indoor scenes have given indoor positioning a larger market. For indoor positioning, the outdoor positioning method cannot be copied, because the widely used Global Positioning System (GPS) positioning has very weak signals in the indoor environment. Common indoor wireless positioning technologies are as follows: based on Wi-Fi ,2, BluetAmong them, the Wi-Fi-based positioning method has a wide range of applications, and the technology has developed from the initial positioning based on RSSI  to the pIn the field of CSI-based indoor positioning, scholars initially believed that multi-path information was interference. Taking the relationship between distance and CSI signal as the starting point, by suppressing the multi-path effect, the relationship between CSI and distance was constructed after preprocessing. As for the positioning method of the ranging model, with the development of CSI in the field of positioning, more scholars have found that multi-path information is not all interference; it can identify fingerprint points with similar distances well and can greatly enhance the discrimination of signals between different fingerprint points. Therefore, fingerprint-based localization methods have gradually become popular.The positioning system based on the ranging model realizes indoor positioning by constructing the relationship between CSI and distance. The main representative is the FILA system. The system uses CSI for the first time to establish a propagation model to achieve indoor positioning. The system “exploits the frequency diversity to compensate the small-scale fading effect” and finally models the relationship between the extracted The fingerprint localization method is to match the test point to the nearest fingerprint point. CSI-MIMO  processeThe current mainstream CSI-based localization method is the fingerprint-based localization method using deep learning. There are still some limitations to this type of approach. We first address two problems: non-adjacent features aggregation and long-distance point mismatching. As shown in CSI feature extraction based on convolutional neural network is still insufficient. In location feature extraction, multi-path information brings higher discrimination to points with similar distances. It is mentioned in ,17 that Multi-path information brings higher discrimination to different points but also makes it hard to map the feature differences between fingerprint points to distances. In other words, it is hard to establish a clear mapping relationship with the distance for the raw CSI features that have not been processed. Therefore, when a mismatch occurs, such methods can match any fingerprint point in the absence of distance constraints. If the algorithm incorrectly matches the test point to the long-distance fingerprint point, the positioning accuracy will be greatly reduced.The change from MLP to CNN has gradually improved the CSI positioning performance. It can be seen that the network feature extraction ability directly affects the positioning performance. At present, the attention mechanism is widely used in neural network architecture. Transformer  proposedIn particular, this article  mentioneIn this paper, a multi-head self-attention mechanism and effective CSI fusion model (MHSA-EC) is constructed. MHSA-EC aims to solve the problem that the CNN model has insufficient ability to extract CSI features and the long-distance point mismatch problem that is easy to occur in the CSI fingerprint-based localization algorithm. MHSA-EC is used to simultaneously perform feature processing and distance information fusion on the CSI information collected from a single location point and construct a joint representation of multi-path information and distance information. The system block diagram is shown in As an end-to-end network model, it is necessary to build a fingerprint database in the offline stage and train MHSA-EC to update the network weights. In the online stage, the trained MHSA-EC is deployed to meet the positioning requirements of online CSI data.1.The algorithm solves the problem of long-distance fingerprint point mismatch in the CSI-based fingerprint positioning algorithm. For the problem of mismatches that are prone to occur at farther distances, we introduce effective CSI as an input to the decision module. Since there is a nonlinear mapping relationship between effective CSI and distance, this signal is introduced to help the decision module to more effectively constrain the position output. The introduction of effective CSI can greatly increase the average positioning accuracy of the system.2.The attention mechanism solves the problem of insufficient CSI feature extraction ability of the CNN network. The multi-path information contained in the subcarriers and arrays in the CSI signal increases the discrimination of CSI features. The CSI feature extraction method based on the CNN network is limited by the receptive field, and it is difficult to aggregate non-adjacent CSI features, resulting in the insufficient ability of the model to extract multi-path information. This paper improves the feature extraction capability of CSI signals by introducing the attention mechanism from a larger network receptive field and a better ability to aggregate non-adjacent features. The model’s ability to extract CSI features determines whether the model can correctly distinguish the CSI features of different fingerprint points. Therefore, the MHSA-EC model with better CSI feature extraction capability can theoretically improve the localization accuracy of the algorithm.3.In addition, this paper also conducts extensive experiments to verify the localization performance of the network in two typical scenes. It also carries out ablation experiments to verify the effectiveness of the network module.The MHSA-EC model proposes the following improvements and innovations for the limitations of CSI positioning:Y represents the received signal, n is the additive white Gaussian noise, X is the transmitted signal, and H represents the channel state information. In practical applications, H can be estimated by the relationship between the received signal and the transmitted signal kth subcarrier. Each subcarrier channel state information kth subcarrier, and kth subcarrier, and kth subcarrier. Effective CSI is used to exploit the frequency diversity to compensate for the small-scale fading effect. While reducing the fading effect, we explore the relationship between the distance between the transmitter and the receiver.OFDM is a bandwidth-limited digital multi-carrier modulation method of wireless communication. Its modulation and demodulation are based on Inverse Fast Fourier Transform (IFFT) and Fast Fourier Transform (FFT), respectively. OFDM has become the most widely used multi-carrier modulation technique, whereas CSI is a sampling of the frequency response in an OFDM system. It can be obtained by inserting a reference signal at the transmitter and estimating the channel at the receiver:Q, K, and V represent query, key, and value respectively, K, X is the input and Q, K, and V respectively.In the field of natural language processing, transformers have excellent performance. The self-attention mechanism is an important method of the transformer model, which can better understand the semantic information by learning the word dependencies within the sentence. Its advantages lies in its low computational complexity, parallel computation, and better learning of long-distance dependencies. Its formula is as follows:i, the collected CSI amplitude can be expressed as:mth link and the nth subcarrier at the i fingerprint point. The link here refers to the connection path between all the receiving ends and the transmitting ends of all the n-numbered subcarriers, so the number of M is equal to the product of the number of transmitter antennas and the number of receiver antennas. Considering the excellent performance of the self-attention mechanism in the sequence classification task, we vectorize the CSI signal at the fingerprint point into a 1*N*M three-dimensional tensor. At the same time, we adopt the normalization method to make the network converge faster during training. The maximum and minimum values of a CSI tensor will be recorded, and the CSI amplitude value of the mth row and nth column will be processed as follows:In the process of fingerprint collection of CSI, for a fingerprint point The normalized CSI tensor is as follows:There is still room for improvement in existing classical CSI-based localization models. Classic models still need improvement in CSI feature extraction. Classical networks such as Confi  and 1d-CLimited by the size of the receptive field, when the network aggregates features, it is