Combination of Formononetin and Sulforaphane Natural Drug Repress the Proliferation of Cervical Cancer Cells via Impeding PI3K/AKT/mTOR Pathway.

Natural substances have been demonstrated to be an unrivalled source of anticancer drugs in the present era of pharmacological development. Plant-based substances, together with their derivatives through analogues, play a significant character in the treatment of cancer by altering the tumor microenvironment and several signaling pathways. In this study, it was investigated whether the natural drugs, formononetin (FN) and sulforaphane (SFN), when combined, assess the efficacy of inhibiting cervical cancer cell proliferation by impeding the PI3K/Akt/mTOR signaling pathway in HeLa cells. The cells were treated with the combination of FN and SFN (FN + SFN) in various concentrations (0-50 µM) for 24 h and then analyzed for various experiments. The combination of FN + SFN-mediated cytotoxicity was analyzed by MTT assay. DCFH-DA staining was used to assess the ROS measurement, and apoptotic changes were studied by dual (AO/EtBr) staining assays. Protein expressions of cell survival, cell cycle, proliferation, and apoptosis protein were evaluated by flow cytometry and western blotting. Results showed that the cytotoxicity of FN and SFN was determined to be around 23.7 µM and 26.92 µM, respectively. Combining FN and SFN causes considerable cytotoxicity in HeLa cells, with an IC50 of 21.6 µM after 24-h incubation. Additionally, HeLa cells treated with FN and SFN together showed increased apoptotic signals and considerable ROS generation. Consequently, by preventing the production of PI3K, AKT, and mToR-mediated regulation of proliferation and cell cycle-regulating proteins, the combined use of FN + SFN has been regarded as a chemotherapeutic medication. Further research will need to be done shortly to determine how effectively the co-treatment promotes apoptosis to employ them economically.

Effects of stocking density on the homeostasis of uric acid and related liver and kidney functions in ducks.

OBJECTIVE: Stocking density (SD) is an important issue in the poultry industry, which is related to the production performance, intestinal health and immune status. In the present study, the effects of SD on the metabolism and homeostasis of uric acid as well as the related functions of the liver and kidney in ducks were examined. METHODS: A total of 360 healthy 56-day-old Shan-ma ducks were randomly divided into the low stocking density (n = 60, density = 5 birds/m2), medium stocking density (n = 120, density = 10 birds/m2) and high stocking density groups (HSD; n = 180, density = 15 birds/m2). Samples were collected in the 3rd, 6th, and 9th weeks of the experiment for analysis. RESULTS: The serum levels of uric acid, lipopolysaccharide and inflammatory cytokines (interleukin-1ß [IL-1ß], IL-8, and tumor necrosis factor-α [TNF-α]) were increased significantly in the HSD group. Serious histopathological lesions could be seen in both the livers and kidneys in the HSD group in the 9th week. The mRNA expression levels of inflammatory cytokines (IL-8 and TNF-α) and related pathway components (toll-like receptor 4, myeloid differentiation primary response gene 88, and nuclear factor-κB) were increased significantly in both the livers and kidneys in the HSD group. The mRNA expression levels of enzymes (adenosine deaminase, xanthine oxidase, phosphoribosyl pyrophosphate amidotransferase, and phosphoribosyl pyrophosphate synthetase 1) related to the synthesis of uric acid increased significantly in the livers in the HSD group. However, the mRNA expression level of solute carrier family 2 member 9, which plays an important role in the excretion of uric acid by the kidney, was decreased significantly in the kidneys in the HSD group. CONCLUSION: These results indicated that a higher SD could cause tissue inflammatory lesions in the liver and kidney and subsequently affect the metabolism and homeostasis of uric acid, and is helpful for guiding decisions related to the breeding and production of ducks.

Adding Granular Activated Carbon and Zerovalent Iron to the High-Solid Anaerobic Digestion System of the Organic Fraction of Municipal Solid Waste: Anaerobic Digestion Performance and Microbial Community Analysis.

