However, the shortage of good hyperspectral data can hinder the accurate assessment of wheat Fv/Fm. In this research, the relationships between wheat canopy Fv/Fm and in-situ hyperspectral vegetation indexes were explored to develop a strategy for accurate Fv/Fm assessment. Fv/Fm had the highest coefficients with normalized pigments chlorophyll ratio index (NPCI) and the medium terrestrial chlorophyll index (MTCI). Both NPCI and MTCI were increased with the increase in Fv/Fm. However, NPCI value ceased to increase as Fv/Fm reached 0.61. MTCI had a descending trend when Fv/Fm value was higher than 0.61. A piecewise Fv/Fm assessment model with NPCI and MTCI regression variables was established when Fv/Fm value was ≤0.61 and >0.61, respectively. The model increased the accuracy of assessment by up to 16% as compared with the Fv/Fm assessment model based on a single vegetation index. Our study indicated that it was feasible to apply NPCI and MTCI to assess wheat Fv/Fm and to establish a piecewise Fv/Fm assessment model that can overcome the limitations from vegetation index saturation under high Fv/Fm value.Chlorophyll fluorescence parameter of F It was the unique approach by which plants gained energy from the environment. There were three basic effects when light struck a leaf surface: absorption, reflection and transmission. The major part of light was absorbed by the chlorophyll used for photosynthesis, and only a small proportion was de-excited via emission with a longer wavelength as fluorescence, or dissipation as heat2. Chlorophyll fluorescence emissions occurred in the red and far-red regions of the plant spectrum (650–800 nm)3. Changes in chlorophyll fluorescence parameters of plant leaves could reflect the changes of environmental factors and their effects on plant photosynthetic physiology to a certain extent4. In many chlorophyll fluorescence parameters, Fv/Fm was used to characterize the conversion efficiency of the light energy of the PS II reaction center, and its numerical changes were of special significance. However, conventional methods of assessing Fv/Fm from field observations, that involved site-specific complicated parameterizations and calculations, made it difficult to apply over large agricultural areas5. These shortcomings could be overcome through the complementary use of hyperspectral measurements of crops, which had several advantages - non-destructive, uniform, could be performed rapidly, and no complicated parameterizations were necessary.Photosynthesis was the most important biological process on earthv/Fm from vegetation indexes (VIs) derived from hyperspectral data, especially remote sensing data, have been reported by several studies10. For instance, some researchers compared the performance of VIs to assess Fv/Fm of legume crops and reached the conclusion that out of the nine kinds of VIs with a close relationship with Fv/Fm, modified soil adjusted vegetation index (MSAVI) performed best11. If ground cover was significant, the impact of the background significantly reduced, and Fv/Fm could be better estimated using normalized difference vegetation index (NDVI). Re-normalized difference vegetation index (RDVI) showed an approximate linear relation to Fv/Fm regardless of ground cover. Hyperspectral remote sensing is an important technique to fulfill real-time monitoring for crop growth status based on its superior performance in acquiring vegetation canopy information rapidly and non-destructively. However, the regression analysis was based on only five points making it statistically uncertain. Other scientists used radiative transfer models to estimate Fv/Fm and found that a linear model based on NDVI produced the best estimate results12.Assessment of Fv/Fm products based on MODIS have been reported in several studies. Coops et al. investigated the increasing availability of time series of Fv/Fm data from MODIS and reported the three dynamic habitat index components varied significantly in their magnitude, principally because of MODIS Fv/Fm estimates being larger than those observed by medium resolution imaging spectrometer (MERIS) Fv/Fm13. In some previous studies, some researchers compared MODIS Fv/Fm with the measurements for sites in the US and found MODIS Fv/Fm was higher than the ground-measured Fv/Fm. Winkel et al. compared Fv/Fm from MODIS with in-situ measurements for a tropical rainforest in Brazil and concluded that MODIS Fv/Fm was reliable for Fv/Fm assessment14. There was a need for an investigation of the performance of VIs in different vegetation ecosystems15.Recently, modeled Fv/Fm -NDVI relationships suffered from a major flaw - NDVI saturated at high leaf area index values16 and, thus a linear model tended to be insensitive to Fv/Fm changes in such cases17. Another issue that needed to be recognized was the scarcity of data for boreal ecosystems. The majority of the above cited studies presented empirical evidence suggesting a functional relationship between Fv/Fm and hyperspectral VIs, and these were mostly focused on forests, grasses (prairies), and some crop types such as rice, wheat and cotton18. There were only few reports on quantitatively estimating Fv/Fm for wheat canopies using VIs from remote sensing data19. Besides, VIs–Fv/Fm relationships differed from one ecosystem type to another ecosystem due to the influences of vegetation type, strong background signals, canopy structure and spatial heterogeneity21. Further, existing remote sensing-based Fv/Fm products lacked adequate ground validation, which was critical for establishing the uncertainty and accuracy of such products so that they could be used for guiding crop production practices23.Models based on linear Fv/Fm -VIs relationships for wheat canopies, using in-situ hyperspectral data collected from a series of field experiments, and aims at determining a practical methodology for estimating Fv/Fm of wheat canopies.This study is motivated by the above-mentioned issues and focuses on exhaustive statistical analyses of Fv/Fm revealed the progressive increase as the growth of wheat crops at different growth stages . Until the blooming stage, Fv/Fm began to increase and reached its maximum value of 0.68. From the blooming stage to the milk stage, Fv/Fm tended to slow, that was saturated.Fges Fig. . An initv/Fm and VIs were observed in 51 cases out of 56 VIs considered  are 0.891, 0.886 and 0.879, respectively. Thus, NPCI, MTCI and NDVI  could be identified as three common VIs relatively well correlated to wheat canopy Fv/Fm, and these were the post probable VIs of choice for estimating Fv/Fm.Statistically significant correlations between Fv/Fm based on a threshold on VI- Fv/Fm correlation  and error statistics (RRMSE) , and with MTCI as while MTCI as the regression variable after the saturation point (Fv/Fm > 0.61).All three VIs in Fig. v/Fm was built according to the range of Fv/Fm value in Fig. v/Fm ≤ 0.61, NPCI should be used to assess Fv/Fm, and the assessment model was y = 1.0616 × −0.076, R2 = 0.929 (p < 0.01); if Fv/Fm > 0.61, MTCI should be used to assess Fv/Fm, and the assessment model was y = 5.5259e−3.529x, R2 = 0.835 (p < 0.01).Based on the aforementioned research results, the piecewise hyperspectral assessment model of Fv/Fm. The estimated and measured Fv/Fm cross-resistance almost coincided with 1:1 relation line shown in Fig. v/Fm values, estimated value might be underestimated. As Fv/Fm increased, estimated values were closer to the measured values. The R2, RRMSE and assessment accuracy values of the piecewise Fv/Fm model were 0.9278, 0.084 and 91.6%, respectively. Compared with the assessment models based on only NPCI, MTCI and NDVI  in Table v/Fm model in different ranges of the Fv/Fm increased by 11.3%, 13.9% and 16.4%, respectively. In conclusion, the piecewise model based on NPCI and MTCI, used to assess Fv/Fm, can not only improve the assessment accuracy, but also solve the saturation problems that occurred in NPCI and NDVI .A total of 60 samples observed from the experiments in 2017 were used to test the hyperspectral VIs-based assessment model of Fv/Fm was primarily controlled by ground cover and leaf area24. Before the jointing stage, Fv/Fm increased significantly , which was captured by the lower rate of Fv/Fm increase. According to agronomic principle of wheat, although the research was lack of Fv/Fm data after the milk stage, it was still available to conclude that leaves started to turn yellow and gradually litter, as the growth period went, and Fv/Fm declined in the combination of wheat’s photosynthetic physiological characteristics. Until full-ripe stage, Fv/Fm was close to 0, because leaves took off green and became withered and died so that they were unable to absorb light energy and the accumulation of dry matter had stopped25.Ftly Fig. , which wv/Fm. Furthermore, many studies indicated that VIs were better correlated to Fv/Fm than the reflectance in single wavebands27, which could be plausibly explained by the fact that VIs could minimize the influence of atmospheric scattering and soil background and enhanced the information of the sensitive wavebands28. Similarly, this study found Fv/Fm to be strongly correlated to the majority of VIs (49 out of 56), with NPCI, MTCI and NDVI  being the best performing VIs. This result is helpful to provide an important technique for the establishment of perfect wheat photosynthetic groups, the improvement of sunlight energy efficiency and the implementation of cultivation control.Significant efforts were presently focusing on the use of VIs in general, and NDVI in particular, for estimating vegetation canopy Fv/Fm gave the lower RRMSE and higher assessment accuracy than NDVI proposed in several studies. Future research should focus on evaluating the performance of the proposed model over wheat crops grown under a variety of conditions, different wheat varieties, as well as other crop types. This will help in refining the model as a useful tool for informing crop management practices. Efforts should also be made to test this model with data from different sources – field-based spectral measurements, as well as current and future satellite data.As compared to the previous studies with NDVI, NPCI and MTCI for estimating the Fv/Fm models. We have addressed this issue by employing the differences in sensitivity of different VIs to Fv/Fm i.e. Fv/Fm had the highest coefficients with NPCI and MTCI. Both NPCI and MTCI were increased with the increase in Fv/Fm. However, NPCI value ceased to increase as Fv/Fm reached 0.61. MTCI had a descending trend when Fv/Fm value was higher than 0.61. A piecewise Fv/Fm assessment model with NPCI and MTCI regression variables was established when Fv/Fm value was ≤0.61 and >0.61, respectively. The model increased the accuracy of assessment by up to 16% as compared with the Fv/Fm assessment model based on a single vegetation index. Our study indicated that it was feasible to apply NPCI and MTCI to assess wheat Fv/Fm and to establish a piecewise Fv/Fm assessment model that can overcome the limitations from vegetation index saturation under high Fv/Fm value.VIs, like NDVI, were often plagued with saturation at high biomass areas, which was a major disadvantage for VIs-F-Yangmai 13, Yangmai 15, Yangmai 16 and Ningmai 9 were used in a field experiment conducted from March to May during the three wheat seasons of 2015, 2016 and 2017 on the Experimental Farm of Yangzhou University, China . The former crop in the field was rice. The soil is yellow brown soil (Alfisolsin U.S. taxonomy), containing 2.23 g kg−1 organic matter, 121.3 mg kg−1available nitrogen, 25.9 mg kg−1 available phosphorus and 83.7 mg kg−1 available potassium in the 0–30 cm soil layer. Canopy spectral parameters were recorded alongside with the quasi-simultaneous measurement of Fv/Fm upon the growing wheat canopies. In order to highlight variations in wheat growth due to biochemical composition changes, three different levels of nitrogen fertilization as urea were implemented, including non-nitrogen fertilization, adequate nitrogen fertilization (450 kg ha−1) and heavy nitrogen fertilization (900 kg ha−1). There are three replicates for each nitrogen level. The plot size was 4 m × 4 m. Local standard wheat cropping management practices pertaining to water, pest, disease and weed were followed. Training data consisted of 95 and 87 samples in 2015 and 2016, respectively, and test data consisted of 60 samples in 2017.Four varieties of wheat 4 calibration panel was used for calculation of hyperspectral reflectance. Vegetation and panel radiance measurements were taken by averaging 20 scans at optimized integration time, with a dark current correction at every spectrometry determination.In 2015, six spectral measurements were carried out in the wheat turning green stage (March 7), jointing stage (March 20), booting stage (April 9), blooming stage (April 25), 15 days after blooming stage (May 9), and milking stage (May 18), respectively. All canopy spectrometry determinations were taken at a vertical height of 1.6 m over the canopy under the cloudless or near cloudless condition between 11:00 and 14:00, using an ASD FieldspecPro spectrometer  fitted with 25° field of view fiber optics, operating in the 350–2500 nm spectral region with a sampling interval of 1.4 nm between 350 nm and 1050 nm, and 2 nm between l050 nm and 2500 nm, and with spectral resolution of 3 nm at 700 nm, 10 nm at 1400 nm, selecting the representative, growth-uniform, pest-free plants, placing the probe of sensor down in measuring. A 40 cm × 40 cm BaSOIn 2016, four spectral measurements with 87 test samples were carried out in the wheat turning green stage (March 9), jointing stage (March 22), blooming stage (April 23), and milking stage (May 20), respectively. In 2017, total three spectral measurements with 60 test samples were carried out in the wheat booting stage (April 11), blooming stage (April 22), and 15 days after blooming stage (May 12), respectively. The other practices in 2016 and 2017 were as same as those in 2015.29. Five-point weighted smoothing method is carried out using Equation  after the completion of each spectrum. First, the dark adaptation clamp was used to adapt the blade to 10 min, and then the initial light energy conversion efficiency of photosystem II (PS II) F30 related to Fv/Fm, leaf area index and chlorophyll (known as an important influence on Fv/Fm absorbed by green vegetation) as the independent variables for establishment of remote sensing assessment models of wheat canopy Fv/Fm. Data from the field experiment in 2015 (95 samples) and 2016 (87 samples) were used to develop the regression models, and data from the field experiments in 2017 (60 samples) were used to evaluate the models.In reference to previous studies, based on spectral characteristics of wheat and combined with the physical meaning of spectral index, a total of 56 VIs were considered Table 30 relatev/Fm relationships were analyzed using a variety of regression models - linear, exponential, logarithmic, and quadratic. Models were ranked based on statistically significant (p < 0.05 or 0.01) correlation coefficients (r in case of linear models) and coefficients of determination (R2 in case of non-linear models). Finally, by plotting the relation figure under the scale 1:1 between estimated and measured Fv/Fm values, the performance of the model was evaluated through the coefficient of determination (R2) and relative root mean squared error (RRMSE) for the assessment of in-situ measured Fv/Fm. The higher the R2 and the lower the RRMSE, the higher the accuracy of the model to assess the Fv/Fm. The RRMSE and assessment accuracy are calculated using Equations ,,v/Fm rel


CellProfiler has enabled the scientific research community to create flexible, modular image analysis pipelines since its release in 2005. Here, we describe CellProfiler 3.0, a new version of the software supporting both whole-volume and plane-wise analysis of three-dimensional (3D) image stacks, increasingly common in biomedical research. CellProfiler’s infrastructure is greatly improved, and we provide a protocol for cloud-based, large-scale image processing. New plugins enable running pretrained deep learning models on images. Designed by and for biologists, CellProfiler equips researchers with powerful computational tools via a well-documented user interface, empowering biologists in all fields to create quantitative, reproducible image analysis workflows. The “big-data revolution” has struck biology: it is now common for robots to prepare cell samples and take thousands of microscopy images. Looking at the resulting images by eye would be extremely tedious, not to mention subjective. Thus, many biologists find they need software to analyze images easily and accurately. The third major release of our free open-source software CellProfiler is designed to help biologists working with images, whether a few or thousands. Researchers can download an online example workflow  or create their own from scratch. Pipelines are easy to save, reuse, and share, helping improve scientific reproducibility. In this release, we’ve added the capability to find and measure objects in three-dimensional (3D) images. We’ve also made changes to CellProfiler’s underlying code to make it faster to run and easier to install, and we’ve added the ability to process images in the cloud and using neural networks (deep learning). We’ve also added more explanations to CellProfiler’s settings to help new users get started. We hope these changes will make CellProfiler an even better tool for current users and will provide new users better ways to get started doing quantitative image analysis. Image analysis software is now used throughout biomedical research in order to reduce subjective bias and quantify subtle phenotypes when working with microscopy images. Automated microscopes are further transforming modern research. Experiments testing chemical compounds or genetic perturbations can reach a scale of many thousands of perturbations, and multidimensional imaging  also produces enormous data sets that require automated analysis. In light of this data scale, computer algorithms must deliver accurate identification of cells, subcompartments, or organisms and extract necessary descriptive features (metrics) for each identified object.Racing to keep up with the advancement of automated microscopy are several classes of biologist-focused image analysis software, such as companion packages bundled with imaging instruments , stand-alone commercial image processing tools , and free open-source packages . Commercial software is often convenient to use, especially when bundled with a microscope. Although cost and lack of flexibility may limit adoption, there is a focus on usability, particularly for applications of interest to the pharmaceutical industry. Still, the proprietary nature of the code in commercial software limits researchers from knowing how their data is being analyzed or modifying the strategy of a given algorithm, if desired.The open-source biological image analysis software ecosystem is thriving . ImageJ CellProfiler, our open-source software for measuring and analyzing cell images, has been cited more than 6,000 times, currently at a rate of more than 1,000 per year. The first version of CellProfiler was introduced in 2005 and published in 2006 . It is wCellProfiler provides advanced algorithms for image analysis, organized as individual modules that can be placed in sequential order to form a pipeline. This pipeline is then used to identify and measure cells or other biological objects and their morphological features. CellProfiler’s modular design and carefully curated library of image processing and analysis modules benefits biologists in several ways:Reproducibility at scale: CellProfiler is designed to produce high-content information for each cell or other object of interest in each image and to apply the same objective analysis in high-throughput, e.g., across thousands or millions of images.Flexible feature extraction: Individual modules measure standard morphological features such as size, shape, intensity, and texture. Customized combinations of modules can extract even more complex information. As such, CellProfiler is commonly used for morphological profiling experiments such as Cell Painting ,8, whichEasy to learn: Each of the 70+ modules includes carefully crafted documentation, curated by both imaging and biology experts, to make image processing more approachable and understandable for the average scientist. Further, each individual setting is explained in practical terms to aid researchers in configuring it. The number of modules and settings is carefully limited to avoid overwhelming users, while a plugin system allows the flexibility of a larger array of contributed modules.Community: CellProfiler has an active community of more than 3,000 people on its online question and answer forum. With more than 15,000 posts, users provide feedback that fuels improvements to CellProfiler, find pipelines related to their area of research, interact with developers, get input on challenging problems, and improve image analysis skills and knowledge by helping other users design solutions.In the CellProfiler 3.0 release, we introduced methods for analyzing 3D images, using deep learning architectures and cloud computing resources, and other improvements to CellProfiler’s usability and capabilities.This new version of CellProfiler has support for analysis of 3D images in many of its modules . AlthougWe developed 3D pipelines to identify cells and subcompartments of cells for a number of experimental situations and sample types across a number of laboratories. We identified nuclei based on a DNA stain  in 3D imTo determine how well the segmented objects agreed with ground truth, CellProfiler’s “MeasureImageOverlap” module was used to calculate the plane-wise Rand index , a perfohttps://github.com/carpenterlab/2018_mcquin_PLOSBio. All raw images, together with ground truth annotations used to test CellProfiler 3.0 performance, are publicly available for further community algorithm development in the Broad Bioimage Benchmark Collection  and Keras-RCNN [https://github.com/broadinstitute/keras-rcnn]) that will provide the foundation for interfaces that allow biologists to annotate, train, and use deep learning models. We expect that over time, these models will reduce the amount of time biologists spend tuning classical image processing algorithms to identify biological entities of interest in images.Finally, deep learning has revolutionized computer vision and other fields in the past few years ,32, and Images were kindly provided by Javier Frias Aldeguer and Nicolas Rivron of Hubrecht Institute for Developmental Biology and Stem Cell Research and Li Linfeng of MERLN Institute for Technology-Inspired Regenerative Medicine. As per Rivron and colleagues , mouse eImages were acquired by collaborators from the Allen Institute for Cell Science, Seattle, as per Roberts and colleagues . Brieflyhttp://www.allencell.org/uploads/8/1/9/9/81996008/sop_for_cellmask-and-nucblue_v1.0_1.pdf. Phenol red–free mTeSR1 is preequilibrated to 37°C and 5% CO2. 