difficult for CNN to aggregate all features in a single-layer network compared to the multi-head self-attention method (MHSA). However, there is a correlation between non-adjacent carriers and antennas in the CSI signal, and mining this correlation is very helpful for capturing multi-path information and improving positioning accuracy. Therefore, the feature extraction ability of the multi-head self-attention mechanism is better.In addition, due to the fluctuation of the CSI signal, the positioning model may encounter the problem of mismatching distant points. The classical model lacks constraints for this problem, resulting in a high maximum positioning error. Introducing a kind of distance information to the model can effectively solve this problem.Based on the above discussion, we design the following network. MHSA-EC includes four modules: feature extraction, statistical module, fusion method, and fully connected decision. The structure is shown in MHSA-EC first uses the multi-head self-attention mechanism to extract the key features of the subcarrier dimension and the antenna dimension, aiming at aggregating the relevant information between the carriers and between the antennas, and calculating the fingerprint characteristics of the current node. Effective CSI, a statistical vector that suppresses multi-path effects, is obtained through frequency diversity. Finally, the fusion module summarizes all the branch inputs to the decision module, and the decision module uses multiple fully connected layers and activation functions to perform fingerprint classification. The input in the network is the CSI amplitude, and the label is the position of the fingerprint point. The parameters are trained and optimized by gradient descent.Considering that the CSI dimension is less and it is difficult to mine potential features, the H self-attention layers, and the output of the self-attention layer whose index is ith eigenvalue. The feature extraction module contains M MHSA layers, and the output of the MHSA layer with index In the MHSA layer, there are c is the wave velocity, n is the path loss fading exponent, and k is the subcarrier index, generally IEEE 802. An 11n standard commercial wireless network card can collect 30 subcarrier numbers, where kth subcarrier. kth subcarrier CSI amplitude. Considering the variable offset that may exist in network training, layer normalization is performed on the Effective CSI is used to compensate for small-scale fading effects, which have a nonlinear relationship with physical distance. In FILA, the effective CSI is related to the distance as follows:This paper considers two ways for the fusion of y is the true probability distribution. By calculating the loss of predicted results and actual results, the optimization direction of the model is guided.The decision module firstly normalizes each dimension of the fusion feature to speed up the convergence of the network. After that, the feature is passed through multiple modules of fully connected layers and nonlinear layers to fit the position of the fingerprint points. For the final position output, the network uses a cross-entropy loss function to guide the network optimization; the formula is:In order to reduce the tedious manual parameter tuning, we choose the Hyperopt tool for automatic hyperparameter selection. Hyperopt uses a form of Bayesian optimization for parameter tuning that allows the user to obtain the best parameters for a given model. Therefore, we define the search space of parameters, where the batch-size search space is {16, 32, 64, 128}, nhead is {2, 3, 4, 5, 6}, and lr is {0.001, 0.0025, 0.0005, 0.00075}. Then, we use Hyperopt for Bayesian optimization to output an optimal positioning network.The positioning stability and accuracy of MHSA-EC are tested in two typical indoor scenes: comprehensive office and laboratory. The comprehensive office scene is larger and has more points, which is conducive to the stability of the test model and the maximum positioning error. In the laboratory, the overall area is small but contains rich multi-path information, so the positioning results here can reflect the model’s ability to extract features. On the acquisition device, both the transmitter and receiver are mobile devices equipped with an Intel 5300 NIC Terminal. Each terminal uses the Ubuntu 16.04 system. At the receiver, fine-grained CSI data are parsed by modifying the driver. The received packets include time stamp, RSSI, number of antennas, noise, CSI, etc.The data set size is 232,000, of which 70% is the training set, 20% is the test set, and 10% is the validation set. There are specific parameters in As shown in As shown in We determine the number of layers of the multi-head self-attention (MHSA) mechanism by comparing the experimental methods. Considering that the amount of data in the antenna branch and the subcarrier branch are the same, we keep the same number of layers on the two branches. The experiments were carried out in the comprehensive office scene. The results are shown in We investigate from the two experimental indicators, the mean absolute error (MAE) and the standard deviation (STD), and it can be seen that the accuracy and stability of the positioning increase significantly with the increase of the number of layers. After continuing to the fourth layer, the increase in the number of layers is not obvious. Based on the comprehensive consideration of model complexity and positioning time, we set the number of multi-head attention layers to three layers.After fixing the network parameters, we study the fusion method of the effective CSI information and features. The experimental scene is also in the comprehensive office. We compared the fusion methods of concatenation and summation. The results are as follows: as can be seen from We compare MHSA-EC with MHSA without effective CSI to show the rithm in  and the rithm in  system. In terms of localization results, MHSA-EC achieves the best localization results due to the introduction of effective CSI. The performance of 1dCNN-CSI and Confi are almost the same. The confidence of 1dCNN-CSI is higher in the range of 0–2 m, but the performance of Confi is better in the range of 8.2–10 m. Although the MHSA network is weaker than 1dCNN-CSI in the range of 3.8–4 m, the overall positioning error is better than the other two mainstream model structures. When the probability reaches 1.0, the positioning error of MHSA-EC is 7.23 m and MHSA is 8.12 m, while Confi is 9.36 m and 1dCNN-CSI is 9.53 m.The result of the comprehensive office scene is shown in the following As shown in In addition, we calculated the average positioning error and positioning standard deviation of the positioning system in different scenes to show the accuracy and stability of the positioning. The results are shown in Experiments show that the MHSA-EC method outperforms other existing methods in localization accuracy. The introduction of the MHSA layer and effective CSI is of great help to the localization effect.The ability to extract CSI amplitude features has a great impact on CSI-based positioning systems. This paper analyzes the difficulty of aggregating long-distance subcarriers in traditional convolutional networks and the problems of long-distance point mismatches. Based on these questions, this paper proposed an indoor localization algorithm fusing the multi-head self-attention mechanism and effective CSI. The algorithm architecture consists of a feature extraction module, a statistical module, a fusion, and a location decision module.First of all, the multi-head self-attention layer is used in feature extraction to achieve a better aggregation effect for features with farther distances. The purpose of this is to better mine the location information contained in the multi-path effect.At the same time, effective CSI is introduced to provide distance constraint information for the location decision module. Finally, the two kinds of information are integrated through the fusion module, and the final position coordinates are output by the decision module. MHSA-EC ensures the stability and accuracy of positioning. The average positioning error in the comprehensive office is 0.71 m, and the average positioning error in the laboratory is 0.64 m.However, this paper only explores the CSI amplitude and does not introduce other signals. Compared with a single signal source, multi-source signals have more abundant location features and can provide complementary information to each other to ensure the accuracy and stability of the positioning algorithm. Therefore, other signal features can be fused with the CSI features extracted by the MHSA-EC algorithm in the future to improve the accuracy and stability of the positioning algorithm.


