A variety of proteins including epigenetic factors are involved in the differentiation of human bone marrow mesenchymal stem cells. These cells also exhibited an epigenetic plasticity that enabled them to trans-differentiate from adipocytes to osteoblasts (and vice versa) after commitment. Further in-depth study of their epigenetic alterations may make sense.Chromatin Immunoprecipitation-PCR (ChIP-PCR) was used to detect the methylation enrichment status of H3K9me2 in the Runx2 promoter, alizarin red and alkaline phosphatase (ALP) staining were used to detect osteogenic differentiation and mineralization ability, western blot and quantitative RT-PCR were used to measure the differential expression of osteogenesis-related proteins and genes. Recombinant Lentivirus mediated gain-of-function and loss-of-function study. The scale of epigenetic modification was detected by laser confocal.Our results showed that compared with human bone marrow mesenchymal stem cells (hBMSCs) without osteogenic differentiation treatment, hBMSCs after osteogenic differentiation significantly promoted osteogenic differentiation and mRNA expression such as JMJD2B/KDM4B, osteogenesis-related genes like Runx2 and FAM210A in hBMSCs cells, suggesting that upregulation of JMJD2B/KDM4B is involved in the promoting effect of osteogenesis. After overexpression and silencing expression of JMJD2B, we found a completely opposite and significant difference in mRNA expression of osteogenesis-related genes and staining in hBMSCs. Overexpression of JMJD2B/KDM4B significantly promoted osteogenic differentiation, suggesting that JMJD2B/KDM4B could promote osteogenesis. In addition, ChIP-PCR showed that overexpression of JMJD2B/KDM4B significantly reversed the methylation enrichment status of H3K9me2 in Runx2 promoter. Furthermore, overexpression of JMJD2B/KDM4B significantly reverses the inhibitory effect of BIX01294 on H3K9me2, suggesting that JMJD2B/KDM4B regulates the osteogenic differentiation of hBMSCs by changing the methylation status of H3K9me2 at the Runx2 promoter.Taken together, these results suggest that JMJD2B/ KDM4B may induce the osteogenic differentiation of hBMSCs by regulating the methylation level of H3K9me2 at the Runx2 promoter. Polydatin The research on the regulation of human bone marrow mesenchymal stem cells(hBMSCs) towards bone formation has been going on for decades. These cells exist in the bone marrow cavity and can differentiate into a variety of cell phenotypes, including osteoblasts, chondroblasts, stromal cells that support hematopoiesis, and adipocytes. The mainstream control methods found in current research can be roughly divided into the following categories: (1) Cell-to-cell regulation. For example, the co-culture of hAMSCs/hBMSCs mixed at the optimal ratio of 3/1 showed significantly higher cell proliferation, antioxidant properties, osteogenic and angiogenic differentiation than hBMSCs or HUVECs alone . (2) Horβ-catenin and prevent ovariectomy-induced bone loss. Some scholars have also discovered that the epigenetic landscape of 3D cell models of human primary articular chondrocytes (hPACs) and human bone-marrow derived mesenchymal stem cells (hBMSCs) exhibits a huge difference in DNA methylation landscape  supplemented with 10% fetal bovine serum (FBS), 50 µM ascorbate, 1% penicillin-streptomycin, and 10 mM β-glycerophosphate in a humidified atmosphere with 5% CO2 at 37 °C. Cells at the third or fourth passage were used in the experiments. For osteogenesis treatment, cells were cultured in human bone marrow mesenchymal stem cells osteogenic medium .Human bone marrow-derived mesenchymal stem cell line was obtained from Saliai Biotechnology . Cells were maintained in Human JMJD2B was amplified using primers 5′- gcatgcATGGGGTCTGAGGACCACG -3′ (sense) and 5′- atgcatTCATCTGCAAGGGTCTTGAGTTG -3′ (antisense) and cloned into LV5-GFP/Puro vector . The clones were confirmed by DNA sequencing. Add the shuttle plasmid and packaging plasmid  containing the target sequence to 293T cells in proportion. The hJMJD2B shRNA was designed according to the shRNA design rules and other literature . Bbs I; 5 cells/ml, inoculate into six-well plates, add 2ml of cell suspension to each well, and store at 37 °C, 5% CO2 conditions Incubate overnight. Incubate overnight until the cell density is about 40% to 60%, and carry out virus infection by 1/2 small volume infection method. Aspirate the original medium in the culture well, add 1/2 volume (1 ml) of α-MEM low-glucose medium, and add polybrene to a final concentration of 5 µg/ml, add the corresponding volume of virus stock solution at MOI = 50, shake well, and put Incubate for 4 h at 37 °C, 5% CO2. After 4 h of incubation, add 1/2 volume (1 ml) of α-MEM low-glucose medium to the culture wells, and incubate overnight at 37 °C under 5% CO2 conditions. On the second day after infection, the virus-containing medium was aspirated, replaced with fresh α-MEM low-glucose medium (without polybrene), and the culture was continued at 37 °C, 5% CO2. After hBMSCs were infected with virus for 72 h, the cells were digested and collected, and the cellular protein and RNA were extracted. The expression of JMJD2B in hBMSCs cells was detected by WB and qPCR. The method is the same as before.Overexpression and silencing recombinant lentiviruses were purchased from Shanghai Hanheng Biotechnology. Collect hBMSCs cells in logarithmic growth phase, count, resuspend cells in complete medium, adjust the cell concentration to 1 ×10Data were collected as previously described in Data were collected as previously described in . Specifically, cells were cultured in osteogenic medium for 1, 3, 7 or 14 days and then fixed in 95% ice-cold ethanol for 15 min. After three washes with ice-cold PBS, cells were stained with alizarin red (Sigma-Aldrich) for 5 min. Alizarin red staining was observed using an Olympus 450-fluorescent inverted phase-contrast microscope .−ΔΔCt method. The PCR reactions were performed in triplicates.Total RNA was isolated from hBMSCs cells using Trizol reagent . All quantitative real-time PCR assays were carried out using three technical replicates and three independent cDNA syntheses. Reverse transcription reaction using cDNA synthesis kit , qRT-PCR was performed using a fluorescence quantitative PCR kit  and gene-specific primers . GAPDH wβ-actin  Incubated overnight at 4 °C. The membrane was washed three times with TBST and incubated with HRP-linked secondary antibody  for 1 h at room temperature.Data were collected as previously described in . Specifically, proteins were isolated from hBMSCs cells using RIPA buffer. Protein samples were separated using 5% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with primary antibody against target protein , and β-aTreatment of cells with ultrasound cross-linking. ChIP was performed using a ChIP Assay Kit  following the manufacturer’s instruction. Briefly, cell lysates were centrifuged at 12,000 rpm for 5 min at 4 °C. Take out 20 µl sample as Input for subsequent testing. Add 70 µl protein A+G Agarose/Salmon Sperm DNA to the remaining nearly 2 ml sample (about 35 µl is precipitation and 35 µl is liquid), and mix slowly or shake at 4 °C for 30 min. The supernatant was collected, and incubated with 0.5–1 µg anti-H3K9me2  and protein A+G Agarose/Salmon Sperm DNA (of which about 30 µl is precipitation and 30 µl is liquid) overnight at 4 °C. The immunoprecipitates were collected and washed three times with wash buffer. Next, the DNA fragments are purified for subsequent PCR experiments. Wash the sample three times with Elution buffer. Add 20 µl of 5M NaCl to 500 µl of supernatant, mix well, and heat at 65 °C for 4 h to remove crosslinks between protein and genomic DNA. For the 20 µl sample obtained as Input, add 1 µl 5M NaCl, mix well, and heat at 65 °C for 4 h. Then add an appropriate amount of EDTA, proteinase K, Tris, chloroform, glycogen, NaAc, and absolute ethanol for further purification, and the obtained DNA precipitate is used for PCR detection of the target gene.5 cells/well. Cells were washed three times with PBS. After treatment, Cells were fixed in 4% paraformaldehyde solution for 15 min at room temperature,followed by fixation with pre-cooled acetone for 15 min. Later, they were washed twice in PBS followed by a 30-minute incubation in 2% bovine serum albuminsolution . Cells were then treated with 0.5% PBS-Triton X-100 for 20 min and incubated with 3% H2O2 for 15 min. The samples were then blocked with 5% serum for 20 min and incubated with JDJM2B  and FAM210A overnight at 4 °C. Then add 200 µL of APC-labeled secondary antibody (1:500) to the sample, and incubate for 1 h at 37 °C, 5% CO2, and saturated humidity. The nucleus was stained with DAPI  for 1 min. Finally, the samples were washed to remove excess solution and mounted on microscope slides for imaging Images were collected using VistarImage 3.0 software  software connected to a Olympus CKX53 inverted microscope. Images were acquired using an UPlanFLN objective at magnification 40 ×.hBMSCS cells were seeded in a 6-well plate at a density of 1 ×10P value less than 0.05 was considered statistically significant.Data are expressed as the mean ±standard deviation. Statistical analysis was performed using SPSS 23.0 software . Comparisons among different groups were performed using one-way analysis of variance followed by Tukey’s post hoc test. A To investigate the effect of osteogenic medium on hBMSCs proliferation, we treated hBMSCs cells at different time points. To further examine the effect of osteogenic medium treatment on osteogenesis and the involvement of histone modification, we determined the mRNA levels of JMJD2B as well as osteogenesis- and myogenesis- genes in hBMSCs cells at 0, 1, 3, 7, 14 days. The results showed that the osteogenic medium significantly upregulated the mRNA expression of Runx2 during the initial (day 3) osteogenic induction process, and furthermore dramatically upregulated the mRNA expression of FAM210A and JMJD2B at 7 and 14 days simultaneously .To further investigate whether osteogenesis affects related protein level. We determined the related protein level in hBMSCs cells treated with osteogenic medium. As shown in To examine whether overexpression of JMJD2B could reverse the effect of silence JMJD2B lentivirus-transfected hBMSCs on osteogenesis, we overexpressed/silenced JMJD2B in hBMSCs cells and then treated the cells with vehicle or osteogenesis Induction Medium. As shown in Then, we measured the mRNA levels of osteogenic markers in hBMSCS cells in response to JMJD2B overexpression/slience. As shown in Considering the critical role of JMJD2B in osteogenesis and the