1X NucBlue solution made in preequilibrated phenol red–free mTeSR1 is spun for 60 minutes at 20,000 g. The 2X and 10X working stocks of CellMask Deep Red lot #1730970 and #1813792, respectively, are made in 1X NucBlue solution. All solutions are kept at 37°C and 5% CO2 until used. The 100 μL and 400 μL of NucBlue solution are added per well of 96-well imaging plates and 24-well imaging plates, respectively, and incubated at 37°C and 5% CO2 for 20 minutes. An equal amount of CellMask Deep Red working stock is added to the wells containing NucBlue solution. Final dye concentrations in the wells are 1X NucBlue and 1X and 5X CellMask Deep Red lots #1730970 and #1813792, respectively. Cells are incubated at 37°C and 5% CO2 for 10 minutes and gently washed with preequilibrated phenol red–free mTeSR1. Fields of view as shown in Three to four days after cells are plated and mature and healthy colonies are observed on 96- and 24-well imaging plates, the cells are stained with NucBlue Live ready probe reagent  and CellMask Deep Red plasma membrane stain  to visualize DNA and plasma membrane, respectively. The protocol is available online: As per Roberts and colleagues , cells whttps://www.gimp.org), an open-source drawing and annotation software. These annotated layers were then exported from GIMP as an image. This outline image is converted to 3D objects via a CellProfiler pipeline , and an object label matrix image is exported, in which each object’s voxels are assigned a unique integer value. These label images are referenced as ground truth.Experienced bioimage analysts drew outlines around nuclear boundaries on each slice of the 3D images and labeled background regions in a different color with GIMP Click here for additional data file.S2 Fighttps://data.broadinstitute.org/bbbc/BBBC032/), as in Images were obtained using PerkinElmer Ultraview VoX spinning disk microscope with a 63× immersion objective (distance between Z-slices = 0.5 μm) and provided by Javier Frias Aldeguer and Nicolas Rivron from Hubrecht Institute, Netherlands. 3D, three-dimensional.Images are available from the Broad Bioimage Benchmark Collection ((JPG)Click here for additional data file.S3 Fighttps://data.broadinstitute.org/bbbc/BBBC033/), as in Images were obtained using a PerkinElmer Ultraview VoX spinning disk microscope with a 63× oil immersion objective (distance between Z-slices = 0.5 μm) and provided by Javier Frias Aldeguer and Nicolas Rivron from Hubrecht Institute, Netherlands, and ground truth was annotated by Li Linfeng from MERLN Institute, Netherlands. 3D, three-dimensional.Images are available from the Broad Bioimage Benchmark Collection ((JPG)Click here for additional data file.S4 Fighttps://data.broadinstitute.org/bbbc/BBBC024/), as in Images are available from the Broad Bioimage Benchmark Collection ((JPG)Click here for additional data file.S5 Fighttps://data.broadinstitute.org/bbbc/BBBC035/), as in The data set was created by Vladimir Ulman and David Svoboda  using MitoGen, part of CytoPacq, to model a Zeiss Axiovert S100 microscope attached to confocal unit Atto CARV with a Micromax 1300-YHS camera with a Plan-Apochromat 40×/1.3 (oil) objective lens,14. 3D, three-dimensional.Images are taken from the Broad Bioimage Benchmark Collection ((JPG)Click here for additional data file.S6 FigThe fraction of nuclei correctly identified relative to their ground truth was assessed for both CellProfiler (solid line) and MorphoLibJ (dashed line) for the results shown in (PNG)Click here for additional data file.S7 Fighttps://data.broadinstitute.org/bbbc/BBBC022/, filename XMtest_B12_s2_w19F7E0279-D087-4B5E-9899-61971C29CB78.tif, see https://github.com/carpenterlab/unet4nuclei. (A) The prediction for the three classes  is calculated for each image. (B) Raw image and nuclei segmentation using ClassifyPixels-Unet and IdentifyPrimaryObjects modules, with objects touching the edge excluded. Pipeline and image available as Image is available from the Broad Bioimage Benchmark Collection ((PNG)Click here for additional data file.S8 FigA data set of seventeen 384-well plates was processed using Distributed-CellProfiler on an AWS cluster. Each plate comprised 3,456 five-channel images . A CellProfiler pipeline was run on each image to identify cells and then extract measurements per cell. In all, 12,415,665 cells were identified, and 2,191 measurements were made per cell. It would have taken more than five months to analyze the data set using a single machine with 16 vCPUs. Using a cluster of 195 such machines on AWS, this data set was processed in less than 21 hours and cost $765 in total. The graph shows the number of pending images over the 21-hour period of processing this data set. The configuration files used to process this data set are provided in (PNG)Click here for additional data file.S9 Fighttps://github.com/CellProfiler/examples and http://cellprofiler.org) for CellProfiler 2.2 and 3.0 on OS X 10.12.6 (2.8 GHz Intel Core i7 and 16 GB 1600 MHz DDR3.). The figure above shows the difference of mean runtimes between CellProfiler 3.0 and 2.2 across 10 identical image sets. CellProfiler 3.0 demonstrates improved or comparable performance to CellProfiler 2.2 in 11 of 16 example pipelines, and these represent the more commonly used applications.. We compared the performance of example pipelines (available at (PNG)Click here for additional data file.S1 TableMacro code constructed in the MorphoLibJ plugin for Fiji software that was used for analysis of 3D image examples presented in (XLSX)Click here for additional data file.S2 TableUnits of time are in seconds. One image representing the 72-hour time point (72 hours) was used to compare CellProfiler 3.0 and Fiji presented in (XLSX)Click here for additional data file.S1 FileMeasurements of mouse blastocyst cells and GAPDH transcript foci created by CellProfiler; these serve as the underlying data for (XLSX)Click here for additional data file.S2 FilePer cell measurements of hiPSCs created by CellProfiler; these serve as the underlying data for (XLSX)Click here for additional data file.S3 FileA file to reproduce the results presented in (ZIP)Click here for additional data file.S4 FileExample pipeline and image needed to run the ClassifyPixels-Unet module.(ZIP)Click here for additional data file.S5 FileThese pipelines and files can be used to run Distributed-CellProfiler; they are specifically configured to run the Cell Painting assay . AWS, Am(ZIP)Click here for additional data file.S6 FileCPU and wall-clock runtimes for individual CellProfiler modules in the example pipelines; the summary of this data can be seen in (XLSX)Click here for additional data file.


Auricularia polytricha followed by chromatographic fractionation of the extract on the ion exchanger DEAE-cellulose and gel filtration on Sephadex-200 to yield two purified fractions: APPI and APPII. The monosaccharide compositions, FT-IR, N-terminal sequences, internal peptide sequences and molecular weights of the two fractions were determined. Furthermore, their hepatoprotective effect on human hepatoma HepG2 cells in vitro and in an animal model of fatty liver disease was evidenced by the findings that APPI and APPII diminished lipid deposit in cells, blood and the liver, increased cellular antioxidant activity and viability, and protected the liver against injury. The mechanistic study revealed that APPI and APPII activated the adiponectin pathway, up-regulated expression of genes controlling free fatty acid (FFA) oxidation, such as AMPK, CPTl, ACOX1 and PPARα genes, enhanced lipid metabolism, preserved hepatic function, promoted the antioxidant defense system and reduced lipid peroxidation. Hence the bioactive compounds of A. polytricha could serve as therapeutic agents in the food and pharmaceutical industries.Fatty liver (FLD) disease is a consequence of metabolic syndrome, which is a health problem worldwide with a phenomenal rise in prevalence. In this study, two hepatoprotective polysaccharide-peptides were extracted from the mushroom  Accumulating data suggest that excessive intake of alcohol and fat induces improper triglyceride metabolism in the hepatocytes2. When synthesis proceeds faster than anabolism, triglycerides accumulate in the liver. The excessive lipid in the hepatocytes, exceeding 5–10% of the liver weight, is the main pathogenetic factor of fatty liver disease (FLD)4. The multiplicity of etiologic factors of FLD, include genetic, dietary factors, insulin resistance, and adipokines5. FLD is a metabolic syndrome, which has become a vital health issue with a phenomenal escalation in prevalence. Fatty liver disease comprises alcoholic fatty liver disease and nonalcoholic fatty liver disease (NAFLD). Alcohol-related liver disease and NAFLD are common chronic hepatic derangements6. The disease affects grown-ups and children alike and is getting more and more rampant in industrialized as well as developing countries. Cirrhosis associated with NAFLD is likely to be a priority for hepatic transplantation in the foreseeable future. The incidence of liver cancer associated with NAFLD shows a rising trend7. Cardiometabolic disorders, dyslipidemia in particular, are often associated with pediatric NAFLD8. Approximately one-third of the Americans have NAFLD with consequent heavy economic and societal burdens9. Treatments for NAFLD are found to be not optimal. Some of the drugs used have side effects10. Hence, it would be highly desirable to devise a management plan to ascertain efficacious natural products for human populations at high risk of developing fatty liver disease in order to forestall or retard the exacerbation of hepatic damage at an early stage. To combat this disease, natural products may have value11.The liver is an important organ in charge of the metabolism of lipids, glucose, proteins, alcohol, drugs, and chemicals12, anti-tumor13, antidiabetic15, renoprotective17, immunomodulatory18 and hepatoprotective20 activities. Polysaccharides isolated from mushrooms have been extensively investigated in the medicinal arena owing to the ready availability of fermentation technology23.Currently, growing attention has been drawn to the exploitation of biomedicines owing to their minimal toxicity and therapeutic efficacy. Polysaccharide, a potential agent that meets the requirement, is found in many vegetables, fruits, edible fungi and other microorganisms. Increasing studies furnish supporting evidence demonstrating that polysaccharides display a diversity of biological activities, encompassing antioxidant26. Polysaccharides from mushrooms exhibit diverse activities: those with hepatoprotective activities have been isolated from Agaricus bisporus, Coprinus comatus, Hypsizigus marmoreus, Oudemansiella radicata, Pholiota dinghuensis, Pleurotus eryngii, Pholiota nameko, Pleurotus djamor, and Russula vinosa37. Polysaccharides with antihyperlipidemic activity have been isolated from Pholiota nameko and Termitomyces albuminosus39.Mushrooms exert a myriad of health promoting actions. Thus mushrooms have captured the attention of many researchers. Two