The air void system purposely introduced by an air-entraining admixture (AEA) is of great significance for the protection of concrete from freeze–thaw damage. Fly ash has been globally used in concrete, while the unburnt carbon in fly ash can adsorb AEA molecules and, thus, increase the AEA demand. Previous studies primarily focused on the air content of fresh fly ash concrete. This paper aimed to explore the stability and distribution of air voids in fly ash concrete at the fresh state. To achieve this goal, eleven different fresh fly ash concrete mixtures with an initial air content of 6 ± 1% were prepared in the laboratory. Samples were taken at various times within 75 min after initial mixing to investigate the air content and air void distribution in fly ash concrete at the fresh state using a super air meter (SAM). The results indicated that there was no significant correlation between loss on ignition (LOI) of fly ash and AEA demand to achieve the initial air content of 6 ± 1%. Class C fly ash concrete tended to have a better air content retention than Class F fly ash concrete. Compared with LOI, AEA demand had a stronger correlation with air content retention. Most of the fly ash concrete mixtures had a satisfactory air void system immediately after mixing, but the SAM number showed an increasing trend over time, suggesting the coarsening of the air void system with time. Concrete is an artificial construction material mainly comprising cementitious materials, aggregate, water, and chemical admixtures in appropriate proportions. Concrete has been widely used in various buildings and infrastructure worldwide. However, as a typical porous media material, concrete is usually prone to deterioration under harsh environmental conditions. For example, due to water invasion, concrete is susceptible to freeze–thaw damage in cold regions. To reduce the freeze–thaw deterioration, the most commonly adopted method in the world is to use air-entrained concrete according to the theoretical hypothesis that the purposely introduced air voids can provide extra space to decrease expansion pressure induced by freezing water ,2.Various test methods have been employed to determine the air void system of concrete. For fresh concrete, some common tests mainly include the ASTM C231  gravimetTo address this need, in the early 1990s, the air void analyzer (AVA) was invented to obtain the distribution of air voids in fresh concrete based on Stoke’s law . The AVAMore recently, a new test device named the super air meter (SAM) was invented to obtain the air void distribution in fresh concrete . The SAMFly ash, a byproduct of coal ignition, has been globally used in concrete over many years considering economic, environmental, and technological benefits. For example, adding fly ash to concrete can reduce the carbon footprint of cement production, and decrease the cement hydration heat in early ages and, thus, the thermal-cracking probability . FurtherThe alkali content in cement usually affects the air void stability in concrete. For example, Dubovoy et al.  reportedHowever, previous studies mainly investigated the air content of fresh fly ash concrete, while the air void stability and distribution have seldom been reported. To this end, this paper aimed to explore the stability and distribution of air voids in fresh fly ash concrete. Eleven types of fly ash from different sources meeting ASTM C618  were use2O + 0.658 K2O) of this study was 0.53% based on A Type I cement meeting ASTM C150  and elevA total of eleven fly ash concrete mixtures were produced in the laboratory. Concrete ingredients including cement, fly ash, coarse and fine aggregate, and tap water with SP and AEA were mixed in an 85 L drum mixer as per ASTM C192  in the l1 at this time was recorded. Both petcocks were then opened and the water was added through one petcock to the bottom chamber until the air-free water flowed out of the other petcock. After that, the second sequential pressures of 14.5, 30, and 45 psi were also applied and the equilibrium between the top and bottom chambers was reached under each pressure. Finally, the equilibrium pressure P2 after finishing the pressure of 45 psi was recorded. The SAM number is calculated as the difference between P1 and P2 (SAM number = P2 − P1). Both air content and SAM number were automatically displayed on the digital gauge. More specific details can be found in the AASHTO TP 118 [Immediately after mixing (0 min), the slump of fresh mixtures was measured as per ASTM C143 . MeanwhiO TP 118 . The calO TP 118 , which wO TP 118 ,26. BothO TP 118 .To analyze the air void stability and distribution in fly ash concrete at the fresh state, both air content and SAM number were measured by the SAM at 25 ± 2 min, 50 ± 2 min, and 75 ± 2 min. To simulate the agitation of fresh concrete in the mixer truck during transportation in practice, the concrete mixtures were mixed for 3 min and then rested for 7 min every 10 min. This method was the same as that adopted by Khayat et al.  to studyThe needed AEA dosage to obtain an initial air content of 6 ± 1% for all the fresh fly ash concrete mixtures is listed in According to SAM tests, the variation in air content and SAM number of fresh fly ash concrete over time after initial mixing was obtained. The air content retention in fresh concrete, defined as the percentage of air content at different times relative to the air content at 0 min, is indicated in As seen in Comparing 2 presented in The correlation between air content retention of fresh fly ash concrete at 25, 50, and 75 min and AEA demand is plotted in The variation in the SAM number of fresh concrete over sampling time is shown in Overall, the SAM test results of this study showed that there was no special difficulty in producing fly ash concrete with a satisfactory air void system right after mixing, which is consistent with previous studies based on the ASTM C457 test . In addi(1)There was no significant correlation between the LOI of fly ash and the AEA requirement for the initial air content of 6 ± 1%.(2)Overall, Class F fly ash concrete had a worse air content retention than Class C fly ash concrete. Compared with LOI, AEA demand had a stronger correlation with air content retention.(3)Most of the fly ash concrete mixtures had an SAM number below 0.2 right after mixing, indicating that it was feasible to produce fly ash concrete with a high-quality air void system, but the SAM number showed an increasing trend with sampling time, which indicated the coarsening of the air void system. Thus, sampling time had a relatively large effect on measuring air content and SAM number for fly ash concrete. The air content and SAM number obtained right after mixing were not necessarily the same as the measured results after 75 min.(4)The correlation between air content in fresh concrete and SAM number showed that increasing the air content could reduce the SAM number and, thus, achieve a lower spacing of air voids.In this study, eleven types of fly ash (five Class C and six class F) were utilized to replace 20% of cement by mass in concrete. Then, eleven fresh fly ash concrete mixtures with an initial air content of 6 ± 1% were produced in the laboratory. The stability and distribution of air voids in fly ash concrete at the fresh state were investigated by a super air meter (SAM). The following main conclusions could be drawn:This study presents a laboratory investigation into the air void stability and distribution in fly ash concrete at the fresh state. It is worthwhile to point out that the air void characteristics in concrete are usually affected by many factors, including types of air-entraining admixture and superplasticizer, alkali content in cement, water–cementitious materials ratio, rheological properties , mixer type, mixing time, pumping, consolidation, placement, and finishing. Therefore, more laboratory tests and field tests on air-entrained fly ash concrete are needed to further verify if similar results will be obtained.


Manufactured sand is one of the effective ways to alleviate the extreme shortage of natural sand in the construction industry. This paper uses granite and limestone manufactured sand to study the effect of high fines content on the durability of high-strength manufactured sand concrete, and analyzes its influence mechanism by combining macro and micro test methods. The results show that the carbonation depth of manufactured sand concrete is the smallest when the fines content is 10%. When the fines content is less than 15%, the chloride and sulfate impermeability of concrete are improved effectively. Through macroscopic and microscopic tests, it is found that the main reason why fines can improve the durability of concrete is the filling effect. Too much fines will inhibit the hydration of cement and adversely affect the durability of concrete. Therefore, the fines content of high-strength manufactured sand concrete should be controlled within 5~15%, and the durability is the best when the fines content is 10%. They are the results of a series of ecological and environmental problems caused by excessive exploitation of non-renewable resources [At present, sustainable development, energy conservation and COesources . River sesources ,4,5. TheManufactured sand is produced by crushing rocks, screening, washing, and other processes . The manMost of the excess fines in manufactured sand is removed by washing with water, but this method of processing fines wastes water resources and increases production costs. Moreover, during the process of washing, not only fines but also particles of 0.15 mm, 0.3 mm, and 0.6 mm or even larger are removed, which is harmful to the packing of the manufactured sand . The lacThe increase of fines content is conducive to the improvement of concrete strength, but with the further increase of fines content, it is not conducive to the development of mechanical properties of manufactured sand concrete when it exceeds the reasonable range . The resAt present, many studies have been done on the effect of fines content on the fluidity, mechanical properties, and durability of manufactured sand concrete ,27,28. HShijing brand P.O42.5R ordinary Portland cement is used. Type C fly ash from Foshan is used, whose chemical composition is shown in The river sand (RS), granite manufactured sand (GMS), and limestone manufactured sand (LMS) are produced locally in Foshan China. They meet the standard of zone II sand, and their fineness moduli are 2.6, 3.0, and 2.7 respectively. The three gradations of sand are shown in According to the Chinese standard , the carThe electric flux test method is carried out according to Chinese standard GB/T 50082-2009. In this specification, it is recommended that concrete containing a large amount of mineral admixtures can be tested for electric flux at 56 days. Generally speaking, the longer the curing time of concrete, the stronger its permeability resistance. Due to the high content of fly ash in the test ratio, hydration takes a long time to complete. Because the fly ash with a large content in the test ratio takes a long time to hydrate completely, the electric flux tests with ages of 28 days, 56 days, and 90 