Miscanthus grass (Miscanthus giganteus), in comparison with traditional fiber sources and their effects on fecal quality, apparent total tract digestibility (ATTD), fecal fermentative end products, and microbiota of healthy adult cats. Four dietary treatments were evaluated, differing in dietary fiber source. The diets were formulated to meet or exceed the AAFCO (2018) nutritional profile for adult cats and contained either cellulose (CO), Miscanthus grass fiber (MF), a blend of Miscanthus fiber and tomato pomace (MF + TP), or beet pulp (BP). The study was conducted using a completely randomized design with 28 neutered adult, domesticated shorthair cats . The experimental period comprised 21 days, and a fresh fecal and a total fecal collection were performed during the last 4 days of the trial period. Daily food intake (DM basis) was similar across all groups (P > 0.05). Additionally, treatment did not affect fecal output (as-is or DM basis), fecal score, or fecal pH (P > 0.05). Cats fed BP had significantly higher total dietary fiber ATTD than all the other treatments (P < 0.05) and the highest concentrations of total short-chain fatty acid, acetate, and propionate (P < 0.05), while butyrate concentrations were similar for all treatments (P > 0.05). Inclusion of dietary fibers was effective in modulating gut microbiota. Cats fed diets containing Miscanthus grass had greater α-diversity than cats fed BP. As no adverse effects on health, fecal quality, or ATTD of macronutrients were observed with the inclusion of 9% Miscanthus grass fiber or fiber blend, the data suggest that Miscanthus grass fiber and fiber blends are viable alternatives to the traditional dietary fiber sources used in commercial extruded feline diets, being most comparable to cellulose.Although dietary fiber is not considered an essential nutrient in a complete and balanced diet for felines, it provides a substrate for fermentation by gut microbiota, thus promoting gastrointestinal health through the production of fermentative metabolites, as well as improving laxation. The aim of this research was to evaluate the novel fiber source,  Since the 1950s, when the term “dietary fiber” was first introduced, several attempts have been made by the scientific community and regulatory bodies to provide a clearer and more encompassing definition of this term . Many stMiscanthus grass (Miscanthus giganteus) is an ingredient that has the potential to act as a novel dietary fiber source in companion animal diets. This grass is largely composed of insoluble fibers, making it compositionally similar to cellulose, and a potential base for blends with more fermentable fiber sources to develop desirable fiber matrices. It also contains naturally occurring xylooligosaccharides that may provide a prebiotic-like effect; however, more research is required to determine this. As an ingredient, Miscanthus grass offers many positive marketing attributes such as “natural,” non-GMO, non-by-product, and potentially organic. Limited data are available on the use of Miscanthus grass in monogastric diets, especially in regard to its effects on gastrointestinal health. Therefore, the goal of this research was to compare the effects of Miscanthus grass fiber to traditional dietary fiber sources and their effects on gastrointestinal tolerance, apparent total tract macronutrient digestibility, and fecal fermentative end products in adult cats fed extruded diets.Miscanthus grass would produce results similar to the diet containing cellulose, with the addition of tomato pomace to the Miscanthus fiber blend leading to results that are intermediate between cellulose and beet pulp diets.The most traditionally used sources of dietary fiber in companion animal diets are cellulose and beet pulp . These in = 7 cats/treatment) (Miscanthus grass fiber (MF), 7% Miscanthus grass fiber plus 2% tomato pomace (MF + TP), or 11% beet pulp (BP) (Four diets were formulated to meet or exceed the AAFCO nutrient profile for adult cats (eatment) . They weulp (BP) .All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee. Twenty-eight neutered, adult domesticated shorthair cats were used in a completely randomized design. At the start of the experiment, all cats were adapted to the CO diet for 7 days. After this control adaptation period, all cats were randomly assigned to one of the four treatment diets and were fed for 21 days to maintain body weight. The CO group consisted of two males and five females ; the BP group, two males and five females ; the MF group, three males and four females ; and the MF + TP group, two males and five females . During the last 4 days of this period, a total fecal and a fresh fecal collection were performed.Cats were group housed for 20 h of the day and individually housed in stainless steel cages for 4 h per day for feeding. Feeding occurred twice a day from 08:00 to 10:00 and 15:00 to 17:00. Cats had free access to water at all times. Food refusals were weighed and recorded after each feeding. Body weights and body condition scores were measured and recorded weekly. Cats were housed in the Edward R. Madigan Laboratory in a climate-controlled room with a 14-h light and 10-h dark cycle. Human socialization periods took place at a minimum of two times per week, and cats had access to behavioral enrichments such as scratching posts.For the duration of the 4-day fecal collection period, cats were housed individually. All feces were collected during this time and composited by cat to determine total fecal output. Each sample also was evaluated for fecal score on a five-point scale , and then samples were stored at −20°C for later analysis to determine the apparent total tract digestibility (ATTD) of macronutrients.A fresh fecal sample was collected from each cat within 15 min of defecation during the 4-day fecal collection period. These samples were also evaluated for fecal pH and score and dry matter. Then, they were aliquoted to determine ammonia, short-chain fatty acid (SCFA), branched-chain fatty acid (BCFA), phenol, and indole concentrations. To determine dry matter (DM) content, duplicates of ~2 g of feces were dried in a forced air oven at 105°C. For determination of fecal ammonia, SCFA, and BCFA concentrations, 3 g of each fresh sample was placed in a Nalgene bottle and mixed with 3 ml of 2 N hydrochloric acid and stored at −20°C for later analysis. Duplicates of 2 g of each fresh sample were placed into plastic test tubes, covered with parafilm, and stored at −20°C for later analysis of phenols and indoles. Fecal samples allocated for microbiota analysis were stored in 2 ml cryovials and stored at −80°C until analysis.via jugular venipuncture. For complete blood count analysis, 1 ml of blood from each cat was placed in EDTA vacutainer tubes and 4 ml was placed in serum separator tubes . Blood analyses were completed by the Clinical Pathology Laboratory at the University of Illinois College of Veterinary Medicine .On 0 and 21 days of the experimental period, cats were fasted overnight, and a blood sample was collected to evaluate blood metabolites and health status. Cats were sedated before collecting 5 ml of blood Experimental diets were subsampled and ground in a Wiley mill  using a size 10-mesh screen resulting in 2 mm average particle size used for proximate laboratory analysis. Total fecal samples were composited for each animal and partially dried in a forced air oven at 57°C. After drying, they were also ground in a Wiley mill to a 2-mm particle size.After the diet and feces samples were prepared, DM and ash content were determined following the AOAC procedures [; methods3PO4 on 80/100 Chromosorb W AW . Nitrogen was used as the carrier gas at a flow rate of 45 ml/min. Oven temperature was set at 125°C, the injection port at 175°C, and the detector port at 180°C. Fecal phenol and indole concentrations were measured using a Thermo Scientific TRACE 1300 Gas Chromatograph coupled with a FID in duplicate according to the modified procedure of Flickinger et al. . A Qubit® 3.0 fluorometer  was used to quantify DNA concentration prior to amplification and sequencing. A Fluidigm Access Array , in combination with Roche High Fidelity Fast Start Kit , was used for the amplification of the 16S rRNA gene. The primers 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACNVGGGTWTCTAAT-3′), targeting a 292-bp fragment of V4 region, were used for amplification  . A FluidP < 0.05. Pooled standard errors of the mean also were obtained using the MIXED model procedure.Data were analyzed using the MIXED Model procedures of SAS version® 9.4 . Animal was used as the random effect, and treatment diet was used as the fixed effect in the statistical model. Data normality was checked using the UNIVARIATE procedure, comparing all treatment least-square means. Experiment-wise error was controlled for using Tukey adjustment. The significance level was set at a probability of P > 0.05) (P > 0.05). Dry matter digestibility ranged from 78.3 to 82.7%. Acid-hydrolyzed fat digestibility of CO (94.5%) was significantly higher compared with that of MF and MF + TP  (P < 0.05) with BP being intermediate (92.6%). The BP diet had the highest TDF digestibility (54.2%) when compared with all the other treatments (average 22.1%) (P < 0.05). Digestible energy , which was calculated by subtracting fecal gross energy from diet gross energy, was higher for CO  than MF+TP  (P < 0.05), with MF and BP being intermediate.The four treatment diets were formulated to contain similar nutrient composition . This wa > 0.05) . AdditioP < 0.05) (P < 0.05), while fecal phenol concentration was not significantly affected (P > 0.05). Fecal ammonia concentration was also similar across all treatments (P > 0.05). BP resulted in the highest concentrations of total SCFA, acetate, and propionate (P < 0.05), while butyrate concentrations were similar for all treatments (P > 0.05). Total BCFA, isobutyrate, and isovalerate concentrations were higher in the MF + TP group than in the CO and BP groups (P < 0.05), with MF being intermediate. A similar trend for valerate concentration was observed with the MF + TP group being higher than the BP group (P < 0.05), with CO and MF groups being intermediate.Cats fed the MF + TP and MF diets had significantly higher total fecal phenol and indole concentrations than CO- and BP-fed cats ( < 0.05) . Fecal iP < 0.05) relative abundances of Firmicutes in relation to the BP treatment. Bacteriodota phylum was increased (P < 0.05) in cats fed BP in contrast with MF. Proteobacteria was increased (P < 0.05) in cats fed BP in contrast with all other dietary groups. A total of 38 families, 102 genera, and 66 species were identified; within those, over 10 and 20 taxonomic groups for family (P < 0.05) among treatments, respectively. The relative abundance of Bacteroidaceae was greater (P < 0.05) for cats fed BP in comparison with those fed CO, but it did not differ for cats fed MF and MF + TP treatments. Cats fed BP also had greater (P < 0.05) relative abundance of Prevotellaceae in contrast with cats fed all the other dietary treatments. The relative abundance of Oscillospiraceae, Butyricicoccaceae, and Anaerovoracaceae were consistently higher (P < 0.05) in cats fed CO, MF, and MF + TP treatments in contrast with the BP treatment, whereas Succinivibrionaceae was consistently lower (P < 0.05) in cats fed those dietary treatments when compared with the BP treatment  in cats fed the CO treatment (4.0%) in