Objectives: To elucidate the potential mechanisms of electroacupuncture (EA) in restoring detrusor-bladder neck dyssynergia (DBND) following suprasacral spinal cord injury (SSCI). Methods: A total of 52 specific pathogen-free (SPF) grade famale Sprague-Dawley (SD) rats (10 – 12 weeks, 250 – 280 g) were randomly assigned to either a sham group (n = 12) or a spinal cord injury model group (n = 40). In the model group, DBND was induced through Hassan Shaker spinal cord transection at T10 level, with 24 rats meeting inclusion criteria and subsequently randomized into DBND group (n = 12) and EA intervention group (DBND + EA group, n = 12). After spinal shock recovery (day 19 after modeling), DBND + EA group received EA treatment at Ciliao (BL32), Zhongji (RN3), and Sanyinjiao (SP6) acupoints for 20 min per session at 10/50 Hz frequencies, once daily for 10 d. Sham and DBND groups received anesthesia only without EA intervention. On day 29 post-modeling, all rats underwent urodynamic assessments, followed by hematoxylin and eosin (HE) staining, tandem mass tag (TMT) proteomics, and Western blot (WB) analysis of detrusor and bladder neck tissues. Differentially expressed proteins (DEPs) were defined as proteins with P < 0.05, unique peptides ≥ 2, and fold change > 1.2 or < 0.83. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed using KOBAS 3.0 (P < 0.01), and protein-protein interaction (PPI) networks were analyzed using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) 11.5 and Cytoscape 3.9.1. Results: Compared with sham group, DBND group showed significantly elevated leak point pressure (LPP) and maximum cystometric capacity (MCC) (both P < 0.01). EA treatment significantly reduced both LPP and MCC compared with DBND group (P < 0.01 and P < 0.05, respectively). HE staining revealed that EA reduced detrusor fibrosis and improved bladder neck inflammation. TMT proteomics identified 30 overlapping DEPs in detrusor and 59 overlapping DEPs in bladder neck when comparing DBND + EA/DBND groups with sham group. In detrusor tissue, KEGG analysis revealed 10 significantly enriched pathways (P < 0.01), including mitogen-activated protein kinase (MAPK) signaling pathway. PPI analysis showed 22 of 30 DEPs were interconnected. In bladder neck tissue, 14 pathways were significantly enriched (P < 0.01), including relaxin signaling pathway, with 51 of 59 DEPs showing interconnections. Both TMT and WB validations demonstrated that compared with sham controls, DBND rats exhibited upregulated collagen type IV alpha 2 chain (Col4a2) and downregulated guanine nucleotide-binding protein G(z) subunit alpha (Gnaz) in detrusor tissue, while EA treatment normalized both proteins (both P < 0.05). In bladder neck tissue, DBND rats showed decreased expression of smoothelin (Smtn) and calcium-activated potassium channel subunit beta-1 (Kcnmb1) compared with sham controls (both P < 0.01), which were both upregulated following EA treatment (P < 0.01 and P < 0.05, respectively). Conclusion EA restores detrusor-bladder neck coordination in DBND through dual-target mechanisms. In detrusor tissue, EA modulates contraction via extracellular matrix remodeling, cyclic adenosine monophosphate (cAMP) signaling pathway regulation, and enhanced adenosine triphosphate (ATP) biosynthesis mediated by neurotransmitters. In bladder neck tissue, EA promotes relaxation by maintaining contractile phenotypes, reducing fibrosis, suppressing smooth muscle excitation, and regulating presynaptic neurotransmitter release. These findings provide mechanistic insights into EA's therapeutic role in managing DBND.

