Absence of calcium-sensing receptor basal activity due to inter-subunit disulfide bridges.

G protein-coupled receptors naturally oscillate between inactive and active states, often resulting in receptor constitutive activity with important physiological consequences. Among the class C G protein-coupled receptors that typically sense amino-acids and their derivatives, the calcium sensing receptor (CaSR) tightly controls blood calcium levels. Its constitutive activity has not yet been studied. Here, we demonstrate the importance of the inter-subunit disulfide bridges in maintaining the inactive state of CaSR, resulting in undetectable constitutive activity, unlike the other class C receptors. Deletion of these disulfide bridges results in strong constitutive activity that is abolished by mutations preventing amino acid binding. It shows that this inter-subunit disulfide link is necessary to limit the agonist effect of amino acids on CaSR. Furthermore, human genetic mutations deleting these bridges and associated with hypocalcemia result in elevated CaSR constitutive activity. These results highlight the physiological importance of fine tuning the constitutive activity of G protein-coupled receptors.

Covalent organic framework/layered double hydroxide composite-coated poly(ether ether ketone) jacket for stir bar sorptive extraction of Sudan dyes.

A novel coating for stir bar sorptive extraction was developed by growing a covalent organic framework, TpPa-1 (derived from phenylenediamine and 1,3,5-trimethylphloroglucinol), onto the surface of Ni-Al layered double hydroxide. Using a poly(ether ether ketone) tube as the supporting substrate, a TpPa-1/layered double hydroxide-coated stir bar was fabricated and demonstrated excellent extraction performance for Sudan dyes. Notably, its extraction efficiency significantly exceeded that of stir bars modified with only TpPa-1 or Ni-Al layered double hydroxide. Based on this innovative coating, a stir bar sorptive extraction-high performance liquid chromatography method was established. This method exhibited low limits of detection (0.04-0.08 ng/mL) for the analysis of Sudan dyes. It also featured a wide linear range (0.25-100 or 200 ng/mL) and demonstrated good repeatability with relative standard deviations ≤6.22%. The recoveries obtained for spiked lake water and chili powder samples were 93.5%-105.2% and 87.8%-100.6%, respectively, demonstrating the practical potential of the developed method for detecting trace Sudan dyes in real samples.

A Potent SOS1 PROTAC Degrader with Synergistic Efficacy in Combination with KRASG12C Inhibitor.

AMG510, as the first approved inhibitor for KRASG12C mutation, has shown promising efficacy in nonsmall-cell lung cancer and colorectal cancer harboring KRASG12C mutation. However, the moderate response rate and the rapid emergence of acquired resistance limit the therapeutic potential of AMG510, highlighting the need for the development of combination strategies. Here, we observed the suppression of RAS-MAPK signaling induced by AMG510 was prolonged and enhanced by SOS1 knockdown. Thus, we design, synthesize, and characterize a potent and specific SOS1 degrader 23. Compound 23 showed efficient SOS1 degradation in KRAS-driven cancer cells and achieved significant antiproliferative potency. Importantly, the combination of 23 with AMG510 suppressed RAS signaling feedback activation, showing synergistic effects against KRASG12C mutant cells in vitro and in vivo. Our findings demonstrated that KRASG12C inhibition plus SOS1 degradation as a potential therapeutic strategy to improve antitumor response and overcome acquired resistance to KRASG12C inhibitor.

Self-Supervised Random Forest on Transformed Distribution for Anomaly Detection.

Anomaly detection, the task of differentiating abnormal data points from normal ones, presents a significant challenge in the realm of machine learning. Numerous strategies have been proposed to tackle this task, with classification-based methods, specifically those utilizing a self-supervised approach via random affine transformations (RATs), demonstrating remarkable performance on both image and non-image data. However, these methods encounter a notable bottleneck, the overlap of constructed labeled datasets across categories, which hampers the subsequent classifiers' ability to detect anomalies. Consequently, the creation of an effective data distribution becomes the pivotal factor for success. In this article, we introduce a model called "self-supervised forest (sForest)", which leverages the random Fourier transform (RFT) and random orthogonal rotations to craft a controlled data distribution. Our model utilizes the RFT to map input data into a new feature space. With this transformed data, we create a self-labeled training dataset using random orthogonal rotations. We theoretically prove that the data distribution formulated by our methodology is more stable compared to one derived from RATs. We then use the self-labeled dataset in a random forest (RF) classifier to distinguish between normal and anomalous data points. Comprehensive experiments conducted on both real and artificial datasets illustrate that sForest outperforms other anomaly detection methods, including distance-based, kernel-based, forest-based, and network-based benchmarks.