contrast with BP (2.4%), and intermediate in cats fed the MF or MF + TP treatments . In contrast, the relative abundance of Prevotella was greater in cats fed BP when compared with cats fed CO (9.2%), MF (5.4%), and MF + TP (6.0%). In addition, the relative abundance of several genera were consistently higher in cats fed CO, MF, and MF + TP, in contrast with BP including Clostridia UCG-014, Butyricicoccaceae UCG-009, Colidextrobacter, Oscillibacter, and Megasphaera. Cats fed BP (4.3%) had greater relative abundance of Succinivibrio than cats fed CO (1.8%) and MF + TP (1.0%), with MF (0.8%) being lowest  in comparison with cats fed CO, MF, and MF + TP treatments. Alpha-diversity was measured as Pielou evenness, Faith's phylogenetic diversity, and Shannon entropy . Similarly, the α-diversity of cats fed BP was lower than cats fed other dietary treatments based on Faith's phylogenetic diversity  than cats fed MF and MF + TP based on Shannon entropy index . Minimal variations in ingredient inclusion rates were necessary to obtain the targeted TDF content of 15%. This level of TDF was chosen to reflect the higher levels of commercial dietary fiber inclusion in order to emphasize any physiological responses to the various fiber ingredients. The chemical composition of the diets fell within a relatively narrow range, with the CO treatment having a slightly higher acid-hydrolyzed fat (AHF) and CP concentration, as well as a gross energy value. This could be due to the slightly higher inclusion level of poultry by-product meal in this diet. Overall, diets were very close to their target TDF content of 15%. Small variations are expected as TDF content can be affected by the plant's growing conditions, time of harvest, and plant maturity, among other things. The BP diet had higher levels of SDF and lower IDF as was expected based on the typical fiber profile of this ingredient  in comparison with Miscanthus grass composition reported by Donadelli and Aldrich as 90% TDF, 7.3% SDF, and 82.7% IDF (DM basis)  , 19. HowMiscanthus grass were added to the formula at a 10% inclusion rate (Miscanthus grass (15.0% TDF), or up to 10% inclusion (13.8% TDF), higher inclusion levels and, subsequently, higher TDF contents may impact palatability and must be considered in the practical utilization of this ingredient (Food intake (g/day) on an as-fed and DM bases did not differ among the treatment groups. Similarly, Donadelli and Aldrich saw no effect on food intake when cellulose, beet pulp, and ion rate . Detweilion rate . This ingredient .Similar fecal scores were observed for all treatments ranging from 1.81 to 2.18 on a five-point scale. Previous research reported similar fecal scores with 8% inclusion of cellulose (11.2% TDF) and 12.5% inclusion of beet pulp 10.6% TDF), 1.8 and 2.3, respectively .6% TDF, –23.Miscanthus grass and observed that cats fed beet pulp  had significantly higher DM and OM coefficients than cellulose  and Miscanthus grass   . Kienzle: 80.5%) . The DM Miscanthus grass being intermediate   . In contctively) , 21, 25.Miscanthus grass fiber (MF and MF + TP). A similar effect was reported by Donadelli and Aldrich  were similar to cellulose (21.8%) in this regard, as they had greater IDF content, which is poorly fermented and, therefore, excreted in higher quantities in the feces. Donadelli and Aldrich reported a similar TDF digestibility coefficient (20.8%) with the inclusion of 10% Miscanthus grass  (P < 0.05)  and propionate (139.0 μmol/g) compared with cats fed diets with no additional fiber, cellulose, or soybean hulls  ( < 0.05) . No stat < 0.05) . While t < 0.05) .Miscanthus grass fiber (MF and MF + TP). While the indole compound can help to maintain gut homeostasis by promoting barrier functions, regulating inflammation, and possibly aid in satiety, it can also be metabolized into indoxyl sulfate, which is a uremic toxin that has been associated with negative health outcomes in humans such as cardiovascular disease and chronic kidney disease  increased total BCFA concentrations compared with cellulose . This coMiscanthus grass fiber and tomato pomace on fecal microbiota has not been evaluated previously. Characterization of the feline gut microbiota has shown that Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria are dominant phyla in adult healthy cats  are involved in the beneficial health effects on the host. These metabolites have been described as post-biotics, a term that is ill-defined by the scientific community , 33. Thethy cats , 42, 43.The phyla Firmicutes, Bacteroidetes (Bacteroidota), and Actinobacteria are considered important producers of metabolites that have direct beneficial effect on gut and host health . A recenMiscanthus grass fiber may support gut health by maintaining microbial richness and evenness in adult cats. However, healthy cats supplemented with either FOS or apple pomace had decreased α-diversity when compared with baseline values , as well as their relative abundance (weighted UniFrac). Microbial diversity has been used as an indicator of gut health, as lower microbial diversity has been associated with clinical conditions including irritable bowel disease and small cell lymphoma in cats . Therefoe values . OverallSerum chemistry and complete blood count analysis were within reference ranges for healthy adult cats. Creatinine was an exception and observed to be slightly higher than the reference range for MF and BP treatments at baseline and for CO, MF, and BP treatments at the end of the trial period. These deviations from the reference range were small and could be due to individual variation among cats. No effect of treatment was observed. Additionally, glucose concentrations were above the normal range. However, this has been observed as a side effect of the sedation used during the blood collection. The results of the serum chemistry and complete blood cell count (data not provided) and the lack of clinical symptoms indicate that the treatments did not result in any negative health outcomes.Miscanthus grass fiber is an advisable dietary fiber for adult felines. The addition of Miscanthus grass fiber and the MF + TP blend had no detrimental effects on animal health, fecal quality, or macronutrient digestibility. Diet inclusion of Miscanthus grass fiber up to 9% (15% TDF) had no negative effect on voluntary food intake, indicating that it had acceptable palatability to the cats. The resulting concentrations of fecal fermentative end products were more similar to those observed in the CO group than in the BP group, as expected from the similarities in the fiber profile of these ingredients. In conclusion, Miscanthus grass can be utilized by the pet food industry as an economical and environmentally conscious ingredient that can provide flexibility in the formulation of diets that aim to maximize the health benefits of dietary fiber. Miscanthus grass fiber can be effectively used as a base ingredient to develop fiber blends in combination with more soluble and fermentable dietary fiber, including prebiotic sources, which might be beneficial to improve SCFA production and modulate gut microbiota. In this study, inclusion of 2% TP in combination with MF resulted in small numerical increases in fecal SCFA concentrations, suggesting that fiber blends can be used to support gut and host health. Inclusion of dietary fibers was an effective strategy to modulate feline gut microbiota. Cats fed diets containing Miscanthus grass fiber had greater α-diversity than cats fed BP. Future studies should further evaluate nutraceutical uses, additional fiber blends, and diet formats, as Miscanthus grass fiber can be a functional ingredient in multiple dietary platforms, including weight management, gut health, and hairball control.The findings from this research indicate that 10.13012/B2IDB-3595148_V1).The data generated for the study are deposited in the Illinois Data Bank repository, accession number (doi: The animal study was reviewed and approved by University of Illinois Animal Care and Use Committee.MdG designed the experiment. SF and FH performed the laboratory analyses. SF and MdG performed the statistical analyses and wrote the manuscript. BS and SR-Z performed the bioinformatics analysis for fecal microbial analysis. All authors revised and provided intellectual input on this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pulmonary fibrosis (PF) is a devastating lung disease, resulting in gas exchange dysfunction until death. The two drugs approved by the FDA, pirfenidone and nintedanib, have obvious side effects. Wen-yu-jin (WYJ), one of the commonly used herbs in China, can treat respiratory diseases. The potential effects and the underlying mechanism of WYJ against PF are unclear.  Employing network pharmacology, molecular docking, and in vivo and in vitro experiments to explore the potential effects and underlying mechanisms of WYJ in the treatment of PF.  Ultra-high pressure liquid chromatography combined with linear ion trap-orbital tandem mass spectrometry  was used to identify compounds of WYJ. We got PF-related targets and WYJ compounds-related targets from public databases and further completed critical targets exploration, network construction, and pathway analysis by network pharmacology. Molecular docking predicted binding activity of WYJ compounds and critical targets. Based on the above results, in vivo and in vitro experiments validated the potential effects and mechanisms of WYJ against PF.  23 major compositions of WYJ were identified based on UHPLC-LTQ-Orbitrap. According to the results of network pharmacology, STAT3, SRC, IL6, MAPK1, AKT1, EGFR, MAPK8, MAPK14, and IL1B are critical therapeutic targets. Molecular docking results showed that most of the compounds have good binding activities with critical targets. The results of in vivo and in vitro experiments showed that WYJ alleviated the process of fibrosis by targeting MAPK and STAT3 pathways.  Network pharmacology, molecular docking, and in vivo and in vitro experiments showed the potential effects and mechanisms of WYJ against PF, which provides a theoretical basis for the treatment of WYJ with PF. Profytokines , 10. Pirytokines , approveytokines , 13. Morytokines , 13–14, Multiple active ingredients and targets of Traditional Chinese Medicines (TCM) arouse the attention of pharmacologists. Previous studies showed that TCM or active ingredients from TCM have protective pulmonic , represeIn silico approaches have been widely used in the field of drug molecule design, improving efficiency and reducing cost , 20. BasIn our study, we established network pharmacology to explore the molecular mechanism of WYJ against PF. Molecular docking was performed to predict the affinity strength between the active compounds and the critical targets and to explore the antipulmonary fibrosis potential of WYJ. Finally, the antifibrosis mechanism of WYJ was further verified via in vitro and in vivo experiments based on the predicted results.WYJ was purchased from Tong Ren Tang Co., Ltd. . The herb was boiled in purified water twice for 1 h each time. We got lyophilized powder in condensation and freeze-dry of liquids and stored it at −80°C for a series of experiments.