Objective:To observe any effect of exercise preconditioning on the levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in the brain tissue of rats after induced cerebral ischemia and reperfusion, and how it might promote angiogenesis.Methods:Thirty-six male Sprague-Dawley rats were randomly divided into a sham-operation group, a model group and an exercise preconditioning group, each of 12. After adaptive running training for 3 days, the exercise preconditioning group ran daily for 30 minutes at 15m/min for 14 days, while the other two groups did not exercise. Middle cerebral artery occlusion and reperfusion were then induced in the model and exercise preconditioning groups using the modified Zea-Longa suture method. Rats in the sham-operation group were only cut open to expose the right carotid artery. Right after the modeling, and again 24 hours later neurological deficit was evaluated using the Zea-Longa score and modified neurological severity scoring (mNSS). Infarct sizes were measured using 2, 3, 5-triphenyl tetrazolium chloride staining. Any morphological changes were noted using hematoxylin and eosin (HE) staining, and the expression of CD31 protein, hypoxia-inducible factor-1α and vascular endothelial growth factor in the ischemic cerebral cortex were quantified immunohistochemically.Results:Right after the modelling, compared with the sham-operation group, the average Zea-Longa scores of the model and exercise groups had increased significantly, but were not significantly different from each other. Twenty-four hours later the average Zea-Longa score, mNSS score and relative cerebral infarction area of the model group had increased significantly compared with the sham-operation group, while the exercise preconditioning group′s averages had decreased significantly. The HE staining showed that compared with the sham-operation group, pathological changes such as loose tissue, reduced number of nerve cells, nucleolysis, and vacuolization of the cerebral cortex on the ischemic side were found in the model group. Compared with the model group, the pathological changes in the exercise preconditioning group were less serious. The levels of CD31 protein, HIF-1α and VEGF in the ischemic cerebral cortexes of the model group had by then increased significantly. But compared with the model group, those levels had increased more in the exercise preconditioning group.Conclusion:Exercise preconditioning can effectively promote angiogenesis after cerebral ischemia and reduce chronic injury. That may be related to the activation of the HIF-1α and/or VEGF signaling pathways.

Objective:To investigate the effect of exercise preconditioning on angiogenesis in ischemic brain tissue in rats with cerebral ischemia-reperfusion injury in the view of VEGF/VEGFR2/dock6 signaling pathway. Method:SD male rats were divided into sham group,model group and exercise preconditioning group by ran-dom number table method,with 18 rats in each group.The sham operation group and the model group were not given any treatment,while the exercise preconditioning group was given adaptive running training for 3 days at a speed of 10 m/min,once a day for 20 minutes each time.After the adaptive training,the exercise preconditioning group was given formal running training for 3 weeks,continuous training for 6 days a week,rest for 1 day,electric treadmill slope of 0°,speed of 15m/min,30min/d.Model group and exercise precondi-tioning group were modified to prepare the middle cerebal artery occlusion(MACO)models by Koizumi thread method,while sham operation group only given skin cutting without thread insertion.Zea longa score and modi-fied neurological severity score(mNSS)were used to score neurological deficit in rats,the relative infarct size of the brain was detected by TTC staining,the morphological changes of the ischemic cerebral cortex was ob-served by HE staining,the expression of CD31 in ischemic cerebral cortex was detected by immunohistochemis-try and the expressions of VEGFA,VEGFR2,Dock6 in ischemic cerebral cortex were detected by western blot. Result:①Zea-Longa scoring:after awaking from anesthetizati,compared with the sham group,the Zea-Longa scores of the other two groups were increased(P＜0.01),and there was no statistical significance in the Zea-Lon-ga scores between the two groups.At 72 hours after reperfusion,compared with the sham group,the Zea-Longa score of the rats in the model group was significantly increased(P＜0.01);compared with the model group,the Zea-Longa score of the rats in the exercise preconditioning group was significantly decreased(P＜0.05).②mNSS scoring:At 72 hours after reperfusion,compared with the sham group,the mNSS score of the rats in the mod-el group was significantly increased(P＜0.01);compared with the model group,the mNSS score of the rats in the exercise preconditioning group was significantly decreased(P＜0.05).③TTC staining:Compared with the sham group,the cerebral infarction volume in the model group was increased(P＜0.01),and compared with the mod-el group,the cerebral infarction volume in the exercise preconditioning group was decreased(P＜0.05).④ HE staining:Compared with the sham group,the model group rats appeared significant pathological changes in the cerebral cortex on the ischemic side.Compared with the model group,the pathological changes of the cerebral cortex on the ischemic side of the rats in the exercise preconditioning group were alleviated.⑤ Immunohisto-chemistry of CD31:Compared with the sham group,the expression of CD31 in the ischemic cerebral cortex of the model group was significantly increased(P＜0.05).The expression of CD31 in the ischemic cerebral cortex of the exercise preconditioning group was further increased(P＜0.05).⑥Western blot of VEGF,VEGFR2 and Dock6:Compared with the sham group,the expressions of VEGF(P＜0.05),VEGFR2(P＜0.05)and Dock6(P＜0.01)in the ischemic cerebral cortex of the model group were significantly increased;compared with the model group,the expressions of VEGF(P＜0.05),VEGFR2(P＜0.05)and Dock6(P＜0.01)in the ischemic cerebral cor-tex of the exercise preconditioning group were further increased. Conclusion:Exercise preconditioning can effectively promote angiogenesis after cerebral ischemia and reduce cerebral ischemia-reperfusion injury,which may be related to the activation of VEGF/VEGFR2/Dock6 signaling pathway.