days are carried out to study the influence of the content of fines on the chloride ion resistance of concrete at the early stage and after hydration. The specimen is a cylinder with a diameter of 100 ± 1 mm and a height of 50 ± 2 mm. 2SO4 solution was used in the test. Na2SO4 was anhydrous sodium sulfate produced by Tianjin Zhiyuan Chemical Reagent Co., Ltd., China. In order to keep the concentration of the solution unchanged, the solution was changed regularly. During the test, the solution was always kept 20 mm higher than the surface of the specimen and the water injection height of the liquid level was kept unchanged.Sulfate continuous immersion test was carried out on concrete samples with age of 30 d, 60 d, 120 d, and 150 d. The size of the sample is 100 mm × 100 mm × 100 mm cube, 3 pieces for each mix. A 5% NaThe compressive strengths of concrete are carried out in accordance with the Chinese standard GB/T 50081-2019 “Test Methods for Physical and Mechanical Properties of Concrete” .The maximum solid concentration of the solid mix comprised of cement, fly ash, fines, fine aggregate, and coarse aggregate with varying amounts of water is taken as the packing density. In this test, the wet packing method is used to measure the packing density of concrete ,33.MIP test can analyze the pore structure inside the hardened concrete. The test uses AutoPore IV 9510 automatic mercury porosimeter, the maximum pressure of the mercury porosimeter is set to 413.69 MPa, and the test range of the aperture is 0.003–1000 μm. The fragments with a size of about 10 mm of the broken specimen at 28 d age after standard curing are taken. Then they are dried in a blast drying oven at 40 °C for 24 h before testing.SEM was used to observe the transition zone of concrete interface and the micro-morphology of hydration products. The instrument is an S-4800 electron microscope produced by Hitachi, Japan. The SEM sample is produced as follows: the concrete specimen is cured for 28 days, and the fragments with a size of about 10 mm inside the specimen are taken after the specimen is broken. The fragments are soaked for 24 h to stop hydration, and finally baked in a blast drying oven at 40 °C for 24 h.2 gas, and the heating rate is 10 °C/min. The instrument model is STA 409 PC synchronous thermal analyzer .TGA is to analyze the changes of substances at different temperatures. According to the weight loss of the substances, it is possible to calculate how much substances are lost. The mass of the test sample is about 20 mg. The temperature rises from 25 °C to 800 °C under the blowing of N3, and the sand to total aggregate ratio was set constant to 44%. According to the actual construction needs, the slump value was set to be 200 ± 20 mm. As can be seen from the water reducer dosage results, a higher fines content results in a lower workability and requires a higher water reducer dosage. With a gradient of 5%, manufactured sands with fines contents of 0%, 5%, 10%, 15%, and 20% were blended to produce concrete samples. A river sand control mix was adopted to study the effect of different fines contents on the performance of C60 manufactured sand concrete after carbonization. The mix proportion is shown in In the test, the water-binder ratio was set constant to 0.3, the total amount of cementitious materials was set constant to 500 kg/mThe carbonation depth results are listed in The carbonation depth of all the mixes in the test is not large. According to Chinese standard “Concrete Durability Inspection and Evaluation Standard” , the sta2 and H2O from entering the concrete, thus improving the carbonization resistance.With the change of fines content, the change of carbonization depth of GMS concrete at different carbonization ages is shown in 2 and H2O enter the concrete from the cracks, resulting in deeper carbonization of concrete. Therefore, under the conditions of the same fines content and the same carbonization environment, the carbonation depth of LMS concrete is larger than that of GMS concrete. The limestone fines plays a filling role in the concrete, which lowers the porosity and increases packing density of the concrete, by which the carbonization resistance of the concrete can be enhanced [It can be seen from enhanced . HoweverAs can be seen from The fact that fines can reduce the electric flux of concrete is also due to the filling effect, which increases the packing density and effectively reduce the amount of chloride ions entering the concrete, thereby improving the resistance to chloride ion penetration of concrete. However, when the fines content is too high, it may adversely affect the resistance to chloride ion of concrete. This is because the volumetric ratio of cement to fines in the concrete decreases, and there are more than enough fines to fill into the voids between cement grain. The packing density is lowered, which results in a decrease in the chloride ion resistance of concrete. Due to the weak conductivity and higher packing density of GMS, the resistance to chloride ion penetration of GMS concrete is higher than that of LMS concrete.When the sulfate concentration in the environment where the concrete is located is higher than the solubility of sulfate itself, crystallization will occur. This causes the expansion of concrete several times, and then causes the concrete to crack and break. In this study, a traditional method was used to investigate the effect of different fines contents on high-strength manufactured sand concrete. First, the change of compressive strength under continuous immersion at 30 d, 60 d, 90 d, and 120 d age was studied. The test results are shown in It can be found that the compressive strength of manufactured sand concrete with different fines content is improved to a certain extent when immersed continuously for 60 days. This is because the strength development with curing age is more significant than the effect of sulfate attack at early age. However, as the immersion continued, the compressive strength began to decline, that is to say, immersing the manufactured sand concrete for more than 60 days would have an adverse effect on the concrete. When the fines content is 10%, the compressive strength of GMS concrete has a maximum value at each immersion age, and the compressive strength reaches 89.2 MPa when immersed for 60 days. The effects of fines content and immersion age have similar effects on the compressive strength. The highest compressive strength is 93.2 MPa when the fines content is 15% and immersion age is 60 days.The change of the compressive strength value of concrete after soaking and standard curing at the same age was studied. It can be seen from It can be seen that the compressive strength of LMS concrete has the similar change as that of GMS concrete at the same age of 90 d and 180 d. The difference is that at the age of 90 days, the reduction ratio of the compressive strength of the LMS concrete with different fines contents is within 2%. At the age of 180 days, the reduction ratio of compressive strength of each mix was more than 17%; the reduction ratios of compressive strength of manufactured sand concrete fines content with 15% and 20% fines content were as high as 24.02% and 20.44%, respectively.42− enters the concrete during the soaking period, and chemically reacts with the cement hydration products to form expansive products such as gypsum and salt crystals, which optimizes the pore structure inside the concrete, thereby improving the compressive strength of the concrete [42− to invade the concrete. Therefore, after the immersion to the later stage, the dense structure inside the concrete is damaged and then the compressive strength decreases [There are two main reasons for the increase of the compressive strength of the two types of manufactured sand concretes with different fines contents after being soaked in sulfate for 60 days. On the one hand, SOconcrete . On the ecreases . An apprThe packing density test was carried out according to the method described above, and the test results are shown in Three mixes with fines content of 0%, 10%, and 20% were selected for 28 d pure slurry MIP test to further study the effect of fines content on the internal pore structure and compactness of concrete, so as to explain the influence mechanism of fines content on the durability of high-strength manufactured sand concrete. In order to study the effect of fines content on the interface between aggregate and cement of high-strength manufactured sand concrete, six mixes with fines content of 0%, 10%, and 20% in GMS and LMS concrete were selected for 1500× magnification scanning electron microscope observation. 2 (CH) content was quantitatively analyzed by thermogravimetric method. From 2O). The calculation result is shown in 2 and AlO3 as the main components will not react with cement and hydration products. However, CaCO3 in limestone fines will react with C3A in cement [In order to study the effect of fines content on cement hydration of high-strength manufactured sand concrete, the Ca(OH)n cement . This won cement , who shoThis paper studies the influence of fines on the durability of high strength manufactured sand concrete such as carbonation resistance, chloride resistance, and sulfate resistance. The macroscopic test method including packing density tests and the microscopic test method including MIP, SEM, and TGA are used for mechanism analysis. The main conclusions are as follows:When the fines content is 5~15%, the high strength manufactured sand concrete has good carbonization resistance. When the fines content is 10%, the carbonization depth is the smallest and the carbonization resistance is the strongest. Compared with LMS concrete, GMS concrete has better carbonation resistance.When the fines content is less than 15%, the electric flux value of the two kinds of manufactured sand concrete decreases with the increase of the fines, and the chloride ion resistance is improved. However, when the fines content exceeds 15%, the chloride ion resistance of manufactured sand concrete will be adversely affected.The compressive strength of GMS and LMS concrete increases after continuous sulfate soaking for 60 days. After 60 days, the compressive strength decreased gradually with the increase of soaking age. At 90 days, the compressive strength of concrete soaked in sulfate is slightly lower than that under standard curing conditions. When the fines content is larger than 15%, the reduction rate of compressive strength of GMS and LMS concrete is larger than 20% after soaking sulfate and standard curing for 180 days.The main reason why fines improve the durability of concrete is that the fines plays a filling role in concrete. Appropriate fines content can increase the packing density of concrete, increase the proportion of harmless holes and less harmful holes, and improve the interface bonding between aggregate and cement.This study demonstrated that an appropriate amount of fines can effectively improve the durability of the manufactured sand concrete, and 10% is the optimal fines content for the highest durability. When the requirements are not high, the fines content can be relaxed to 15%, but it is better not to exceed 15%.