Novel Sulfonylurea-Based NLRP3 Inflammasome Inhibitor for Efficient Treatment of Nonalcoholic Steatohepatitis, Endotoxic Shock, and Colitis.

The NLRP3 inflammasome is a critical component of innate immunity involved in the pathophysiology of various inflammatory diseases. In this study, we designed and synthesized a series of NLRP3 inflammasome inhibitors based on MCC950. Specifically, we optimized the furan moiety, which is considered to be potentially associated with drug-induced liver injury. The representative inhibitor N14, 4-(2-(dimethylamino)ethyl)-N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)benzenesulfonamide, not only maintains the NLRP3 inhibitory activity of MCC950 with IC50 of 25 nM but also demonstrates improved tolerability in human hepatic cells line and mouse primary hepatocytes. In addition, N14 exhibits superior pharmacokinetic properties, with an oral bioavailability of 85.2%. In vivo studies demonstrate that N14 is more effective than MCC950 in multiple NLRP3-related animal model diseases, including nonalcoholic steatohepatitis, lethal septic shock, and colitis. Our research has provided a lead compound that directly targets the NLRP3 inflammasome and can be developed as a novel therapeutic candidate for NLRP3-driven diseases.

Discovery of BWA-522, a First-in-Class and Orally Bioavailable PROTAC Degrader of the Androgen Receptor Targeting N-Terminal Domain for the Treatment of Prostate Cancer.

We report small molecular PROTAC compounds targeting the androgen receptor N-terminal domain (AR-NTD), which were obtained by tethering AR-NTD antagonists and different classes of E3 ligase ligands through chemical linkers. A representative compound, BWA-522, effectively induces degradation of both AR-FL and AR-V7 and is more potent than the corresponding antagonist against prostate cancer (PC) cells in vitro. We have shown that the degradation of AR-FL and AR-V7 proteins by BWA-522 can suppress the expression of AR downstream proteins and induce PC cell apoptosis. BWA-522 achieves 40.5% oral bioavailability in mice and 69.3% in beagle dogs. In a LNCaP xenograft model study, BWA-522 was also proved to be an efficacious PROTAC degrader, resulting in 76% tumor growth inhibition after oral administration of a dose of 60 mg/kg. This study indicates that BWA-522 is a promising AR-NTD PROTAC for the treatment of AR-FL- and AR-V7-dependent tumors.

Deep Into the Domain Shift: Transfer Learning Through Dependence Regularization.

Classical domain adaptation methods acquire transferability by regularizing the overall distributional discrepancies between features in the source domain (labeled) and features in the target domain (unlabeled). They often do not differentiate whether the domain differences come from the marginals or the dependence structures. In many business and financial applications, the labeling function usually has different sensitivities to the changes in the marginals versus changes in the dependence structures. Measuring the overall distributional differences will not be discriminative enough in acquiring transferability. Without the needed structural resolution, the learned transfer is less optimal. This article proposes a new domain adaptation approach in which one can measure the differences in the internal dependence structure separately from those in the marginals. By optimizing the relative weights among them, the new regularization strategy greatly relaxes the rigidness of the existing approaches. It allows a learning machine to pay special attention to places where the differences matter the most. Experiments on three real-world datasets show that the improvements are quite notable and robust compared to various benchmark domain adaptation models.

Design, Synthesis, and Biological Evaluation of Androgen Receptor (AR) Antagonist-Heat Shock Protein 90 (Hsp90) Inhibitor Conjugates for Targeted Therapy of Castration-Resistant Prostate Cancer.

Androgen deprivation in cases of castration-resistant prostate cancer (CRPC) leads to adverse effects, including loss of muscle and bone mass and gain of subcutaneous fat. The tumor-specific suppression of androgen receptor (AR) signaling, while not global, may reduce side effects. We present a class of small-molecular conjugates consisting of an AR antagonist linked to a heat shock protein 90 (Hsp90) inhibitor. We demonstrate that the high accumulation of Hsp90 on the surface of CRPC cells allows uptake of conjugates and increases the enrichment of drugs in the tumor cells. After penetrating prostate cancer cells, the conjugates not only inhibit AR function by the antagonist component but also bind to Hsp90 and suppress the AR protein level. Compared to AR antagonists, these conjugates showed improved tumor-targeting ability and enhanced potency against Enzalutamide-resistant 22Rv1 cells.