essential role of histone modification in hBMSCs differentiation regulation, we examined the effect of JMJD2B overexpression/slience on the methylation enrichment status of H3K9me2 in the Runx2 promoter in hBMSCs exposed to osteogenic medium, we wanted to determine whether the altered transcriptional activity of JMJD2B may be attributed to differences in DNA binding. To do this we compared the chromatin association of H3K9me2 with the Runx2 promoter using Chromatin Immunoprecipitation (ChIP). LV5-JMJD2B significantly increased H3K9me2 occupancy at the Runx2 promoter. However, Si-JMJD2B decreased H3K9me2 recruitment to Runx2 promoter . These rAs shown in To further investigate whether JMJD2B activates H3K9me2 binding levels, we measured the mRNA levels of Runx2 and JMJD2B in hBMSCs cells in response to H3K9me2 inhibition. As shown in DNA is the essence of heredity. This view has almost become a genetic truth. The rise of epigenetics has changed this phenomenon to a certain extent. People are surprised to find that the emergence of new traits is not necessarily the result of changes in DNA sequences. The environment may induce changes in biological traits, and this change can also Inherited to the next generation. Epigenetic refers to genetic changes in gene expression without altering the DNA sequence, including DNA methylation, histone modification and small non-coding RNA related regulation . Lysine To verify the effect of JMJD2B on expression and osteogenic response, we treated hBMSCs with osteogenic medium. We found that mRNA/protein/fluorescence signal expression of JMJD2B was significantly upregulated in response to osteogenic medium stimulation, along with significantly facilitated/suppressed osteogenic differentiation in the following gain/loss of function study, suggesting that osteogenic medium promotes osteogenesis possibly by upregulating JMJD2B-mediated histone demethylation of osteogenesis-related chromatin. The canonical transcription factor Runx2 facilitates bone formation. Of note, JMJD2B-mediated histone demethylation is essential for Runx2 expression. Thus, we hypothesized that osteogenic medium promotes osteogenesis possibly through upregulating JMJD2B-mediated histone demethylation of H3K9me3/H3K9me2 in Runx2 promoter and that inhibitory expression of H3K9me2 might suppress the effects of osteogenic medium. Indeed, our results showed that overexpression/silence of JMJD2B increased/reduced the enrichment status of H3K9me2 in Runx2 promoter, in addition, inhibitory expression of H3K9me2 reversed the promoted effects of osteogenic medium on osteogenic differentiation as well as mRNA/protein expression of Runx2,which could be compensated by overexpression of JMJD2B. Together, these events facilitate osteogenic differentiation of hBMSCs, as evidenced by enhanced ALP and alizarin red staining and mRNA expression of osteogenic markers in hBMSCs. JMJD2B-mediated histone demethylation plays an essential role in the activation of Runx2 expression. Dysregulation of JMJD2B blocks the activation of Runx2 expression.In conclusion, osteoinduction promotes JMJD2B expression in hBMSCs. Overexpression of JMJD2B reversed the inhibitory effects of negative inhibition of silent lentivirus on osteogenic differentiation as well as mRNA expression of osteogenic markers in hBMSCs. JMJD2B regulates the methylation enrichment status of H3K9me2 in the Runx2 promoter to influence the osteogenic differentiation and Runx2 expression in hBMSCs. These findings suggest that overexpression of JMJD2B is as a potential therapeutic approach to regulate the osteogenic differentiation of hBMSCs through activating histone demethylation of H3K9me3/H3K9me2 in the Runx2 promoter.10.7717/peerj.13862/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.13862/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.13862/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.13862/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.13862/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.13862/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.13862/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.13862/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.13862/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.13862/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.13862/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.13862/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.13862/supp-13Supplemental Information 13Click here for additional data file.10.7717/peerj.13862/supp-14Supplemental Information 14Click here for additional data file.


Older adults with type 2 diabetes are at higher risk of developing common geriatric syndromes and have a lower quality of life. To prevent type 2 diabetes in older adults, it’s unclear whether the health benefits of physical activity (PA) will be influenced by the harms caused by increased exposure to air pollution during PA, especially in developing countries with severe air pollution problem. We aimed to investigate the joint effects of PA and long-term exposure to air pollution on the type 2 diabetes in older adults from China.10, PM2.5 and PM1) were collected from open products. The joint effects were assessed by the marginal structural mean model with generalized propensity score.This cross-sectional study was based on the China Multi-Ethnic cohort (CMEC) study. The metabolic equivalent of PA was calculated according to the PA scale during the CMEC baseline survey. High resolution air pollution datasets  µg/m3, 40.45 (15.66) µg/m3 and 27.62 (6.51) µg/m3, respectively. With PM10 < 92 µg/m3, PM2.5 < 61 µg/m3, and PM1 < 36 µg/m3, the benefit effects of PA on type 2 diabetes was significantly greater than the harms due to PMs when PA levels were roughly below 80 MET-h/d. With PM10 ≥ 92 µg/m3, PM2.5 ≥ 61 µg/m3, and PM1 ≥ 36 µg/m3, the odds ratio (OR) first decreased and then rose rapidly with confidence intervals progressively greater than 1 and break-even points close to or even below 40 MET-h/d.A total of 36,562 participants aged 50 to 79 years were included in the study. The prevalence of type 2 diabetes was 10.88%. The mean (SD) level of PA was 24.93 (18.60) MET-h/d, and the mean (SD) level of PMOur findings implied that for the prevention of type 2 diabetes in older adults, the PA health benefits outweighed the harms of air pollution except in extreme air pollution situations, and suggested that when the air quality of residence is severe, the PA levels should ideally not exceed 40 MET-h/d.The online version contains supplementary material available at 10.1186/s12877-022-03139-8. Diabetes is a metabolic disorder caused by environmental and genetic factors and has been considered as one of the major contributors to the global burden of disease , 2. NearResearch has consistently demonstrated that the adoption of physical activity (PA) can prevent diabetes –10, espeSome studies have revealed the joint effects of PA and air pollution on cardiovascular disease, lung function/respiratory disease and mortality –28, but We aimed to investigate the joint effects of PA and long-term exposure to air pollution on type 2 diabetes in older adults exposed to heavy air pollution from China, where air pollution and the prevalence of type 2 diabetes both pose a grave public health concern , 33.This cross-sectional study was based on the China Multi-Ethnic cohort (CMEC), documented in detail elsewhere . In brien = 4993 for Aba; n = 7780 for Lasa). We then excluded 9372 participants who had changed their place of residence within three years prior to the baseline survey, and excluded 2739 participants diagnosed with type 2 diabetes before exposure assessment. We further excluded 672 participants who self-reported having cancers, mainly lung, oesophageal, stomach, liver, prostate and cervical cancers, as well as 2775 pregnant women and 161 participants self-reported having tuberculosis. We then selected adults over 50 years of age and excluded 1237 participants due to incomplete information on air pollution exposure, PA, health outcomes and other covariates. A total of 36,562 adults aged 50 to 79 years were included in this study.The participant selection procedure is shown in the Data Supplement Fig S. Tibetan10 (particulate matter with an aerodynamic diameter of 10 µm or less), PM2.5 (particulate matter with an aerodynamic diameter of 2.5 µm or less) and PM1 (particulate matter with an aerodynamic diameter of 1 µm or less) dataset were collected from open products, which were estimated using a newly developed space–time extremely randomized trees (STET) model based on the newly released MODIS Collection 6 MAIAC 1-km AOD products, meteorological variables, pollution emissions, land cover, surface topographic data and population data [10, PM2.5 and PM1, respectively.The high-resolution (1 km) and high-quality PMion data –40. The 10, PM2.5 and PM1 concentrations to each participant based on their geocoded residential address and calculated the 3-year average exposure concentrations before the baseline survey. Figure 10, PM2.5 and PM1 concentrations by participants’ address locations.We assigned the estimated annual PMThe information of PA for each participant was collected through the questionnaire during the baseline survey. Both the PA intensity and duration one year preceding the survey were obtained. The PA intensity was presented by the corresponding metabolic equivalent values (MET). This study assigned different MET to various physical activities –43, and 1c ≥ 6.5% (48 mmol/mol). Those criteria were based on the recommendations of the American Diabetes Association [The diabetes status of the participants was defined by the following criteria: (1) self-reporting of taking any antidiabetic medication  or (2) fasting plasma glucose ≥ 126 mg/dL (7.0 mmol/L), or (3) HbAociation .In this study, we included the following covariates in the main analysis: age (year), sex , marital status , education , annual household income, body mass index , smoking status , passive smoking status (yes or no), alternative Mediterranean diet (aMED) score , self-reCausal inference methods are gradually being used in environmental epidemiology because of better control of confounding, and avoiding ethical issues , 48. AmoIn this study, let d Imbens , we exteLet  the GPS , the bi-The above balancing property implies that given the bi-dimensional GPS, covariates mentclass2pt{minimThe overall workflow of the IPTW method was presented in supplementary Fig S, and theeriments .10, PM2.5 and PM1) on type 2 diabetes, which combined the bi-dimensional GPS and the generalized additive model [A marginal structural mean model  was