Methionine restriction and selenium supplementation are recommended because of their health benefits. As a major nutrient form in selenium supplementation, selenomethionine shares a similar biological process to its analog methionine. However, the outcome of selenomethionine supplementation under different methionine statuses and the interplay between these two nutrients remain unclear. Therefore, this study explored the metabolic effects and selenium utilization in HepG2 cells supplemented with selenomethionine under deprived, adequate, and abundant methionine supply conditions by using nuclear magnetic resonance-based metabolomic and molecular biological approaches. Results revealed that selenomethionine promoted the proliferation of HepG2 cells, the transcription of selenoproteins, and the production of most amino acids while decreasing the levels of creatine, aspartate, and nucleoside diphosphate sugar regardless of methionine supply. Selenomethionine substantially disturbed the tricarboxylic acid cycle and choline metabolism in cells under a methionine shortage. With increasing methionine supply, the metabolic disturbance was alleviated, except for changes in lactate, glycine, citrate, and hypoxanthine. The markable selenium accumulation and choline decrease in the cells under methionine shortage imply the potential risk of selenomethionine supplementation. This work revealed the biological effects of selenomethionine under different methionine supply conditions. This study may serve as a guide for controlling methionine and selenomethionine levels in dietary intake. Selenium has been considered an essential trace element for humans since the late 1950s ,3. HowevAs a major selenium species in human dietary sources, selenomethionine (SeMet) is produced in plants and fungi and abundant in certain selenium-enriched protein products. Compared with inorganic selenium, SeMet is more recommendable in dietary selenium supplementation . SeMet i3-Se group of SeMet is more hydrophobic than the CH3-S group of Met [Following selenocysteine (Sec), SeMet is the second most discussed selenium species in proteins and enzymes; it is inserted into human proteins via the random substitution of methionine and gene coding . Physiolp of Met . CertainFew studies have explored the interplay between SeMet and Met as well as their biopotencies under various relative amounts. Based on a limited number of studies, the incorporation of SeMet in JNK stimulatory phosphatase-1 in vitro would not significantly affect the expression and activity of this enzyme . In greeIn recent decades, the MR diet has been widely recommended because of its health benefits, such as repressing cancer growth , improviWith the rapid development of omics technology in the past decades, exploring the systematic biological effect of SeMet at the molecular level has become possible. In this work, the effects of SeMet supplementation on different Met supply levels were mainly obtained with the aid of NMR-based metabolomics, which is endowed with powerful reproducibility, quantitative ability, and capability for identifying compounds and deducing structures of unknowns . HepG2 c2PO4·2H2O, K2HPO4·3H2O and methanol  were obtained from Sinopharm Chemical Reagent Co., Ltd. . D2O and 3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP-d4) were obtained from Cambridge Isotope Laboratories .L-selenomethionine and L-methionine were obtained from Sigma-Aldrich . NaHHepG2 cells were obtained from ATCC and cultured in regular Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS) . After cells in culture dishes reached 40–50% confluence, the regular medium was replaced with different DMEM culture mediums listed below, randomly.Met-free DMEM was customized at Boster Biological Technology Co., Ltd. . All treatments were prepared by adding different amounts of Met and SeMet into Met-free DMEM with 10% FBS. The contents of both free Met and total Met in FBS were quantitated by the national standard method (GB 5009.124-2016) with an automatic amino acid analyzer . Correspondingly, SeMet or Met, 200 μM SeMet, 200 μM Met, 200 μM SeMet and 200 μM Met, 400 μM Met, and 400 μM SeMet and 200 μM Met were supplemented in the media of the MR, MR_SeMet, control (its ingredients are the same as the standard DMEM), control_SeMet, Met, and Met_SeMet groups, respectively. For cellular metabolites and total selenium analysis, cells were washed three times with phosphate buffer (pH 7.4) to remove the excess medium after being exposed for 48 h. Phosphate buffer was discarded after centrifugation, and the cell pellets were stored in a −80 °C refrigerator  before use.After cells were seeded in 96-well plates with regular DEME medium for 12 h, the mediums were replaced with that containing different concentrations of Met and SeMet. The viability of HepG2 cells exposed to different SeMet and Met concentrations for 12, 24, and 48 h was measured using the MTT Cell Proliferation and Cytotoxicity Assay Kit  in accordance with the manufacturer’s instructions. Each treatment was performed with three biological replicates.3 in a Microwave Digestion System . After being diluted to 2% HNO3 solution, the total selenium concentrations in the cells were measured through inductively coupled plasma mass spectrometry  using external calibration. The operational parameters of ICP-MS were as follows: nebulizer Ar gas, 0.95 L/min; auxiliary Ar gas, 1.2 L/min; plasma Ar gas, 15 L/min; RF power, 1400 W; dwell time, 50 ms; and lens voltage, 7.2 V. Each treatment was performed with three biological replicates.For the determination of total selenium, HepG2 cells were digested with concentrated HNO2O (2:1 v/v) solution and subjected to three freeze–thaw cycles. Sonication was performed at 4 °C for 15 min, with the repeat cycle of 1 min sonication and 1 min pause. The supernatant was collected after centrifugation at 12,000× g for 10 min, and the remaining cell residues were further extracted twice using the above procedure. The supernatant from the three extractions was pooled together and lyophilized after methanol removal using a SpeedVac . The cell extracts were dissolved in the phosphate buffer  containing 0.001% TSP-d4. After centrifugation at 16,000× g for 10 min at 4 °C, the supernatants were transferred into 5 mm NMR tubes for NMR analysis.Cell pellets were homogenized in 600 μL of methanol/H1H NMR spectra of cell extracts were acquired using noesygppr1d sequence on a Bruker Avance III 800 MHz NMR spectrometer equipped with a cryogenic probe . Water presaturation was achieved by irradiation of water resonance. All 1H NMR spectra were multiplied by an exponential function with a line broadening factor before conducting the Fourier transformation. The spectra were referenced to the methyl resonance signal (δ 0.000) of TSP. For the signal assignment, 2D NMR spectra, including 1H-1H correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) and J-resolved spectra, 1H-13C heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC), were acquired under previously described parameters and processing information [ormation .The spectral region δ 0.5–9.5 was integrated into regions with an equal width of 0.004 ppm using an AMIX software package . Region δ 4.606–5.166 was discarded to avoid the effect of water saturation. Each integral bucket was then normalized to the sum of the total integrals prior to statistical analysis.p < 0.05).Multivariate data analyses were performed on SIMCA-P+ software . Principal component analysis (PCA) was first conducted on the mean-centered NMR data to generate an overview of the data distribution and detect potential outliers. Orthogonal projection to latent structure with discriminant analysis (OPLS-DA) was subsequently applied to the unit variance scaled data . The quap < 0.05).The model was interpreted by back-transformed loadings incorporated with color-coded correlation coefficients with Matlab script . The color of the correlation coefficient indicates the contribution of the metabolite to class differentiation. In this study, a correlation coefficient |r| greater than 0.755 was considered to indicate statistical significance . The primers for six selenoprotein genes  and the reference gene (GAPDH) are listed in GAPDH and was determined using the Delta Ct method [Cells were harvested after 48 h of incubation, and total RNA was extracted with Trizol  in accordance with the manufacturer’s protocol. Reverse transcription was performed on the ABI 7900HT system  using a First-Strand cDNA Synthesis kit . Quantitative RT-PCR was performed in triplicate using SYBRt method . Each trg for 10 min, and the supernatant was used for the