The emerging role of oxidative stress in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic immune-mediated condition that affects the digestive system and includes Crohn's disease (CD) and ulcerative colitis (UC). Although the exact etiology of IBD remains uncertain, dysfunctional immunoregulation of the gut is believed to be the main culprit. Amongst the immunoregulatory factors, reactive oxygen species (ROS) and reactive nitrogen species (RNS), components of the oxidative stress event, are produced at abnormally high levels in IBD. Their destructive effects may contribute to the disease's initiation and propagation, as they damage the gut lining and activate inflammatory signaling pathways, further exacerbating the inflammation. Oxidative stress markers, such as malondialdehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OHdG), and serum-free thiols (R-SH), can be measured in the blood and stool of patients with IBD. These markers are elevated in patients with IBD, and their levels correlate with the severity of the disease. Thus, oxidative stress markers can be used not only in IBD diagnosis but also in monitoring the response to treatment. It can also be targeted in IBD treatment through the use of antioxidants, including vitamin C, vitamin E, glutathione, and N-acetylcysteine. In this review, we summarize the role of oxidative stress in the pathophysiology of IBD, its diagnostic targets, and the potential application of antioxidant therapies to manage and treat IBD.

HucMSC-Ex alleviates DSS-induced colitis in mice by decreasing mast cell activation via the IL-33/ST2 axis.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disease that poses challenges in terms of treatment. The precise mechanism underlying the role of human umbilical cord mesenchymal stem cell-derived exosome (HucMSC-Ex) in the inflammatory repair process of IBD remains elusive. Mucosal mast cells accumulate within the intestinal tract and exert regulatory functions in IBD, thus presenting a novel target for addressing this intestinal disease. METHODS: A mouse model of Dextran Sulfate Sodium (DSS)-induced colitis was established and hucMSC-Ex were administered to investigate their impact on the regulation of intestinal mast cells. An in vitro co-culture model using the human clonal colorectal adenocarcinoma cell line (Caco-2) and human mast cell line (LAD2) was also established for further exploration of the effect of hucMSC-Ex. RESULTS: We observed the accumulation of mast cells in the intestines of patients with IBD as well as mice. In colitis mice, there was an upregulation of mast cell-related tryptase, interleukin-33 (IL-33), and suppression of tumorigenicity 2 receptor (ST2 or IL1RL1), and the function of the intestinal mucosal barrier related to intestinal tight junction protein was weakened. HucMSC-Ex treatment significantly reduced mast cell infiltration and intestinal damage. In the co-culture model, a substantial number of mast cells interact with the epithelial barrier, triggering activation of the IL-33/IL1RL1 (ST2) pathway and subsequent release of inflammatory factors and trypsin. This disruption leads to aberrant expression of tight junction proteins, which can be alleviated by supplementation with hucMSC-Ex. CONCLUSION: Our results suggest that hucMSC-Ex may reduce the release of mast cell mediators via the IL-33/IL1RL1 (ST2) axis, thereby mitigating its detrimental effects on intestinal barrier function.

Review of METTL3 in colorectal cancer: From mechanisms to the therapeutic potential.

N6-methyladenosine (m6A), the most abundant modification in mRNAs, affects the fate of the modified RNAs at the post-transcriptional level and participants in various biological and pathological processes. Increasing evidence shows that m6A modification plays a role in the progression of many malignancies, including colorectal cancer (CRC). As the only catalytic subunit in methyltransferase complex, methyltransferase-like 3 (METTL3) is essential to the performance of m6A modification. It has been found that METTL3 is associated with the prognosis of CRC and significantly influences various aspects of CRC, such as cell proliferation, invasion, migration, metastasis, metabolism, tumor microcirculation, tumor microenvironment, and drug resistance. The relationship between METTL3 and gut-microbiota is also involved into the progression of CRC. Furthermore, METTL3 might be a viable target for CRC treatment to prolong survival. In this review, we comprehensively summarize the function of METTL3 in CRC and the underlying molecular mechanisms. We aim to deepen understanding and offer new ideas for diagnostic biomarkers and therapeutic targets for colorectal cancer.

Insight into the regulatory mechanism of m6A modification: From MAFLD to hepatocellular carcinoma.