μm). Mobile phase A was water with 0.1% formic acid while mobile phase B was acetonitrile. The column temperature was set to 35 and the flow rate was 0.3 mL/min. Elution conditions were summarized as follows: 0–3 min (5%-5%B), 3–45 min , and 45.1–50 min (5%-5%B).We used processed WYJ for in vitro and in vivo experiments. The compounds of processed WYJ were reduced or changed compared with the herbal plant's library. Therefore, we used LC/MS analysis to clarify the compounds of WYJ. The coupling of the UHPLC System (Thermo Fisher Scientific) equipped with a binary pump, an auto sampler, a column thermostat, and DAD detector, and LTQ-Orbitrap XL (Thermo Fisher Scientific) equipped with an electrostatic ionization source (ESI) was used For LC/MS experiments. Data was controlled and processed by Xcalibur software (Thermo Fisher Scientific). The column used in the LC analysis was Thermo Scientific Hypersil BDS C18  and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform . Compounds-related protein names were converted to the gene names using UniProt (https://www.uniprot.org/).According to the compounds identified by UHPLC-LTQ-Orbitrap, we obtained these compound-related targets from the SwissTargetPrediction database  (http://https://www.genecards) and OMIM database [https://omim.org/). After combining the targets obtained from different databases using the same keywords, we got the targets of PF by taking the intersection of targets with different keywords.We used “pulmonary fibrosis (PF),” “idiopathic pulmonary fibrosis (IPF),” or “interstitial lung disease (ILD)” as keywords to obtain PF-related targets from GeneCards database v5.0  (https:/database  (https:/https://www.string-db.org/) [WYJ-related targets and PF-related targets were intersected to obtain gene symbols. Using the string database (db.org/) , the prohttps://david.ncifcrf.gov/) [P < 0.05. Compound-target-pathway network explored the connection of compounds of WYJ, key targets, and important signaling pathways and was visualized using Cytoscape.To determine the biological meaning behind key targets, pathway enrichment was analyzed by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database of David v6.8 (rf.gov/) , 33. Thehttps://pubchem.ncbi.nlm.nih.gov/). All compounds were optimized using the MM2 force field, and saved in.pdbqt format as docking ligands in AutoDock Tools 1.5.6 software (ADT) [http://www.rcsb.org/). Proteins should be human proteins with one or more cocrystallized ligands and crystal structures with small “resolution” value. PyMoL software was utilized to remove water molecules and original ligands [The active compounds were molecularly docked with the core proteins based on the results of network pharmacology. The specific operations are as follows: (1) ligands molecule preparation. We downloaded the core compound structures from the Pubchem database (re (ADT) . (2) Rec ligands . AutoDocBleomycin (BLM) can induce the formation of a mouse model of PF, and mouse lungs have lung pathological changes similar to those of patients with PF. 75 male mice  were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.  and were adaptively reared for one week. After the mice were anesthetized, 15 mice in the control group were given normal saline via the oropharynx, and the rest of the mice were given BLM via the oropharynx to establish the PF model. The dose of BLM was 5 mg/kg. The BLM-induced PF model was randomized into four groups: BLM treatment group (model), BLM with WYJ high concentration treatment group (WYJ-H); BLM with WYJ medium concentration treatment group (WYJ-M), and BLM with WYJ low concentration treatment group (WYJ-L).According to the previous literature and preliminary experimental results, WYJ-H, WYJ-M, and WYJ-L groups were given WYJ 1.2, 0.6, and 0.3 g/kg doses, respectively. The control group and the model group were treated with normal saline. Treatments were started 12 days after the establishment of the PF model, once a day, for 14 days. We sacrificed all mice on day 26 and stored lung tissue at −80°C for different experiments.We recorded the body weight (BW) and the whole lung weight (PW) after mice were sacrificed. Pulmonary indexes (PI) were calculated using the equation PI = PW/BW ∗ 100.μg/lung.The lung tissue was processed according to the instructions of the Hydroxyproline Kit . HYP is the main marker of the development of PF. The level of HYP in the lung tissue was measured by the absorbance of 550 nm and the result was expressed with Lung tissues fixed with 10% formalin were embedded in paraffin and sectioned. Sections were stained with hematoxylin and eosin (H&E) and Masson . Inflammation and collagen deposition of the sections were observed and assessed with an optical microscope.2 and 37°C. Cells were passaged at a confluence of 80%–90% using standard trypsinization techniques. HPF cells stimulated with TGF-β1 (10 ng/mL) were compared with cells that were grown in the same conditions but without stimulation of TGF-β1.HPF is a normal human fibroblast cell line from the National Infrastructure of Cell Line Resource . HPF cells were cultured in medium (DMEM) with 10% FBS and 1% PS and maintained at the cell incubator with 5% CO4 cells/well and treated with or without WYJ . In each well, 200 μL media was added with 10% CCK-8 solution after 48 h and then incubated at 37 for 1-2 h. The absorbance of the well was measured at 450 nm using a microplate reader .We assessed the viability of HPF cells pretreated with WYJ using Cell Counting Kit-8 . HPF cells were seeded into a 96-well plate in the density of 2 × 10−△△Ct method. Gene primer sequences are shown in Total RNA extracted from HPF cells or mouse lung tissues using Trizol reagent  was purified with RNeasy column . The cDNA was obtained from purified RNA following the manufacturer's instructions of a reverse transcription kit . Full using QuantStudio6 Flex, gene expression analysis was performed following instructions of QuantiFast SYBR Green PCR kit . Fold changes of relative gene expression were calculated using the 2μg/lanes was loaded and separated by SDS-PAGE gel electrophoresis. After SDS-PAGE, proteins were transferred to the polyvinylidene fluoride (PVDF) membranes . Membranes were blocked with 5% fat-free milk dissolved with Tris-buffered saline with Tween 20 (TBST) for 120 min at room temperature. Rinsed with TBST, these membranes were incubated with primary antibodies at 4°C overnight. On the second day, blots were rinsed with TBST and then incubated with secondary antibody for 1 h. Blots were visualized by gel imager. GAPDH was used for loading positive control and the results were analyzed using Image J.Proteins obtained from lung tissue or cells were further analyzed with Bicinchoninic Acid  for protein concentration. Protein at 30–50 α-SMA , anti-Collagen 1 , anti-p-MAPK , anti-MAPK , anti-p-STAT3 , anti-STAT3 , anti-GAPDH , anti-rabbit IgG , and anti-mouse IgG .Antibodies: anti-P values <0.05 are adopted as statistically significant.One-way ANOVA was performed using GraphPad Prism software (version 8.0.2). Using UHPLC-LTQ-Orbitrap to detect and identify chemical compounds of WYJ , a totalBased on the network pharmacology methods above, 436 WYJ-related targets were obtained from SwissTargetPrediction and TCMSP after removing duplicate items. Information about WYJ-related targets is provided in Supplementary STRING databases were used to explore the underlying interactions of 124 potential targets. There were 124 nodes and 1716 edges of the PPI network visualized by Cytoscape . Accordi48 key targets obtained from PPI were selected to investigate the biological processes (BP) and mechanisms of WYJ on PF treatment. Results showed that BP were involved in PF treatment , includiCompound-target-pathway network explored the connection of compounds of WYJ, key targets, and 20 pathways. The integrated compound-target-pathway network comprised 69 nodes and 258 edges . STAT3, RMSDs between the cocrystal structures and their original ligands were less than 2 Å, which indicated the method for docking was reasonable (Supplementary-Molecular Docking). We found that the affinity energies of most small molecules docking with proteins were less than −5.0 kcal/mol (Supplementary-Molecular Docking). Heat map of molecular docking is shown in the β1 was observed during the development of PF [β1 mRNA in lung tissue of the PF model , and HYP are the main markers of the development of PF [It has developed many animal models to study the pathogenesis and treatment of different diseases in humans. The BLM-induced PF mouse model has been widely accepted as a model for studying PF. This model could cause a pulmonary pathological change, which is like that of patients with lung fibrosis , 50. Comnt of PF –10, 51. β1 can induce lung fibroblasts to differentiate into myofibroblasts [β1 to induce HPF and observed whether WYJ can inhibit HPF proliferation and differentiation. We found that WYJ significantly reduced the expression of fibrosis markers  in HPF cells induced by TGF-β1. These results are consistent with the results of the mouse model.Various lung cell types  are involved in the occurrence and development of PF under pathological conditions . Howeverroblasts . Therefoα) are essential members of the MAPKs family. MAPKs signaling cascades regulate processes, including cell cycle progression, cell migration, cell survival, and differentiation [β is achieved through a two-step process. The activation of the MAPK pathway participates in the synthesis of the inactive 31 kDa precursor (pro-IL-1β), and the inflammation induces the production of the active/mature form of IL-1β (17 kDa) [β not only is an effective inducer of TGF-β1 [β pathway [β signaling, EGFR and SRC also mediate the phosphorylation and activation of STAT3 [Multiple targets and multiple pathways regulate the occurrence of PF. So, only targeting one target for treatment will not eliminate the pathological activation of other targets in PF. Therefore, we used network pharmacology to screen out 9 critical targets of WYJ in the treatment of PF. MAPK1 (ERK2), MAPK8 (JNK1), and MAPK14  , 57. IL-f TGF-β1  but also pathway . EGFR is pathway –62. Inhi pathway . KEGG an pathway . Phospho pathway , 65, 66. pathway , 68. In of STAT3 –72. IL6,of STAT3 , 63, 73.The molecular docking results described above are helpful for a basic understanding of the mechanism of drug action. If the affinity energy is less than −5 kcal/mol, it indicates that the target has certain binding activity with the compound. We found that the affinity energies of most small molecules docking with proteins were less than −5.0 kcal/mol. We performed further analysis of the interaction between the target proteins and the compounds. The comparative analysis showed that positive control drug nintedanib showed good binding activity, but some of the active compounds of WYJ  showed higher binding activity with the core gene target to some extent, which also provides direction for the development of new drugs and subsequent research.In conclusion, our study identified that WYJ alleviated pulmonary fibrosis. According to the results of network pharmacology and molecular docking, the pharmacological mechanism of WYJ in the treatment of PF was further validated by experiment. The inhibitory effect of WYJ on PF may function mainly by inhibiting MAPK and STAT3 signaling pathways, which provide new insights for the treatment of WYJ at PF. The method of exploring the pharmacological mechanism of WYJ used in our study provides a novel approach to explain the pharmacological basis of other herbs. Network pharmacology has limitations. More experiments are needed to verify the reliability anti-fibrotic effect of WYJ.