Anaerobic digestion (AD) performance and microbial dynamics were investigated in a high-solid anaerobic digestion (HSAD) system of the organic fraction of municipal solid waste (OFMSW). 1, 5, 10, and 15% (w/w, dry weight of the OFMSW) of granular activated carbon (GAC) and zerovalent iron (ZVI) were added to the HSAD system. The results showed that adding ZVI and GAC can improve the methane yield of the OFMSW. Notably, R-(GAC + ZVI) exhibited the highest cumulative methane yield of 343.0 mL/gVS, which was 57.1% higher than that of the R-control. At the genus level, the dominant bacteria included norank_f__norank_o__MBA03, norank_f__norank_o__norank_c__norank_p__Firmicutes, Fastidiosipila, norank_f__Rikenellaceae, and Sphaerochaeta, while Methanoculleus, Methanobacterium, and Methanosarcina were the dominant archaea. The highest relative abundance of norank_f__norank_o__norank_c__norank_p__Firmicutes was 30.8% for the R-(GAC + ZVI), which was 71.4% higher than that of the R-control. The relative abundance of Methanoculleus and Methanobacterium for the R-(GAC + ZVI) and the R-control group accounted for 79.0 and 90.8% of the total archaeal abundance, respectively. Additionally, the relative abundance of Methanosarcina was 10.6% for R-(GAC + ZVI), which was higher than that of the R-control (1.1%). After the addition of GAC and ZVI, the electron transfer capacity of the HSAD system was enhanced, resulting in promoted methane production. Thus, the simultaneous addition of GAC and ZVI to the HSAD system can be an effective strategy to promote the cumulative methane yield of the OFMSW.

A compendium of genetic regulatory effects across pig tissues.

The Farm Animal Genotype-Tissue Expression (FarmGTEx) project has been established to develop a public resource of genetic regulatory variants in livestock, which is essential for linking genetic polymorphisms to variation in phenotypes, helping fundamental biological discovery and exploitation in animal breeding and human biomedicine. Here we show results from the pilot phase of PigGTEx by processing 5,457 RNA-sequencing and 1,602 whole-genome sequencing samples passing quality control from pigs. We build a pig genotype imputation panel and associate millions of genetic variants with five types of transcriptomic phenotypes in 34 tissues. We evaluate tissue specificity of regulatory effects and elucidate molecular mechanisms of their action using multi-omics data. Leveraging this resource, we decipher regulatory mechanisms underlying 207 pig complex phenotypes and demonstrate the similarity of pigs to humans in gene expression and the genetic regulation behind complex phenotypes, supporting the importance of pigs as a human biomedical model.

Transcriptomic Analysis on Pectoral Muscle of European Meat Pigeons and Shiqi Pigeons during Embryonic Development.

In avian muscle development, embryonic muscle development determines the number of myofibers after birth. Therefore, in this study, we investigated the phenotypic differences and the molecular mechanism of pectoral muscle development of the European meat pigeon Mimas strain (later called European meat pigeon) and Shiqi pigeon on embryonic day 6 (E6), day 10 (E10), day 14 (E14) and day 1 after birth (P1). The results showed that the myofiber density of the Shiqi pigeon was significantly higher than that of the European meat pigeon on E6, and myofibers with a diameter in the range of 50~100 µm of the Shiqi pigeon on P1 were significantly higher than those of European meat pigeon. A total of 204 differential expressed genes (DEGs) were obtained from RNA-seq analysis in comparison between pigeon breeds at the same stage. DEGs related to muscle development were found to significantly enrich the cellular amino acid catabolism, carboxylic acid catabolism, extracellular matrix receptor interaction, REDOX enzyme activity, calcium signaling pathway, ECM receptor interaction, PPAR signaling pathway and other pathways. Using Cytoscape software to create mutual mapping, we identified 33 candidate genes. RT-qPCR was performed to verify the 8 DEGs selected-DES, MYOD, MYF6, PTGS1, MYF5, MYH1, MSTN and PPARG-and the results were consistent with RNA-seq. This study provides basic data for revealing the distinct embryonic development mechanism of pectoral muscle between European meat pigeons and Shiqi pigeons.

DNA Demethylation of Myogenic Genes May Contribute to Embryonic Leg Muscle Development Differences between Wuzong and Shitou Geese.

Skeletal muscle development from embryonic stages to hatching is critical for poultry muscle growth, during which DNA methylation plays a vital role. However, it is not yet clear how DNA methylation affects early embryonic muscle development between goose breeds of different body size. In this study, whole genome bisulfite sequencing (WGBS) was conducted on leg muscle tissue from Wuzong (WZE) and Shitou (STE) geese on embryonic day 15 (E15), E23, and post-hatch day 1. It was found that at E23, the embryonic leg muscle development of STE was more intense than that of WZE. A negative correlation was found between gene expression and DNA methylation around transcription start sites (TSSs), while a positive correlation was observed in the gene body near TTSs. It was also possible that earlier demethylation of myogenic genes around TSSs contributes to their earlier expression in WZE. Using pyrosequencing to analyze DNA methylation patterns of promoter regions, we also found that earlier demethylation of the MyoD1 promoter in WZE contributed to its earlier expression. This study reveals that DNA demethylation of myogenic genes may contribute to embryonic leg muscle development differences between Wuzong and Shitou geese.