In recent years, there has been a significant increase in the incidence of metabolic-associated fatty liver disease (MAFLD), which has been attributed to the increasing prevalence of type 2 diabetes mellitus (T2DM) and obesity. MAFLD affects more than one-third of adults worldwide, making it the most prevalent liver disease globally. Moreover, MAFLD is considered a significant risk factor for hepatocellular carcinoma (HCC), with MAFLD-related HCC cases increasing. Approximately 1 in 6 HCC patients are believed to have MAFLD, and nearly 40 % of these HCC patients do not progress to cirrhosis, indicating direct transformation from MAFLD to HCC. N6-methyladenosine (m6A) is commonly distributed in eukaryotic mRNA and plays a crucial role in normal development and disease progression, particularly in tumors. Numerous studies have highlighted the close association between abnormal m6A modification and cellular metabolic alterations, underscoring its importance in the onset and progression of MAFLD. However, the specific impact of m6A modification on the progression of MAFLD to HCC remains unclear. Can targeting m6A effectively halt the progression of MAFLD-related HCC? In this review, we investigated the pivotal role of abnormal m6A modification in the transition from MAFLD to HCC, explored the potential of m6A modification as a therapeutic target for MAFLD-related HCC, and proposed possible directions for future investigations.

Exploring the impact of computer game playing on cognitive function, Alzheimer's disease risk, and brain-derived neurotrophic factor levels: Basic evidence from Mendelian randomization.

Introduction: The potential positive impact of computer game playing on cognitive function and its potential role in reducing the risk of Alzheimer's disease (AD) has been suggested. However, current observational studies have certain limitations. We utilized Mendelian randomization (MR) alongside extensive genome-wide association study (GWAS) data to examine the relationship between computer game playing, cognitive function, risk of AD, and levels of brain-derived neurotrophic factor (BDNF). Methods: We collected datasets on computer game playing, cognition function, risk of AD, and BDNF level from the IEU Open GWAS project. Causal effects were assessed using various MR methods, including inverse variance weighted (IVW), weighted median, MR-Egger, simple mode, and weighted mode. To ensure the accuracy of the results, sensitivity analyses were conducted. Results: Our analysis revealed a significant association between computer game playing and cognitive function (ß = 0.801, 95% CI: 0.351, 1.328, P = 0.001). There was no statistically significant association between computer game playing and either BDNF level or risk of AD (ß = -0.112, 95%CI: -1.315, 1.091, P = 0.855; OR = 1.000, 95% CI: 1.004, 0.997, P = 0.891, respectively). We further confirmed the reliability of our evidence through the MR-Egger intercept test, MR-PRESSO global test, Cochran's Q test, and funnel plots. Conclusion: The results of our study indicate that engaging in computer game playing may confer a safeguarding influence on cognitive function. This underscores the potential advantages associated with computer gaming. Nevertheless, given the constraints inherent in our research, further investigation is warranted to substantiate our findings and delve into the underlying mechanisms.

ALKBH5 regulates arginase 1 expression in MDSCs and their immunosuppressive activity in tumor-bearing host.

Myeloid-derived suppressor cells (MDSCs) are closely related to the occurrence and development of many cancers, but the specific mechanism is not fully understood. It has been found that N6-methyladenosine (m6A) plays a key role in RNA metabolism, but its function in MDSCs has yet to be revealed. In this study, we found that MDSCs in mice with colorectal cancer (CRC) have significantly elevated levels of m6A, while ALKBH5 expression is decreased. Overexpression of ALKBH5 can reduce the immunosuppressive function of MDSCs in vivo and in vitro, and attenuates the protumorigenic ability of MDSCs. Mechanism study found that the overexpression of ALKBH5 in MDSCs reduced the m6A modification level of Arg-1 mRNA, and then weakened the stability of Arg-1 mRNA and protein expression. These data suggest that the decreased expression of ALKBH5 in CRC tumor mice may promote the expression of Arg-1, enhance the immunosuppressor function of MDSCs, and promote tumor growth. These findings highlight that ALKBH5 may regulate the function of MDSCs in tumor-bearing mice and may be a new target for immunotherapy. This research provides a new perspective for our understanding of the role of MDSCs in cancer development, and also brings new hope for cancer treatment.