The objective of this study is to investigate the effect of chemokine CXCR4 on the apoptosis of gastric cancer cells through IL-6/STAT3 signaling pathway and to explore the related mechanisms.Fluorescence quantitative PCR and Western blot were performed to detect the mRNA and protein expression levels of the chemokine CXCR4 in human gastric cancer tissues and adjacent non-cancerous tissues(PCT).Next,CXCR4 knockdown and overexpression were achieved by transfecting SGC7901 gastric cancer cell line with lentiviral vectors.TUNEL staining was used to evaluate the apoptosis of SGC7901 cells,while MTT assay was employed to measure cell proliferation.Western blot was conducted to determine the expression of apoptosis-related proteins Bax and Bcl2.Further,enzyme-linked immunosorbent assay(ELISA)was employed to measure the secretion levels of inflammatory cytokines.Real-time fluorescent quantitative PCR(RT-PCR)was utilized to quantify the expression of IL-6 mRNA in the IL-6/STAT3 signaling pathway,and Western blot was performed to analyze the expression of STAT3-Ser727 protein.In addition,after knocking down CXCR4 in SGC7901 cells,IL-6/STAT3 signaling pathway agonist lipopolysaccharide(LPS)was transfected,while in CXCR4-overexpressing SGC7901 cells,IL-6/STAT3 signaling pathway inhibitors angoline or bruceantinol were transfected.Then TUNEL staining was used to assess cell apoptosis,and Western blotting was performed to examine the expression levels of apoptosis-related proteins Bax and Bcl2 in these cells.Data showed that the expression of immune chemokine CXCR4 was increased in gastric cancer tissues,as compared with adjacent non-cancerous tissues.Single-cell gel electrophoresis analysis indicated that knockdown or overexpression of CXCR4 do not induce DNA damage in SGC7901 cells.TUNEL staining,MTT cell proliferation assay and Western blotting demonstrated that knockdown of CXCR4 in SGC7901 cells promoted the apoptosis in SGC7901 cells,while overexpression of CXCR4 inhibited the apoptosis.ELISA showed that knockdown of CXCR4 in SGC7901 cells promotes the expression of pro-inflammatory factors IL-1β and TNF-α,while inhibited the expression of anti-inflammatory factors IL-10 and TGF-β.Conversely,overexpression of CXCR4 demonstrated opposite effects.Finally,the activation of the IL-6/STAT3 signaling pathway significantly reduced the apoptosis induced by knocking down CXCR4 in iSGC7901 cells,whereas the inhibition of IL-6/STAT3 signaling pathway can significantly suppressed the induction of SGC7901 cells proliferation induced by CXCR4 overexpress.In conclusion,immunochemokine CXCR4 regulates gastric cancer cell apoptosis and inflammatory cytokines secretion through IL-6/STAT3 signaling pathway.

BACKGROUND: Given the general unavailability, common adverse effects, and complicated administration of tetracycline, the clinical application of classic bismuth quadruple therapy (BQT) is greatly limited. Whether minocycline can replace tetracycline for Helicobacter pylori ( H . pylori ) eradication is unknown. We aimed to compare the eradication rate, safety, and compliance between minocycline- and tetracycline-containing BQT as first-line regimens. METHODS: This randomized controlled trial was conducted on 434 naïve patients with H . pylori infection. The participants were randomly assigned to 14-day minocycline-containing BQT group (bismuth potassium citrate 110 mg q.i.d., esomeprazole 20 mg b.i.d., metronidazole 400 mg q.i.d., and minocycline 100 mg b.i.d.) and tetracycline-containing BQT group (bismuth potassium citrate/esomeprazole/metronidazole with doses same as above and tetracycline 500 mg q.i.d.). Safety and compliance were assessed within 3 days after eradication. Urea breath test was performed at 4-8 weeks after eradication to evaluate outcome. We used a noninferiority test to compare the eradication rates of the two groups. The intergroup differences were evaluated using Pearson chi-squared or Fisher's exact test for categorical variables and Student's t -test for continuous variables. RESULTS: As for the eradication rates of minocycline- and tetracycline-containing BQT, the results of both intention-to-treat (ITT) and per-protocol (PP) analyses showed that the difference rate of lower limit of 95% confidence interval (CI) was >-10.0% (ITT analysis: 181/217 [83.4%] vs . 180/217 [82.9%], with a rate difference of 0.5% [-6.9% to 7.9%]; PP analysis: 177/193 [91.7%] vs . 176/191 [92.1%], with a rate difference of -0.4% [-5.6% to 6.4%]). Except for dizziness more common (35/215 [16.3%] vs . 13/214 [6.1%], P = 0.001) in minocycline-containing therapy groups, the incidences of adverse events (75/215 [34.9%] vs . 88/214 [41.1%]) and compliance (195/215 [90.7%] vs . 192/214 [89.7%]) were similar between the two groups. CONCLUSION: The eradication efficacy of minocycline-containing BQT was noninferior to tetracycline-containing BQT as first-line regimen for H . pylori eradication with similar safety and compliance. TRIAL REGISTRATION ClinicalTrials.gov, ChiCTR 1900023646.