Substance bioconversion, hydrolases activity, and metagenomic analysis to unravel the enhanced biomethanation of corn stover with urea-hydrothermal pretreatment.

Corn stover (CS) is a promising feedstock for producing biomethane, that can replace diminishing fossil fuels. However, the recalcitrant structure of CS resulted in low degradability in anaerobic digestion (AD). Numerous studies investigated the pretreatment of CS before AD, but the insight mechanism of biomethanation enhancement is not fully revealed. Therefore, this study advanced low-temperature urea-hydrothermal pretreatment of CS, and the biomethane production, substance bioconversion, hydrolase activity, and metagenomic analysis were conducted to unravel the intrinsic mechanisms of pretreatment for the enhanced biomethanation. The results showed that the pretreatment improved 11.5% of the specific surface area of CS, providing 111.5% higher total volatile fatty acids and 19.9% higher reducing sugars than the control, potentially enriching more anaerobic microorganisms. As a result, the pretreated CS achieved 19.1% higher biomethane yield, 9.1% higher volatile solid removal rate, and 3 days shorter digestion time. The mass balance and microbial community succession analysis indicated that the pretreatment reinforced the biomethane conversion from carbohydrate, which was attributed to the rapid enrichment of hydrolytic acidification bacteria (g__unclassified_o__Bacteroidales) (33.2%) and mixotrophic archaea (Methanosarcina) (72.3%). Meanwhile, the activity of cellulase and xylanase was enhanced up to 23.7% and 66.7%. Metagenomic analysis revealed that the combined pretreatment of CS promoted methanogenesis by enhancing various CAZymes secretion (such as oligosaccharide-degrading enzymes), and functional genes expression of hydrolytic, acidification and acetate-methane pathways at days 1-5. The study indicated that the combined pretreatment could influence microbial composition and function by changing the physicochemical properties of the CS, thereby improving methanogenic performance.

Combined modifications of CaO and liquid fraction of digestate for augmenting volatile fatty acids production from rice straw: Microbial and proteomics insights.

The modification sequence of chemical (CaO) and biological (liquid fraction of digestate, LFD) for augmenting volatile fatty acids (VFAs) production from rice straw was investigated in this study. The coupling order of the modifiers influenced acidification performance, and simultaneous modification (CaO-LFD) was superior to other modes. The highest VFAs production was obtained in CaO-LFD, 51% higher than that in the LFD-first additional modification. The CaO-LFD demonstrated the highest selectivity of acetate production, accounting for 79% of the total VFAs. In addition, CaO-LFD modification changed the direction of the domestication of fermentative bacteria and increased populations of the key anaerobes (Atopostipes sp.) responsible for acidification. The synergistic effect of CaO and LFD was revealed, namely, the effective function of CaO in degrading recalcitrant rice straw, the promotion of transport/metabolism of carbohydrates and acetogenesis by LFD.

A multi-tissue atlas of regulatory variants in cattle.

Characterization of genetic regulatory variants acting on livestock gene expression is essential for interpreting the molecular mechanisms underlying traits of economic value and for increasing the rate of genetic gain through artificial selection. Here we build a Cattle Genotype-Tissue Expression atlas (CattleGTEx) as part of the pilot phase of the Farm animal GTEx (FarmGTEx) project for the research community based on 7,180 publicly available RNA-sequencing (RNA-seq) samples. We describe the transcriptomic landscape of more than 100 tissues/cell types and report hundreds of thousands of genetic associations with gene expression and alternative splicing for 23 distinct tissues. We evaluate the tissue-sharing patterns of these genetic regulatory effects, and functionally annotate them using multiomics data. Finally, we link gene expression in different tissues to 43 economically important traits using both transcriptome-wide association and colocalization analyses to decipher the molecular regulatory mechanisms underpinning such agronomic traits in cattle.

Comparative transcriptome in large-scale human and cattle populations.