The TE result was significantly higher in those with risk factors for liver fibrosis than in those without risk factors . There was a positive correlation between the PIIINP level and body weight (p = 0.028), cumulative MTX dose (p = 0.007), RA activity (p = 0.028) and diabetes mellitus (DM) (p = 0.001). There was a positive correlation between the ELF-1 score and age (p < 0.001), cumulative MTX dose (p = 0.007) and RA activity (p < 0.001). The PIIINP level and ELF-1 score are not organ specific, and readings may vary depending on RA activity. TE is organ specific and can be performed by a skilled ultrasonographer might be useful to assess actual liver condition.Methotrexate (MTX) is recommended as a first-line treatment for rheumatoid arthritis (RA). There are no strict guidelines regarding monitoring for liver damage in RA patients. This study aimed to evaluate noninvasive diagnostic procedures in assessing liver fibrosis in RA patients. Ninety-six RA patients were recruited for this study. The procollagen III N-terminal peptide (PIIINP) serum level was measured in all patients. The Enhanced Liver Fibrosis score (ELF-1) was calculated for 82 patients. Transient elastography (TE) was performed in 91 patients, those examined were divided into two groups: a study and control group, comprising patients with and without risk factors for liver fibrosis, respectively. The TE result correlated only with the body mass index—BMI (The online version contains supplementary material available at 10.1007/s00296-021-05059-z. Methotrexate (MTX) is an essential disease-modifying antirheumatic drug (DMARD) used in rheumatoid arthritis (RA) therapy due to its efficacy, relatively good tolerance and low price-to-efficacy ratio , 2. The In the European League against Rheumatism (EULAR) recommendations for the management of RA from 2019, there is no information about monitoring for the side effects of therapy or how to manage them. The authors of those recommendations assumed that the clinicians prescribing the drugs would be aware of any potential toxicity , 6.While liver biopsy is a common method for assessing liver damage, the procedure is connected with a considerable risk of pain, bleeding and other complications. Therefore, to determine the level of liver fibrosis while avoiding the aforementioned complications, as recognized alternatives, the Enhanced Liver Fibrosis (ELF) test and transient elastography (TE) were performed in our study.The ELF test is a simple blood test used to assess liver fibrosis. It was applied in our patients along with other routine blood tests. The ELF panel includes three serum biomarkers of extracellular matrix (ECM) fibrogenesis and fibrous protein degradation: hyaluronic acid (HA), procollagen III N-terminal peptide (PIIINP) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1). The three included biomarkers are combined in an algorithm for calculation of the ELF score, which correlates to the liver fibrosis level found on liver biopsy . In addiTE is an imaging technique used to assess liver fibrosis by measuring the propagation of low-frequency ultrasound 50 Hz). The velocity of ultrasound waves is determined by the stiffness of the tissue, with faster propagation in harder tissues. TE is a quick test and can be done repeatedly. American elastography guidelines for the treatment of psoriasis recommend using TE as the standard method for assessing MTX-induced liver toxicity 0 Hz. The, 9.The aim of this research was to evaluate the usefulness of noninvasive methods of liver examination, acknowledged in assessing liver fibrosis in viral hepatitis or psoriasis, including transient ultrasonography (TE) and determination of serum markers , in patients with RA receiving methotrexate.It is a retrospective study, focusing on non-invasive diagnostic procedures acknowledged in diagnosing liver fibrosis in diseases, such as viral hepatitis or psoriasis.We recruited 96 patients (72 women and 24 men), all of whom were patients of Rheumatology Clinic associated with Department and Clinic of Rheumatology in Wroclaw University Hospital. In the study we analyzed data regarding patients biometrics, methotrexate treatment and its effect on treated patients. Initial data was collected from medical history and completed on the recruitment visit. During that visit in a short questionnaire, we collected lacking physical data from all patients and information regarding the treatment schedule, as follows: age; weight; body mass index (BMI); duration of methotrexate therapy; cumulative dose of methotrexate; concomitant therapy; comorbidities, and alcohol intake. During the physical examination, the number of painful and swollen joints was counted. On the basis of the aforementioned parameters and Patients Global Health scale, RA activity was evaluated using the Disease Activity Score for 28 joints (DAS-28) scale. Statistical data of the examined patients are presented in Table Transient elastography examinations were all performed with a Siemens Acuson S2000 by the same examinator (MG). For examination, the patient was in a supine position with the right upper limb under the head. Measurements were taken from the right lobe of the liver through the intercostal space. The results were measured in kilopascals. The distinguishing point of liver fibrosis was established at 7.1 kPa and 13 kPa for liver cirrhosis which corresponds with METAVIR F2 and F4 degrees in vast majority of analyzed studies , 10–14. For further analysis of transient elastography result patients were divided into two groups according to whether they had risk factors for liver damage. We selected diabetes mellitus (DM), obesity (BMI over 30) and preexisting liver disease  as the most important risk factors. Having at least one risk factor was the criterion to be selected for the study group, while the control group comprised patients with no risk factors. Statistical data of patients in these groups are presented in Table The selection criteria were as follows: age over 18 years; confirmed diagnosis of rheumatoid arthritis; ongoing treatment with methotrexate; and signed written consent. Active viral hepatitis, alcohole abuse (> 7 units per week) and malignant neoplastic disease disqualified patients from participation.U test was used in nonparametric statistics. A multivariate linear regression was performed to distinguish the parameters defining the ELF-1 and PIIINP scores. In the model, we used variables that showed a linear correlation with the assessed laboratory markers of fibrosis: for both parameters, we used MTX treatment time, cumulative MTX dose and the DAS28 result; in addition, we analyzed body weight for PIIINP and age for ELF-1.Statistical analysis was performed using Statistica ver. 10.0. Pearson’s correlation coefficient was used to evaluate linear correlations and Spearman’s correlation coefficient was performed to assess non-linear interconnections. Mann–Whitney p = 0.5); ELF score (p = 0.56); cumulative MTX dose (p = 0.33). No other common risk factors for liver fibrosis correlated with the TE result: DM ; concomitant therapy with hepatotoxic drugs .Ninety-one patients underwent transient elastography examination. Eight of them had equivocal results. Only 23 patients (27.7%) had no signs of fibrosis on the performed scans, and 17 (20.5%) had a result suggesting liver cirrhosis. Due to the large group of participants with the TE result of 13 kPa and above, its statistical evaluation was performed. Out of 17 patients 12 (70.6%) were women, 7 (41.2%) had BMI above 30, 11 (64.7%) had concomitant therapy with other hepatotoxic drug with 5 (29.4%) taking statins, 9 (2.9%) had preexisting liver disease and 5 (29.4%) had DM. Mean age of that group was 62.53, mean cumulative MTX dose was 5679.4 mg. The TE result correlated only with the BMI . More obese patients tended to have more advanced liver fibrosis. There was no correlation of the TE result with any other parameter assessed in our study: PIIINP serum level . In both groups, there was no correlation between the TE result and the cumulative dose of MTX .Eighty-three patients with unequivocal results were divided into two groups depending on whether they had risk factors for liver injury . The study group consisted of 42 patients, and the control group consisted of 41 patients. There was a statistically significant difference between the two groups , duration of MTX therapy , cumulative dose of methotrexate  and RA activity assessed by the DAS-28 score . The mean PIIINP level measured in blood samples from patients weighing less than 80 kg was 7.76, and that measured in blood samples from patients weighing over 80 kg was 10.33. For further analysis, the cumulative dose threshold was set at 4000 mg, which corresponds with the recommendation for performing elective liver biopsy. The mean PIIINP level in the serum of patients with a cumulative dose less than 4000 mg was 7.86 ng/ml, and that in patients with higher MTX intake was 9.72 ng/ml. Patients with low RA activity (DAS-28 score < 3.2) had a mean PIIINP level of 7.61, and those with high RA activity (DAS-28 score > 5.1) had a PIIINP level of 11.32 ng/ml. DM was an independent risk factor for higher PIIINP levels (p = 0.001). In diabetic patients, the median PIIINP level was 11.38 ng/ml, compared with 7.15 in nondiabetic patients. To better define the PIIINP result a multivariate linear regression was performed. In the initial model we analyzed all aforementioned parameters and after subsequent removal of irrelevant variables we acquired statistically important model  with DAS 28 (p = 0.009) and MTX treatment duration (p = 0.012) as describing parameters.The PIIINP serum level was measured in 96 patients receiving MTX. Forty-three of them (44.8%) had elevated values. There was a weak positive but statistically important correlation between the PIIINP level and body weight , duration of MTX treatment  and cumulative dose of methotrexate . RA activity had strong, positive interconnection to ELF-1 score value . The mean ELF score in patients older than 65 was 10.35, and that in patients younger than 65 was 9.41. The same threshold for the cumulative dose of methotrexate as in the analysis of the PIIINP level was assumed in the analysis of the ELF score. The mean ELF score in patients with cumulative doses below 4000 mg was 9.51, while it was 9.85 in patients with cumulative doses over 4000 mg. However, a greater difference was seen with greater cumulative doses of methotrexate . The most relevant correlation was observed between RA activity and the ELF score. In patients with low RA activity, the mean ELF score value was 8.95, and in patients with high RA activity, it was 10.55. Correlation of both PIIINP and ELF-1 with examined parameters is presented in Table R2 = 0.465; p < 0.001) with age (p = 0.002), MTX treatment duration (p = 0.013) and DAS28 (p < 0.001) as describing parameters.The ELF score was determined in 82 patients in our study. Only two of them were at a low risk of hepatic fibrosis, with ELF scores < 7.7, and 33 patients were at a high risk, with ELF scores > 9.8. There was a weak, positive but statistically significant correlation between the enhanced liver fibrosis score and parameters, such as age  [Transient elastography, as a noninvasive method used to measure liver stiffness, is broadly used to assess liver fibrosis, especially in patients with viral hepatitis . New gui (F0–F1) , 9. In o (F0–F1) , 14, 17.In most studies, an interconnection between the cumulative dose of MTX and the TE result was not observed , 14–20. PIIINP is a coproduct of collagen fiber metabolism that may reflect the intensity of the process of fibrosis in humans. In a number of publications, serum PIIINP measurements were utilized to assess the liver toxicity of methotrexate in psoriatic patients –28; on tThere was a significant linear correlation between the PIIINP level and cumulative methotrexate dose in our study, which is in agreement with the results of other studies , 24. TheRheumatoid arthritis activity measured by the DAS-28 scale was an important risk factor for an elevated PIIINP serum level in our study. This observation corresponds with the results of other studies concerning RA as well as psoriasis , 26, 28.Because PIIINP is one of three biomarkers that are used to calculate the enhanced liver fibrosis score, there is a straight correlation between them. However, the ELF score is regarded as more specific to liver fibrosis . NeverthThe main limitations of our study are to small examined group to determine strong, unequivocal conclusions and the lack of histopathological verification of study results, as we performed only noninvasive procedures. However, the evaluated diagnostic procedures are recognized in screening for liver fibrosis in viral hepatitis, NAFLD, and psoriasis, with AUC/ROC calculations measured and confirmed to be significant , 10. In Methotrexate plays a fundamental role in RA treatment and requires proper monitoring for side effects of therapy. Liver biopsy carries a potential risk, which is why new methods for hepatotoxicity assessment are needed. Serum biomarkers, such as the PIIINP level and ELF score, may be analyzed in standard blood samples; however, they are not organ specific, and readings may vary depending on RA activity. Another disadvantage is that it presents the actual process of fibrogenesis, and the results may be negative, even in end-stage cirrhosis. Transient elastography is an organ-specific examination that if performed by a skilled ultrasonographer might provide useful information regarding the actual liver condition. This examination technique is acknowledged as a standard procedure in monitoring for MTX-induced hepatotoxicity in psoriatic patients or assessing the liver condition in patients with viral hepatitis. In spite of aforementioned restrictions of both methods it is necessary to highlight their usefulness in screening of liver damage, especially transient elastography.Supplementary file1 (DOCX 15 KB)Below is the link to the electronic supplementary material.