important activities are hepatoprotective and antihyperlipidemic activitiesAuricularia polytricha, alternatively referred to as wood ear or Jew’s ear, belonging to Auriculariaceae family, is a culinary-medicinal fungus which manifests a multitude of activities. Modern research indicates that A. polytricha is a kind of healthy mushroom with a high carbohydrate content (about 630 g/kg in dried fruiting bodies)40. The polysaccharides from the fruiting bodies have multiple pharmacological functions with potential for clinical application and are devoid of toxicity and significant side effects. After oral administration a soluble polysaccharide from A. polytricha exhibited antihypercholesterolemic activity in rats41. An ethanolic extract of mycelial culture exhibited antioxidant and tyrosinase inhibitory activities42. An aqueous extract of fruiting bodies demonstrated hepatoprotective effect against paracetamol in rats43. An aqueous extract was devoid of cytotoxicity toward normal kidney NRK-52E cells44 but displayed antiproliferative effect on COLO-205 colon cancer cells45. Polysaccharides with anticancer activity in S180 sarcoma bearing mice46, antimutagenic activity against the alkylating agent cyclophosphamide47, and antiproliferative, cell cycle arresting and apoptotic activities toward A549 human lung cancer cells48 have been isolated from A. polytricha. A hot water extract manifested hypoglycemic effects49. Hot water extracts inhibited platelet aggregation with a mechanism independent of cyclic AMP51. Thermostable constituents suppressed activity of beta secretase which liberates toxic β-amyloid peptide in the brain and protects against neurodegenerative diseases such as Alzheimer’s disease52. Polysaccharides with different activities have been reported from A. polytricha48. Three anticancer polysaccharides AAPS-1, AAPS-2, and AAPS-3 with molecular weights of 162, 259, and 483 kDa46 and an antimutagenic 930 kDa salt-soluble polysaccharide47 from A. polytricha have been reported.A. polytricha effectively lowered the levels of total cholesterol, LDL-cholesterol and triglycerides41 in the blood circulation, indicating that the polysaccharide could regulate the metabolism of lipids, and is exploitable for alleviation of FLD and liver injury. Nevertheless, to date, no detailed studies have been carried out on the characterization, hepatoprotective effect and mechanism of polysaccharides or polysaccharide-peptides from A. polytricha. Thus we commenced this investigation on purification and characterization of bioactive polysaccharide-peptides, exploring their hepatoprotective effect and mechanism with the intent to ascertain novel bioactive constituents utilizable in the food and pharmaceutical industries.Our previous studies indicated that polysaccharide prepared from A. polytricha fruiting bodies was firstly resolved on DEAE-cellulose. According to the charge difference, two peaks, D1 and D2, eluted with 0 and 0.2 mol/L NaCl solution respectively, were detected by using the phenol–sulfuric acid method  and A. polytricha polysaccharide-peptide II (APPII), respectively  is an important method to predict the structures of natural macromolecules such as polysaccharide-peptides. To more precisely characterize polysaccharide-peptide fractions, the characteristic absorption of polysaccharides was performed in the region 4000–600 cmPPI Fig. , the intCordyceps militaris. The peptide sequence GTPSSYIDNLTFPK of APPII manifested considerable homology with immunomodulatory protein from Flammulina velutipes. Another peptide sequence of APPII, ELATGQNGFGYAGSSFHR, demonstrated pronounced resemblance to peptidyl-prolyl cis-trans isomerase from Lentinula edodes.The amino acid sequences of APPI and APPII at the N-terminal were DLYEVVEGEI, and VPSSMVVVVG, respectively. Results from internal amino acid sequence analysis showed that two peptide sequences of APPI, namely VQNVGNGVLLGFHGR and HQTSGDQVTSSTQHSFR, were strikingly similar to mannose-binding lectin from HepG2 cells of the injury model were exposed to different concentrations of FFA and ethanol, and cell viability was evaluated with the 3--2,5-diphenyltetrazolium bromide (MTT) assay. It was found that cells could retain around 60% viability when 0.5 mmol/L FFA and 1.8% (v/v) ethanol were used to induce hepatic injury. Triglyceride (TG) deposits in the cells were revealed by the Oil-red O staining method.6. Thus, activities of extracellular aminotransferases can reflect the status of liver cells, with higher values reflecting liver damage. It was shown that the activities of extracellular alanine and aspartate aminotransferases (ALT and AST) of the model group were elevated relative to those of the control group, implying that pathogenetic factors of FFA and ethanol inflicted damage on the cells. However, APPI and APPII mitigated this damage, and the elevated activities of alanine and aspartate aminotransferases consequently declined (P < 0.05), as shown in Table The protective effects of APPI and APPII against cell injury induced by pathogenetic factors was assessed by various assays comprising the MTT test, together with assays of intracellular TG, total superoxide dismutase (SOD), and extracellular aspartate transaminase (AST) and alanine aminotransferase (ALT) activities. It was shown that APPI and APPII were not cytotoxic to HepG2 cells in the concentration range of 0–100 μg/mL Fig. . The celde novo lipid synthesis8. When drugs stimulate the adipocytes, adiponectin is secreted and conjugated with Adipor 2 receptors on the surface of the hepatocytes. The complex activates AMPK gene expression, induces genes including CPTl and ACOX1 in FFA oxidation pathway upregulation. Meanwhile, AMPK induces activation of transcription factor PPARα and accelerates FFA metabolism.Adiponectin is an adipocyte-derived cytokine. Adiponectin and its receptor 2 abundant in human hepatocytes, constitute a complex, and play a central role in the pathogenesis of liver disorders. Adiponectin, which has an anti-steatotic effect on liver cells, acts mainly through the AMPK and PPARα pathway. It promotes oxidation of FFA and suppresses gluconeogenesis, entry of FFA and Adipor 2 gene was significantly down-regulated in the model group, as well as AMPK, CPTl, PPARα and ACOX1, which deactivate FFA oxidation process and accelerate FFA accumulation in hepatocytes or hepatic tissue. It was disclosed that the TG concentration of the model group rose. It was found that Adipor 2 gene was up-regulated in APPI and APPII groups, the same took place regarding AMPK, CPTl, PPARα and ACOX1. The results indicated that APPI and APPII could stimulate adipocytes to secrete adiponectin and activate the FFA metabolic pathway, reducing lipid accumulation.In order to validate the mechanism of APPI and APPII, five key genes have been selected for qRT-PCR analysis Fig. . The resFLD is the leading cause of chronic liver disease worldwide. Unhealthy high fat diets and sedentary habits result in the high prevalence of FLD. The rats were randomized to receive either standard laboratory diet (SLD) or high cholesterol and high fat diet (HFD) for 8 weeks. The biochemical analysis showed that HFD induced a rise in the levels of TG, total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), and the activities of ALT and AST in serum  in ALT and AST activities were observed in APPI and APPII-treated groups similar to the simvastatin- treated group Table . It indiHFD is associated with fat accumulation in liver. As shown in Fig. Histopathological examination was used to evaluate the hepatoprotective effects of APPI and APPII on HFD-elicited acute liver lesion. Figure A. polytricha fruiting bodies with an extraction rate approximating 20% by using the following conditions: extraction with 28 mL water/g mushroom at 95 °C for 4 h. The polysaccharide preparation exerted an antihypercholesterolemic action in diet-induced hypercholesterolemic rats and depressed the plasma cholesterol level by about one-third at the doses of 4.5 and 9 mg/kg/d. In the present investigation the polysaccharide was fractionated into two polysaccharide-peptides APPI and APPII have similar monosaccharide moieties and FITR spectra but differ in molar ratio of the monosaccharides and molecular weight. APPII also differs from APPI in exhibiting absorption around 1420.96 cm−1 probably indicating deforming vibrations of the C-H bond and absorption at 1375.60 cm−1 and 1250.16 cm−1 signifying the presence of a carboxyl group and a sulfate radical. Although APPI and APPII possess distinct N-terminal and internal partial amino acid sequences, both of them resemble other mushrooms in these sequences. In APPI, two of the internal partial amino acid sequences closely resemble those of Cordyceps militaris mannose-binding lectin. In APPII, two of the internal partial amino acid sequences manifest pronounced similarity to those of Flammulina velutipes immunomodulatory protein and Lentinula edodes peptidyl-prolyl cis-trans isomerase, respectively.In our earlier study a soluble polysaccharide was prepared from Auricularia auricular polysaccharides simulated hydrolysates, derived from A. auricular which is a species very closely similar to A. polytricha, were composed of arabinose, galactose, glucosamine, glucose, xylose and mannose with the molar ratio of 1.91: 0.67:0.23:1.00: 0.52: 2.8959. A. auricular polysaccharide was composed of arabinose, galactose, glucosamine, glucose, rhamnose and mannose with the molar ratio of 0.93:0.91:4.32:1:37.5360. APPI isolated in the present study was composed of arabinose, galactose, glucose, xylose and mannose in a molar ratio of 1:4.4:15.4:38.3:46.2, while the corresponding molar ratio for APPII was 1:71.5:99.2: 10: 5.1. Glucosamine was lacking in APPI and APPII. The data showed that they were all different in the composition of the sugar moiety.