BACKGROUND: Cross-species comparison of transcriptomes is important for elucidating evolutionary molecular mechanisms underpinning phenotypic variation between and within species, yet to date it has been essentially limited to model organisms with relatively small sample sizes. RESULTS: Here, we systematically analyze and compare 10,830 and 4866 publicly available RNA-seq samples in humans and cattle, respectively, representing 20 common tissues. Focusing on 17,315 orthologous genes, we demonstrate that mean/median gene expression, inter-individual variation of expression, expression quantitative trait loci, and gene co-expression networks are generally conserved between humans and cattle. By examining large-scale genome-wide association studies for 46 human traits (average n = 327,973) and 45 cattle traits (average n = 24,635), we reveal that the heritability of complex traits in both species is significantly more enriched in transcriptionally conserved than diverged genes across tissues. CONCLUSIONS: In summary, our study provides a comprehensive comparison of transcriptomes between humans and cattle, which might help decipher the genetic and evolutionary basis of complex traits in both species.

Determining Optimal Temperature Combination for Effective Pretreatment and Anaerobic Digestion of Corn Stalk.

Temperature is one of the important factors affecting both chemical pretreatment and anaerobic digestion (AD) process of corn stalk (CS). In this work, the combined ways between pretreatment temperature (40 °C and 60 °C) and AD temperature (35 °C and 55 °C) were selected to investigate the AD performance for sodium hydroxide (NaOH) pretreated CS. Three organic loading rates (OLRs) of 1.6, 1.8 and 2.0 g·L-1·d-1 were studied within 255 days using continuously stirred tank reactors (CSTR). The results revealed that biogas yields of CS after pretreated were higher than that of untreated groups by 36.79-55.93% and 11.49-32.35%, respectively. When the temperature of NaOH pretreatment changed from 40 °C to 60 °C, there was no significant difference in enhancing the methane yields during the three OLRs. The mesophilic AD (MAD) of CS pretreated with 2% NaOH under 40 °C and 60 °C conditions produced 275 and 280 mL·gvs-1 methane yield at OLR of 1.6 g·L-1·d-1. However, as the OLR increased, the methane yield of CS under thermophilic AD (TAD) condition was further higher than under MAD condition. Furthermore, from the perspectives of energy balance and economic analysis, AD of 40 °C-treated CS recovered more energy and TAD is less expensive. Therefore, temperature of 40 °C was considered as an appropriate for pretreatment whether in mesophilic or thermophilic AD system. On the other hand, TAD was chosen as the optimal AD temperatures for higher OLRs.

Comparative transcriptomics in the hypothalamic-pituitary-gonad axis of mammals and poultry.

The hypothalamic-pituitary-gonad (HPG) axis is vital for reproductive activities in vertebrates. The large-scale comparative analyses of gene expression in the HPG axis across vertebrates have not been carried out yet. Here we collected 175 high-quality RNA-seq samples of hypothalamus, pituitary, ovary and testis from eight species (four mammals and four poultry) to compare transcriptome in the HPG axis, and to detect key pathways and related genes associated with reproduction. We demonstrated the distinguished difference in gene expression of the HPG axis between mammalian and avian species by a series of bioinformatics analysis, including gene differential expression, the phylogeny analysis of gene expression, and their functional annotations. We revealed two pathways, i.e., neuroactive ligand-receptor interaction and calcium signaling pathway, which play important roles in animal reproduction. In these two pathways, we detected 17 differentially expressed genes shared in 4 tissues, while 13, 27, and 27 were specifically differentially expressed genes in hypothalamus, pituitary and ovary, respectively. Our study on the comparative transcriptomics in the HPG axis across species will provide novel knowledge for exploring the molecular mechanism underlying reproductive traits in animals.

Effects of organic loading rate and pretreatments on digestion performance of corn stover and chicken manure in completely stirred tank reactor (CSTR).

The performance, system stability, and microbial community response in anaerobic co-digestion (AcoD) of corn stover (CS) and chicken manure (CM) were investigated by running completely stirred tank reactor (CSTR) under controlled organic loading rate (OLR). Prior to anaerobic digestion (AD), potassium hydroxide (KOH) or liquid fraction of digestate (LFD) was applied to pretreat CS, respectively. The results showed that the daily biogas production (DBP) in co-digestion showed a gradual increasing trend with an increase in the OLR from 65 g TS·L-1 to 100 g TS·L-1. The daily methane production per g volatile solids (DMP-VS) in co-digestion increased by 23.0%-27.1%, 18.7%-18.8%, and 17.5%-18.0% at the OLRs of 65, 80, and 100 g TS·L-1, respectively, upon pretreatment with KOH or LFD, as compared to that in co-digestion CSTR without any pretreatment. In addition, all co-digestion CSTRs were operated in stable state. Approximately half of the total carbon in the substrates was recovered in the form of a biogas product, with the carbon mass balance being impacted by the OLR as well as pretreatment. The diversity as well as function of the microbial community varied in response to different OLRs and pretreatment methods. The majority of bacterial genera were strongly correlated with operational parameters. The study indicates that management of OLR and selection of proper pretreatment method could enhance the efficiency and productivity of CS and CM co-digestion in CSTR.