consve model . The modThe variable ion term .10 were 40 50, 65, 70, 88, 92, 104, 107, 120 µg/m3; the nine levels of PM2.5 were 20, 25, 35, 48, 52, 54, 61, 65, 70 µg/m3; and the nine levels of PM1 were 18, 21, 23, 26, 32, 33, 36, 38 and 42 µg/m3.To present the results clearly without using 3D plots, we investigated the exposure–response relationship between PA and type 2 diabetes at different concentrations of PMs. Participants exposed to the lowest PMs and lowest PA levels were the reference group. The choice of different PMs concentrations was based on its distribution and the WHO recommendations. Specifically, the nine levels of PMSensitivity analyses were performed to assess the robustness of joint effects: (1) the above main analyses were repeated after excluding subjects taking any antidiabetic medication because their intentional lifestyle changes may produce estimate bias, such as doing more exercise to prevent exacerbation of the disease; (2) the 2-year average exposure of air pollution and 4-year average exposure of air pollution were employed to evaluate the possible impact of different exposure windows.All statistical analysis were performed in R software, version 3.4.0.vs. 25.21), and higher levels of long-term exposure to pollutants at their residence.A total of 36,562 participants were included in the study, and the prevalence of type 2 diabetes was 10.88%. Table 10, PM2.5 and PM1 were indeed wide, ranging from 33.26 µg/m3 to 165.19 µg/m3, 18.24 µg/m3 to 105.29 µg/m3 and 15.49 µg/m3 to 53.57 µg/m3, respectively , the potential detrimental effects due to augmented exposure to air pollution during PA could outweigh the protective effects of PA, especially when PA levels were roughly above 40 MET-h/d.To our knowledge, this is the first study to investigate the joint effects of air pollution and physical activity on type 2 diabetes in older adults exposed to widely varying levels of air pollution exposure. Based on the pattern shown in the results, this study indicated that with PMExercise is known to reduce the risk of type 2 diabetes, and daily moderate- or high-intensity exercise is likely optimal to enhance insulin activity . The benSo far, the trade-off between the potential harmful effects caused by augmented exposure to air pollution during PA and the health benefits of increased PA remains unclear. Some studies has revealed that there was no significant interaction effects between air pollution and PA on hypertension , lung fu10 exposure was 50 (5.69) µg/m3 [2.5 exposure was 26.1 (7.3) µg/m3 [10 and PM2.5  levels are associated with an approximately 22% and 24% higher long-term mortality risk relative to the AQG level  [However, those studies were conducted in developed countries or regions, with low or moderate air pollution exposure settings , such as9) µg/m3  or PM2.53) µg/m3 , in whicctively) . We foun2.5 concentrations of 66 µg/m3, the tipping points, beyond which additional PA will not lead to more health benefits, were 1 h per day for cycling, and 6.25 h per day for walking [2.5 concentrations, i.e. 65 µg/m3, our results showed a tipping point of about 21 MET-h/d, implying about 6.36 h per day for walking . Therefore, more research is needed to investigate the distribution of PA in China and to explore whether the WHO guidelines are appropriate for the Chinese context, so as to derive suitable PA guidelines for China.In addition, WHO recommended older adults should do at least 150–300 min of moderate-intensity aerobic PA per week, which would mean 15–30 MET-h/week if the moderate-intensity PA was set at 6 MET . AlthougCausal inference based on the IPTW has been widely popular and applied in observational studies, but there is a lack of IPTW studies on the joint or interaction effects of two continuous variables. This study proposed a bi-dimensional GPS, and then applied the IPTW method to investigate the joint effects of two continues exposure on health, which is a very common and inevitable situation in real life. Although some assumptions of causal inference are untestable, our study showed that the findings were reliable and robust by the exclusion of participants with self-reported disease.10, PM2.5, or PM1 exposure and to obtain stable and precise estimates. Finally, a spatiotemporal model was used to estimate the concentrations of ambient PM10, PM2.5 and PM1 at a high resolution (1 × 1 km2), and indoor air pollution was included in our analysis.This study has several important strengths. First, most studies have been conducted in North America and European countries with good air conditions , 22, 28,10, PM2.5, and PM1 exposure with diabetes. Second, although the cross-sectional study design was a limitation for our study, we tried to use a study design to minimize the problem of inversion of cause and effect, such as excluding participants diagnosed with type 2 diabetes 3 years earlier and including only participants with at least 3 years of stable residence. Finally, we adopted a questionnaire to collect information related to exposure, such as smoking, drinking, PA, indoor pollution situation and dietary habits; thus, recall bias cannot be avoided, and misclassification might have occurred.This study also has a few limitations. First, we did not distinguish between indoor and outdoor PA. Thus, we could not exclusively examine the joint associations of outdoor PA and ambient PMIn conclusion, these findings suggest that for the prevention of type 2 diabetes in older adults, the health benefits of PA could outweigh the harms caused by air pollution, except in extreme air pollution situations, and that PA levels should not exceed 40 MET-h/d when the air quality of residence is severe. More robust research on the dose–response relationship is warranted to validate our findings with a cohort study design in future research.Additional file 1.


SMCHD1, DNMT3B, or LRIF1. Genetic diagnosis of FSHD is challenging because of the complex procedures required.Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder characterized by asymmetric muscle wasting and weakness. FSHD can be subdivided into two types: FSHD1, caused by contraction of the D4Z4 repeat on chromosome 4q35, and FSHD2, caused by mild contraction of the D4Z4 repeat plus aberrant hypomethylation mediated by genetic variants in We applied Nanopore CRISPR/Cas9-targeted resequencing for the diagnosis of FSHD by simultaneous detection of D4Z4 repeat length and methylation status at nucleotide level in genetically-confirmed and suspected patients.We found significant hypomethylation of contracted 4q-D4Z4 repeats in FSHD1, and both 4q- and 10q-D4Z4 repeats in FSHD2. We also found that the hypomethylation in the contracted D4Z4 in FSHD1 is moderately correlated with patient phenotypes.Our method contributes to the development for the diagnosis of FSHD using Nanopore long-read sequencing. This finding might give insight into the mechanisms by which repeat contraction causes disease pathogenesis.The online version contains supplementary material available at 10.1186/s12967-022-03743-7. DUX4 is a causative gene for FSHD and is located within an approximately 3.3 kb repeat sequence, referred to as D4Z4, which comprises 1–100 repeat units (RUs) on the subtelomeric regions of chromosomes 4 and 10. Chromosome 4 has two haplotypes distal of the D4Z4 repeat, 4qA and 4qB, where only the 4qA allele contributes to FSHD development, due to the presence of a polyadenylation signal in the most distal D4Z4 RU [Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal disease characterized by muscle weakness that initially manifests in the face, shoulder, and upper arms, followed by asymmetric involvement of other muscles . DUX4 is D4Z4 RU , 3.SMCHD1, DNMT3B, or LRIF1, which each encode epigenetic modifiers [FSHD has two types, FSHD1 and FSHD2, both caused by genetic defects leading to aberrant DUX4 expression in skeletal muscle , while Fodifiers –8. Epigeodifiers  DNA methodifiers –12. CpG odifiers , 13, 14;Southern blotting, bisulfite sequencing, molecular combing, and next-generation sequencing are currently used for genetic diagnosis of FSHD , but theHere, we applied Nanopore CRISPR/Cas9-targeted resequencing (nCATS) to measure the number of D4Z4 RUs and their methylation status in patients with FSHD. We specifically analyzed D4Z4 RUs derived from 4qA and measured the CpG methylation rate in each RU. D4Z4 RUs from 10q were also analyzed.4Cl, 10 mM KHCO3, 1 mM EDTA, pH 7.4) on ice for 15 min, followed by centrifugation  . After a repeat EL buffer wash, pellets were suspended in 3 ml NL buffer , followed by addition of 1% SDS and proteinase K and incubation at 37 °C overnight. DNA lysis solution was added with 1 ml 5 M NaCl, followed by phenol/chloroform extraction and ethanol precipitation. DNA pellets were suspended in TE buffer.Peripheral blood lymphocytes (10 ml) were combined with 30 ml EL buffer . To generate Cas9 ribonucleoprotein complexes (RNPs), annealed 1 μM tracrRNA-crRNA pool (CR1/CR2/CR3/CR4) and 0.5 μM HiFi Cas9 were incubated at room temperature (around 23 °C) for 30 min. Genomic DNA (2 μg) was dephosphorylated with Quick Calf Intestinal Phosphatase  at 37 °C for 10 min, followed by 80 °C for 2 min. For Cas9 RNP cleavage and dA-tailing, dephosphorylated genomic DNA samples were treated with Cas9 RNPs, Taq polymerase , and dATP  at 37 °C for 30 min, followed by 72 °C for 5 min. For native barcode ligation, native barcoding expansion 1–12  were ligated to cleaved and dA-tailed genomic DNAs using Blunt/TA Ligase Master Mix  at room temperature for 10 min, followed by purification with Agencourt AMPure XP Beads  on a magnet. AMII adapters were ligated to barcoded genomic DNA using Quick T4 DNA ligase  at room temperature for 10 min, followed by purification with AMPure XP Beads on a magnet. The DNA library from Cas9-targeted native barcoding was primed into a MinION Flow Cell (FLO-MIN106D) on a MinION Mk1C and sequencing was performed for 20–21 h.The crRNA design tool, CHOPCHOP , was useBases were called from Fast5 files using Guppy to generate Fastq files. Alignment to the reference sequence, which contains 10 D4Z4 RUs and flanking sequences from 3950 bp upstream of CR1 to 251 bp downstream of CR4, was conducted using Minimap2. Reference sequences were constructed using SnapGene software . For DNA methylation analysis, sense- and antisense-strand reads from the 4qA and 10q loci were re-aligned to the corresponding reference sequences and then Nanopolish was performed . ReferenFor CAS9 cleavage, we designed two types of guide RNA each for the p13E-11 CR1/CR2) and A-haplotype (CR3/CR4) regions; the distal guides, CR3 and CR4, specifically recognized