Extracellular vesicle-encapsulated miR-10a-5p derived from MDSCs restrains germinal center B cells in experimental Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a progressive systemic autoimmune disease characterized by chronic inflammation of the exocrine glands, resulting in damage to the salivary and lacrimal glands. Our group and other researchers have reported that myeloid-derived suppressor cell-derived extracellular vesicles (MDSC-EVs) could attenuate the progression of autoimmune disease by impairing T-cell function. However, the effect of MDSC-EVs on B-cell function and the underlying mechanism remains largely unknown. In this study, we found that MDSC-EVs significantly attenuated the progression of experimental Sjögren's syndrome (ESS). Moreover, treatment with MDSC-EVs via intravenous injection markedly reduced the percentage of germinal center (GC) B cells in ESS mice. In vitro, MDSC-EVs could directly suppress the generation of GC B cells and the expression of B cell lymphoma 6 (Bcl-6) in B cells under GC B-cell-polarizing conditions. Mechanistically, miR-10a-5p carried by MDSC-EVs regulated the differentiation of GC B cells by targeting Bcl-6, and inhibition of miR-10a-5p in MDSC-EVs significantly reversed the effect of MDSC-EVs involved in alleviating the development of ESS. Taken together, our findings demonstrated that miR-10a-5p carried by MDSC-EVs inhibited the generation of B cells by targeting Bcl-6 and eventually alleviated the progression of ESS, which may provide novel therapeutic targets for the treatment of pSS.

Single-cell RNA sequencing and traditional RNA sequencing reveals the role of cancer-associated fibroblasts in oral squamous cell carcinoma cohort.

Chronic inflammation of the alveolar bones and connective tissues supporting teeth causes periodontal disease, one of the most prevalent infectious diseases in humans. It was previously reported that oral cancer was the sixth most common cancer in the world, followed by squamous cell carcinoma. Periodontal disease has been linked to an increased risk for oral cancer in some studies, and these studies have found a positive relationship between oral cancer and periodontal disease. In this work, we aimed to explore the potential correlation between oral squamous cell carcinoma (OSCC) and Periodontal disease. The single-cell RNA sequence analysis was applied to explore the genes that were closely associated with cancer-associated fibroblasts (CAFs). the head and neck squamous cell carcinoma. The Single sample Gene Set Enrichment Analysis (ssGSEA) algorithm was applied to explore the scores of CAFs. Subsequently, the differentially expressed analysis was applied to explore the CAFs-related genes that play a key role in the OSCC cohort. The LASSO regression analysis and the COX regression analysis were applied to construct the CAFs-based periodontal disease-related risk model. In addition, the correlation analysis was used to explore the correlation between the risk model and clinical features, immune-related cells, and immune-related genes. By using the single-cell RNA sequence analysis, we successfully obtained the biomarkers for the CAFs. Finally, we successfully obtained a six-CAFs-related genes risk model. The ROC curve and survival analysis revealed that the risk model showed good predictive value in OSCC patients. Our analysis successfully provided a new direction for the treatment and prognosis of OSCC patients.

Netrin-1 Promotes the Immunosuppressive Activity of MDSCs in Colorectal Cancer.

Myeloid-derived suppressive cells (MDSC) inhibit antitumor immunity and confer a survival advantage for tumor evasion. Tumor cells also support MDSC expansion and recruitment by secreting multiple growth factors and cytokines, but the mechanisms by which tumors affect MDSC function are not completely understood. Here, we found that the neuronal guidance protein netrin-1 was selectively secreted by MC38 murine colon cancer cells, which could enhance the immunosuppressive activity of MDSCs. MDSCs predominantly expressed one type of netrin-1 receptor, adenosine receptor 2B (A2BR). Netrin-1 interacted with A2BR on MDSCs to activate the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway, which ultimately increased CREB phosphorylation in MDSCs. Furthermore, netrin-1 knockdown in tumor cells inhibited the immunosuppressive activity of MDSCs and restored antitumor immunity in MC38 tumor xenograft mice. Intriguingly, high netrin-1 in the plasma correlated with MDSCs in patients with colorectal cancer. In conclusion, netrin-1 significantly enhanced the immunosuppressive function of MDSCs through A2BR on MDSCs, thus promoting the development of tumors. These findings highlight that netrin-1 may regulate the abnormal immune response in colorectal cancer and may become a potential target for immunotherapy.