−1 characteristic of hydroxyl groups with stretching vibration, and a peak at 2934.88 cm−1 due to C-H stretching vibration. The absorption peak at 1250.16 cm−1 revealed that the polysaccharide-peptide APPII possessed a sulfate radical. In the study of Zhang et al.61, the FT-IR spectrum of A. auricular polysaccharide also displayed a band in the region of 3411.1 and 3410.5 cm−1 corresponding to the hydroxyl stretching vibration of the polysaccharide and at 2923.3 and 2921.3 cm−1 corresponding to a weak C–H stretching vibration at 1258 cm−1 due to an asymmetrical S=O stretching vibration. Although there were similarities the spectra of the aforementioned polysaccharides were not identical.The FT-IR spectrum of APPI exhibited a broad peak at 3400.42 cmA. polytricha, which was composed of phenolics, tannins and polysaccharides, was found to reduce lipid storage, inflammation, proinflammatory cytokines and oxidative stress in rats provided with a high-fat diet containing 10% lard. Upon completion of the treatment which lasted for 12 weeks. It was observed that changes brought about by the high-fat diet including the hepatosomatic index, serum activities of aminotransferases, triglyceride, serum levels of free fatty acid, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, very low density lipoprotein cholesterol, antioxidant vitamins C and E, malondialdehyde, and the proinflammatory cytokines interleukin-6 and tumor necrosis factor-alpha, were at least partially reversed by treatment with the A. polytricha polysaccharide-peptides62. Alterations in hepatic triglyceride, cholesterol, malondialdehyde, antioxidant vitamins E and C, and antioxidant enzymes were also attenuated62. Intake of a Ganoderma lucidum preparation enriched in polysaccharide-peptides and triterpenoids by human subjects restored the hepatic condition to normal from the initially mild fatty liver as evidenced by abdominal ultrasonography and reduction in plasma activities of aminotransferases63. However, it is known that phenolics and tannins also displayed similar activities64. Thus, the aforementioned actions of the aqueous extract of A. polytricha62 were probably attributed to each of its constituents: phenolics, tannins and polysaccharides, and the action of the Ganoderma lucidum preparation63 was partly due to triterpenoids66.With regard to its effect on lipid metabolism, an aqueous extract of in vitro as well as in vivo. In the in vitro system employing ethanol and a mixture of palmitic and oleic acids to induce hepatotoxicity, the resulting elevated level of intracellular triglyceride and activity of extracellular aspartate and alanine aminotransferase were suppressed by APPI and APPII, and the decreased cellular superoxide dismutase activity was upregulated by the two polysaccharide-peptides. APPI and APPII increased the viability of the hepatocytes. It appeared that APPII was more potent in inhibiting triglyceride accumulation in the hepatocytes. However, this was not true regarding the effects on hepatocyte viability, superoxide dismutase activity, and aminotransferases released into the culture medium. APPI had a more potent effect on Adipor 2, CPTl, and PPARα whereas APPI had a higher activity on AMPK and ACOX1.In the present investigation both APPI and APPII exhibited a lipid lowering effect in vivo system, the high fat diet brought about a rise in the circulatory titers of triglyceride, total cholesterol, low density cholesterol, aspartate aminotransferase and alanine aminotransferase but a decline in plasma high density cholesterol. APPII was more potent than APPI in its antihypercholesterolemic and antihypertriglyceridemic and LDL lowering activities. APPI was more potent than APPII in raising plasma HDL cholesterol activity. APPI and APPII were similar in their activity in preventing hepatic triglyceride accumulation and secretion of aspartate aminotransferase and alanine aminotransferase into the blood stream. The hepatocytes after treatment with APPI and APPII were similar in appearance under the microscope.In the A. auricula very closely similar to A. polytricha also reduced the serum concentrations of total cholesterol and low-density lipoprotein cholesterol in mice receiving a diet enriched in cholesterol and improved lipoprotein lipase activity and total antioxidant capacity67. A water-soluble crude polysaccharide from A. auricular mycelia grown under solid-state fermentation lowered serum concentrations of triglyceride, total cholesterol, and low-density lipoprotein cholesterol in a high fat diet induced hyperlipidemic mice60. A. auricular polysaccharides simulated hydrolysates from the dried fruiting bodies induced a decline in the serum concentrations of triglyceride and low-density lipoprotein cholesterol without affecting high-density lipoprotein cholesterol and total cholesterol in streptozotocin -induced diabetic rats59. The alcohol extract of A. auricula-judae provided to mice on a high fat diet reduced plasma lipids and hepatic enzymes and downregulated expression of adipogenic/lipogenic genes  in 3T3-L1 cells68. An alcohol extract of A. auricula enriched in polyphenolics lowered serum total cholesterol and upregulated high-density lipoprotein cholesterol level and fecal bile acid excretion. However, it was devoid of any action on serum low-density lipoprotein cholesterol and triglycerides and fecal neutral cholesterol excretion59. The effects of Auricularia cornea polysaccharides and enzymatic-extractable polysaccharides displayed antioxidant and reactive oxygen species scavenging activities in vitro and exerted a protective action on alcohol-induced liver pathology in mice. The polysaccharides produced their hepatoprotective effects by suppressing lipid peroxidation, facilitating alcohol metabolism, and downregulatiing the expression of inflammatory mediators and preventing the alcohol-induced histopathological alterations69.Polysaccharides from 74. Adiponectin reduces insulin resistance, prevents excess hepatic lipid accumulation, inhibits inflammation and fibrosis, and exerts a hepatoprotective action77. It also protects against cerebrovascular, cardiovascular, and chronic renal diseases80. It is a target in the therapy of obesity, cardiovascular and inflammatory ailments, nonalcoholic steatohepatitis, diabetes and neurodegenerative diseases80. Efforts are made to upregulate adiponectin by therapeutic medications and/or changes in lifestyle77.Adiponectin is anti-inflammatory and insulin-sensitizing adipokine. Adiponectin deficiency linked with an inflammatory state is present in obesity, severe chronic hepatitis C-related steatosis, non-alcoholic fatty liver diseases such as hepatic steatosis and non-alcoholic steatohepatitis, and cancerdb/db mice after consumption of ethanol-soluble extract of Panellus serotinus82. Supplementation with Agaricus blazei reduces insulin resistance and improves circulatory adiponectin level in patients with type 2 diabetes Murill83. β-glucan-rich polysaccharides derived from Pleurotus sajor-caju upregulated adiponectin expression84. The ethyl acetate fraction of Hericium erinaceus downregulated lipopolysaccharide -elicited decline of adiponectin mRNA in 3T3-L1 fat cells in coculture with RAW264 macrophages85. An aqueous extract of Antrodia cinnamomea reversed the action of a high-fat diet on body weight gain, inflammatory cytokines and adiponectin production in mice86. Adiponectin promotes fatty acid oxidation in skeletal muscle79. The purified polysaccharide- peptides acquired from A. polytricha act via genes affect fatty acid oxidation. Peroxisome proliferator-activated receptors (PPARs) are metabolic regulators of lipid and lipoprotein levels which are divided into α, β/δ and γ subtypes. The PPAR-α agonists suppress triglyceride levels. PPAR-γ agonists demonstrate potent hypoglycemic but weaker triglyceride lowering activity. PPAR-α/δ agonists display antihyperglycemic, and triglyceride lowering activities. They are promising for the therapy of atherogenic dyslipidemias and NAFLD87. PPARs are paramount to energy homeostasis of the entire body and as fatty acids sensors in several human lipid metabolic diseases88. Extracts, polysaccharides and other compounds from mushrooms inhibit fat production by acting via PPAR γ and/or PPAR α. This study is the first to reveal the action of mushroom polysaccharide peptides through PPAR α.Mushroom extracts exhibit a stimulatory effect on adiponectin production. An elevation of the circulatory adipokine level, decline in levels of markers associated with hepatic damage, and activities of key enzymes catalyzing fatty acid biosynthesis, together with attenuation of liver enlargement and triglyceride accumulation, were observed in A. polytricha and elucidation of the mechanism involved represents the first of its kind on purified polysaccharide-peptides. The A. polytricha polysaccharide peptides are of considerable interest and promising for development into therapeutic agents in view of the array of health-enhancing activities of adiponectin.The present report on the hepatoprotective and fatty liver alleviating activities of purified polysaccharide-peptides acquired from A. polytricha fruiting bodies were collected in Beijing, China, and were ground to produce a fine powder. Monosaccharides , DEAE-cellulose, MTT, oleic acid and palmitate were products of Sigma-Aldrich (USA). Superdex-200 column was obtained from General Electric Company , and HepG2 cell line was purchased from ATCC (American Type Tissue Culture Collection). Dulbecco’s modified Eagle’s minimum essential medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), trypsin solution, penicillin and streptomycin were products of Invitrogen (USA). The assay kits for protein content, TG, ALT, AST, and SOD were purchased from Nanjing Jiancheng Bioengineering Institute . The other chemicals and solvents employed were of analytical reagent grade and obtained from Sinopharm Chemical Reagent Co., Ltd. .A. polytricha following published procedures41. Briefly, the dry powder was extracted thrice, for 4 h each time, by employing 40 volumes of hot water (90 °C). The extracts were combined and concentrated in a rotary evaporator. The concentrated solution was deproteinated using Sevag reagent 89. Ethanol (100%) was added to the deproteinated solution which was then allowed to stand at 20 °C overnight, and the precipitated polysaccharide-peptide (APP) was obtained by centrifugation. Subsequently, a solution of the APP in distilled water was fractionated on a DEAE-cellulose column (1 cm × 30 cm) equilibrated with distilled water. The column was eluted sequentially with 0, 0.2 and 1 mol/L NaCl solution successively at a flow rate of 2.0 mL/min. The unadsorbed peak D1 and adsorbed peak D2, with the carbohydrate content measured with the phenol-sulfuric acid method, were enriched in polysaccharide. The D1 and D2 fractions were collected, concentrated, and further fractionated on an FPLC-Superdex 200 10/300 column in 0.2 mol/L NH4HCO3 (pH 8.5) buffer using an AKTA Purifier , respectively to obtain bioactive polysaccharide-peptides APPI and APPII.Crude polysaccharide-peptides were extracted from et al.90.Monosaccharide compositions of APPI and APPII were determined with a gas chromatography mass spectrometer (GC-MS). The sample was hydrolyzed following the method of Yu −1. The dried sample was ground with spectroscopic grade potassium bromide (KBr) powder and then pressed into 1- mm pellets.The IR spectra of APPI and APPII were obtained by using a Fourier transform infrared spectrophotometer  within the wave number range of 4000 to 400 cm3 and 0.05%NaN3 as the mobile phase.The purity and molecular weights of APPI and APPII were assessed with HPGPC technique using HPLC on a TSK GMPWXL column. The samples were injected and eluted at 0.6 mL/min with 0.1 mol/L NaNO91.The polysaccharide-peptides band excised from the SDS-PAGE gel was transferred to a polyvinylidenedifluoride (PVDF) membrane by Western blotting and then stained with Coomassie brilliant blue R-250. Analysis of the stained band was performed with the automated Edman degradation methodThe polysaccharide-band of SDS-PAGE gel was recovered and dispatched to Tsinghua University  for partial amino acid sequence analysis. Sequence homology with known sequences was searched using the BLAST/NCBI database.et al.92. The hepatocyte injury model was established as follows. HepG2 (liver cancer) cells were cultured in Dulbecco’s Modified eagle medium (DMEM) medium containing 10% (v/v) fetal bovine serum (FBS), 100 IU/mL penicillin and 100 mg/L streptomycin, at 37 °C in a humidified atmosphere of 5% (v/v) CO2. Cells were then seeded onto 96-well plates at a concentration of 8 × 103 cells/well, and allowed to incubate for 12 h before addition of FFA and ethanol. Incubation was then conducted for 24 h. Afterwards, the MTT assay was performed to measure cell viability. As a control, PBS instead of pathogenetic factors (FFA and ethanol) was added to the wells.FFA and ethanol were considered as the