the 4qA and 10q loci, but not 4qB , 2 (13 RUs), and 3 (10 RUs and 12 RUs)  or failed to determine repeat lengths based on restriction fragment sizes (Samples 9–15) Table . Using nInterestingly, we also detected a genomic deletion, as an atypical cause of rearrangement of D4Z4 repeats. Samples 13 and 14 each generated one read with an intermediate size between 2 and 3 RUs. Sequence analysis revealed that both reads contained a deletion spanning 1.3 kb from 469 bases proximal to the most proximal D4Z4 RU to 859 bases within it  separately  than that between D4Z4 repeat length and age at onset (R2 = 0.401). Although the correlation coefficient between CpG methylation and age at hospital inspection was not high (R2 = 0.306), there was a tendency toward correlation, in that CpG methylation rate < 10% was associated with hospital inspection at a younger age (≤ 20 years old), while CpG methylation rates of 10–20% were associated with that at > 40 years old.Epigenetic changes in the contracted D4Z4 repeats on chromosome 4qA have been observed previously and are considered to be associated with the development of FSHD1 , 13, 14.In general, nCATS could be applicable to any other genetic disorders. In particular, it has an advantage on diagnosis of repeat-associated disorders, such as Huntington disease, spinal cerebellar ataxia, neuronal intranuclear inclusion disease, oculopharyngeal distal myopathy and others, in which the causative genetic variation cannot be identified by short read sequencing. In fact, it has been applied for analysis of some tandem repeat disorders, fragile X syndrome and myotonic dystrophy –23. NanoIn this study, we developed a direct sequencing system using nCATS to analyze clinical samples from patients with FSHD. Our method is more efficient and can collect more detailed information than conventional method. Conventional method for diagnosis of FSHD is carried out by multiple Southern blots for detection of the size of 4q-derived D4Z4 repeat and haplotyping 4q, and by bisulfite sequencing for measurement of the CpG methylation rate. In contrast, our method enables us to simultaneously identify the number and the methylation rate of D4Z4 repeat unit and the haplotype derived from 4qA. Our system has several advantages. First, long read sequencing can be applied to analysis of a similar DNA fragment size range to that detected by Southern blotting. Second, CRISPR/CAS9 enrichment allows barcoding sequencing of five samples simultaneously, saving time and cost. Third, single-molecule sequencing technology provides genetic information at the base level and can determine the number of RUs, even in samples that have mutated restriction enzyme sites, which prevent determination of RU number by the standard Southern blotting method. Finally, the nCATS system allows simultaneous detection of CpG methylation and D4Z4 RUs numbers, providing information about local epigenetic modification of D4Z4 repeats, due to the application of single-molecule sequencing of unamplified genomic DNA molecules derived from individual nuclei, without any bias.DUX4C, FRG2, DBE-T, and myogenic enhancers, are deleted, suggesting that their role in FSHD pathogenesis requires reevaluation [Along with successful determination of D4Z4 RU numbers in patients, we also detected atypical rearrangement of D4Z4 repeats. As shown in Figs. aluation .DUX4 gene in the normal 4qA allele, while contraction of D4Z4 repeats causes hypomethylation of distal D4Z4 similar to proximal D4Z4 in the 10q locus, leading to DUX4 expression and consequent development of FSHD1. Indeed, mean CpG methylation rate of the most distal RUs and disease onset in patients was well-correlated. A larger study of the relationships among methylation rate, D4Z4 contraction, and clinical phenotypes is needed. To this end, we aim to overcome the limitation of decreased acquisition of sequencing reads from alleles with more than 10 RUs.The most important finding in our study was detection of DNA methylation rates across entire contracted and normal expanded D4Z4 repeat sequences from the 4qA and 10q loci. As shown in Fig. The nCATS method has limitations. First, the number of sequencing reads containing mildly contracted D4Z4 repeats (11–13 RUs) detected was quite low, particularly as only a few reads were obtained from the normal 10q locus, and no reads were obtained from some samples. The reasons why we could not obtain read from chromosome 10 in all samples and the number of reads in various samples are different are; (1) the difficulty to purify intact high molecular weight DNA, because the longer DNA might tend to be subject to degradation, (2) the difficulty to obtain longer DNA fragments beyond 13 RUs, because we used only the reads harboring full-length D4Z4 repeat in our analysis, (3) the efficacy of CAS9 cleavage of hypermethylated DNA, because distal D4Z4 were extremely higher methylation rates. Technical improvements in terms of preparation of genomic DNAs are required to overcome this shortcoming. Second, our method does not isolate reads derived from 4qB. Although the lack of analysis on 4qB is not likely to affect our conclusion, the epigenetic status in 4qB could be meaningful information as reference data for methylation rate of 4qA-derived D4Z4.In this study, we successfully determined the hypomethylation of D4Z4 RUs in individual 4qA fragments in FSHD. The hypomethylation in the contracted D4Z4 in FSHD1 provides a good explanation why the shortening of D4Z4 repeats is associated with severe phenotypes in patients and it induces abnormal DUX4 expression which leads to developing FSHD. For a further improvement, we need to have a large cohort of patients and controls in the future, which might give a clue for complete understanding of the pathomechanism of FSHD.Additional file 1: Fig. S1. Characteristic sequences detected by nCATS. Sequences of representative (A) 4qA- and (B) 10q-derived reads obtained from the indicated samples. The XapI/non-XapI and BlnI/non-BlnI sites in the most distal D4Z4 RU are shown. In Samples 8, 14, and 15, the XapI, XapI and non-BlnI, and non-XapI sites, respectively, in the second most distal D4Z4 RU are shown, due to the difficulty in identifying restriction sites. Table S1. Lengths of reads derived from the 4qA locus in each patient. Table S2. Lengths of reads derived from the 10q locus in each patient. Table S3. Methylation rates across all D4Z4 RUs at the 4qA and 10q loci.


The molar compositions of the synthesized copolymers were determined using FTIR and 1H NMR-spectroscopy, elemental analysis, and conductometric titration. It was found that in the radical copolymerization reaction, the DMAPMA monomer was more active than the DADMAC monomer; for this reason, the resulting copolymers were always enriched in the DMAPMA monomers. The study of the influence of the DADMAC–DMAPMA copolymer on structure-formation in the bentonite suspension showed that this copolymer significantly increased the static shear stress (SSS) of the system. In this case, the structure-forming properties of the copolymer depended on the pH of the medium. The lower the pH level, the better the structure-formation was in the suspension in the presence of the copolymer. The study of antibacterial activity showed that the DADMAC–DMAPMA copolymer had a biocidal effect against sulfate-reducing bacteria (CRB) at a concentration of not less than 0.05 wt.% and can be used to inhibit the growth of this bacteria.The copolymer of N,N-diallyl-N,N-dimethylammonium chloride and N-[3-(Dimethylamino)propyl]methacrylamide (DADMAC–DMAPMA) was synthesized by radical polymerization reaction in an aqueous solution in the presence of the initiator ammonium persulfate (NH In the oil and gas industry, the successful drilling of wells largely depends on the correct choice of drilling fluid ,2. The dDrilling fluids are divided into the following types: water-based muds (WBMs), oil-based muds (OBMs) and foam drilling fluids ,5,6,7. ATo perform the above basic functions, drilling fluids must have some necessary rheological properties , stability to temperature and pressure changes, resistance to the action of contaminating components , and they should prevent fluid loss additives ,11,12. IBentonite clay, added to the drilling fluid, provides the necessary viscosity in the system, volume structure formation, thixotropic properties of the suspension, and the formation of a thin filtration layer on the surface of the well wall. A filtration layer reduces the risk of collapse of the well wall and decreases water filtration through the well walls into the formation (reservoir water absorption).The filtration characteristics of a water-based drilling fluid have a significant impact on wellbore stability. The penetration of water into porous formations reduces the stability of the wellbore and can lead to serious problems, such as a stuck pipe and the collapse of formations ,13,14.The viscosity and thixotropic nature of the drilling fluid ensure the retention of cuttings particles in the suspension during a temporary (forced) stop of the drilling process, i.e., prevent sedimentation of drill cuttings particles on the bit of the drilling rig.In many cases, the addition of natural and synthetic water-soluble polymers to the clay drilling fluids ensures the formation of a thin filter cake on the wellbore wall, which sharply reduces water filtration through the wellbore walls into the formation and increases the stability (strength) of the wellbore wall ,17,18,19To improve the rheological and filtration properties of the drilling fluids, the following natural polymeric compounds were used: carboxymethyl cellulose and polyanionic cellulose ,21,22, xThe main advantage of natural polymer compounds is their availability and relatively low cost. However, natural polymeric additives to drilling fluids based on polysaccharides have a serious drawback—their high biodegradability under the action of various microorganisms ,34. As aTo suppress the vital activity of microorganisms, different substances with a bactericidal effect, such as phenols, formaldehyde, dialdehyde , ketones , and polAnother serious problem in the oil and gas industry is microbiological corrosion of drilling rigs, and oil and gas constructions ,39,40. DBiocidal polymers are often used to inhibit chlorine-resistant bacteria (CRB) ,44,45. HIn this regard, the synthesis of new water-soluble polymer surfactants from available industrial monomers, which simultaneously have structure-forming and biocidal properties, is one of the urgent tasks.In this work, a new polymeric surfactant has been synthesized, which has a structure-forming and biocidal effect.The solution of the monomer N,N-diallyl-N,N-dimethylammonium chloride (DADMAC) , monomer N-[3-(Dimethylamino)propyl]methacrylamide (DMAPMA)  and 1,4-Dioxane  were purchased from Sigma Aldrich (USA).3 (purity 99.8 wt%) and ammonium persulfate, (NH4)2S2O8 (purity 99.7 wt%) were kindly provided by “LaborPharma” Ltd. . Acetone (purity 99.9 wt%) was purchased from “Labchimprom” Ltd. . Argon (purity 99.995 wt%) was purchased from “Ikhsan TechnoGas” Ltd. . Distilled water with a conductivity of 2.4 mS/cm (20 °C) was used in all experiments.AgNO2O-1.00; MgO-3.59; Al2O3-22.46; SiO2-58.78; SO3-0.06; Cl-0.02; K2O-0.59; CaO-3.99; TiO2-0.79; Cr2O3-0.01; Fe2O3 8.60 and others–0.2 /[M2]; f = [m1]/[m2]; [M1] and [M2] are mole fractions of DMAPMA and DADMAC in the initial monomers’ mixture, respectively; [m1] and [m2] are mole fractions of DMAPMA and DADMAC in the resulted copolymer, respectively; y = (F/f)⋅(f − 1); x = F2/f; r1 and r2 are reactivity ratios of DMAPMA and DADMAC, respectively.The reactivity ratio is an important parameter for describing the copolymerization mechanism and composition of copolymers. The monomer reactivity ratio can be determined from the initial monomer ratio and resulting copolymer composition by the Mayo–Lewis (1) and Fineman–-Ross Equations (2) ,50:r2 = Structure formation in the bentonite suspensions in the presence of a copolymer was determined using a rotational viscometer with manual control of 6 speeds RC-35D . For the study, 30 wt.