Effect of wheat straw pretreated with liquid fraction of digestate from different substrates on anaerobic digestion performance and microbial community characteristics.

The effects of liquid fraction of digestate (LFD) pretreatment on anaerobic digestion (AD) performance and microbial community characteristics were estimated. Prior to AD, LFD (LFDSM, LFDFW, and LFDWS) collected separately from three continuously stirred tank reactors (CSTRs) using swine manure (SM), food waste (FW), and wheat straw (WS) as the mono-substrate was applied to pretreat WS. The results showed that AD with LFD pretreatment resulted in biomethane yields of 240.2-277.9 mL·gVS-1, a 33.57%-54.54% improvement over the yield of the control, and also produced a time saving of 32.26%-46.77%. The pretreatment parameters were optimized for LFD pretreatment. The enhancement effect was in the order of LFDFW > LFDSM > LFDWS. Simultaneously, the cellulose, hemicellulose and lignin contents in the WS and their characteristics (surface properties, crystallinity index, etc.) varied accordingly. The function of the microbial community was strengthened during the pretreatment stage, but the structure of the microbial community had a clear response to the LFD source substrates. Bacteroidetes was the most dominant phyla and was positively correlated with the hydrolysis rate. Consequently, the LFD from the different substrates used as pretreat agents could improve the AD performance of WS.

Current development and perspectives of anaerobic bioconversion of crop stalks to Biogas: A review.

As one of the most abundant biomass resources, crop stalks are great potential feedstock available for anaerobic digestion (AD) to produce biogas. However, the specific physical properties and complex chemical structures of crop stalks form strong barriers to efficient AD bioconversion. To overcome these problems, many efforts have been made over the past few years. This paper reviewed recent research in the evolving field of anaerobic bioconversion of crop stalks and was focused on three critical aspects affecting AD performance: various pretreatment methods and their effects on the improvement of crop stalk biodegradability, determination of specific AD operation parameters for crop stalks, and development of AD technologies. Finally, recommendations on the future development of crop stalk AD were proposed.

The theory on and software simulating large-scale genomic data for genotype-by-environment interactions.

BACKGROUND: With the emphasis on analysing genotype-by-environment interactions within the framework of genomic selection and genome-wide association analysis, there is an increasing demand for reliable tools that can be used to simulate large-scale genomic data in order to assess related approaches. RESULTS: We proposed a theory to simulate large-scale genomic data on genotype-by-environment interactions and added this new function to our developed tool GPOPSIM. Additionally, a simulated threshold trait with large-scale genomic data was also added. The validation of the simulated data indicated that GPOSPIM2.0 is an efficient tool for mimicking the phenotypic data of quantitative traits, threshold traits, and genetically correlated traits with large-scale genomic data while taking genotype-by-environment interactions into account. CONCLUSIONS: This tool is useful for assessing genotype-by-environment interactions and threshold traits methods.

CAF Proteins Help SOT1 Regulate the Stability of Chloroplast ndhA Transcripts.

Protein-mediated RNA stabilization plays profound roles in chloroplast gene expression. Genetic studies have indicated that chloroplast ndhA transcripts, encoding a key subunit of the NADH dehydrogenase-like complex that mediates photosystem I cyclic electron transport and facilitates chlororespiration, are stabilized by PPR53 and its orthologs, but the underlying mechanisms are unclear. Here, we report that CHLOROPLAST RNA SPLICING 2 (CRS2)-ASSOCIATED FACTOR (CAF) proteins activate SUPPRESSOR OF THYLAKOID FORMATION 1 (SOT1), an ortholog of PPR53 in Arabidopsis thaliana, enhancing their affinity for the 5' ends of ndhA transcripts to stabilize these molecules while inhibiting the RNA endonuclease activity of the SOT1 C-terminal SMR domain. In addition, we established that SOT1 improves the splicing efficiency of ndhA by facilitating the association of CAF2 with the ndhA intron, which may be due to the SOT1-mediated stability of the ndhA transcripts. Our findings shed light on the importance of PPR protein interaction partners in moderating RNA metabolism.