Pan-cancer analysis reveals distinct clinical, genomic, and immunological features of the LILRB immune checkpoint family in acute myeloid leukemia.

Leukocyte immunoglobulin (Ig)-like receptor Bs (LILRBs), a family of type I transmembrane glycoproteins, are known to inhibit immune activation. Here, we comprehensively evaluated the molecular, prognostic, and immunological characteristics of LILRB members in a broad spectrum of cancer types, focusing on their roles in acute myeloid leukemia (AML). We showed that LILRBs were significantly dysregulated in a number of cancers and were associated with immune-inhibitory phenotypes. Clinically, high expression of LILRB1-LILRB4 predicted poor survival in six independent AML cohorts. Genetically, LILRB1 was associated with more mutational events than other LILRB members, and multiple genes involved in immune activation were deleted in LILRB1 high patients. Epigenetically, LILRB4 was significantly hypomethylated and marked by MLL-associated histone modifications in AML. Immunologically, LILRBs were positively associated with monocytic cells, including M2 macrophages, but were negatively associated with tumor-suppressive CD8 T cells. Importantly, patients with higher LILRB expression generally showed a better response to immune checkpoint blockade (ICB) in five independent immunotherapy cohorts. Our findings reveal critical immunological and clinical implications of LILRBs in AML and indicate that LILRBs may represent promising targets for immunotherapy of AML.

Clinical and prognostic relevance of CXCL12 expression in acute myeloid leukemia.

BACKGROUND: Accumulating studies have been made to understand the association between CXC chemokine ligand-12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) and acute myeloid leukemia (AML). However, large-scale data analysis of potential relationship between CXCL12 and AML remains insufficient. METHODS: We collected abundant CXCL12 expression data and AML samples from several publicly available datasets. The CIBERSORT algorithm was used to quantify immune cell fractions and the online website of STRING was utilized for gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The statistical analysis and graphical work were mainly performed via the R software. RESULTS: CXCL12 expression was extremely down-regulated in AML. Clinically, low CXCL12 expression was correlated with higher white blood cells (WBCs) (P < 0.0001), more blasts in bone marrow (BM) (P < 0.001) and peripheral blood (PB) (P < 0.0001), FLT3-internal tandem duplications (FLT3-ITD) (P = 0.010) and NPM1 mutations (P = 0.015). More importantly, reduced CXCL12 expression predicted worse overall survival (OS) and event-free survival (EFS) in all AML, non-M3-AML, and cytogenetically normal (CN)-AML patients in three independent cohorts. As for immune cell infiltration, high CXCL12 expressed groups tended to harbor more memory B cells and plasma cells infiltration while low CXCL12 expressed groups exhibited more eosinophils infiltration. GO enrichment and KEGG pathways analysis revealed the potential biological progress the gene participating in. CONCLUSIONS: CXCL12 is significantly down-regulated in AML and low CXCL12 expression is an independent and poor predictor of AML prognosis. CXCL12 expression level correlates with clinical and immune characteristics of AML, which could provide potential assistance for treatment. Prospective studies are needed to further validate the impact of CXCL12 expression before routine clinical application in AML.

Case-Control Study on TNFRSF6B Gene Polymorphism and Susceptibility to Gastric Cancer in a Chinese Han Population.

PURPOSE: To explore the relationship between rs2297440 and rs2297441 polymorphisms of TNFRSF6B gene and susceptibility to gastric cancer. METHODS: A hospital-based case-control study was conducted. A total of 577 gastric cancer cases and 678 normal controls were recruited. Their genotypes were determined using the SnapShot method. RESULTS: The smoking rate in the case group (34.49%) was higher than that in the control group (27.29%). For TNFRSF6B rs2297440, among people <62 years old, the risk of gastric cancer in TC people was 1.84 times that in TT people. Among the non-drinking people, the risk of gastric cancer in the CC type was 0.66 times that in the TT+TC type. Among the drinking population, the risk of gastric cancer in the TC type was 1.67 times that in the TT type, and the risk in the TC+CC type was 1.70 times that in the TT type. As for TNFRSF6B rs2297441, in males and non-drinkers, the risk of gastric cancer in the AG type was less than that in the GG type. No matter how old the patient is, the risk of gastric cancer in the AA type was less than that in the AG+GG type. CONCLUSION: A correlation exists between smoking and gastric cancer. For TNFRSF6B rs2297440, the TC genotype may be a risk factor for gastric cancer in people <62 years old. In the non-drinking population, the homozygous mutant of CC may be a protective factor for gastric cancer. In the drinking population, TC type may be a risk factor, whereas the TC+CC type dominated by C may be a protective factor. For TNFRSF6B rs2297441, the AG genotype may be a risk factor for gastric cancer in males and non-drinkers. The AA homozygous mutant may be a protective factor for gastric cancer.