determination of total glutathione. For the determination of GSSG, a GSH clearance reagent was added to the samples prepared above, and the vortex was conducted immediately. Then 4 µL of GSH scavenging reagent per 100 µL of the sample was added to the solution. The reaction was performed at 25 °C for 60 min. The absorbance values of this solution at 412 nm were measured by a microplate reader after adding the total glutathione assay working solution.Cellular reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were measured using GSH and GSSG assay kits  in accordance with the manufacturer’s instructions. Cells for kit assay were washed with PBS and collected by centrifugation. Precipitated protein removal reagent M was added to cells; samples were then subjected to two rapid freeze–thawing processes using liquid nitrogen and a 37 °C water bath. After an ice bath for 5 min, cells were centrifuged at 10,000× g for 10 min at 4 °C, and the supernatant (S1) was used for supernatant (S2) preparation and total NADP+ and NADPH determination. An amount of 100 µL of the S1 was heated in a water bath at 60 °C for 30 min to decompose NADP+. After centrifugation for 5 min, the S2 in the tube was transferred for NADPH determination. An amount of 50 μL of supernatant S1 and S2 was transferred into a 96-well plate as the samples to determine total NADP+ and NADPH, and NADPH, respectively. WST-8-based colorimetric detection for NADP+ and NADPH quantitation was applied in this method [Oxidized nicotinamide adenine dinucleotide phosphate (NADP+) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were determined using their assay kits  in accordance with the manufacturer’s instructions. NADP+ and NADPH in cells were extracted using the extract solution in the kit. Centrifugation was performed at 12,000× s method . Both GSp < 0.05.Statistical analysis was performed using Graph Pad Prism 8.0 software . Results were analyzed using Student’s t-test or one-way ANOVA, and differences between the two means were considered statistically significant at Despite the 10% FBS contributing some free Met at a concentration of 4.70 μM in the medium, the viability of the HepG2 cells in the MR group was highly restrained. Their proliferation rate was slower than those of the cells under SeMet and Met supplementation . The sup1H NMR spectra of HepG2 extracts  by using an NMR-based metabolomic approach. A total of 37 intracellular metabolites were identified in two typical extracts , the sigextracts . Among tPCA was performed using the integral matrices of normalized NMR signals to generate an overview of the metabolic differences between all samples. Component 1 accounting for 62.9% of the variation and component 2 accounting for 23.5% of the variation helped further distinguish the sample groups. The score plot based on the first two principal components of the PCA model  showed tp-value of CV-ANOVA.OPLS-DA models that can filter out irrelevant orthogonal signals were established to assess the metabolic responses of HepG2 cells to SeMet supplementation under restricted, adequate, and abundant Met conditions. In the OPLS-DA models, pairwise comparisons between NMR profiles of cells incubated with an additional 200 μM SeMet and those incubated with 0 μM Met (MR group), 200 μM Met (control group), and 400 μM Met (Met group) were carried out, respectively. The overall trends were similar to those observed in the PCA model but more obvious in the OPLS-DA score plots A,C,E. ThThe loading plots of OPLS-DA constructed between the MR and MR_SeMet groups  revealedAfter being exposed to SeMet under different Met supply conditions for 48 h, the total selenium content of HepG2 cells was determined using ICP-MS to further explore the uptake and metabolism efficiency of SeMet under multiple Met supply conditions. As shown in GPX1, GPX2, GPX4, TXNRD1, TXNRD2, and SELENOP. Results showed that SeMet supplementation at 200 μM promoted the transcriptional activities of selenoprotein genes in the HepG2 cells. However, MR inhibited the effect of selenoprotein transcription when compared with the other groups. The transcription levels of the observed selenoprotein genes remained stable under adequate or abundant Met supply. Furthermore, SeMet supplementation with Met depletion affected the transcription of GPX1, GPX2, TXNRD1, and TXNRD2 within certain limits.For intracellular biological activities involved in selenium utilization, the relative transcription levels of several highly expressed selenoprotein genes in the liver cells were monitored using qRT-PCR. TXNRD1 and TXNRD2 gene transcription.GSH, GSSG, NADP+, and NADPH are important substrates that participate in biochemical reactions catalyzed by selenoenzymes of glutathione peroxidases (GPXs) and thioredoxin reductases (TRXs). GSH/GSSG and NADP+/NADPH levels were measured using commercialized kits to verify the enzymatic efficiency of GPXs and TRXs, and characterize the redox status of HepG2 cells under different SeMet and Met supply conditions. As illustrated in SeMet can be incorporated into proteins in place of Met . In a MeA metabolomic approach was employed to evaluate the impact of SeMet supplementation on cell metabolic phenotypes under different Met supply conditions. Given the cluster distributions in Compared with adequate and abundant Met supply, SeMet supplementation under Met depletion caused the most significant disturbance in metabolism pathways, including but not limited to the TCA cycle, glutamine metabolism, and choline metabolism. The amount of intermediates in the TCA cycle was higher in the cells with SeMet supplementation under Met depletion than in the other groups. The transamination–decarboxylation possibly became hyperactive without the Met buffering, and alpha-ketobutyrate possibly flowed into the TCA cycle through propanoate metabolism. Along with phosphocholine, GPC is a choline derivative and one of the two major forms of choline storage in the cytosol. A significant decline in choline and GPC levels has been suggested as a signal implying the defect of SeMet supplementation under MR because choline is essential in maintaining membrane structural integrity and promoting transmethylation metabolism and fatty acid metabolism . CholineIn the present study, the impact of SeMet on lactate, glycine, citrate, and hypoxanthine levels could be attributed to SeMet supplementation under adequate and abundant Met supply. Lactate is a hallmark of the Warburg effect; its decline is a positive signal in cancer therapy because a high level of lactate could act as an oncometabolite, immunosuppressant, and metabotoxin . LactateThe uptake of selenium is highly affected by cell lines, selenium species, and concentrations ,49. As sGPX1, GPX2, GPX3, TXNRD1, TXNRD2, and SELENOP was upregulated with SeMet supplementation. However, the expression of these selenoproteins decreased in the MR group, and SeMet supplementation in this condition hardly elevated the expression of GPX1, GPX2, TXNRD1, and TXNRD2 to the same levels as the cells supplied with adequate Met. Combined with the results of the metabolomic study, the gap in post-transcriptional activity between SeMet supplementation under MR and sufficient Met conditions was assumed to be the consequence of a certain Sec shortage because more SeMet was required in supporting cellular metabolism in the absence of Met. Hypoxanthine, which is a prevalent metabolite in the purine salvage pathway, stabilizes the RNA folding structure of riboswitches, thereby providing a mechanism for regulating gene expression in response to an increase in intracellular concentrations of the metabolite [Aside from participating in cellular metabolism, selenium is also translated into selenoproteins or selenoenzymes that are catalytically active in redox processes. To clarify the potential correlation of redox status with SeMet supplementation under different Met supply, a series of selenoprotein genes were investigated because of their overwhelming roles in the oxidant defense system and selenium transportation. Enhanced selenoprotein expression could be observed in virous biological models with selenium supplementation. In the present study, the expression of tabolite . The incGSH/GSSG and NADP(+)/NADPH are substrates that participate in the reactions catalyzed by selenoenzymes GPXs and TRXs, respectively. These ratios are reliable markers for evaluating biochemical parameters representative of the redox states . In accoNMR-based metabolomics, ICP-MS, and qRT-PCR were employed to unveil the metabolic phenotype, selenium uptake, and selenoprotein expressions of liver cells supplemented with SeMet under Met-deprived, adequate, and abundant conditions. SeMet supplementation elevated the levels of most amino acids and decreased the levels of creatine, Asp, and some nucleoside diphosphate sugar. SeMet supplementation under Met depletion caused remarkable selenium accumulation and the most significant disturbance in metabolites, implying a potential health risk. No toxicological effect was observed even after 48 h of treatment. Notably, the metabolic disturbance induced by SeMet declined with increasing Met supply, except for some changes in lactate, glycine, citrate, and hypoxanthine levels, which could be attributed to SeMet supplementation under adequate and abundant Met supply. The results of this work highlighted the differences in cellular response of SeMet supplementation affected by the Met status. This study could drive people’s awareness toward following the dietary intake amounts of Met and SeMet. Future studies could evaluate the toxicological effect of SeMet supplementation under MR conditions in animal models, and omics are recommended as the technique options.