Inhibition of Astrocytic Carbohydrate Sulfotransferase 15 Promotes Nerve Repair After Spinal Cord Injury via Mitigation of CSPG Mediated Axonal Inhibition.

Nerve tissue regeneration is a significant problem. After neural diseases and damage such as spinal cord injury (SCI), the accumulation of chondroitin sulfate proteoglycans (CSPG) comprising axonal inhibitory glycosaminoglycan chains in the microenvironment is a major barrier that obstructs nerve repair. Interfering with the production of glycosaminoglycans, especially the critical inhibitory chains, could be a potential therapeutic strategy for SCI, which is, however, poorly defined. This study identifies Chst15, the chondroitin sulfotransferase controlling the generation of axonal inhibitory chondroitin sulfate-E, as a therapeutic target of SCI. Using a recently reported small molecular Chst15 inhibitor, this study investigates the effects of Chst15 inhibition on astrocyte behaviors and the associated consequences of in vivo disruption of the inhibitory microenvironment. Deposition of CSPGs in the extracellular matrix and migration of astrocytes are both significantly impaired by Chst15 inhibition. Administration of the inhibitor in transected spinal cord tissues of rats effectively promotes motor functional restoration and nerve tissue regeneration by a mechanism related to the attenuation of inhibitory CSPGs, glial scar formation and inflammatory responses. This study highlights the role of Chst15 in the CSPG-mediated inhibition of neural recovery after SCI and proposes an effective neuroregenerative therapeutic strategy that uses Chst15 as a potential target.

A Rab10-ACAP1-Arf6 GTPases cascade modulates M4 muscarinic acetylcholine receptor trafficking and signaling.

Membrane trafficking processes regulate the G protein-coupled receptor activity. The muscarinic acetylcholine receptors (mAChRs) are highly pursued drug targets for neurological diseases, but the cellular machineries that control the trafficking of these receptors remain largely elusive. Here, we revealed the role of the small GTPase Rab10 as a negative regulator for the post-activation trafficking of M4 mAChR and the underlying mechanism. We show that constitutively active Rab10 arrests the receptor within Rab5-positive early endosomes and significantly hinders the resensitization of M4-mediated Ca2+ signaling. Mechanistically, M4 binds to Rab10-GTP, which requires the motif 386RKKRQMAA393 (R386-A393) within the third intracellular loop. Moreover, Rab10-GTP inactivates Arf6 by recruiting the Arf6 GTPase-activating protein, ACAP1. Strikingly, deletion of the motif R386-A393 causes M4 to bypass the control by Rab10 and switch to the Rab4-facilitated fast recycling pathway, thus reusing the receptor. Therefore, Rab10 couples the cargo sorting and membrane trafficking regulation through cycle between GTP-bound and GDP-bound state. Our findings suggest a model that Rab10 binds to the M4 like a molecular brake and controls the receptor's transport through endosomes, thus modulating the signaling, and this regulation is specific among the mAChR subtypes.

Lethal activity of BRD4 PROTAC degrader QCA570 against bladder cancer cells.

Bladder cancer is the most common malignancy of the urinary system. Efforts to identify innovative and effective therapies for bladder cancer are urgently needed. Recent studies have identified the BRD4 protein as the critical factor in regulation of cell proliferation and apoptosis in bladder cancer, and it shows promising potential for pharmacologic treatment against bladder cancer. In this study, we have evaluated the biological function of QCA570, a novel BET degrader, on multiple bladder cancer cells and explore its underlying mechanisms. QCA570 potently induces degradation of BRD4 protein at nanomolar concentrations, with a DC50 of ∼ 1 nM. It decreases EZH2 and c-MYC levels by transcriptional suppression and protein degradation. Moreover, the degrader significantly induces cell apoptosis and cycle arrest and shows antiproliferation activity against bladder cancer cells. These findings support the potential efficacy of QCA570 on bladder cancer.

Effects of different proportions of stevia stalk on nutrient utilization and rumen fermentation in ruminal fluid derived from sheep.