% suspension of the bentonite clay was prepared by stirring at 300 rpm for 10 min in water. Then, the prepared suspension was left for 24 h for the complete swelling of the bentonite clay. The next day, the suspension was treated with the required volume of copolymer solution and stirred at 100 rpm for 3 min. After treating the suspension with the copolymer, the concentration of the suspension was always equal to 20 wt.%. A copolymer solution with a concentration of 0.1 wt.% was prepared the day before. The desired copolymer concentration was prepared by diluting the stock solution.The static shear stress (SSS) of the bentonite suspensions was measured at room temperature, 1 min (SSS-1) and 10 min (SSS-10) after the treating of the suspension with the copolymer solution. The SSS is one of the most important drilling fluid parameters. It indicates the rate of the formation and strength of the gel forming in suspension at rest. The higher the SSS, the larger the size of the particles that are in suspension and that do not settle to the bottom of the well.The antimicrobial action of the DADMAC–DMAPMA copolymer, with different molar compositions, on sulfate-reducing bacteria (SRB) was tested at the “Scientific and Production Center of Microbiology and Virology, LLP” . The effect of the copolymer samples on the growth of SRB was studied in three concentrations (wt%): 0.01, 0.05 and 0.1.For the study, 1 mL of the culture liquid of SRB was added to the test tubes. Then, the tubes were filled with a nutrient medium with a copolymer dissolved in the appropriate concentration. In the control sample, SRB was cultivated without copolymer. When SRB grows, a black film with a metallic outflow on the inner surface of the tube is observed.4)2S2O8 as an initiator. The total molar concentration of monomers in the mixture was equal to 5 mol/L. After purging with argon for 20 min, the ampoule containing the monomers was sealed and placed in a water bath heated at 343 K. Then, the reaction mixture was heated for 180 min. After this, the resulting copolymer was precipitated from the reaction environment and repeatedly purified with a mixture of 1,4-dioxane and acetone (50:50 vol.%). At the end, the purified copolymer was dried under a vacuum at 313 K until a constant weight was achieved. The percentage conversion (y) of the monomers was obtained by gravimetry .The copolymer of N,N-diallyl-N,N-dimethylammonium chloride and 3-[(dimethyl)aminopropyl]methacrylamide (DADMAC–DMAPMA copolymer) was synthesized in the ampoule by radical copolymerization of DADMAC with DMAPMA in an aqueous environment at different initial molar ratios of monomers in the presence of (NHcR).Determination of the copolymerization reaction rate  ::54]:The relatively low reactivity of the DADMAC monomer can be explained by the complex structure of this monomer and the presence of a positive charge in the macrochain. Like-charged monomers repel each other when they approaching, which makes it difficult for them to interact with each other closely enough to react. The relatively low reactivity of the DADMAC monomer in radical copolymerization reactions with dimethylacrylamide, acrylonitrile, itaconic and fumaric acids, and 4-vinylpyridine was noted in several works ,62,63,64c) and conversion  (after 240 min) of the reaction were determined  and conversion  (after 240 min) of the reaction were determined  for T = 343 K and the initial reaction mixture [DADMAC]:[DMAPMA] = 50:50 mol %.Thus, we can conclude that the copolymerization rate equation for the DADMAC–DMAPMA copolymer is 4)2S2O8 was obtained. The copolymerization rate equation for this system was Rp = k × [M]2.23 × [I]0.70, where [DADMAC]:[AA] was 4:1.Exponent numbers for a total monomer concentration greater than 1.0 have been found in many studies. In the paper by Liu and co-workers , the rea4)2S2O8) was obtained in [p = k × [M]2.6 × [I]0.60 .The polymerization rate equation for the copolymer DADMAC–VEMEA (Vinyl Ether of Monoethanolamine) at 65 °C (initiator-(NHained in : Rp = k 4)2S2O8) as an initiator and obtained the following overall rate equation: Rp = k × [M]2.9 × [I]0.8.Jaeger W, et al. , studied2S2O8-Na2SO3 redox system at 40 °C in an aqueous solution and obtained the following polymerization rate equation: Rp = k × [Na2S2O8]0.55 × [Na2SO3]0.56 × [M]1.14 for m(DADMAC):m(AA) = 1:2 .Zhang W, et al. , studied4)2S2O8-Na2SO3 redox complex in an aqueous solution were studied. At 45 °C for the monomer molar ratio n(DADMAC):n(AA): 1:9; 2:8; 3:7; 4:6 and 5:5, the following copolymerization rate equations were obtained, respectively: Rp1 = k × [M]2.61 × [IO]0.51 × [IR]0.52; Rp2 = k × [M]2.70 × [IO]0.50 × [IR]0.53; Rp3 = k × [M]2.73 × [IO]0.50 × [IR]0.56; Rp4 = k × [M]2.77 × [IO]0.51 × [IR]0.59 and Rp5 = k × [M]2.84 × [IO]0.51 × [IR]0.61  2.63 × [I]0.40.The authors of , studiedAccording to ,63, the cR = k × [M]3.48 × [I]0.033, we can calculate the rate constant (k) of the copolymerization reaction for the system DADMAC–DMAPMA. From −1, the value cR = 0.068 mol × L−1 × min−1 corresponds to the value [M] = 4 mol × L−1. Then we have 0.068 mol × L−1 × min−1 = k × [4 mol × L−1]3.48 × [0.022 mol × L−1]0.033; k = 1.03 × 10−5 L2.51 × mol−2.51 × s−1.Now, using the resulting equation: −1, the value cR = 0.067 mol × L−1 × min−1 corresponds to the value [I] = 0.5 wt% ≈ 0.022 mol × L−1. Then we have 0.067 mol × L−1 × min−1 = k × [4 mol × L−1]3.48 × [0.022 mol × L−1]0.033; k = 1.02 × 10−5 L2.51 × mol−2.51 × s−1.From k = 1.015 × 10−5 L2.51 × mol−2.51 × s−1.Thus, the average value of the rate constant for the copolymerization reaction of DADMAC with DMAPMA at 70 °C in the presence of ammonium persulfate is 4)2S2O8) was studied and obtained the following value: k = (0.85÷2) × 10−5 L2.2 × mol−2.2 × s−1.In the paper , the kin2O2 as an initiator. It was found that the rate of the copolymerization reaction depended on the temperature, and at 60 °C, it was equal to 4.8 × 10−5 mol × L−1 × s−1.The authors of , studied2S2O8) was studied. It was found that by increasing the initiator concentration from 1.3 × 10−2 to 1.75 × 10−2 mol × L−1, the rate of the copolymerization reaction increased from 1.13 × 10−4 to 1.28 × 10−4 mol × L−1 × s−1.In other papers ,65, the In order to determine a possibility of using the DADMAC–DMAPMA copolymer as a structure-forming agent for controlling the viscosity (thixotropic properties) of clay drilling fluids, we studied the static shear stress after 1 (SSS-1) and 10 min (SSS-10) aging of a bentonite clay suspension of the Tagan deposit (West Kazakhstan) in the presence of poly-DADMAC and DADMAC–DMAPMA copolymers with different compositions . AnalysiAccording to ,67, the An increase in the content of DMAPMA in the copolymer raises the hydrophobicity of the macromolecule, which leads to enhanced stability of the copolymer–bentonite particles’ association ,70.The increase in the suspension’s SSS in an acidic medium can be explained as follows. In the acidic medium, a protonization of the amino groups of the DMAPMA monomers takes place, and as a result, the positive charge density  of the DADMAC–DMAPMA copolymer significantly increases. Due to this, the electrostatic interaction of the copolymer chains with the surfaces of the bentonite particles is enhanced.The DADMAC–DMAPMA copolymers synthesized from mixtures of monomers with the molar composition [DMAPMA]:[DADMAC] = 80:20 (A), 50:50 (B), and 20:80 (C), were chosen as biocidal materials.The effect of copolymer samples on SRB growth was studied at three concentrations: 0.1% (1), 0.05% (2), and 0.01% (3). The development of SRB was observed on the fifth day of cultivation. When SRB grows, a black film with a metallic outflow on the inner surface of the tube appears. It was found that the samples of DADMAC–DMAPMA copolymer (1) and (3) completely (100%) inhibited the development of SRB at concentrations of 0.05% and 0.1%; however, at a concentration of 0.01%, the development of SRB was observed. Sample (2) completely inhibited the development of SRB, only at a concentration of 0.1%. The biocidal effect of the samples was observed throughout the test period (for 15 days).According to ,72,73,74Therefore, it can be stated that DADMAC–DMAPMA copolymers obtained from a mixture with a molar composition of [DMAPMA]:[DADMAC] = (80÷20):(20÷80) have antibacterial activity against SRB at a concentration of at least 0.05 wt%.4)2S2O8. The composition and structure of the synthesized copolymers were determined by FTIR, 1H NMR spectroscopy, elemental analysis, and conductometric titration. It was found that the DMAPMA monomer is more active than the DADMAC monomer in the radical copolymerization reaction; thus, the DADMAC–DMAPMA copolymers were always enriched in DMAPMA monomer.In this research, DADMAC–DMAPMA copolymers with different molar compositions were synthesized by free radical copolymerization at 343 K in an aqueous solution in the presence of an initiator:  of clay drilling fluids.It was also found that the DADMAC–DMAPMA copolymer with a wide molar composition had an antibacterial activity at a concentration of not less than 0.05 wt% and can be used to inhibit the growth of the corrosion-dangerous bacteria, SRB.