pathogenetic factors which induce hepatocyte injury. FFA used was a mixture of palmitic and oleic acids, prepared according to the method described by Garcia 4 cells/well, for preparation of the injury models before addition of APPI and APPII at optimal doses. Following incubation for 48 h, the cells and culture medium in all treatment groups were collected to evaluate the repairing ability. Intracellular protein, cellular TG and the activities of SOD from cells were quantified using commercial assay kits, and the activities of ALT and AST in the culture medium were assayed with colorimetric assay kits, all in accordance with the manufacturers’ protocols. Histological analysis of cellular lipid was determined with the Oil-red O stain method.To determine hepatoprotective effects, injured HepG2 cells were treated with APPI and APPII at various concentrations for 48 h. Upon termination of the incubation, viability of the cells was measured with the MTT assay. The optimal doses, obtained by using cell viability determination, were considered as effective concentrations to analyze the mechanism of APPI and APPII. HepG2 cells were seeded onto 6-well plates at a concentration of 3 × 10To analyze the therapeutic pathways of APPI and APPII, the expression levels of lipid metabolism genes in the adiponectin pathway were determined by qRT-PCR. Extraction of total RNA from the samples was conducted by using TRIzol reagent  and reversely transcribed with oligo (dT) using EasyScriptTM First-Strand cDNA Synthesis SuperMix , in accordance with the manufacturer’s protocol. qRT-PCR was performed following the protocol of the Maxima SYBR Green/ROX qRT-PCR Master Mix  employing an ABI 7500 . The GAPDH RNA level was used as an endogenous control for mRNAs. The qRT-PCR procedure comprised pre-denaturation at 95 °C for 5 min and 40 cycles which consisted of exposure to 95 °C for 30 seconds, and to 60 °C for 1 min. The relative expression level was computed by the 2-ÄÄCt method. Five independent experiments were performed.Male Wistar rats (220–280 g) obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, were utilized in this study. All experimental protocols were approved by the University Safety Office and Animal Experimentation Ethics Committee at The Chinese University of Hong Kong and China Agricultural University. All animal experiments were carried out in accordance with the approved guidelines of the Animal Care and Use Committee of The Chinese University of Hong Kong and China Agricultural University. The normal control group was fed with SLD, while the other groups received a HFD including 2% cholesterol and 25% pig fat.Rats were randomly allocated in seven groups with six rats in a group. The normal control group was fed SLD, while the other six groups received a HFD including 2% cholesterol and 25% pig fat for eight weeks. The six HFD groups were treated as follows: (i) the model group (no treatment), (ii) low-dose APPI group (50 mg/kg/d), (iii) high-dose APPI group (100 mg/kg/d), (iv) low-dose APPII group (50 mg/kg/d), (v) high-dose APPII group (100 mg/kg/d), (vi) positive group of simvastatin treatment (2 mg/kg/d). APPI, APPII and simvastatin were administered intragastrically for 4 weeks after an 8-weeks HFD model foundation. Rats in the normal control group and model control group an equal volume of normal saline instead. Free access to food and water was allowed. The rats were weighed weekly, and fresh food was provided daily.After the final drug treatment, the animals were sacrificed by cervical dislocation, and blood was collected for biochemical analysis. The liver was removed and weighed. A small piece of the liver was fixed in 10% buffered formalin solution for histological processing, and the remainder was frozen at −80 °C till biochemical analysis.The hepatic tissues were preserved in 4% paraformaldehyde at 4 °C for 24 h before embedding in paraffin for sectioning. The tissue sections were prepared and stained with hematoxylin and eosin (H&E). Hepatic histology was observed using an Olympus microscope, IX71 .Liver tissue was homogenized using phosphate buffer saline. Following centrifugation at 10,000 r/min for 40 min at 4 °C, the supernatant was saved for biochemical evaluation. TC and TG levels as lipid indexes, and ALT and AST activities as hepatic injury indexes were assayed using commercial kits following the manufacturer’s instructions.All experiments in all of the bioassays were conducted in triplicate. Data are shown as mean ± standard deviation (SD). Statistical analysis was conducted with one-way analysis of variance (ANOVA) using SPSS software . Data were assessed by using ANOVA and P ≤ 0.05 was considered to be statistically significant.A. polytricha and elucidation of the mechanism involved represents the first of its kind on purified polysaccharide-peptides. The A. polytricha polysaccharide peptides are of considerable interest and promising for development into a therapeutic agent in view of the array of health-enhancing activities of adiponectin.The present report on the hepatoprotective and fatty liver alleviating activities of purified polysaccharide-peptides acquired from Supplementary information


The purpose of this study was to examine the relationships between individual parent stressors  and adolescent obesity in African American adolescents.  Data were from a cross section convenience sample of 273 African American parent-child dyads (ages 11–19) from Washtenaw County, Michigan. A subset of 122 dyads who completed parent and child questionnaires were included in this analysis. Parent stressors were assessed using the Crisis in Family Systems Revised (CRISYS-R) questionnaire. Height, weight, and waist circumference were measured by trained staff; height and weight were converted to BMI. Multivariate linear regression models were used to examine the relationships between individual parent stressors and adolescent BMI and waist circumference. β = 1.20(0.47), p=0.01) and waist circumference (β = 2.86(1.18), p=0.02). Parental appraisal of stressors related to safety in the community as “difficult to get through” was positively associated with adolescent BMI (β = 0.39(0.14), p=0.006) and waist circumference (β = 1.00(0.35), p=0.005). These relationships remained significant when adjusting for behavioral and psychosocial covariates. There were no significant relationships observed between other parent stressors and adolescent BMI or waist circumference.  Parental exposure to stressors related to safety in the community was positively associated with adolescent BMI (NCT02938663. These findings suggest parents' exposure and appraisal of stressors related to community safety are associated with increased adolescent obesity in African American youth. Longitudinal, larger-scale studies are needed to better understand the mechanisms by which community safety may increase obesity risk in this ethnic minority pediatric population. This trail is registered with  Health and Behaviour in Teenagers Study, Van Jaarsveld and colleagues noted that higher levels of psychological stress over 5 years of follow-up was associated with higher waist circumference and body mass index (BMI) in a multiethnic sample of 4,065 adolescents (ages 11–16) [National, Heart, Lung, and Blood Institute Growth and Health Study, Tomiyama and colleagues observed that higher levels of psychological stress over 10 years of follow-up predicted larger increases in BMI in 2,379 African American and White adolescent girls (ages 10–19) [Childhood obesity is a major public health concern, particularly among African American youth. Data from the National Center for Health Statistics estimated that 22% of African American youth were classified as obese compared to 14% of non-Latino White youth [s 11–16) . In the Children's Health Study, Shankardass and colleagues examined the impact of parental psychological stress on BMI in 4,078 children (ages 5–10 years) over four years of follow-up [Southeastern Pennsylvania Household Health Survey, Parks and colleagues determined that the number of stressors experienced by parents was related to child obesity in a sample of 2,119 parents/caregivers and their children (ages 3–17) [All Babies in Southeast Sweden project [p < 0.01) and longitudinally  [While research has shown a consistent relationship between parent stress and child weight status, few have explored the independent roles of specific stressors on adolescent obesity in African American youth. In the ollow-up . Study pDifferent types of parent stressors are likely to influence adolescent obesity in a variety of ways. For example, financial stressors—changes in income and inability to obtain household goods—could contribute to the types of food parents are able to buy, leading to low-cost, but high-calorie foods in the home. In the same way, financial stressors may make purchasing physical activity equipment or paying fees associated with sports team participation a challenge . Stress As few studies have examined parent stressors comprehensively, the aim of this study was to examine parent stressors from eleven different content areas including financial, legal, career, relationships, safety in the home, safety in the community, medical issues pertaining to self, medical issues pertaining to others , home issues , authority , prejudice, and their influence on adolescent BMI and waist circumference among African American adolescents. We hypothesized that both exposures to and appraisals of each type of stressor experienced by the parent would predict higher BMI and waist circumference in their children.Adolescents were recruited from neighboring communities in southeast Michigan to participate in the Health and Culture Project, a two-component study investigating social, psychological, and cultural predictors of obesity , 14–16. n = 85), high schools (n = 31), after-school programs (n = 46), community centers (n = 30), and the general community in Washtenaw County, Michigan (n = 81), via flyers and word of mouth. Data collection occurred at the University of Michigan Childhood Disparities Research Laboratory or off-site at a school, after-school program, or community center. In total, 122 African American adolescents  who completed questionnaires assessing food intake and had at least 4 days of valid accelerometer data were included in this analysis. Participants were excluded for the following reasons: 20 child participants did not self-identify as African American or Black, 19 child participants were outliers for caloric intake , and 109 caregivers did not complete the stress questionnaire [n = 122) were compared with those who were excluded (n = 151), both groups were similar in BMI, height, weight, and waist circumference . Included participants, however, were older  compared with excluded participants. Written assent (for adolescents under age 17) and written consent from older adolescents and parents were obtained before participating. This study was reviewed and approved by the University of Michigan Institutional Review Board.Two hundred seventy-three adolescent participants, ages 11–18 years, were recruited from middle schools  . Waist cParent stressors were measured using the Crisis in Family Systems Revised (CRISYS-R) questionnaire which has been validated for use in a multiethnic sample including African American parents . This 63Adolescent's sex and age were assessed via child self-report. Moderate-to-vigorous