This work surveyed how the Mach band effect was mentioned in the dental literature and provided a qualitative assessment of diagnostic relevance and interfaces covered. PubMed, Scopus, and Google Scholar were queried in mid-Jan 2022. The search string was (“mach band effect” OR “mach effect”) AND . All publications returned by the searches were screened. Exclusion criteria included irrelevance  and not written in English. Reference lists of returned publications were manually searched to identify potentially missed papers. For each included publication, the following parameters were recorded: any presentation of radiographic images showing a Mach band effect, direct investigation of the effect, relevance to which structural interfaces, diagnostic relevance, and in which parts of the publication Mach band effect was mentioned. Seventy-seven publications were included and analyzed. The majority of the publications mentioned the Mach band effect in the Discussion section about its diagnostic relevance to caries detection at the enamel-dentinal junction and the interface between restorative material and tooth structure. Eight of them presented radiographic images showing a Mach band effect. Three of them investigated the Mach band effect. Dental publications seldom covered the Mach band effect. When they covered it, most of them only mentioned it in the Discussion section without actually investigating it. The Mach band effect was first described by Austrian physicist Ernst Mach in 1865 . The appThe Mach band effect seems to be common and could occur in many clinical situations ,9. In thhttps://harzing.com/resources/publish-or-perish (accessed on 20 January 2022)). All three literature databases searched all fields of the publications , but Google Scholar yielded far more publications due to its broadest literature coverage as well as its ability to gain access to the full text of the publications  AND . To enable batch export, Google Scholar was queried with a software called Publish or Perish , whereas the others were review/opinion papers , dissertations , and academic books . The first publication could be traced back to 1983 by Berry Jr.  in the other teeth on the patient’s bitewings…” [“ewings…” Since the Mach band effect is an optical illusion, it is possible to “mask” or circumvent it. As Berry Jr.  suggesteTherefore, one clinically relevant aim of this work was to remind clinicians to be aware of the Mach band effect during radiographic image interpretation, which was not limited to resemblance to caries or marginal defect of a restoration, but also to a variety of scenarios listed above. Moreover, as Buckle et al.  explaineThis literature survey only considered English-language publications. Readers should be aware that the findings might not be readily applicable to the non-English literature.This literature survey identified fewer than a hundred dental publications that mentioned the Mach band effect. Very few of them presented radiographic images that illustrated the Mach band effect or actually investigated the Mach band effect. Instead, the majority of the publications mentioned the Mach band effect in the Discussion section about its diagnostic relevance to caries detection at the enamel-dentinal junction and the interface between restorative material and tooth structure. Beside restorative/cariology relevance, the Mach band effect was also mentioned with relevance to implant dentistry and endodontology. However, no study was found to adopt a masking technique to eliminate the Mach band effect as the observers interpreted the images, which has been suggested by earlier works ,28. Dent


Irrational prescription of antibiotics is an ongoing global public health concern, leading to antibiotic resistance. Understanding the prescribing pattern of antibiotics is important to tackling mal-prescription and antibiotic resistance. We aimed to investigate the pattern and factors affecting outpatients’ antibiotic prescribing by family physicians in Primary Health Care (PHC).A cross-sectional study was conducted in 19 PHC facilities in Alborz province. Prescribing pattern of antibiotics was evaluated among 1068 prescriptions by family physicians. Prescribing pattern of antibiotics included prescriptions containing antibiotics, the number of antibiotics per prescription, type, name of antibiotic, and mal-prescription. Multiple logistic regression analysis was used to estimate the adjusted odds ratios and 95% confidence intervals.P < 0.05). In 59.31% of antibiotic prescriptions at least one of the scientific criteria was not fulfilled. In the final analysis, after adjusting for the potential confounders, field experts of physicians , female sex , and winter season  were found associated factors with antibiotic prescribing.Overall, 57% of the prescriptions had ≥ 1 antibiotic and the average number of antibiotics per prescription was 1.27. Amoxicillin was the commonly prescribed antibiotic. There was a significant relationship between age, sex, type of health insurance, work experience of the physician, and seasons with antibiotic prescribing (The average number of antibiotics per prescription and the percentage of irrational prescriptions were relatively high in this study. There is need to improve antibiotic prescribing patterns among family physicians working in primary health care. Irrational use and mal-prescription of antibiotics are a major and ongoing global public health problem, leading to antibiotic resistance both in developing and developed countries that deserve more attention by health systems and policymakers , 2, 3. RIrrational prescribing of antibiotics and the prompt and constant spread of antimicrobial-resistant organisms are major threats to our ability to successfully treat most contagious diseases. In the lack of development of new generations of antibiotic drugs, appropriate use of current antibiotics is required to guarantee the long-term availability of effective therapy for microbial infections , 7, 8, 9In Iran, the Primary Health Care (PHC) is the first and nearest line of basic and essential health services which is provided by family physicians and other healthcare providers in the all cities and rural areas. Findings indicated that the irrational prescription of antibiotics and the prevalence of self-medication are leading to antibiotic resistance and side effects in Iran , 15. It Rational prescribing of antibiotic is crucial to tackling antibiotic resistance. Among PHC family physicians, the pattern and the associated factors of antibiotic prescribing are poorly understood in Iran, especially after expanding the family physicians plan to urban areas. We aimed to evaluate family physicians prescribing pattern of antibiotic to outpatients in primary health centers of the Alborz province, Iran.A cross-sectional study was conducted in 19 PHC facilities/community health centers from September 2018 to September 2019 in Alborz province, Iran. We evaluated the family physicians' antibiotic prescribing patterns. Prescribing patterns of antibiotics included prescriptions containing antibiotics, the number of antibiotics per prescription, name, form, type of antibiotic, and consumption method, and mal-prescriptions. Family physicians are the chief healthcare providers in the Iranian PHC. The Iranian health system is based on the referral system; the first level is primary level of health services to access all urban and rural populations provided by family physicians and other health service providers. Patients are referred to the second and third levels by family physicians when patients