The effect of heat stress on proliferation, synthesis of steroids, and gene expression of duck granulosa cells.

Granulosa cells (GCs) play an important role in the development of follicles. In this study, we investigate the impact of heat stress at 41°C and 43°C on duck GCs' proliferation and steroids secretion. And, the transcriptomic responses to heat treatment were examined using RNA-sequencing analysis. Digital gene expression profiling was used to screen and identify differentially expressed genes (fold change ≥ 2 and Q value < 0.05). Further, the differential expression genes (DEGs) were classified into GO categories and KEGG pathways. The results show that duck GCs blocked in the G1 phase were increased on exposure to heat stress. Meanwhile, the expression of proliferative genes, which were essential for the transition from G1 to S phase, was inhibited. At the same time, heat stress inhibited the estradiol synthesis of GCs by decreasing CYP11A1 and CYP19A1 gene expression. A total of 241 DEGs including 181 upregulated and 60 downregulated ones were identified. Transcriptome result shows that heat shock protein and CXC chemokines gene were significantly activated during heat stress. While collagenases (MMP1 and MMP13) and strome lysins (MMP3) were downregulated. And, the hedgehog signaling pathway may be a prosurvival adaptive response under heat stress. These results offer a basis for better understanding the molecular mechanism underlying lay-eggs-less in ducks under heat stress.

Gonadotropin inhibitory hormone downregulates steroid hormone secretion and genes expressions in duck granulosa cells.

The mechanisms by which GnIH regulates the steroid synthesis pathway in duck granulosa cells remain poorly understood. In this study, we measured steroid hormone secretion by ELISA and reproduction-associated gene expression by quantitative real-time Polymerase Chain Reaction (qPCR) in duck granulosa cells treated with different concentrations of GnIH (0, 0.1, 1, 10, and 100 ng/mL) for 24 h. The genome-wide expression profiles of GnIH-treated cells (0 and 10 ng/mL) were evaluated by high-throughput RNA sequencing. Compared with untreated cells, the secretion of the steroid hormones E2, E1, P4, and T was downregulated, with that of E1 and P4 reaching statistical significance (P<0.05); in contrast, the secretion of ACV and INH was significantly upregulated (P<0.05) after treatment with 10 and 100 ng/mL GnIH. The expression of encoding steroidogenic proteins and enzymes genes (STAR, CYP11A1, CYP17A1, CYP19A1, and 3-ß-HSD) and encoding gonadotropin receptors genes (FSHR, LHR) were significantly declined (P<0.05) in the 10 and 100 ng/mL GnIH treatments. Transcriptome sequencing identified 348 differentially expressed genes (DEGs), including 253 upregulated and 95 downregulated genes. The DEGs were mainly involved in cell growth and death, immune response, and steroid biosynthesis pathways. We identified four novel DEGs (MROH5, LOC113840576, SDR42E1, and LOC113841457) with key roles in the regulation of steroid hormone biosynthesis. Our study revealed changes in gonadal steroid hormone secretion and steroid biosynthesis pathway-related gene expression in duck granulosa cells under the inhibitory effect of GnIH. These data contribute to our understanding of the molecular and genetic mechanisms underlying reproduction in ducks.

Biomethane Yield, Physicochemical Structures, and Microbial Community Characteristics of Corn Stover Pretreated by Urea Combined with Mild Temperature Hydrotherm.

The corn stover (CS)'s compact structure makes it challenging for microorganisms to use in anaerobic digestion (AD). Therefore, improving CS biodegradability has become a key focus in AD studies. Methods are being targeted at the pretreatment of CS, combining advanced urea with mild temperature hydrotherm pretreatment to study its effect on promoting the AD process of CS. The biomethane yield, physicochemical structure, and microbial community characteristics were investigated. CS samples were assigned into groups differed by a range of pretreatment times (from 24 to 96 h) and set at a temperature of 50 °C with a 2% urea addition. Results revealed that the 72-h group obtained the highest biomethane yield of 205 mL/g VS-1, volatile solid (VS) and total solid (TS) removal rates of 69.3% and 47.7%, which were 36.7%, 25.3% and 27.5% higher than those of untreated one, respectively. After conducting several analyses, results confirmed the pretreatment as a method for altering CS microstructures benefits biomethane production. The most resounding differences between pretreated and untreated groups were observed within a microbial community, an integral factor for improved AD performance. This study serves to confirm that this specific pretreatment is an effective method for enhancing biomethane production in CS.