Recently, the emergence of immunotherapy has revolutionized traditional tumour treatment. However, effective treatments for patients exhibiting αPD-1 resistance are still lacking. In our study, a combination of cytosine–phosphate–guanine oligodeoxynucleotides (CpG-ODNs), anti-OX40 and cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) injection in situ systematically generated a robust antitumour immune response in TC1 and B16 cells, which are αPD-1-resistant malignancies. More precisely, this method activates both adaptive and innate immunity. Additionally, in situ vaccination with CpG/αOX40/cGAMP fully activates the production of cytokines. However, the combination of αPD-1 does not improve the efficacy of triple therapy, prompting further questions. Collectively, the combination of CpG/αOX40/cGAMP causes the regression of various αPD-1-resistant tumours through the full mobilization of innate and adaptive immunity. In addition, we explored the therapeutic effect of triple therapy on the αPD-1-sensitive cell line CT26. The results showed that triple therapy could significantly enhance the therapeutic effect of αPD-1, and some mice even achieved complete tumour regression after the combined application of αPD-1 and triple treatment.The online version contains supplementary material available at 10.1007/s00262-021-03095-z. The incidence and mortality rates of cancer are increasing gradually and are primary factors threatening human health. According to global cancer statistics, in 2018, approximately 18.1 million new cases of cancer were diagnosed worldwide, and approximately 9.6 million deaths occurred [In recent years, the advent of immunotherapy has opened a novel chapter in the diversified treatment of various tumours, providing new hope for patients with cancer, particularly for some patients with advanced cancers. Currently, tumour immunotherapy is mainly divided into four categories according to the method: non-specific immune modulators, adoptive cell therapy, immune checkpoint inhibitors and tumour vaccines. Among them, the most common immunotherapy is PD-1/PD-L1 immune checkpoint inhibitors, which have been administered to many patients with cancer and have achieved a high response rate and lasting remission, significantly improving the survival rate of patients with recurrent or refractory Hodgkin's lymphoma –4, non-s+ T cells, and the combination of CpG with agonistic anti-OX40 antibody further enhances the antitumour effect of immune cells, systematically shrinking the tumours in mice, particularly lymphoma [In addition, in situ vaccination is a promising antitumour treatment method in which immune-enhancing agents are directly injected into the tumour site to stimulate the antitumour immune response throughout the body by inducing antigen release, promoting antigen presentation, activating immune effector cells, etc. . A growilymphoma . Temizozlymphoma . Accordilymphoma . Based olymphoma , 16. In lymphoma . HoweverAn extensive preliminary study and screening were performed to identify an in situ vaccine with good efficacy against αPD-1-resistant tumours. Notably, the combination of cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), a STING agonist and CpG/αOX40 exerted a powerful vaccine effect; specifically, systemic tumour growth was significantly slowed, and surprising results were observed. In addition, in situ vaccination with CpG/αOX40/cGAMP increased the infiltration of immune cells in distant tumours and throughout the body, fully mobilized the production of cytokines and simultaneously activated adaptive and innate immunity  were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. All mice were housed under specific pathogen-free conditions and maintained at a constant temperature (22 ± 1 °C) and humidity (60–70%). In addition, all animals were provided ultrapure water and clean food and housed on 12 h light/dark cycles. All animal experimental procedures were conducted in accordance with guidelines for the care and use of laboratory animals from the National Institutes of Health and were approved by the Animal Experimental Ethics Committee of Wenzhou Medical University.2.The TC1, B16 and CT26 cell lines were purchased from the Cell Bank of Chinese Academy of Science . They were cultured in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) (Sigma), 100 μg/mL penicillin and 100 μg/mL streptomycin in a humidified incubator at 37 °C with 5% COCpG-ODN 2395 was provided by Synbio Technologies . The agonistic anti-OX40 antibody was provided by BioXcell . 2′3’-cGAMP was purchased from InvivoGen (Cat.# tlrl-nacga23m). CD4-, CD8- and NK-depleting antibodies were purchased from BioXcell (USA).5 TC1/B16 cells (50 μL of PBS) on day 0. When the tumour volume reached 10–20 mm3 on day 6 or 7, the mice were randomly divided into 2 groups: the PBS and αPD-1 groups. PBS or αPD-1 (100 μg/mouse) was administered by intraperitoneal injection every 2 days for 3 injections.For the construction of a mouse model for the αPD-1 treatment experiment, the left flank of each C57BL/6 mouse was subcutaneously injected with 2 × 105 TC1/B16 cells (50 μL of PBS) (primary tumour) on day 0, and the same number of TC1/B16 cells was injected subcutaneously on the other side on day 2. On day 7, when the TC1 tumour volume reached 10–20 mm3, the mice were randomly divided into the following 5 groups: PBS, CpG, cGAMP, CpG/αOX40 and CpG/αOX40/cGAMP. CpG (50 μg/mouse), αOX40 (30 μg/mouse) and cGAMP (10 μg/mouse) were administered by in situ injection only into the primary tumour every 2 days for 3 injections. When the B16 tumour volume reached 5–10 mm3, the treatment began.For the mouse model of systemic tumour, the left flank of each C57BL/6 mouse was subcutaneously injected with 2 × 10+ T cells, CD8+ T cells or NK cells were successfully knocked out. The tumour dimensions were measured with Vernier callipers every 2 or 3 days, and the tumour volume was calculated using the following equation: length × width2/2 (mm3). Mice were killed when the tumour volume reached 1500 mm3.For the cell depletion experiment, anti-CD4 , anti-CD8  or anti-NK  mAbs were intraperitoneally injected at a dose of 200 μg/mouse one day before therapy. When the treatment began, each mouse was injected with 100 μg of the anti-CD4, anti-CD8 or anti-NK mAbs every 2 days for 5 injections. Finally, a flow cytometry analysis of blood samples was performed to validate whether CD45 CT26 cells into the left flank of Balb/C mice (primary tumour) and 2 days later injected the same number of CT26 cells into the right side (distant tumour). Mice were randomly divided into 4 groups—the PBS group, αPD-1 group, CpG/αOX40/cGAMP triple group and CpG/αOX40/cGAMP/αPD-1 quadruple group—and treated on day 5 with the same drug dose as previously described. The tumour size and body weight of the mice were observed every 2–3 days. The mice were killed when the tumour volume reached 1500 mm3.To explore the efficacy of the triple drug in the PD-1-sensitive cell line CT26, we subcutaneously injected 4 × 106 cells) was stained with CD45-APC-A750, CD3e-FITC, CD4-APC, CD8-PC7, CD3-APC, CD11c-FITC and MHC II-PE antibodies and incubated at 4 °C in the dark for 30 min. After washing with staining buffer, a CyFLEX flow cytometer (Beckman Coulter) was used to conduct flow cytometry. The voltage was regulated by a negative control and single dye tube. The final data were analysed using CytExpert software.Mouse spleen tissues were completely ground in the filter and digested with a tumour dissociation kit (Miltenyi Biotec Inc.) at 37 °C for 1 h, and the suspension was filtered through 40 μm sieves to obtain a single-cell suspension. Next, the single-cell suspension was placed in 0.02% saline for approximately 20 s and then neutralized with an equal volume of 0.16% saline to lyse the erythrocytes. One hundred microlitres of the single-cell suspension  after incubation at room temperature for 2 h, and the supernatant was collected to analyse the changes in cytokine levels in the treated mice. The serum levels of Th1 , Th2  and Th17 (IL-17A) cytokines were detected using a CBA kit according to the manufacturer’s instructions.P < 0.05 was considered significant.All statistical analyses were performed using GraphPad Prism 7.0 software and SPSS 16.0. All data are presented as means ± SEM. Two-way ANOVA was used to examine the differences between groups, and Mice bearing TC1/B16 tumours were treated with αPD-1 (100 μg/mouse) 3 times, but no difference in tumour volume was observed between the treatment group and the PBS group Fig. a–b. TherA mouse model of αPD-1-resistant tumours was established, and all the treatments were only administered to the primary tumour to study the systemic efficacy of an in situ vaccine in αPD-1-resistant tumours Fig. a. For miP < 0.01. The survival time of mice in the other treatment groups was not significantly different from that of the PBS group . Regardless of what cell type was knocked out, triple therapy still exerted a strong antitumour effect on the treated side of the tumours, and no difference in the tumour volume was observed compared with the triple therapy group without antibody knockout , Th2  and Th17 (IL-17A) cytokines were noticeably increased, particularly in the triple therapy group, including both immunostimulatory cytokines and immunosuppressive cytokines  to maintain and strengthen the antitumour response of CTLs. On the other hand, even in the absence of CD8+ T cells, CD4+ T cells also directly kill tumour cells through an IFN-γ-dependent mechanism [+ T cells, CD8+ T cells and NK cells were all knocked out, indicating that both adaptive immunity and innate immunity played an essential role. Previous studies have found that an intratumoural injection of CpG/cGAMP also induces adaptive immunity and innate immunity, which further confirmed our findings [In situ injection of antitumour drugs is a practical strategy for cancer immunotherapy , 22; in bination . However” tumour , 24. Altechanism –28. In tfindings .+ T cells and inhibits the progression of B16-F10 melanoma with lung metastasis [., CpG/αOX40 therapy increases the expression of the cytokines IL-12, TNF and IFN [In addition, triple therapy fully mobilized various cytokines, including Th1, Th2 and Th17 cytokines, which play important roles in regulating innate immunity and adaptive immunity. Th1 cytokines mainly include IL-2, IFN-γ, TNF and IL-12, which promote the activation and proliferation of cytotoxic T cells and dominate cellular immunity , 30. Meatastasis . Howevertastasis –43. AccoFinally, our study found that a new combination therapy also substantially suppressed the progression of tumours that failed to respond to PD-1 immune checkpoint inhibitors. Currently, two types of tumour immunotherapy are usually used: one is to relieve the inhibitory effect of cancer cells on immune cells by administering drugs, such as αPD-1/αPD-L1 and αCTLA-4 antibodies, an approach often compared to "releasing the brakes", 45; theCompared with other tumour immunotherapies, such as adaptive cell therapy, immune checkpoint inhibitors and tumour vaccines, the in situ injection of CpG/αOX40/cGAMP triggered an effective antitumour immune response while limiting the risk of systemic exposure and associated toxicity, which not only alters the tumour microenvironment at the injection site but also produces a systemic immune response and suppresses the development of distant tumours. Of course, our research also has some limitations. First, in situ injections are only achieved when the tumour is sufficiently large and accessible, which leads to the practical limitations of tumour treatment. Second, our analysis of the antitumour mechanism of triple therapy was not performed at a sufficient depth, and the reasons why αPD-1 does not enhance the curative effect of CpG/αOX40/cGAMP are not clear; therefore, the escape mechanism requires further exploration in future studies.In summary, we designed an effective and safe drug combination. In situ CpG/αOX40/cGAMP injection generates a systemic antitumour immune response and simultaneously activates adaptive and innate immune responses, which is expected to overcome the bottleneck of ineffective treatment with PD-1/PD-L1 immune checkpoint inhibitors and provide benefits to certain cancer patients.In general, our study found that in situ vaccination with CpG/αOX40/cGAMP can be an effective treatment in a murine model of anti-PD1-resistant tumours. This method can not only activate both adaptive and innate immunity but also fully activate the production of cytokines, including Th1, Th2 and Th17 cytokines, exerting a non-negligible "heating" effect on the tumour immune microenvironment Supplementary file2 (PDF 283 KB)Supplementary file2 (DOCX 13 KB)Below is the link to the electronic supplementary material.