To describe the objective and design of the Fujian Eye Study and to introduce the general characteristics and vision condition of this study.The Fujian Eye Study (FJES) is a population-based cross-sectional survey on the public eye health status of residents over 50 years old in the entire Fujian Province of Southern China, which contains both urban and rural areas and coastal and inland regions. 10,044 participants were enrolled using a two-stage cluster sampling design and underwent a questionnaire and a series of standard examinations both physical and ocular. The main subgroups of data collection included age, sex, region, refractive error, education background, income, eating habits, smartphone usage in the dark, complaints of eye discomfort, history of chronic diseases, consumption of tobacco, alcohol, or tea.8211 (81.8%) participants were finally included and were divided into urban populations (4678 subjects) and rural populations (3533 subjects) and coastal residents (6434 subjects) and inland residents (1777 subjects); 4836 participants were female. The mean age was 64.39 (SD 8.87) years . 227 (3.33%) had vision impairment (VI), 195 (2.87%) had low vision and 14 (0.21%) were blind. The mean presenting near visual acuity (PNVA) was 0.28 (0.17), the mean presenting distance visual acuity (PDVA) was 0.61 (0.30), and the mean best corrected visual acuity (BCVA) was 0.82 (0.28).The FJES collected detailed questionnaire information and overall ocular and physical examinations, which provide the opportunity to identify risk factors and images of VI and eye diseases and to evaluate their associations with chronic diseases and basic personal information. Population-based studies could usually deliver the evidence for findings and hypotheses formulated on the basis of hospital-based investigations and provide new exploration directions and practical basis for basic experimental research. Previous major population-based studies in the field of Ophthalmology were mostly from United States , 2, AustThe Fujian Eye Study (FJES) is a population-based cross-sectional on-site survey of eye health projects in public health of more than 10,000 residents in Fujian Province, southeastern China, which was part of the national “active health and aging science and technology response” key project. Our team crossed over 20,000 km and performed a field survey of more than 50 towns that covered both urban and rural areas and coastal and inland regions. This is the first ophthalmologic epidemiological survey of an entire coastal province of China to date. Previous eye surveys in China were in northern areas , 13, urbChiCTR2100043349) and the study protocol was approved by the Ethics Committee of Xiamen Eye Center affiliated with Xiamen University (Acceptance number: XMYKZX-KY-2018-001), and written informed consent was obtained from all participants. All residents in a community are officially registered at the local mayor’s office. Using this register as the sampling frame, all residents of the communities who were aged over 50 years were eligible for the study. We used a two-stage cluster sampling design where the clusters were first selected with probability proportional to cluster number, and then subjects are randomly sampled inside selected clusters. According to the data of the National Bureau of Statistics and Fujian Provincial Department Of Finance in 2017 (http://czt.fujian.gov.cn/zfxxgk/fdzdgknr/czzjgl/zjfpwj/201803/t20180329_4563507.htm), the permanent residents of Fujian Province was approximately 38.74 million and there were 2153 communities. Our finally total sample was 10,044 subjects, so we randomly selected 54 communities in an equal proportion manner, including 33 urban communities and 21 rural communities. Then we randomly selected subjects based on the register from each community according to the community size. Figure The FJES was performed from May 2018 to October 2019 as an ophthalmologic epidemiologic on-site survey on a random sample of coastal and inland, urban and rural Southern Chinese adults, reflecting the current eye health status of local Chinese people. The location of the study is illustrated in Fig. I.Officially registered at the local mayor’s officeII.Age over 50 yearsIII.Live and work in the county right nowIV.Good mental state, and able to cooperate with the examinationI.Not officially registered at the local mayor’s officeII.Age less than 50 yearsIII.Not live or work in the county right nowIV.Permanent migrantV.Have severe mental disease which can not cooperate the inspectionn = deff×μα2 × p × (1-p)/d2. The present study can achieve a precision of 0.05 (d), considering confidence interval of 95% , μα2 of 1.96, design effect of 2, relative error of 0.15 and d = r × p. The sample size was targeted to achieve an adequate precision around estimates of prevalence and to allow for risk factor analyses to be carried out. As the prevalence of the main eye diseases in cross-sectional baseline surveys were estimated to be greater than 2.0% [The following calculation formula was used to estimate the sample size: han 2.0% –20. Basehan 2.0% , the perThe FJES provides exhaustive longitudinal information including sociodemographic and administrative data , physical data , heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), blood index) and ocular data ; presenting distance visual acuity (PDVA); refractive state; best corrected visual acuity (BCVA); intraocular pressure (IOP); slit lamp examination and fundus examinations ) data. The process of on-site inspection is shown schematically in Fig. PDVA and BCVA were measured using E Standard Logarithmic Visual Acuity Chart (GB 11533—1989) at a distance of 5 m. For results less than 0.1, PDVA and BCVA at a distance of 1 m were tested. If that was not possible, finger counting, hand movement, and light perception were tested.NVA was measured by logarithmic visual acuity chart at a distance of 30 cm, followed the WHO definitions of VI as BCVA in better eyes of < 20/60 or worse ) and blindness as BCVA in better eyes worse than 20/400 ), defined PNVI as PNVA worse than 20/50  and defined presbyopia as PNVA worse than 20/50 and BCVA better than 20/40 ).The Lens Opacities Classification System III (LOCS III) was used to evaluate the type of cataract. It consisted of six slit-lamp images for grading nuclear colour (NC) and nuclear opalescence (NO), five retroillumination images for grading cortical cataracts (C), and five retroillumination images for grading posterior subcapsular (P) cataracts .A fundus photograph of each eye was taken using a scanning laser device , which has a resolution of 20 μm and is able to capture the fundus even through an undilated pupil. Fundus photography was used to take at least two fundus photographs following the operation rules, one centred on the macular disc and one centred on the optic disc.Multicolour OCT  was used for high-resolution imaging of the optic disc and central retina in both eyes. Prior to imaging, autokeratometry data were entered to correct for ocular magnification effects. The instrument’s eye-tracking software was used to minimize the effects of eye movements. If a lesion was visible, the lesion site was identified with multi-slice scanning images. The protocol for Multicolour OCT imaging for each eye was as follows .Forty-nine-line raster scan of a 15° × 10° area. The average of nine frames for each B-scan was used to improve quality.Peripapillary retinal nerve fibre layer (RNFL) thickness measurements. A circular B-scan of the peripapillary RNFL was taken along a 3.5 mm-diameter (~ 12°) circle.Optic nerve head radial and circle (ONH-RC) scan. Forty-eight equidistant (7.5° spaced) radials and three circle B-scans were obtained. Each radial B-scan was averaged from 25 frames and spanned 4.7 mm. The three circular B-scans were 3.5 mm (~ 11.5°–12.5°), 4.1 mm (~ 13.5°–14.5°) and 4.7 mm (~ 15.5°–16.5°) in diameter, and each was averaged from 100 frames. Prior to starting the scans, the ONH-RC programme automatically detected the foveal and Bruch’s membrane opening positions. The examiner checked and, if necessary, manually corrected the positions of these landmarks.Thirty-one-line raster scan of a (30° × 25°). Each B-scan was averaged from nine frames.Enhanced depth imaging of the macular. Each B-scan spanned approximately 8.6 mm (~ 30°), and the average of 100 frames for each scan was recorded for analysis.Before the on-site survey, all technicians and clinicians recruited were trained uniformly and needed to finish an examination, and each survey examination was required to be fixed consistently with the same technician. During our on-site survey, participants were asked to complete all the tests before they can get the final diagnosis report, in order to improve the response rate. In the aspect of data collation, double entry with EpiData v3.1  was used to check the data to ensure the correctness of the data.2) tests were used to compare proportions. Multiple regression models were used to examine the relation between visual acuity measurements and selected sociodemographic characteristics. Logistic regression was applied for the comparison of binary parameters versus categorical parameters and for the comparison of binary parameters versus continuous normally distributed parameters. Linear regression was applied for the comparison of normally distributed parameters. Logistic regression was used to examine the correlation degree of each group. The statistical correlations was reported as the correlation coefficient r and statistical strength of correlations was described using odds ratio (OR). Confidence intervals  are presented. All described associations were derived from the multivariable statistical analysis, unless indicated otherwise. All P-values were two sided and less than 0.05 were considered statistically significant.Stata/SE statistical software  was used to analyse the data. Data are provided as the mean ± standard deviation (SD). Only one randomly selected eye per subject was obtained for statistical analysis of visual acuity unless intraindividual intereye differences were evaluated. Analysis of variance (ANOVA) was applied to compare the mean among groups of normally distributed parameters. Chi-square  aged ≥50 years were eventually included, and 4836 (58.9%) were female. 