Background: Stevia straw is a byproduct of sugar crop stevia. It is a good feed material because of richness in nutrients and active substances (steviosides and flavonoids). However, due to improper utilization such as piling, burning and so on, it became a large amount of wasted straw resources and lead to environmental pollution. Methods: We added 0%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0% and 1.5% of stevia stalk to study the effects of different stevia stalk concentrations on nutrient utilization and rumen fermentation in sheep (based on sheep diet). In vitro fermentation method was used, with 17 repetitions for each treatment. All fermentation substrate based on sheep diet with different stevia stalk concentrations were fermented for 2 h, 6 h, 12 h, 24 h and 48 h, then the gas production, dry matter degradability (DMD), crude protein degradability (CPD), neutral detergent fiber degradability (NDFD), acid detergent fiber degradability (ADFD), pH, ammonia nitrogen (NH3-N) and volatile fatty acids (VFAs) were determined. Results: The results showed that at different fermentation time, the change trend of gas production in each teatment was basically same, but the maximum occurred in 1.0% treatment at 48 h. The DMD, CPD, NDFD and ADFD of sheep diets increased with fermentation time increasing, especially the CPD48h, NDFD48h and ADFD48h of diets in 0.8%, 1.0% and 1.5% treatments were significantly higher than those in control (P < 0.05). The pH of fermentation substrate in each treatment remained within the normal range of 6.21∼7.25. NH3-N24h-48hin 0.8%, 1.0% and 1.5% treatments were higher than that in control. At 6 h-12 h, the total acid content of 0.8% and 1.0% treatments were significantly higher than those of other treatments (P < 0.05), it reached the highest in 1.0% treatment. According to overall evaluation, effect ranking of stevia stalk on sheep nutrient utilization was as follows: 1.0% >0.8% >1.5% >0.4% >0.6% >0.2%. Overall, 1.0% stevia stalk could promote nutrient degradation and sheep rumen fermentation.

Strategies for activity analysis of single nucleotide polymorphisms associated with human diseases.

Genome-wide association studies (GWAS) have identified a large number of single nucleotide polymorphism (SNP) sites associated with human diseases. In the annotation of human diseases, especially cancers, SNPs, as an important component of genetic factors, have gained increasing attention. Given that most of the SNPs are located in non-coding regions, the functional verification of these SNPs is a great challenge. The key to functional annotation for risk SNPs is to screen SNPs with regulatory activity from thousands of disease associated-SNPs. In this review, we systematically recapitulate the characteristics and functional roles of SNP sites, discuss three parallel reporter screening strategies in detail based on barcode tag classification, and recommend the common in silico strategies to help supplement the annotation of SNP sites with epigenetic activity analysis, prediction of target genes and trans-acting factors. We hope that this review will contribute to this exuberant research field by providing robust activity analysis strategies that can facilitate the translation of GWAS results into personalized diagnosis and prevention measures for human diseases.

One-pot synthesis of magnetic sulfonated covalent organic polymer for extraction of protoberberine alkaloids in herbs and human plasma.

A novel magnetic sulfonated covalent organic polymer was prepared for magnetic solid-phase extraction of protoberberine alkaloids. The magnetic sulfonated covalent organic polymer was rapidly synthesized under mild conditions. The physicochemical properties of the prepared materials were characterized by Fourier-transform infrared spectrometry, transmission electron microscopy, and X-ray photoelectron spectroscopy. Several extraction parameters were systematically investigated, including desorption time, pH of sample solution, acetonitrile content, acetic acid content in the eluent, extraction time, and sample volume. By coupling magnetic solid-phase extraction and high-performance liquid chromatography, an efficient and sensitive method for the extraction and determination of protoberberine alkaloids in complex samples was developed. The proposed method showed great linearity (r > 0.9989), low limits of detection (0.2-0.3 ng/ml), and high precision (relative standard deviations ≤ 5.74%). The proposed method was further applied to the analysis of protoberberine alkaloids in Cortex phellodendri and human plasma samples. The recoveries were 91.50%-110.31% with relative standard deviations less than 6.63% in Cortex phellodendri and 96.12%-111.20% with relative standard deviations lower than 5.56% in plasma samples.

GPI-anchored glutathione S-transferase as marker allows affinity sorting of transfection-positive cells.