We sought to validate findings for CpG sites within several of the top hit genes,AHRR,CYP1A1, andGFI1,using targeted quantitative bisulfite pyrosequencing. Comparing results from cord blood specimens of tobacco smoke-exposed to unexposed newborns, we confirmed significance at all previously identified CpG sites tested, including one inAHRR (p=0.007), three inCYP1A1 (p<0.0001), and one inGFI1 (p=0.008). These assays also captured novel differentially methylated CpGs located near the identified sites that were not included in the prior array-based studies . These results validate the prior findings and provide a simplified and more economical approach to analysis of CpG sites for expanded use as biomarkers ofin uterotobacco smoke exposure.Maternal exposure to tobacco smoke during pregnancy has been associated with many negative child health outcomes. Tobacco smoke exposure alters DNA methylation in the developing embryo/fetus and may be a mechanism that increases risk of later life disease. Previous studies have identified CpG sites in umbilical cord blood that are associated with Gestational exposureto tobacco smoke is associated with low birth weight , airway hyperreactivity associated with asthma , and neurodevelopmental effects such as attention deficit hyperactivity disorder . These outcomes have been linked to epigenetic alterations, including changes in DNA methylation .The Developmental Origins of Health and Disease (DOHaD) hypothesis is based on anecdotal and empirical findings that early life exposures are associated with later life susceptibility to disorders and diseases (Barker 1990). Our susceptibility toin uterotobacco smoke exposure  and identified 26 significant CpG probes. A number of these are in coding regions of the growth factor independent 1 transcription repressor (GFI1) and the aryl-hydrocarbon receptor repressor (AHRR) genes, in addition to a region upstream of the cytochrome P450 gene,CYP1A1. Further studies have validated these results using the same or similar array-based methods . Here we sought to validate these findings using bisulfite pyrosequencing assays to measure DNA methylation in umbilical cord blood leukocytes from infants who were unexposed or exposed to tobacco smoke during pregnancy.A previous study utilizing the Infinium HumanMethylation450 (450k) BeadChip reported differential methylation related toFigure 1A-1C. The 146 bp PCR amplicon forAHRRcontains three CpGs, one of which corresponds to probe cg05575921. The 226 bpCYP1A1 amplicon contains six CpG sites, including CpGs corresponding to probes cg22549041, cg11924019, and cg18092474. The 61 bpGFI1amplicon is 61 bp long and contains six CpG sites, one of which corresponds to probe cg09935388.The sequence and positions of the specific CpGs analyzed are presented inAHRR: R2=0.97, p=0.0028;CYP1A1: R2 >0.96, p=0.0029;GFI1: R2>0.92, p=0.003).Pyrosequencing assays were tested in duplicate or triplicate with mixtures of Qiagen’s Epitect fully methylated and unmethylated human DNAs containing 0%, 25%, 50%, 75% or 100% methylated DNA. Measured methylation was plotted against expected methylation levels. Goodness of fit assays for each CpG showed the assays measured incremental increases in DNA methylation as the amount of input methylated DNA increased across the range of possible values (AHRRcg05575921 (p=0.0007) ,CYP1A1 cg11924019, cg22549041, and cg18092474 (p<0.0001) , andGFI1cg09935388 (p=0.008) .The assays were then used to measure methylation in human umbilical cord blood DNAs from the Newborn Epigenetics STudy (NEST) cohort. Differential methylation was substantiated for the HumanMethylation450 probe CpGs forAHRR, sites p1 and p2 showed an average decrease in the exposed group of 3.9% (p=0.0009) and 4.9% (p<0.0001), respectively. ForCYP1A1, sites p2, p3, and p4 showed an average increase in the exposed newborns of 8.4% (p<0.0001), 6.7% (p<0.0001), and 6.0% (p=0.0006), respectively. TheGFI1assay identified only one additional CpG site as significantly differentially methylated. Site p5 exhibited a 9.1% (p<0.02) decrease in the exposed group.An advantage of targeted analyses by pyrosequencing is the ability to assess adjacent CpG sites that may not be represented on the 450k platform. Forin uterotobacco smoke exposure using array-based platforms . We found significantly different levels of methylation between unexposed and exposed infant cord blood at identified sites forAHRR,CYP1A1, andGFI1. We also showed significant differences in methylation at adjacent CpG sites that were not represented on the arrays used in the prior studies. These CpGs may collectively be useful as biomarkers forin uterotobacco smoke exposure using a relatively cost-efficient and scalable platform.Using an independent quantitative method, we corroborated the findings from previously published studies of cord blood showing altered DNA methylation in association withAHRRandCYP1A1are involved with the aryl-hydrocarbon receptor (AhR) pathway, which is known to facilitate changes in gene expression after toxicant exposure. Specifically, this pathway is known to mediate toxicity of polycyclic aromatic hydrocarbons (PAHs), several of which are found in cigarette smoke (Nguyen and Bradfield 2008). AlthoughGFI1has not been previously functionally associated with tobacco smoke exposure, it is involved with fundamental development processes such as hematopoiesis, inner ear and pulmonary neuroendocrine cell development, cellular proliferation, differentiation, and apoptosis, and pre-mRNA splicing control as well as immune system response .A limitation of the study was that we used self-reported exposure rather than cotinine levels for some cases in which these data were missing. There is a tendency for participants to underestimate exposure, which is either intentional under-reporting due to societal expectations or a lack of awareness of secondhand exposure. The overall effect of under-reporting would diminish the differences between groups, as the group classified as unexposed may contain exposed individuals.In conclusion, we have shown concordance between the previously reported 450k BeadChip findings and those from bisulfite pyrosequencing of an independent cohort. Our results provide the foundation for implementation of these relatively low-cost assays for defined analysis of these regions in other human cohort studies seeking to define exposure effects on these vulnerable regions of the genome.Samples. This research was approved by the Duke Institutional Review Board (protocol Pro00043033). Cord blood specimens were selected based on self-reported smoking status during pregnancy . These specimens were derived from the Durham, NC-based Newborn Epigenetics STudy (NEST), a mother-infant prospective cohort designed to determine the influence of early life exposures on the epigenome and health-related outcomes [see ].AHRR—42 unexposed, 58 exposed;CYP1A1—43 unexposed, 59 exposed;GFI1—41 unexposed, 50 exposed. Of these, maternal blood collected at enrollment for ~88% underwent measurement for cotinine, a major metabolic breakdown product of nicotine. For 12 women with missing cotinine values, self-report of tobacco exposure was used for exposure status classification. There was 87% congruence between self-reported exposure and exposure status from cotinine levels for the 820 women with plasma cotinine measurements in the parent study .Pregnant women were recruited from 2005 to 2011. Cord blood DNA was extracted from leukocytes separated by centrifugation from whole blood collected in vacutainer tubes containing EDTA. Samples were included in the analyses if we obtained clean PCR amplicons, the numbers of which were as follows:DNA Methylation Analysis. Genomic DNA was purified using Puregene Reagents (Qiagen) and 800 ng was modified with sodium bisulfite using the Zymo EZ DNA Methylation kit (Zymo Research). Pyrosequencing assays were designed to determine methylation levels at the previously identified significant CpG sites inAHRR,CYP1A1, andGFI114. All assay designs included adjacent CpGs not included on the array platform used in prior studies. Primers (Sigma) were designed using Qiagen Assay Design Software 1.0.6.AHRRPCR primers: F, 5’-TGG GGA TTG TTT ATT TTT GAG AG-3’; R, 5’-[biotin]AAA AAA CCC TAC CAA AAC CAC TC-3’.CYP1A1PCR primers: F, 5’-[biotin]ATG GGA GGT GAG GGG ATT-3’; R, 5’-CCC CAA TAC CAT TTA ACA TAA C-3’.GFI1PCR primers: F, 5’-AGG GAG TTA AGT GGT TAG ATA A-3’; R, 5’-[biotin] TAT ACC CAC AAC ACT CCA ATT C -3’. PCR was performed in 8-well strip tubes with total reaction volumes of 10 µL per well: 1 µL (~20 ng) BS-treated DNA and 9 µl master mix containing 5 µL PyroMark PCR buffer, 0.6 µL 25mM MgCl2, 1 µL 10x Coral Load (Qiagen), 0.12 µL of both primers at 10 µM (Sigma), and 2.16 µL molecular grade water. PCR conditions were:AHRR, 95°C for 15 minutes; then 55 cycles of 94°C for 30s, 58°C for 30s and 72°C for 30s; final extension 72°C for 10 minutes;CYP1A1, 95°C for 15 minutes; then 55 cycles of 94°C for 30s, 55°C for 30s and 72°C for 30s; final extension of 72°C for 10 minutes;GFI1: 95°C for 15 minutes; then 5 cycles of 95°C for 30s, 66°C for 30s and 72°C for 30s; 5 cycles of 95°C for 30s, 63°C for 30s and 72°C for 30s; 5 cycles of 95°C for 30s, 60°C for 30s and 72°C for 30s; then 45 cycles for of 95°C for 30s, 57°C for 30s, and 72°C for 30s with final extension of 72°C for 10 minutes.AHRR,5’-TTG TTT ATT TTT GAG AGG GTA-3’;CYP1A1, 5’- CCA AAA AAA AAA AAA TTA TAT T-3’; andGFI1: 5’-TTA AGT GGT TAG ATA AGG AT-3’. Validation of pyrosequencing assays was performed using EpiTect DNAs (Qiagen). Pyrosequencing was performed on a Qiagen Pyromark Q96 MD instrument and percent methylation was determined using Qiagen PyroMark CpG software 1.0.11.Sequencing primers were:Statistical analysis. All statistical analysis was conducted using GraphPad Prism version 7.03. Data from the unexposed and exposed samples were tested for normal distribution using the D’Agostino and Pearson normality test, and statistical differences between the two groups were determined using the Welch’s t-test for normally distributed data or Mann-Whitney test for data with a non-normal distribution.