need special health services [We randomly selected family physicians' prescriptions from County PHC health centers. Given that all family physicians' drug prescriptions (paper) were archived, a total of 1068 prescriptions (267 prescriptions in each season) were randomly selected. All prescriptions were available, therefore in the sampling process, the unreadable or prescriptions with poor information were excluded and the next prescriptions were replaced. More details of the sampling process and the study method are presented in Fig. p = 0.50) [α = 0.05, 95% confidence interval and 20% compensate for poor and ineligible prescriptions and to provide an accurate estimation.The sample size was calculated based on the previous study in Iran ( = 0.50)  and by cIncorrect or mal-prescription of antibiotics was assessed based on four scientific criteria including a) dose per consumption, b) dose per day, c) duration of therapy and d) possible interaction with other antibiotics or drugs. The prescription was considered incorrect if one or more of the above criteria were not fulfilled. Assessment of the prescriptions was performed by a high expert pharmacist (more than 10 years’ experience) who trained for the study methods and he was not involved in the study or analysis of the outcome measure. The assessment was based on Martindale: The Complete Drug Reference. For this purpose, firstly the symptoms and prescribed antibiotics according to the type of diagnosis (severity of the disease) were extracted from the prescriptions and health records by the clinician and the pharmacist. To do this, after randomly selecting the prescriptions, the patient's medical record in detailed information related to that prescription was also reviewed and evaluated. To this end, the incorrect prescription was evaluated based on the four mentioned standard criteria.Based on the standard guidelines, each drug and disease has a standard dose per day, duration of therapy, interaction with other drugs or antibiotics in accordance with various ages, genders, weight and disease. The appropriateness of antibiotic prescriptions were assessed regarding the related guideline and the mentioned four criteria, then the incorrect prescriptions were identified for each prescription.Same number of 276 prescriptions were selected in each season of the year. The dose of all drugs and antibiotics per each consumption, day, duration, possible interaction based on the type of disease stage, and diagnosis were determined by the Complete Drug Reference. The dose of antibiotics (different format) calculated according to milligram (mg) for edible  drugs  and milliliter (ml) for suspension, ampoule and infusion drugs. The number of drops and the speed of liquid infusion were calculated with this formula: A checklist was used for data collection. In addition to the basic and demographic characteristics of family physicians and outpatients, variables and information such as the name and type of the prescribed antibiotics, form and usage method of drugs, consumption way based on the amount of use for each time, duration of the treatment course, times of use each day, possible interaction with antibiotics or drugs, rate of combination therapy, number and total price of drug items, type of physicians’ graduation (private or government universities), family physicians occupation/position, and the working experience (years) were extracted from prescriptions and medical records. The government medical universities are classified on 3 levels based on the annual scientific report of the Iranian Ministry of Health and Medical Education.p = 0.751) and experience of the physicians (p = 0.688). Chi-square (χ2) test was used to compare categorical variables, and the independent T-test was used for comparison of normal quantitative variables [p-value less than 0.2 were analyzed with multiple logistic regression using the backward stepwise method [P-value < 0.05 was considered significant in all of the tests.SPSS software  was used for data analysis. The Kolmogorov–Smirnov test was carried out for checking the normality of the variables such as age . Winter season was associated with an increased odds of antibiotic prescribing .Table Among all evaluated prescriptions, the majority of outpatients 640 (60.0%) were female, while the proportion of received antibiotics was more in male than female (63.3 vs. 52.5%). According to the type of health insurance, outpatients who had social security health insurance booklets had the majority of prescriptions (more than 50%). We found there is a relation between family physicians’ experience and antibiotic prescription. The work experience (≤ 2 years) of family physicians was associated with an increased probability of antibiotic prescription . The mean age of outpatients who received antibiotics was 32.4 ± 21.4 years. There was a statistically significant association between patients who had received and no received antibiotics (26.07 ± 19.3) and (40.7 ± 21.2), respectively  was higher than prescriptions without antibiotics . A relationship was found between price, number of drugs, and prescriptions with antibiotics  is similar to the result of an Indian study that reported 55%,  but thisThe present study indicated that the high field experience of family physicians, sex, and seasons are the most important factors associated with antibiotic prescribing in this County. Similar results were found by Safaeian et al. in Isfahan Province of Iran . Yet, thThe average rate of prescribed drug number in this study was 3.47 per prescription, this finding is almost in agreement with the Iranian protocol of family physicians' average prescriptions which is 3.5 number of drugs . HoweverAnother related factor to the antibiotic prescribing in the present study was the impact of the seasons so that the number of antibiotic prescriptions increased as the cold season came. The mean age of outpatients in winter was lower than warm seasons in this study. This finding is in agreement with national studies , 31. It One of the most important criteria for evaluating the correct and rational prescribing of antibiotics is compliance with international valid guidelines and up-to-date medical science. Our findings showed that the majority of the prescribed antibiotics did not follow the correct scientific method in our study area. This defect may be a major determinant of the development of antibiotic resistance and may be a threat to human life.In this study, incorrect and unscientific antibiotic prescriptions was assessed based on four criteria including antibiotic dosage per consumption, daily doses, duration of therapy and interaction with other antibiotics or drugs. Our findings showed that the majority of antibiotic prescriptions by family physicians no fulfilled four correct criteria. This finding has been observed in other studies in Iran and other countries –36 whileIn the present study, injection form of antibiotics was prescribed more than other forms with 22.27%. Although this amount was reported at 49% in a study in Urmia city of Iran  which isThe price of antibiotics in proportion to the total price of drugs of each prescription was 33.7% and for the prescriptions that included antibiotics was 56.4%. This is a high number in comparison to the results of the study from Urmia, Iran with 35%  and the In this study, we evaluated the pattern of family physicians' antibiotic prescribing to outpatients in PHC system of Iran. Although we carried out multiple logistic regression analysis to estimate adjusted ORs in the association between antibiotic prescribing and related factors, it may be affected by many contextual, educational, cultural and qualitative variables  other thOur findings indicated that family physicians' antibiotic prescribing is relatively high. Nonetheless, not only the average number of antibiotics per prescription is high, but also incorrect prescribing of antibiotics needs to be adjusted. The findings of this study can be useful in tackling the pattern of irrational and mal-prescribing of antibiotics in Iranian PHC by family physicians. There is a need to improve the level of knowledge and skill of antibiotic prescribing patterns among family physicians working in primary health care.Qualitative and holistic studies are suggested for a better understanding and reasons for the high pattern of antibiotic prescribing by family physicians.The study highlights the need for high-quality, and evidence-based interventions to improve rational antibiotic prescribing by family physicians. This study also showed the need for coping strategies that may assist to decrease antibiotic mal-prescription.