4678 (57.0%) were from urban area, and 3533 (43.0%) were from rural area. 6434 (78.4%) were from coastal region, and 1777 (21.6%) were from inland region. The response rates were 80.2 and 84.0% for the urban population and rural population, respectively, and 80.3 and 87.6% for the coastal population and inland population, respectively. Mean age was 64.39 (SD 8.87) years . The proportion of age group population in this study was similar to that in China on the whole. Per Capital Annual Net Income in this rural area is 16,335 Yuan, approximately $2419 US, which is higher than the average annual income  per Capital of those living in rural areas throughout Mainland China, and 39,001 Yuan in urban area, approximately $5776 US, which is also higher than the average annual income  per Capital of those living in urban areas throughout Mainland China [In the FJES, 4836 were female, and 3375 were male. Among them, 4776 females and 3287 males had PDVA test results, 4257 females and 2566 males had BCVA results, and 4836 females and 3192 males had PNVA results. The mean PNVA was 0.28 (0.17), the mean presenting VA was 0.61 (0.30) and 0.23 (0.27) logMAR units, and the mean BCVA was 0.82 (0.28) and 0.08 (0.19) logMAR units. Table The FJES enrolled 8211 participants from May 2018 to October 2019. The participant baseline characteristics and ocular characteristics were reported. Compared with several cross sectional study before , our stuAs the study reported, the percentage of blindness and VI in our study was 0.21 and 3.33%, respectively, which was lower than the global data from a Lancet review. The review showed the percentage of blindness and VI was approximately 0.49 and 3.69%, respectively, until 2015 . BesidesIn this study, VI was significantly correlated with biological and sociodemographic factors, including age, urban and rural regions, coastal and inland areas, educational background, income and refractive error. Sex was not statistically significantly associated with VI after taking into account the interdependency of the parameters.Encouragingly, many innovative results were found, such as the difference in VI between coastal and inland regions, a higher DVI rate in the inland population, a higher combined VI rate in both rural and inland populations, and NVA improvement in the tea-drinking population. Dramatic changes have taken place in residents’ lifestyle with the coronavirus disease-19 (COVID-19) outbreak. In particular, near vision at all ages has been affected significantly due to the penetration of electronic products when people reduce outdoor social activities. As a result, future epidemiological research may involve changes to models or performance. Our study only examined eye health status before the epidemic and provides a pre-epidemic sample for future research. The project is the first domestic epidemiological study for eye diseases covering coastal and inland areas in both urban and rural regions and provides evidence for the establishment of policy making and control strategy for eye diseases. The FJES can facilitate collaborations in clinical practice for ophthalmologists and cardiologists or endocrinologists.There are also some interesting findings. For example, compared with the inland population, the coastal population had less myopia and better vision function overall. Potential differences in economic level, education level, transportation, environmental and lifestyle factors, such as the coastal diet preference for seafood and higher UV levels , 28 in cThe FJES has several strengths and some differential features with regard to other information resources. First, a key strength of the FJES database is the detailed questionnaire information and overall ocular and physical examinations collected in more than 50 towns of all nine cities in an entire province of southern China. Second, many cross-sectional studies have provided a description of VA, but most studies have reported NVA and DVA separately. This study integrated both NVA and DVA and explored the influence of geographic factors, including urban and rural and coastal and inland areas, which have not previously been covered. Third, most comparisons in previous studies were based on various baselines among different studies, whereas the data analysis and comparison in this study used a single baseline, making the correlation more convincing. Fourth, we used advanced measurement instruments, such as multicolour OCT and nonmydriatic colour fundus photography. Furthermore, we designed various subgroups to provide more original information, which can update and complement worldwide epidemiological surveys in ophthalmology.There are some limitations of this study. First, there may be information biases due to absent registration (data completeness). Second, the examination was not perfect. Because of time limitations, we only performed DVA correction , and we did not perform NVA correction. Unfortunately, we also did not perform ocular biometry, which can obtain structural parameters such as the refractive power of the cornea, the depth of the anterior chamber, the axial length of the eyeball and the thickness of the lens. Third, data quality may be a strength in some databases but also a weakness for certain data, such as the incompleteness of NVA and DVA data, which could slightly affect the integrity and accuracy of the final diagnosis results.The FJES is a population-based cross-sectional on-site survey on the public eye health status of Chinese residents in Fujian province. The rich data collected from the study provide the opportunity to identify risk factors and associations of VI and eye diseases with chronic diseases and basic personal information. This project will be meaningful for guidance in eye health-related policy-making.


Listeria monocytogenes is a major pathogen contributing to foodborne outbreaks with high mortality. Nisin, a natural antimicrobial, has been widely used as a food preservative. However, the mechanisms of L. monocytogenes involved in nisin resistance have not yet to be fully defined. A mariner transposon library was constructed in L. monocytogenes, leading to the identification of 99 genes associated with the innate resistance to nisin via Transposon sequencing (Tn-seq) analysis. To validate the accuracy of the Tn-seq results, we constructed five mutants  in L. monocytogenes. The results revealed that yqgS and lafA, the lipoteichoic acid-related genes, were essential for resistance to nisin, while the gtcA and lmo1464 mutants showed substantially enhanced nisin resistance. Densely wrinkled, collapsed surface and membrane breakdown were shown on ΔyqgS and ΔlafA mutants under nisin treatment. Deletion of yqgS and lafA altered the surface charge, and decreased the resistance to general stress conditions and cell envelope-acting antimicrobials. Furthermore, YqgS and LafA are required for biofilm formation and cell invasion of L. monocytogenes. Collectively, these results reveal novel mechanisms of nisin resistance in L. monocytogenes and may provide unique targets for the development of food-grade inhibitors for nisin-resistant foodborne pathogens.IMPORTANCEListeria monocytogenes is an opportunistic Gram-positive pathogen responsible for listeriosis, and is widely present in a variety of foods including ready-to-eat foods, meat, and dairy products. Nisin is the only licensed lantibiotic by the FDA for use as a food-grade inhibitor in over 50 countries. A prior study suggests that L. monocytogenes are more resistant than other Gram-positive pathogens in nisin-mediated bactericidal effects. However, the mechanisms of L. monocytogenes involved in nisin resistance have not yet to be fully defined. Here, we used a mariner transposon library to identify nisin-resistance-related genes on a genome-wide scale via transposon sequencing. We found, for the first time, that YqgS and LafA (Lipoteichoic acid-related proteins) are required for resistance to nisin. Subsequently, we investigated the roles of YqgS and LafA in L. monocytogenes stress resistance, antimicrobial resistance, biofilm formation, and virulence in mammalian cells. Listeria monocytogenes is an intracellular foodborne pathogen, leading to a severe invasive infection called listeriosis after consumption of contaminated food  and LafB (Lmo2554) are required for the formation of Glc-DAG and Gal-Glc-DAG, respectively, as an important bacterial cell wall component, is a zwitterionic polymer and is typically composed of polyglycerolphosphate backbone chain tethered to the membrane by a glycolipid  . In L. mpectively. YqgS  with or without subinhibitory concentration of nisin (200 μg/mL). Subsequently, DNA was extracted from samples, and we amplified transposon junctions and attached adaptor sequences to construct a DNA library for sequencing. The sequencing results indicated that each sample had at least 10 million Tn-seq readings and no biased insertion sites of the transposon were identified. The distribution of transposon was in an even and high-density pattern, and 43,793 unique insertion sites were identified throughout the genome (Supplementary File 2). Finally, analysis of Tn-seq results was carried out to identify genes associated with nisin resistance.To identify genome-wide genes associated with nisin resistance, a ftsW), peptidoglycan lytic protein P45 (spl), glycolipid and polyglycerolphosphate lipoteichoic acid synthesis (yqgS and lafABC), d-alanyl-teichoic acids biosynthesis (dltA), lysinylation of phospholipids (mprF), penicillin-binding protein (pbp4), d-alanyl-alanine synthetase (ddl), and UDP-glucose 4-epimerase . Ten regulators genes were identified, including seven putative transcriptional regulators , two 2-component response regulators (liaR and virR), and a recombination regulator (recX).We identified 99 genes, and the number of transposon insertions was significantly different among groups in the presence or absence of nisin (Benjamini-Hochberg < 0.05), which were involved in nisin resistance  and B. NvirA and anrAB) were useful to confer nisin resistance in L. monocytogenes, which were regulated via two-component systems  were identified. The DNA repair genes are essential for DNA damage caused by antimicrobial treatment (polC), NAD-dependent DNA ligase (ligA), and single-strand DNA-binding protein (lmo2523). Moreover, eight genes involved in the biosynthesis of secondary metabolites were identified, six genes  of which were clustered in the porphyrin and chlorophyll metabolism pathway. And five susceptibility genes are located in the atpABCDEFG operon , which is involved in membrane bioenergetics such as ATP synthase  were linked to cell envelope, which encoded diacylglycerol kinase and wall teichoic acid glycosylation protein GtcA, respectively. The other five genes with significant fold decrease are annotated as monovalent cation/H+ antiporter subunit C (lmo2380) and hypothetical proteins . Overall, the transposon library screening has comprehensively identified genes that confer resistance or are important for the mechanisms of action of nisin at the genome-wide scale.Seven genes were identified as contributing to nisin sensitivity (Table S3). That is, the corresponding transposon mutants were significantly enriched in the presence of nisin compared with the untreated group. Of those, two genes , which were identified with high fold changes and significant P-values. The VirRS signal transduction system is known to determine the nisin resistance, yet, whether the deficiency in the other four genes changes nisin sensitivity has not been investigated. Under nisin-free conditions, the growth rate or cell density in the stationary phase of mutants was not significantly changed compared with the wild-type strain (yqgS mutant was grossly affected in the presence of nisin (650 μg/mL), and nisin (750 μg/mL) also had a severe effect on the growth of lafA mutant  and lipoteichoic acid backbone (yqgS) synthesis, indicating that Lipoteichoic acid is essential for nisin