Cell transfection efficiency is still a limiting factor in gene function research. A method that allows isolation and enrichment of the transfection-positive cells is an effective solution. Here, we report a transfection-positive cell sorting system that utilizes GPI-anchored GST (Glutathione S-transferase) as a plasmid marker. The Glutathione S-transferase fusion protein will be expressed and displayed on the cell surface through GPI anchor, and hence permits the positive cells to be isolated using Glutathione (GSH) Magnetic Beads. We prove that the system works efficiently in both the adherent Lenti-X 293T cells and the suspension K-562 cells. The affinity cell sorting procedure efficiently enriched positive cells from 20% to 98% in K-562 cells. The applications in gene knockdown and overexpression experiments in K-562 cells dramatically enhanced the extent of gene alteration, with the gene knockdown efficiency increasing from 7% to 60% and the gene overexpression level rising from 47 to 253 times. This Glutathione S-transferase affinity transfection-positive cell sorting method is simple and fast to operate, large-instrument free, low cost, and hence possesses great potential in gene function study in vitro.

Biochar combined with organic and inorganic fertilizers promoted the rapeseed nutrient uptake and improved the purple soil quality.

Biochar is a kind of organic matter that can be added into soil to improve soil quality. To study the effect of biochar combined with organic and inorganic fertilizers on rapeseed growth and purple soil fertility and microbial community, a completely randomized block design was designed with three levels of biochar (B0: no biochar, B1: low-rate biochar, B2: high-rate biochar); two levels of inorganic fertilizers (F1: low-rate inorganic fertilizer; F2: high-rate inorganic fertilizer); and two levels of organic fertilizers (M1: no organic fertilizer; M2: with organic fertilizer). All combinations were repeated three times. The combined application of biochar and organic and inorganic fertilizers could improve soil pH, soil fertility and soil microbial community richness: The pH of B1F2M1 increased 0.41 compared with the control, the nitrogen, phosphorus and potassium content increased by 103.95, 117.88, and 99.05%. Meanwhile, soil microbial community richness was also improved. Our research showed that biochar could promote the Nutrient Uptake of rapeseed, and the combined application of biochar with organic and inorganic fertilizers could improve soil fertility and increase microbial diversity. Low-rate biochar combined with organic fertilizer and low-rate inorganic fertilizer was the most suitable application mode in rapeseed production in purple soil area of Southwest China.

A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag.

Gene delivery efficiency is an essential limit factor in gene study and gene therapy, especially for cells that are hard for gene transfer. Here we develop an affinity cell sorting system that allows efficient enrichment of gene transfer-positive cells. The system expresses an enhanced green fluorescent protein (EGFP) fused with an N-terminal high-affinity Twin-Strep-Tag (TST) that will be anchored to the cell membrane at the out-surface through a glycosylphosphatidylinositol (GPI) membrane-anchoring structure. The EGFP permits microscopy and flow cytometry analysis of the gene transfer-positive cells, and the TST tag at the N terminal of EGFP allows efficient affinity sorting of the positive cells using Strep-Tactin magnetic beads. The cell sorting system enables efficient isolation of gene transfer-positive cells in a simple, convenient, and fast manner. Cell sorting on transfected K-562 cells resulted in a final positive cell percentage of up to 95.0% with a positive cell enrichment fold of 5.8 times. The applications in gene overexpression experiments could dramatically increase the gene overexpression fold from 10 times to 58 times, and in shRNA gene knockdown experiments, cell sorting increased the gene knockdown efficiency from 12% to 53%. In addition, cell sorting in CRISPR/Cas9 genome editing experiments allowed more significant gene modification, with an editing percentage increasing from 20% to 79%. The gene transfer-positive cell sorting system holds great potential for all gene transfer studies, especially on those hard-to-transfect cells.

Serum Extracellular Vesicles Attenuate Cardiomyocyte Injury Induced by Hypoxic/Reoxygenation by Regulating miR-1229-5p.

Ischemic heart disease and the resulting heart failure remain the leading causes of death and disability worldwide. This study aimed to investigate the role of miR-1229-5p in serum extracellular vesicles (EVs) mediated myocardial protection by constructing a hypoxia/reoxygenation model (HR) in H9c2 cells. Cardiomyocytes were cultured and divided into different treatment groups: control group, HR group, serum-EVs group, and serum-EVs + miR-1229-5p inhibitor group. The expression levels of miR-1229-5p were detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The changes in cell proliferation and apoptosis were detected by MTT assay and flow cytometry. The myocardial injury-related indicators, cardiac troponin I (cTnI), creatinine kinase MB (CK-MB), and lactate dehydrogenase (LDH), were measured by enzyme-linked immunosorbent assay (ELISA). Finally, the luciferase reporter assay was used to verify the miR-1229-5p target. The proliferation of myocardial cells in the HR group was reduced, the number of apoptotic cells was increased, and myocardial injury indicators concentration was decreased. Transfection of miR-1229-5p inhibitor under serum-EVs treatment reduced the protective effect of serum-EVs on myocardial cell injury, decreased cell proliferation, increased the number of apoptotic cells, and increased myocardial injury indicator concentration. Additionally, FOXO4 may be the target of miR-1229-5p. Our data suggest that serum-EVs alleviate HR-induced cardiomyocyte injury by regulating miR-1229-5p/FOXO4.