However, OS was significantly worse in patients with T2mN0M0 than T2sN0M0 when the actual surgical proportion existed after PSM1 (p < 0.001). OS was similar between T2mN0M0 and IIIB regardless of whether PSM1 (p = 0.907) or PSM2 (p = 0.699) was performed. The surgical treatment was verified to associate with prognosis. Conclusions: The survival benefit by surgical treatment was existed in Stage II but not in Stage IIIB iCCA patients. The OS for T2mN0M0 will approach that of T2sN0M0 if the surgical proportion is gradually increased.Background: This study aimed to determine the role of surgical treatment in patients with stage II intrahepatic cholangiocarcinoma (iCCA). Methods: Data were extracted from the Surveillance, Epidemiology, and End Results (SEER) database. We divided stage II iCCAs into solitary tumors with vascular invasion (T2sN0M0) and multiple tumors with/without vascular invasion (T2mN0M0) according to the criteria of AJCC v.8. The Kaplan–Meier method and log-rank test were used to evaluate differences in overall survival (OS). We performed two propensity score-matching analyses with (PSM2) or without (PSM1) surgical treatment. Results: 667 and 778 iCCA patients with stage II and IIIB were recruited. After PSM2, there was no survival difference in stage II iCCA patients in hypothetical conditions with similar surgical proportions ( Intrahepatic cholangiocarcinoma (iCCA) is the second most common malignant tumor of the liver, accounting for 10% to 15% of all liver cancers . AlthougIn 2018, the eighth edition of the American Joint Committee on Cancer (AJCC) staging system (AJCC v.8) proposed a new standard for stratifying patients with iCCA, predicting the prognosis, and guiding treatment decisions . In AJCCHowever, we found that the survival difference was existed in stage II iCCA patients in clinical practice, especially in patients with multiple tumors with vascular invasion. It seemed like a shorter overall survival (OS) time existed in these patients compared with patients with solitary tumors. This was contrary to the conclusion of AJCC v.8. To identify the cause of this problem and explore the controversial issues surrounding stage II disease, we used the Surveillance, Epidemiology, and End Results (SEER) database in the United States (US) to investigate the prognostic differences between multifocal disease and solitary lesions in patients with stage II iCCA in an effort to provide a reasonable explanation for this controversy.In this study, stage II iCCAs were classified into the following two groups: (1) solitary tumors with vascular invasion and without lymph node or distant metastasis (T2sN0M0) and (2) multiple tumors with/without vascular invasion and without lymph node or distant metastasis (T2mN0M0). The primary objective of this study was to confirm whether the classification of T2N0M0 in AJCC v.8 is accurate. The secondary objective was to examine the previous classification of T2mN0M0 as stage IVa and determine the survival prognosis.Data were obtained from the SEER database, in which all patients with primary iCCA from 2004 to 2015 were identified. We used the SEER-18 database; its data come from 18 United States regions, accounting for about 27.8 percent of the total population. The inclusion criterion was a diagnosis of iCCA classified with the International Classification of Diseases for Oncology, third edition codes . TNM information was retrieved based on the following codes: derived AJCC T , derived AJCC N , derived AJCC M , and CS extension (2004–2015). In the project of CS extension (2004–2015), the codes corresponding to each group were as follows: T2sN0M0 , T2mN0M0 , and stage IIIB . The specific meaning of CS extension (2004–2015) is shown in The 1-, 3-, and 5-year overall survival (OS) of each group was estimated according to the Kaplan–Meier method, and survival curves were compared using the log-rank test. For stage II iCCAs, univariate and multivariable Cox proportional hazard models were used to investigate the association of demographics and clinical characteristics with OS. Categorical variables were compared using the chi-square test or Fisher’s exact test, and continuous variables were compared using the t-test. Propensity score matching (PSM) was performed using 1:1 fixed-ratio nearest-neighbor matching between T2sN0M0 and T2mN0M0. This is a technique that simulates a randomized experimental study in an observational data set in order to estimate a causal effect for key variables. So, we could use PSM to identify the prognostic role of independent factors in iCCA patients. In this study, we performed PSM twice (PSM1 and PSM2) for each group. The matched confounding factors for the PSM1 analysis included age, race, sex, and tumor size, and surgical treatment was added for the PSM2 analysis. In addition, a subgroup analysis according to sex , age (<70 and ≥70 years) and types of surgery  was performed. PSM was also performed using 1:1 fixed-ratio nearest-neighbor matching between T2mN0M0 and stage IIIB (including T4N0M0 and any TN1M0). All P values were two-tailed, and values of <0.05 were considered statistically significant. All statistical analyses were performed using SPSS version 26.0.0 .p = 0.026). However, there was no significant difference in age, ethnicity, or sex between the two groups, and most patients with iCCA were Caucasians (75.3%). Among 233 patients with T2sN0M0, 51.1% underwent surgical treatment. However, among 434 patients with T2mN0M0, only 28.6% underwent surgical treatment (p < 0.001).The clinical characteristics of all enrolled patients with T2N0M0 iCCAs (according to AJCC v.8) are summarized in p = 0.034). However, there was no significant difference in age, ethnicity, sex, or surgical treatment between the two groups.The clinical characteristics of patients with T2mN0M0 and stage IIIB are summarized in p < 0.001; hazard ratio [HR], 1.61; 95% confidence interval [CI], 1.36–1.9)    was significantly lower than that among patients with T2sN0M0  (log-rank .36–1.9) A. The su.36–1.9) . The log52–2.37) B. That m98–1.52) C. These p < 0.001; HR, 1.58; 95% CI, 1.28–1.96) and ≥70-year age group , T2mN0M0 showed worse survival than T2sN0M0  and ≥70 years    B,E. Afte52–3.32) . However86–1.90) C,F.p = 0.007; HR, 1.39; 95% CI, 1.09–1.76) and men     (The entire study population comprised 351 women and 316 men before PSM . T2mN0M049–2.42) A,D. Afte49–2.42) . Similar73–3.27) B,E. Afte73–3.27) . T2sN0M073–1.35) F. In men08–2.01) C. These p = 0.110), lobectomy group (p = 0.603) and extended lobectomy group (p = 0.382), T2sN0M0 and T2mN0M0 had similar OS     D. There 85–1.16) E. After 83–1.14) F. It seep = 0.040), surgical treatment , and multiple lesions  were independently associated with OS , larger tumor size (>5 cm), and multiple lesions were significantly associated with worse OS . Race we with OS .The AJCC v.8 provides a novel staging system for iCCA, and the staging of iCCA has thus gained significant attention . HoweverBefore PSM and after the PSM1 analysis in the present study, the survival of patients with T2sN0M0 was significantly better than that of patients with T2mN0M0. However, after the PSM2 analysis , no significant difference in OS was noted between the T2mN0M0 group and the T2sN0M0 group. Obviously, the key variable in these groups is surgical treatment. So we believe that this discrepancy was mainly caused by the difference in the ratio of surgical treatment between the two analyses. The rate of surgical treatment is higher for T2sN0M0 than for T2mN0M0 iCCA, and this was confirmed in our study. Additionally, it has been proven that surgical treatment can improve the survival of patients with iCCA . HoweverWe also compared T2mN0M0 and stage IIIB iCCAs. Our study indicated that before PSM and after the PSM1/PSM2 analyses, the survival of T2mN0M0 was similar to that of stage IIIB iCCAs. This result indicates that the survival benefit of surgical treatment in stage IIIB is unobvious. It is obvious that surgical treatment is not recommended as a priority in guidelines. In addition, it is worth noting that for men, even if the proportion of surgical treatment is similar , survival is still worse for T2mN0M0 than T2sN0M0 iCCA. This phenomenon indicates the presence of sex-related survival differences in T2N0M0 iCCA. Georgios et al.  suggesteIn terms of surgical treatment, we studied whether survival varies based on different types of surgery. A study by Angelico et al. showed that active surgical procedures are reasonable in selected iCCA patients with vascular involvement, and different types of surgery give patients acceptable surgical results. However, it is not clear whether T2mN0M0 iCCAs can gain similar surgical benefits to T2sN0M0 iCCAs in different types of surgery. Our study indicated that T2mN0M0 and T2sN0M0 had similar OS in a wedge or segmental resection, lobectomy, and extended lobectomy. This showed that among the three types of surgery, T2mN0M0 iCCAs can obtain comparable surgical benefits as T2sN0M0 iCCAs.Our multivariate analysis showed that age, surgical treatment, and multiple lesions were significantly associated with OS. This was also suggested by Raoof et al. , who repOur study cleverly used PSM twice to make a lot of meaningful findings. Our study confirmed that the surgical benefits of T2mN0M0 are comparable to T2sN0M0, and the same results were obtained even under different types of surgery. More importantly, our study pointed out that the prognosis of T2mN0M0 was worse than T2sN0M0, and the key factor leading to the poor prognosis of T2mN0M0 was its lower rate of surgical treatment. All these results are different from a study by Linlin Yin et al. . HoweverThe prognosis of T2N0M0 with multiple tumors and T2N0M0 with a solitary tumor is similar only when the proportion of patients undergoing surgery is similar. In fact, the prognosis of T2N0M0 with multiple tumors is significantly worse than that of T2N0M0 with a solitary tumor and similar to that of stage IIIB iCCA. However, patients who have T2N0M0 with multiple tumors can substantially benefit from surgical treatment; thus, increasing the proportion of surgical treatment in this population can improve the overall prognosis.