resistance but not to bacitracin in L. monocytogenes.In order to validate the Tn-seq results, we constructed five mutants  and transmission electron microscopy (TEM) scans of the nisin-treated bacterial cultures. No significant difference is observed between the wild-type and mutant strains as displayed in ch point  and 5. OyqgS and ΔlafA mutants, the surface charge of the mutant and wild-type strains was determined by measuring the binding of cytochrome c. As expected, we observed higher levels of cytochrome c binding with the ΔyqgS and ΔlafA mutants than wild-type strain  on bacterial growth in different strains. The resistance of Δ to acid  and salt to acid  was slig to acid . These rtenuated . OverallL. monocytogenes pathogenicity. To test this assumption, we infected the RAW264.7 murine macrophage-like cells with different strains  and determined the number of intracellular bacteria as a function of time. No obvious differences were detected in adherence from all the strains  and lipoteichoic acid backbone (yqgS) synthesis.In this study, 99 genes associated with nisin resistance have been identified in to nisin , 28, 55.to nisin , 56–59. ss-links , 61. ThelisRK, liaRS, and virRS) perform important functions in innate lantibiotic resistance  were identified as nisin-resistance genes, the three resistance genes of which were previously described including virA  were correlated with resistance against nisin, which also were verified experimentally in previous studies  have not been identified. The signal transduction system LisK and the glutamate decarboxylase GadD1 determined the sensitivity of L. monocytogenes LO28 to nisin  were deleted to validate the accuracy of Tn-seq analysis. We have demonstrated the role of GtcA and Lmo1464 in nisin sensitivity, and these mutants showed significantly enhanced resistance to nisin. However, the detailed mechanism still needs to be investigated.The reidentification of locus previously shown to be related to nisin resistance confirms the reliability of the screening, but two known nisin-resistance genes  and synthase LtaS (Lmo0927) are responsible for LTA backbone synthesis  are essential for L. monocytogenes resistance to nisin but not to bacitracin. LTA was previously reported to be involved in nisin resistance of Streptococcus bovis and S. aureus  are required for biofilm formation, surface charge maintenance, and intracellular multiplication, which also significantly contribute to the resistance of cell envelope-acting antimicrobials and unfavorable conditions. These findings may provide novel targets for the development of food-grade inhibitors for nisin-resistant foodborne pathogens.In conclusion, this study provided comprehensive identification of genes required for nisin resistance, which revealed novel mechanisms of nisin resistance in L. monocytogenes strain EGDe (American Tissue Culture Collection) and the Escherichia coli strains EC1000 were used in this study. Unless otherwise noted, the bacteria were cultured in BHI (Hopebiol) at 37°C with shaking. Under necessary conditions, the concentrations of antibiotics used are as follows: gentamicin 25 μg/mL for L. monocytogenes and E. coli, chloramphenicol 5 μg/mL for L. monocytogenes, spectinomycin 100 μg/mL for L. monocytogenes and E. coli. All strains and plasmids used in this study are listed in Table S1.The L. monocytogenes. The mutant library was generated according to previously described methods  for overnight culture. Then three experimental replicates of approximately 2 × 107 CFU were inoculated into 100 mL BHI or 100 mL BHI with 200 μg/mL nisin (Aladdin), which were incubated at 37°C for 16 h and then further processed for Tn-seq as described previously ; RPTAM = (number of reads mapped to a gene × 106)/. The statistical analysis on the RPTAM values was performed by Cyber-T. Genes that significantly contributed to either nisin susceptibility or nisin resistance were determined when the Benjamini-Hochberg (BH) corrected P-value was < 0.05 and the difference in abundance of the transposon mutant during growth in BHI with or without nisin was > 5. Tn-seq data was provided in supplementary file 2.Tn-seq data analysis was performed as previously described. In short, barcodes were split using the Galaxy platform and 16-nucleotide fragments of each read were mapped to the L. monocytogenes, with a method based on the Cre-lox recombination system  on SmaI site of the pWS3 vector with NovoRec plus One step PCR Cloning Kit (novoprotein), resulting in plasmid pWS3- yqgS which used EC1000 as the cloning host. The recombinant plasmid with homologous arm and gentamicin maker was introduced in L. monocytogenes EGDe by electroporation. Positive clones were inoculated in prewarmed BHI medium without antibiotics and grown overnight at 37°C to get marked deletion mutants. To obtain the markerless mutants, the shuttle plasmid pWS3-erm-cre was electrotransformed into the marked mutants. Then cell culture was plated on BHI agar plates and incubated at 37°C to get the markerless mutations, verified by PCR and sequencing.Plasmid pWS3 was used to generate mutations through a process of double homologous recombination in n system . The upslafA, gtcA, and lmo1464 genes in L. monocytogenes to get the corresponding markerless mutations. Primers used in this study are listed in Table S1. All plasmid constructs and gene deletions were confirmed by DNA sequencing.The same strategy was used to remove the L. monocytogenes strains (wild-type and mutants), overnight cultures were diluted 100-fold in fresh BHI and BHI containing appropriate nisin or bacitracin. The absorbance of 630 nm (A630 nm) was measured every 1 h for 16 h. The same strategy was used to draw growth curves of L. monocytogenes wild-type, ΔyqgS, and ΔlafA mutants in unfavorable conditions.To draw growth curves of L. monocytogenes strains were inoculated into the fresh BHI medium at a ratio of 1:100 and grew to exponential phase (OD600nm = 0.6 to 0.8), and then spun down and resuspended in phosphate buffer saline (PBS) to OD600nm = 0.65, next nisin  was added. Viable bacterial CFU counts were conducted after 2 h.Overnight cultures of L. monocytogenes strains were grown to exponential phase (OD600nm = 0.8), followed by nisin (800 μg/mL) treatment in experimental groups for 1 h, after which the bacteria cultures were spun down and fixed with 2.5% glutaraldehyde in PBS for more than 4 h. The samples were washed three times in PBS, postfixed with 1% OsO4 for 1 h, and washed three times. Then the dehydration of samples used a graded series of ethanol . The samples were dehydrated by alcohol for 20 min, and dehydrated in Hitachi Model HCP-2 critical point dryer. Later we can coat with gold-palladium in Hitachi Model E-1010 ion sputter for 4 to 5 min and observe in Hitachi Model SU-8010 SEM. The samples were transferred to absolute acetone for dehydration, placed in 1:1 mixture of absolute acetone and the final Spurr resin mixture, then transferred to 1:3 mixture of absolute acetone and the final resin mixture and to the final Spurr resin mixture for overnight. Specimens were heated at 70°C, then sectioned in LEICA EM UC7 ultratome, and sections were stained by uranyl acetate and alkaline lead citrate for 5 to 10 min respectively and observed in Hitachi Model H-7650 TEM.The L. monocytogenes strains were inoculated into the fresh BHI medium and grew to exponential phase (OD600nm = 0.6 to 0.8), then washed twice in 20 mM MOPS buffer (pH 7.0) and adjusted to OD600nm = 0.7. Bacterial aliquots were concentrated in half volume of 0.5 mg/mL equine cytochrome c dissolved in 20 mM MOPS buffer. After 10 min of incubation in the dark, the bacteria were centrifuged  and the supernatant liquid was transferred to a 96-well plate. The absorbance was measured at 530 nm. The absorbance of samples only containing MOPS buffer was recorded as 100% binding, and the value of samples containing cytochrome c but lacking bacteria was recorded as 100% binding.Bacterial surface charge was performed as described . Overnig6 CFU/mL. Then 200 μL of this bacterial liquid was absorbed into 96-well plate and incubated at 37°C for 48 h. The biofilm was quantified with the crystal violet (CV) assay  assay . Briefly6 CFU were added to 2 mL BHI with confocal dish and incubated at 37°C for 48 h. Cultures were discarded and dishes were washed thrice with PBS. Next, 200 μL SYTO9/PI dye solution was added to the culture dishes and treated for 30 min in the dark. After washing the dishes with PBS once, they were observed under a Confocal Laser Scanning Microscope LSM700.Fluorescence staining was used to observe the formation of biofilm under confocal microscope. 4 × 10The antibiotic disk diffusion assays were performed as previously studied , 86. Bri2O2 was performed as mentioned above. It should be noted that 15 μL H2O2 (33%) was added to a 6-mL blank disk and left to dry for an hour. Subsequently, the H2O2 disks were placed onto the corresponding BHI plates and incubated at 37°C for 24 h to measure the size of inhibition zone.The size of inhibition zone of HL. monocytogenes strains were grown to exponential phase (OD600nm = 0.6 to 0.8), washed three times in dulbecco's modified eagle medium (DMEM), and diluted to MOI ≈25. Bacterial suspensions were transferred to macrophages and incubated for 1 h at 37°C, then washed three times and added fresh DMEM with gentamicin (25 μg/mL) for 1.5 h. Cells were lysed in 0.1% Triton X-100 and serially diluted for counting in BHI at different time points .Invasion and intracellular multiplication assays in RAW were performed as described . The L. t test. The differences were considered statistically significant at the 95% level of confidence (P < 0.05).To ensure the significance of the results, the data were analyzed using Student’s L. monocytogenes in Tn-seq have been submitted in the NCBI server (Accession No. SRR17303189).Illumina sequencing reads of