Effects of Biochar With Inorganic and Organic Fertilizers on Agronomic Traits and Nutrient Absorption of Soybean and Fertility and Microbes in Purple Soil.

Biochar is a kind of organic matter that can be added into the soil as a soil amendment to improve its quality. What are the effects of using biochar on purple soil and soybeans? Can the use of biochar reduce the use of fertilizers? This is our concern. Therefore, we carried out this study. The objectives of our study were to evaluate the effects of biochar, inorganic and organic fertilizer application on plant growth, chlorophyll content, photosynthetic gas exchange, and yield of soybean as well as fertility and microbial community in purple soil, and to appraise the possible reduction rate of inorganic fertilizer under the biochar application. A pot experiment was conducted with three levels of biochar, two levels of inorganic fertilizer, and two levels of organic fertilizer in a randomized complete block. The results indicated that the low rate of biochar together with half rate of inorganic fertilizer and organic fertilizer increased the plant growth of soybean. Meanwhile, the chlorophyll content, root growth, and yield of soybean were increased by 16.61, 197.73, and 96.7%, respectively, with biochar compared with no biochar. The high rate of biochar with half rate of inorganic fertilizer and organic fertilizer can promote the exchange of photosynthetic gas in soybean, and the photosynthetic rate increased by 45.25% compared with the blank control. At the full pod stage, the nitrogen content, phosphorus content, and potassium content of the whole plant under the high rate of biochar were 28.35, 13.65, and 28.78%, respectively, higher than that of the blank control. The application of biochar increased nitrogen, phosphorus, and potassium uptake of soybean. The high rate of biochar with half rate of inorganic fertilizer and organic fertilizer can improve soil nutrient content and soil microbial community. Compared with no biochar treatments, total organic carbon (TOC) increased by 740.28%, and cation exchange capacity (CEC) increased by 54.17%. Phospholipid fatty acid (PLFA) increased by 65.22%, and all kinds of soil microorganisms increased to varying degrees. In conclusion, the application of biochar can reduce the use of organic and inorganic fertilizers, improve the agronomic traits and yield of soybean, and play a positive role in soil nutrients and soil microorganisms.

The prognostic and clinicopathological roles of microsatellite instability, PD-L1 expression and tumor-infiltrating leukocytes in familial adenomatous polyposis.

BACKGROUND: Microsatellite instability, programmed death-ligand 1 and tumor-infiltrating leukocytes are prognostic biomarkers in colorectal cancer but unknown toward familial adenomatous polyposis. AIM: To investigate the prognostic and clinicopathological roles of microsatellite instability, programmed death-ligand 1 and tumor-infiltrating leukocytes in familial adenomatous polyposis. METHODS: Clinical data and paraffin embedded tissues from 45 familial adenomatous polyposis patients were collected. Microsatellite instability was detected by immunohistochemistry and polymerase chain reaction. Programmed death-ligand 1 was detected by immunohistochemistry. Tumor-infiltrating leukocytes comprising CD8+ T cells, M1 and M2 tumor associated macrophages, CD56bright and CD56dim natural killer cells were analyzed using multiple fluorescence immunohistochemistry. RESULTS: Microsatellite instability high was noted in 6 samples but not associated with overall survival or progression-free survival. Programmed death-ligand 1 is negative on tumor cells but positive on tumor-infiltrating leukocytes, and positive programmed death-ligand 1 expression on tumor-infiltrating leucocytes is associated with overall survival. Low CD56bright natural killer cell infiltration was associated with longer progression-free survival and was an independent prognostic factor in FAP. CONCLUSION: For familial adenomatous polyposis, microsatellite instability high can be found but has no correlation with prognosis; programmed death-ligand 1 on tumor-infiltrating leukocytes is related with overall survival; CD56bright natural killer cell is an independent prognostic factor associating with longer progression-free survival.