The M2 macrophage marker CD206: a novel prognostic indicator for acute myeloid leukemia.

Hematological malignancies possess a distinctive immunologic microenvironment compared with solid tumors. Here, using an established computational algorithm (CIBERSORT), we systematically analyzed the overall distribution of 22 tumor-infiltrating leukocyte (TIL) populations in more than 2000 bone marrow (BM) samples from 5 major hematological malignancies and healthy controls. Focusing on significantly altered TILs in acute myeloid leukemia (AML), we found that patients with AML exhibited increased frequencies of M2 macrophages, compared to either healthy controls or the other four malignancies. High infiltration of M2 macrophages was associated with poor outcome in AML. Further analysis revealed that CD206, a M2 marker gene, could faithfully reflect variation in M2 fractions and was more highly expressed in AML than normal controls. High CD206 expression predicted inferior overall survival (OS) and event-free survival (EFS) in two independent AML cohorts. Among 175 patients with intermediate-risk cytogenetics, the survival still differed greatly between low and high CD206 expressers (OS; P < .0001; 3-year rates, 56% v 32%; EFS; P < .001; 3-year rates, 47% v 25%). When analyzed in a meta-analysis, CD206 as a continuous variable showed superior predictive performance than classical prognosticators in AML (BAALC, ERG, EVI1, MN1, and WT1). In summary, M2 macrophages are preferentially enriched in AML. The M2 marker CD206 may serve as a new prognostic marker in AML.

[Construction, expression and immunogenicity of EV71 multiepitope-mGITRL eukaryotic plasmid].

AIM: To construct an enterovirus 71(EV71) multiepitope-mGITRL eukaryotic plasmid and study its immunogenicity in BALB/c mice. METHODS: We first designed and synthesized VP1' epigene containing two B cells and two T cells epitopes of VP1, and amplified mGITRL gene by PCR. The VP1' epigene and mGITRL gene were then cloned into the expression vector pIRES to construct the recombination plasmid pIRES-VP1'-mGITRL. The recombination plasmid was transfected into COS7 cells by liposome-mediated method. The protein expressions of VP1' and mGITRL were detected by Western blotting. BALB/c mice were immunized with pIRES-VP1'-mGITRL plasmid, and its serum antibody titer was measured by ELISA. RESULTS: The recombination plasmid pIRES-VP1'-mGITRL was successfully constructed as demonstrated by sequencing. Western blot analysis indicated that the VP1'-mGITRL fusion protein was expressed in COS7 cells and muscle cells. After BALB/c mice were immunized with this plasmid, we detected the high titer of anti-VP1 antibody in serum. CONCLUSION: VP1'-mGITRL fusion protein can be highly expressed in COS7 cells and muscle cells by the construction and transfection of the recombination plasmid pIRES-VP1'-mGITRL, and it could elicit the dramatic immune response in mice.

Responsive polyelectrolyte multilayers assembled at high ionic strength with an unusual collapse at low ionic strength.

Responsive polyelectrolyte multilayers (PEMs) of poly(diallyl dimethyl ammonium chloride) (PDADMAC) and poly(styrene sodium sulfonate) (PSS) with thicknesses between 350 and 400 nm for 11 deposited polyelectrolyte layers were fabricated assembling the polyelectrolytes at 3 M NaCl. When the 3 M NaCl bulk solution is replaced by water, the PEMs release water, approximately a 46% of the total mass, and experience a thickness reduction of more than 200 nm. Changes in thickness and water content are fully reversible. The film recovers its original thickness and water content when it is exposed again to a 3 M NaCl solution. A responsive polymer film is achieved with the capability of swelling at high ionic strength and collapsing in water with variations in thickness of hundred of nanometers.