physical activity (MVPA) and sedentary activity was assessed over a 7-day period for each participant via accelerometry . Participants were asked to wear the device snugly on their right hip using an adjustable belt. At home, they were advised to remove the accelerometers during bathing, contact sports, water-based activities, and sleeping. Participants were also asked to complete a nonwear and sleep diary. The frequency at which the devices collected raw data was 30 Hz. The raw data obtained from the accelerometer were retrieved and integrated into 10-second epochs using ActiLife software, version 6.11.8. At least 4 days of valid (>600 min/day) accelerometer data were required for MVPA and sedentary activity to be recorded. Puyau cut-points, most commonly used for adolescent populations, were used to derive moderate-to-vigorous activity intensity . These cp > 0.05). To examine the relationship between parent stressors and adolescent BMI, two models were run for each stressor. The first model included the exposure score as the dependent variable, and the second model included the appraisal score as the dependent variable. Both age-adjusted and multivariate linear regression models were used to examine the relationship between parent stressors and BMI with adolescent sex, age, dietary intake, sedentary time, MVPA, and parent employment status included as covariates. The same analyses were then run to examine the relationship between parent stressors and waist circumference. The significance level for all analyses was α < 0.05. Analyses were conducted using Stata Special Edition 14.0.Before analysis, data were evaluated for normality, and natural log transformations were made to the following variables: dietary intake, sedentary time, and MVPA. Spearman's correlations were used to explore the associations between the parent stressor variables . Multiple imputation was used to account for missing data in both adolescents and parents. Most study variables had <10% missing data. The highest fraction of missing data was MVPA (29.2%). Participants with imputed data did not significantly differ by age, BMI, waist circumference, height, or weight from participants without imputed data . The weakest correlation was seen between medical issues (self) and legal issues . Most correlations between parental stressor exposures were weak (r = 0.05–0.40).r = 0.54, p < 0.05). The weakest correlation was seen between home safety and authority . Most correlations between parental stressor appraisals were weak (r = 0.05–0.40).β = 0.90(0.46), p=0.05). When sex, dietary intake, sedentary time, MVPA, and parent employment were accounted for, the association between stressors related to safety in the community and adolescent BMI became significant (β = 1.20(0.47), p=0.01). Exposure to stressors related to safety in the community was positively associated with adolescent waist circumference for both the age-adjusted model (β = 2.55(1.19), p=0.03) and the multivariate-adjusted model (β = 2.86(1.18), p=0.02).β = 0.31(0.13), p=0.02) and multivariate-adjusted model (β = 0.39(0.14), p=0.006). Appraisal of stressors related to safety in the community was positively associated with adolescent waist circumference in the age-adjusted model (β = 0.94(0.34), p=0.006) and the multivariate-adjusted model (1.00(0.35), p=0.002). Appraisal of stressors related to parent medical issues was positively associated with adolescent waist circumference in the age-adjusted model (β = 1.43(0.70), p=0.045); however, this relationship was no longer significant after adjusting for sex, dietary intake, sedentary activity, MVPA, and parent employment status .This study explored different parent stressors and their relation to adolescent obesity in a sample of African American parent-child dyads. While previous studies assessed BMI as an indicator of adolescent obesity, this study examined both adolescent's BMI and waist circumference as previous data suggest stress plays a greater role in central adiposity (measured via waist circumference) than BMI . ConsistNational Institute of Child Health and Human Development Study of Early Child Care and Youth Development [Previous research supports our findings that parent stress related to safety in the community are associated with increased adolescent obesity. Lumeng et al. reported that parental perceptions of their neighborhood as unsafe was associated with a greater risk of being overweight by age 7 in a multiethnic sample of 768 children from the elopment . Previouelopment , 37. Cecelopment . Cecil-Kelopment . Yet, inIn the age-adjusted models, parent stressors related to personal medical issues were positively associated with adolescent waist circumference. These findings are consistent with Isasi and colleagues who included parent health in overall measures of stress and found a positive association between parent stress and adolescent obesity . GaraskyThere were no significant relationships between adolescent BMI or waist circumference with the remaining stressors. It is important to note that our sample had a higher level of educational attainment compared to African Americans nationally (61% had at least a bachelor's degree vs 18%) which may have served as a protective factor in this cohort , 32. EduThe strengths of this study include the focus on African American adolescents (a group at increased risk for childhood obesity), controls in the analyses for appropriate variables, objective measures of adolescent MVPA and sedentary time using accelerometers, the use of multiple measures of adolescent obesity (BMI and waist circumference), and the inclusion of a wide array of parent stressors using a validated measure. Limitations included a small convenient sample and a cross-sectional design that relied on self-reported data and the use of a food frequency questionnaire (FFQ) to measure adolescent caloric intake. High fractions of missing data were noted for adolescent physical activity; however, multiple imputation was used to account for these missing data. Additionally, the study used a wide range of ages (11–18) and had a high proportion of female participants (62%). Finally, the generalizability of this study may be limited to African American youth residing in Southeast Michigan given the higher level of household education reported in this group. Despite these limitations, we observed a strong and consistent relationship between parent stress related to safety in the community and adolescent waist circumference.Moving to Opportunity study [The present study suggests that parent stressors related to community safety can play an important role in shaping adolescent weight status among African American youth. Additionally, addressing the broader financial and safety concerns present in African American neighborhoods may also be useful in overcoming the potential risks imposed by an unsafe neighborhood on adolescent obesity. Indeed, findings from the ty study  demonstr


Patients with chronic kidney disease have a greater risk of cognitive impairment. Cerebral uremic solute accumulation causes uremic encephalopathy; however, the association of protein-bound uremic toxins on cognitive function remains unclear. The present study aimed to investigate the association of two protein-bound uremic toxins, namely indoxyl sulfate (IS) and p-cresyl sulfate (PCS), on cognitive function in patients receiving hemodialysis (HD) for at least 90 days. Circulating free form IS and PCS were quantified by liquid chromatography/mass spectrometry. Mini-Mental State Examination (MMSE) and Cognitive Abilities Screening Instrument (CASI) were used to evaluate cognitive function. In total, 260 HD patients were recruited with a mean age of 58.1 ± 11.3 years, of which, 53.8% were men, 40% had diabetes, and 75.4% had hypertension. The analysis revealed that both free IS and free PCS were negatively associated with the CASI score and MMSE. After controlling for confounders, circulating free IS levels persisted to be negatively associated with MMSE scores  and CASI scores , mainly in the CASI domains of long-term memory, mental manipulation, language ability, and spatial construction. However, there was no correlation between free PCS and total MMSE or total CASI scores after controlling for confounders. In conclusion, circulating free form IS, but not PCS is associated with lower cognitive function test scores in HD patients. Thus, a further study is needed to evaluate whether a decrease in free IS levels can slow down cognitive decline in HD patients. Dementia further worsens the adverse outcomes, leading to mortality, hospitalization, disability, and dialysis withdrawal3. Patients with CKD not only have the same risk factors for dementia as the general population4, but also have additional nephrogenic risk factors5. Previous studies have addressed the role of cerebrovascular disease, anemia, and hyperparathyroidism in dementia development in patients with CKD or ESKD5, but the association of uremic toxins on cognitive function remains unclear. The accumulation of uremic solutes in the blood and the brain may affect cognitive function6. Uremic toxins are classified into three groups based on their protein binding property and molecular weight: protein-bound solutes, middle molecules, and small water-soluble solutes7. Although hemodialysis (HD) eliminates small water-soluble uremic toxins, the remaining middle-molecule and protein-bound uremic toxins interfere with cognitive function by disrupting the brain barrier8. Similarly, peritoneal dialysis clearance of protein-bound uremic toxins is lower than the clearance of small solutes12, with residual renal function accounting for most protein-bound uremic toxin removal in peritoneal dialysis patients10.Dementia is characterized by progressive loss of daily life functions and the development of psychiatric symptoms. Patients with chronic kidney disease (CKD) or end-stage kidney disease (ESKD) have a higher rate of cognitive impairment, with approximately threefold higher dementia prevalence than similar aged individuals in the general population13. IS may trigger cerebral dysfunction through the induction of inflammation and oxidative stress, which are initiated by astrocytes and microglia14, further reducing neuronal viability and contributing to neurodegeneration15. However, only a few studies have evaluated the association between protein-bound uremic solute levels and cognitive impairment, and the results were inconclusive17. Therefore, we investigated the association of IS and PCS with cognitive function in HD patients.Indoxyl sulfate (IS) and p-cresyl sulfate (PCS), known as protein-bound uremic toxins, are difficult to eliminate via dialysis because they tightly bind to albumin in the blood; consequently, the IS level in brain and plasma in patients with CKD is high compared with control subjectsIn silico data, the prediction was analyzed by the online BBB server (http://www.cbligand.org/BBB/predictor.php) using the admetSAR database18. The BBB predictor predicts the ability of a compound to pass the BBB and was built using support vector machine algorithms for the four types of fingerprints  of 1593 reported compounds19. Absorption, Distribution, Metabolism, and Toxicity (ADMET) properties were calculated based on the admetSAR database that offers various chemical data with known ADMET properties.The brain is safeguarded from exogenous compounds