Colorectal cancer (CRC) is the leading cause of cancer deaths around the world. It is necessary to identify patients with poor prognosis or with high risk for recurrence so that we can selectively perform intensive treatments such as preoperative and/or postoperative chemotherapy and extended surgery. The clinical usefulness of inflammation-related prognostic biomarkers available from routine blood examination has been reported in many types of cancer, e.g., neutrophil–lymphocyte ratio (NLR), lymphocyte–C-reactive protein ratio (LCR), platelet–lymphocyte ratio (PLR), lymphocyte–monocyte ratio (LMR), and so on. Moreover, some scoring systems based on circulating blood cell counts and albumin concentration have been also reported to predict cancer patients’ prognosis, such as the Glasgow prognostic score (GPS), systemic inflammation score (SIS), and prognostic nutritional index (PNI). The optimal biomarker and optimal cutoff value of the markers can be different depending on the cancer type. In this review, we summarize the prognostic impact of each inflammation-related marker in CRC. Colorectal cancer (CRC) is one of the main causes of cancer death in the world. Despite recent improvements in multidisciplinary approaches, including surgery, chemotherapy, and radiotherapy, the mortality rate of CRC is still high, especially in patients with distant metastasis or postoperative recurrence even after curative surgery. Development of the optimal biomarker useful for the prediction of recurrence or poor prognosis is clinically important in order to identify patients who can benefit from intensive treatment including chemotherapy, chemoradiotherapy, and extended surgery.The cancer-associated systemic inflammatory response is one of the critical indicators of tumor progression. Numerous previous studies have reported serum systemic inflammatory markers that can be useful for the prediction of prognosis, e.g., neutrophil–lymphocyte ratio (NLR), lymphocyte–monocyte ratio (LMR), platelet–lymphocyte ratio (PLR), Glasgow prognostic score (GPS), lymphocyte–C-reactive protein ratio (LCR), systemic inflammation score (SIS), and prognostic nutritional index (PNI) . These mHowever, the best combination and optimal cutoff value can be different depending on the cancer type. Moreover, various anti-cancer treatments can differently affect the systemic inflammation status. Therefore, it is mandatory to focus on a certain cancer type so that we can evaluate the prognostic impact of inflammation-related markers.Previously, we reported that the combination of GPS and NLR could effectively predict the prognosis of CRC patients who underwent curative resection . In addi9/L) were associated with better disease-free survival (DFS) [The cancer-associated systemic inflammatory response is often correlated with an increase in circulating neutrophil counts. Neutrophils secrete cytokines and chemokines, which play important roles in cancer progression. Yang et al. reported that a high serum neutrophil count itself was independently associated with poor overall survival (OS) and progression-free survival (PFS) in metastatic CRC patients with wild-type RAS . On the al (DFS) .n = 5336) following curative resection (Stage I-III) and reported that NLR > 2.72 was an independent predictor for OS and DFS [The neutrophil–lymphocyte ratio (NLR) is one of the most robust biomarkers to predict prognosis in various types of cancer. Proctor et al. analyzed 12,118 patients including 1413 CRC patients, and indicated that NLR was a significant surrogate marker for OS and cancer-specific survival (CSS) . There wThe patient population enrolled in each study was also different, i.e., CRC patients who underwent curative resection, metastatic CRC (Stage IV), colorectal liver metastasis (CRLM) following liver resection, CRC with peritoneal carcinomatosis, and so on. For example, Dell’Aquila et al. analyzed 413 metastatic CRC who received FOLFOXIRI or FOLFIRI plus bevacizumab enrolled in a TRIBE trial  and repoMost of the papers evaluated “preoperative NLR”, while some assessed NLR in a different way. Yasui et al. stratified 568 CRC patients into three groups depending on inflammation status: preoperatively low , preoperatively high but postoperatively low , and persistently high (elevated group) . They inOn the other hand, Thiagarajan et al. analyzed 436 CRC patients with peritoneal carcinomatosis, and evaluated NLR on several postoperative timings  . They reSome previous reports indicated that NLR could be predictive for recurrence patterns after liver resection against CRLM. Giakoustidis et al. reported that the patients with preoperatively elevated NLR (>2.5) had a higher risk for extrahepatic/multifocal recurrence . SimilarThus, numerous papers have proven that NLR can be used as a significant predictor for the survival of CRC patients, although the characteristics of the patients enrolled and the optimal cutoff values of NLR varied from report to report.Albumin is one of the acute-phase proteins and decreases in response to inflammation. Moreover, a low albumin concentration reflects cancer-induced malnutrition and can have negative impact on prognosis.The GPS, introduced by Forrest et al., is a well-known inflammation-related marker that includes serum C-reactive protein (CRP) levels and serum albumin levels . GPS is A number of previous studies have revealed the postoperative prognostic potential of GPS . As mentGPS is also useful for metastatic CRC. Kobayashi et al. collected data from 63 CRLM patients who underwent curative resection and reported that GPS 1 or 2 was an independent prognostic factor for CSS . SimilarGPS has been modified by numerous recent reports so that it could be more sensitive for the prediction of prognosis; this is often called “modified GPS” (mGPS) . For exaOn the other hand, Inoue et al. defined mGPS as follows: CRP ≤ 0.5 mg/dL and albumin ≥ 3.5 mg/dL was a score of 0, CRP > 0.5 mg/dL or albumin < 3.5 mg/dL was a score of 1, and CRP > 0.5 mg/dL and albumin < 3.5 mg/dL was a score of 2. They indicated the prognostic potential of this mGPS by analyzing 245 Stage IV and recurrent CRC patients treated with chemotherapy .Thus, there were a number of different mGPSs, and the optimal definition is still controversial.Other than GPS, there are also several albumin-related prognostic markers. For example, the prognostic nutritional index (PNI), first reported by Onodera et al. , is a weWe have recently reported the novel prognostic marker “LA”, which means “lymphocyte count × albumin level (g/dL) . We retrWang et al. analyzed 877 CRC patients who underwent curative resection (Stage I-III) and built a scoring system, “NLR–albumin”. They reported that the combination of high NLR (≥2.39) and hypoalbuminemia <39.75 g/L) was a negative prognostic factor for OS in the multivariate analysis . Hong et g/L was Furthermore, Shibutani et al. showed the usefulness of ALI (advanced lung cancer inflammation index) for CRC patients . ALI wasThe albumin–globulin ratio (AGR) is also reported as contributive for the prediction of cancer-related prognosis. Low AGR reflects the combination of hypoalbuminemia and hyperglobulinemia. Hyperglobulinemia is also correlated to systemic inflammation, which can promote cancer development. Li et al. investigated 5336 CRC patients who underwent curative resection (Stage I-III) and indicated that AGR was an independent factor of OS and DFS . SimilarIn the tumor microenvironment, monocytes play an important role in tumor progression. Monocytes differentiate into tumor-associated macrophages (TAMs) and can contribute to tumor infiltration and metastasis. An increase in serum monocyte count can reflect the activity of TAMs.3) was significantly associated with poor OS and DFS in a cohort of 270 pathological T3N0M0 rectal cancer patients who underwent curative resection [3) was independently associated with poor CSS in a cohort of 97 CRLM patients who underwent liver resection [Zhang et al. reported that an elevated monocyte count  has been evaluated in several previous reports ; that isChan et al. evaluated 1623 CRC patients who underwent curative surgery  and found that elevated LMR >2.38) was independently associated with better OS, although NLR and PLR were not . They al was inde9/L and monocyte count ≤0.80 × 109/L were allocated a score of 0, patients with lymphocyte count <1.5 × 109/L and monocyte count >0.80 × 109/L were assigned a score of 2, and the others were assigned a score of 1. They reported that both LMR and LMS were significantly associated with OS and CSS in multivariate analysis. On the other hand. Chan et al. evaluated the perioperative change in inflammation markers in CRC patients and found that the patients with consistently high LMR (>2.86) both before and after surgery had significantly better prognosis [Dolan et al. compared the prognostic value between composite ratios and cumulative scores of inflammation markers . They derognosis .SIS is a comparatively new prognostic marker that was first introduced by Chang et al. in patients with renal cell carcinoma . SIS conCRP is one of the most useful parameters to evaluate the inflammation status of cancer patients preoperatively or postoperatively. In many countries, however, CRP is not routinely tested in general clinical settings, although not in Japan; CRP is evaluated in almost all cases in a routine blood examination in Japan. CRP itself has been reported as a prognostic marker for CRC patients. For example, Koike et al. evaluated 300 CRC patients and reported that preoperative high CRP (>0.5 mg/dL) was an independent predictor of prognosis .The combination of higher CRP and hypoalbuminemia can be also a sensitive biomarker for cancer-related prognosis. Matsuoka et al. evaluated 133 Stage III CRC patients who underwent curative resection and found that postoperative C-reactive protein–albumin ratio (CAR) (≥0.035) was significantly associated with worse OS and RFS . Ide et n = 373) and validated the result in the validation cohort (n = 104) [Lymphocyte–C-reactive protein ratio (LCR) is the combination of lymphocyte count and CRP, and is also a robust prognostic marker. Suzuki et al. investigated 16 inflammation-related markers, namely NLR, LMR, PLR, CAR, PNI, LCR, neutrophil–albumin ratio (NAR), monocyte–albumin (MAR), platelet–albumin ratio (PAR), neutrophil × monocyte, neutrophil × platelet, neutrophil × CRP, monocyte × platelet, monocyte × CRP, platelet × CRP, and LA, for 1303 CRC patients who underwent curative resection (Stage II–III) . They inn = 104) . They prMost of the studies about CRP-related makers adopted preoperative parameters. However, as mentioned above, Yasui et al. analyzed the inflammation status between before and after surgery, and showed that postoperative, but not preoperative, inflammation-based markers  were significantly associated with OS and RFS . The besAs with neutrophils, platelets are also a typical blood cell component responsible for the inflammatory response, and thrombocytosis is often observed in solid tumor patients with chronic inflammation ,94. In t9/L) was independently associated with poor OS [9/L) could be a significant predictor for poor OS and CSS in Stage IV CRC patients [In several previous reports, serum platelet count was reported to be a prognostic factor in CRC. Ishizuka et al. analyzed 453 CRC patients  who underwent surgery and reported that an elevated platelet count (>300 × 10 poor OS . Similarpatients .Platelet–lymphocyte ratio (PLR) has been reported as a well-known prognostic marker in CRC . A high Kim et al. investigated 1868 CRC patients and reported that high PLR (≥160) and high NLR (≥3.0) were independent predictors for poor OS and DFS in Stage III and IV cases, although not in Stage I and II cases . Erstad Mercier et al. recently introduced a new prognostic marker, the platelet–neutrophil–lymphocyte ratio (PNLR) . High PNAccumulating evidence has shown the usefulness of inflammation-related biomarkers in CRC patients. In this review, we divided the markers into five categories: neutrophil-related markers, albumin-related markers, monocyte-related markers, CRP-related markers, and platelet-related markers. Of note, the characteristics of the enrolled patients and the cutoff values of each inflammation-related marker varied widely from study to study. Multicenter prospective studies will be required to find the optimal cutoff values.