[Identification of different corrosion covering the surface of Chinese ancient lead glazed potteries].

In the present paper, different corrosions of three pieces of Chinese ancient lead glazed potteries from different burial circumstances were analyzed by using XRD, FTIR, EDXRF, RS etc, and the analyses indicate that the main crystalline phase of corrosion of HYT is cerussite (PbCO3), that of SYT is calcium-lead hydroxyapatite [Pb(10-x) Ca(x) (PO4)(OH)2 (x < 2.7)] and the yellow area of the erosion is attributed to a certain mount of iron oxide, that of TSC is the cerussite (PbCO3) and calcium-lead hydroxyapatite [Pb(10-x)Ca(x) (PO4) (OH)2 (x < 2.7)], and the brown-black area of the erosion product is the result of the existence of little mount of iron and carbon black.

[Preliminary study on the function of potteries of Liangzhu Culture].

Liangzhu Culture is an important late neolithic culture of China, but there are different opinions on the function of some potteries of Liangzhu Culture, for example vat and Ding. In the present paper, for discussing the function of these potteries, the content of Cl, Ca and P of some shard samples of vat and Ding from Liangzhu site were analyzed by using EDXRF line scanning technology. The results show that the content of element Cl in the outside and inside parts of the bottom of vat sample is not different from that of other contemporaneous archaic potteries, and that the distribution of element Cl in the outside part of the bottom of vat sample is the same as that in the inside part of the bottom, so it is concluded that the function of vat from Liangzhu Culture is not related to the production or storage of salt, but with regard to the true function of this pottery more works remain to be done. The analytical results also show that the content of two elements, Ca and P, in the bottom of the other pottery, Ding, is obviously higher than that in vat and other contemporaneous archaic potteries, and that the content of two elements, Ca and P, in the outside part of the bottom of Ding sample is higher than that in the inside part of the bottom. So it is concluded that Ding from Liangzhu Culture is a kind of cooking ware and its main function is meal cooking.

[A primary Raman microscopic study of the turquoise and its role in provenance-tracking].

The authors analyzed four modern turquoises from Hubei province and Anhui province by using the Raman microscopic with the samples are gathered on the spot. According to the study the authors discovered that the Raman spectra of the Hubei turquoises with different color but with the same backgrounds of mineral resource and the formation cause of mineral resource and in the same formation line of turquoise mineral resource have little difference. On the contrary, there is a strong difference in the 900-100 cm(-1) region of the Raman spectra between the turquoises from Hubei province and the turquoise from Anhui province which has remarkable different backgrounds of mineral resource and the formation cause of mineral resource. At the same time the authors studied two ancient turquoises to discuss the feasibility of using the Raman spectra of turquoises, the provenance of which is known, as the fingerprint directions to track the provenance of ancient turquoises.

[Spectral analysis of ceramic-painting pigments from Taosi site].

Based on the analysis of Raman, IR spectroscopy and XRD methods, the structure of the different pigments and bond in red pigment in the ceramic from Taosi site in Xiangfeng county, Shanxi province was analyzed. It is very prominent that both red and white pigments have been well preserved. The red pigment was identified as HgS, while white pigment is CaCO3, and the bond in red pigment is CaCO3, which was made from white lime, and the reasons for its formation is because of carbon dioxide in air, which was absorbed by white lime over long history. Moreover, it was indicated that the Raman and IR spectra are more effective for identifying the ancient pigments in very few quantities than XRD. Furthermore, the fact that quartz was unfound in vermilion, suggested that the technique for synthetic vermilion might have been known in 4 000 years ago in Taosi site.

[Study on colored drawing technique of Qionglai kiln by SRXRF].

The colored drawing technique of Qionglai kiln is a debate problem in the archaeology and ancient ceramic research. In the present paper, SRXRF linescan technology was used to study the distribution mode of the colorific elements on the cross-sections of samples. The analytical result indicates that there existed two kinds of decoration techniques of colored drawing, i. e. the techniques of the underglaze color and oveglaze color coexisted in that time.