through the blood-brain barrier (BBB). The potential of a toxin to enter the brain is an essential parameter when considering the toxicity, therefore, a BBB predictor was designed to determine whether a compound can cross the BBB. From August 2016 to January 2017, patients were recruited at Kaohsiung Medical University Hospital to establish the HD cohort. Participants with maintenance dialysis for more than 90 days (N = 341) were eligible. In total, 286 individuals were screened using neuropsychological tests. Participants who refused to undergo blood tests or had missing data regarding protein-bound uremic toxin (IS and PCS) measurement (N = 26) were excluded from the final analysis  were also conducted.For all participants, demographic data , dialysis duration, medical history, and biochemical data were extracted from routine electronic healthcare records. Hypertension was defined as blood pressure ≥140/90 mmHg or antihypertensive drug use; diabetes was defined as HbA1C ≥6.5% or antidiabetic drug use. Participants’ history of coronary artery disease and cerebrovascular disease was obtained based on physicians’ diagnosis. Blood samples of patients were obtained at a single midweek dialysis session after overnight fasting through vascular access before their scheduled HD session. Biochemical data of HD patients were obtained at the beginning of the HD session within 30 days before cognitive testing.21 and Cognitive Abilities Screening Instrument (CASI)23 For free form PCS and IS free analyses, each human serum samples (300 μL) was packed into a centrifugal filter device  and centrifuged for 30 min at 13,300 g at 4°C. Then, the supernatant was carefully transferred into another Eppendorf tube and evaporated with a spin vacuum instrument. The lyophilized samples were re-dissolved with 100 µL 30% acetonitrile (MeCN) with 0.1% formic acid, then 100 µL of the solution was added 10 µL IS-d4  and filtrated with 0.22 µm polytetrafluoroethylene (PTFE) filters for mass spectrometer analysis. The tandem mass spectrometry system was equipped with micro electrospray ionization (ESI) ion source, coupled with an Acella 1250 Ultra-high-performance liquid chromatography (UHPLC) analytical system  with 3.0 kV setting. The samples were separated using a Shiseido HPLC CAPCELL PAK C18 MGII column  after injection into the UHPLC system via the Acella 1250 autosampler. The mobile phases contained 0.1% (v/v) FA (A) in water and 0.1% (v/v) FA (B) in acetonitrile with a 250 mL/min flow rate. The linear gradient was set as follows: 30%, 30–60%, 60–98%, 98%, 98–30%, 30% in 2/6/3/2/0.1/6.9 minutes respectively (B). The negative ion mode with a voltage of 2.5 kV was set up as detection mode of the mass spectrometer, the temperature for the vaporizing and capillary temperatures was 300 °C and 350 °C, and the pressure setting for sheath gas and aux gas were 35 and 10 with a collision pressure of 1.5 and collision energy of 22 V. The quantification was evaluated by survey scan mode multiple reaction monitoring (MRM) transitions 212 > 80 and 212 > 132 for IS and 187 > 80 and 187 > 107 for PCS, using Xcalibur software .p values set at 0.2 in the models for independent variables to enter and stay, subsequently p values < 0.05 in the final elimination step. The covariates adjusted for in the model were age, sex, education level, depression scale (CES-D), comorbidities , clinical data , and HD duration. The results are reported as the β coefficient with a 95% confidence interval (CI). In addition, ROC-derived AUC cutoff values were used as cutoffs for statistical analyses rather than using an arbitrary cutoff level. The results are reported as the odds ratio (OR) with 95% CI. Two-tailed p < 0.05 was considered statistically significant. STATA version 15 and SAS version 9.4 were used to perform statistical analyses.Data are shown as mean ± standard deviation or percentages as appropriate. The association of cognitive function test scores with IS or PCS was evaluated using linear regression models controlling for age, sex, and education level. To identify the independent determinants of IS and PCS, multivariate linear regression analysis with forward stepwise selection of variables was performed to assess the relationship between uremic toxins levels and MMSE/CASI scores, with in silico simulation analysis, the BBB prediction score was applied to investigate the BBB penetration ability of IS or PCS. If the BBB score is >0, the compound can be considered as BBB+. The BBB predictor classified IS and PCS as BBB+ compounds  Table . FurtherWe evaluated the association between protein-bound uremic toxins and cognitive function test scores  in HD participants using a regression model. IS was negatively correlated with the scores of MMSE, CASI, and CASI subdomains of long-term memory, mental manipulation, orientation, abstract thinking, language, and spatial construction Table , model 1p = 0.023), CASI scores , and the scores of CASI domains of long-term memory , mental manipulation , language ability , and spatial construction  (Table p = 0.035) and spatial construction  in the multivariate linear regression analysis. , circulating free form IS levels were persistently negatively associated with MMSE scores  and in age and sex-adjusted logistic regression model .The cutoff value of IS on cognitive impairment was analyzed by ROC curves and was 1.45 μg/ml. Higher circulating IS levels (>1.45 μg/ml) were associated with cognitive impairment compared to a lower IS level in the unadjusted logistic regression model . Another metabolomic study could not find an association between IS and cognitive impairment in HD patients due to the lack of power17, therefore, we enrolled 260 HD patients in the present study to investigate the correlation between IS and cognitive function. We evaluated the cognitive function based on MMSE and CASI to determine the association between uremic toxins and cognition. The different impact of IS and PCS on cognitive dysfunction in hemodialysis patients could be explained by the increased protein binding ability of IS which was associated with the increased free IS levels. However, the protein binding ability of PCS decreased with increasing free moiety concentrations25. Furthermore, it has been suggested that the affinity of PCS for human serum albumin binding is moderate at 25 °C and weak at 37 °C  using microcalorimetry analysis26, thus, PCS binding to albumin appears to be less strong than previously reported26. The higher protein binding ability is the main cause of poor removal of these protein-bound toxins by HD, therefore, these differences between IS and PCS may play a role in the observed cognitive impairment differences between IS and PCS.Cognitive function impairment was found in HD patients compared with subjects without kidney disease in the present study and previous meta-analysis6. HD can partially clear protein-bound uremic toxins produced by the gut microbiome27, depending on the affinity and conjugates to albumin. IS normally crosses the BBB via organic anion transporter 3 (OAT-3), so may accumulate in the brain due to dysfunctional OAT-324. IS levels were higher in the brain and circulation among patients with CKD compared with non-CKD subjects13. Furthermore, increased IS levels in the blood may impair the brain transporters’ ability to remove other solutes, as IS has been shown to inhibit related transporters in kidney and liver cells28. Several direct pathophysiology mechanisms of IS-related neurotoxicity exist. IS stimulates the aryl hydrocarbon receptor (AhR), known as a ligand-activated transcriptional factor29, prolonged activations of which could cause endothelial dysfunction, thereby contributing to neurotoxicity31. Furthermore, IS direct influences glial and astrocyte function, causing neuronal damage14. In the presence of IS, astrocyte cells increase the release of reactive oxygen species and decrease the activation of nuclear factor-like 2 and the expression of heme oxygenase-1 and NADPH dehydrogenase quinone 114. IS also induces apoptosis of human astrocytes via oxidative stress and mitogen-activated protein kinase pathway inhibition32. Additionally, IS may induce inflammatory and oxidative stress mediator production from glial cells, which actively contribute to various dementia forms33, and complex neuron-glia interaction disturbances are important in the pathophysiological mechanism for neurological disorders, including neurodegeneration34. Glial cell dysfunction can deteriorate neuronal function. Moreover, IS induces neuron cell death in a dose-dependent manner14.Protein-bound uremic toxins, such as IS, may play a role in cognitive function decline through direct toxicity or by other putative factors, such as vascular calcification, endothelial dysfunction, oxidative stress, and inflammation35. Furthermore, IS activates AhR and promotes oxidative stress in astrocytes and further implicates sensorimotor and cognitive dysfunction35. Considering the direct neuronal toxicity of protein-bound uremic toxins, IS may be involved in cognitive impairment in patients with CKD through vascular toxin effects and direct toxic effects on cells, especially in those without obvious cerebrovascular disease. IS increases oxidative stress leading to histological brain alterations, and inflammatory markers were found in the IS-injected mice model14. Moreover, IS increased inflammation and oxidative stress in primary central nervous system cells through nuclear factor-kappa B and AhR, and IS induced neuron death15. Furthermore, in the presence of AST-120, pro-oxidant and pro-inflammatory responses reduced in astrocytes treated with patients’ serum15. In an animal study, cisplatin-administered rats showed increased IS concentrations in the brain tissue, with associated increases in nephrotoxicity and disturbances in the circadian rhythm of the transcription of the clock gene rPer236. The aforementioned results support the present study findings regarding IS neurotoxicity and that higher circulating levels of free form IS lead to poor cognitive function scores.AhR is expressed in the hippocampus, cerebral cortex, and cerebellum10. Thus, these findings may difficult to apply for patients with peritoneal dialysis because of the different uremic toxin concentration in patients with peritoneal dialysis37. Second, a cross-sectional study could not be used to imply causality. To gain clarity, longitudinal studies with longer follow-up durations are required. Third, to ascertain the cognitive impairment etiology, brain images were unavailable in this study. Finally, the IS concentration in the cerebrospinal fluid is possibly more crucial than circulating levels with respect to cognitive function, but this was not assessed in this study.The current study is not without limitations. First, all subjects in this study received HD, not peritoneal dialysis. In peritoneal dialysis patients with significant residual renal function, more IS and PCS are removed in the urine than in the dialysateCirculating free form IS but not PCS was associated with poor cognitive function (MMSE and CASI) in patients undergoing HD. In addition, IS was negatively associated with CASI domains of long-term memory, mental manipulation, language ability, and spatial construction. Additional studies are needed to investigate whether IS elimination in patients with advanced CKD delays cognitive decline.Supplementary Tables and figures.