Humans ; Bismuth/therapeutic use* ; Metronidazole/therapeutic use* ; Esomeprazole/pharmacology* ; Minocycline/pharmacology* ; Helicobacter pylori ; Potassium Citrate/therapeutic use* ; Anti-Bacterial Agents ; Tetracycline/adverse effects* ; Helicobacter Infections/drug therapy* ; Drug Therapy, Combination ; Amoxicillin

Objective:To investigate the clinical characteristics of patients with malignant tumors and immune checkpoint inhibitors (ICI) related multisystem adverse events as well as therapeutic efficacy of ICI.Methods:The general data, immune-related adverse events (irAE) type, onset time, severity and ICI efficacy of patients with malignant tumors who developed irAE after receiving ICI in China-Japan Friendship Hospital between January 2019 and November 2021 were retrospectively analyzed. All patients were divided into multisystem irAE group and single system irAE group according to whether patients with more than 1 organ or system developed irAE for once. The occurrence of irAE was summarized, and the clinical characteristics of patients were compared. Progression-free survival analysis was not performed owing to the pause of immunotherapy caused by some irAE, so the efficacy of ICI was evaluated by using ICI treatment duration (TD).Results:A total of 47 patients with malignant tumors and irAE were included in this study, with 70 times of irAE in total. The median onset time was 90 d (35 d, 196 d). Among them, 12 patients (25.53%) developed multisystem irAE (32 times of irAE in total); the other 35 patients (74.47%) developed single system irAE (38 times of irAE in total). Cutaneous toxicity for 7 times, thyroid toxicity for 7 times and pulmonary toxicity for 5 times were the most frequent among multisystem irAE group; pulmonary toxicity for 13 times, thyroid toxicity for 12 times and cutaneous toxicity for 5 times were the most frequent among single system irAE group. There were no statistically significant differences in the proportion of patients stratified by age, gender, the combination of other treatments and different body mass between the two groups (all P > 0.05). The median follow-up time was 20 months (9-40 months). The median TD of ICI was 16.00 months (95% CI 3.62-31.22 months) in multisystem irAE group and 4.60 months (95% CI 4.12-11.30 months) in single system irAE group; TD in multisystem irAE group was longer than that in single system irAE group, and the difference was statistically significant ( HR = 0.413, 95% CI 0.202-0.844, P = 0.038). Conclusions:The efficacy of ICI in patients with malignant tumors and multisystem irAE is better than that in those with single system irAE. It suggests that the better efficacy of ICI may be associated with greater risk of irAE. There is no significant difference in the clinical features between multisystem irAE and single system irAE.

Objective:To analyze the effect of transitional care model (TCM) mode combined with resistance breathing training on hypoxic reactivity of respiratory center in elderly obese obstructive sleep apnea-hypopnea syndrome(OSAHS).Methods:Totally 78 patients with OSAHS who met the criteria were selected from the geriatric department of Nanjing Drum Tower Hospital from January 2018 to December 2020 and divided into observation group and control group by random digits table method, with 39 cases in each group; the control group was intervened by basic nursing combined with resistance breathing training mode, and the observation group was intervened by TCM mode on the basis of the control group. Before nursing and 1 month after discharge, Pittsburgh Sleep Quality Index (PSQI), Short-Form 36-item Health Survey (SF-36), Montreal Cognitive Assessment (MoCA) were used to evaluate the sleep quality, quality of life and cognitive function of the patients. Besides, FVC, FEV 1, FEV 1/FVC were also tested before nursing and 1 month after discharge. Results:One month after discharge, the daytime dysfunction, use of sleep drugs, habitual sleep efficiency, subjective sleep quality, sleep disorder, sleep latency, sleep duration and total score of PSQI in the observation group were significantly lower than those in the control group (the control group: 2.27 ± 0.34, 2.03 ± 0.31, 2.09 ± 0.23, 1.85 ± 0.28, 2.11 ± 0.28, 1.40 ± 0.24, 2.12 ± 0.41, 13.87 ± 0.56; the observation group: 1.63 ± 0.33, 1.22 ± 0.29, 1.63 ± 0.29, 1.12 ± 0.31, 1.35 ± 0.34, 1.09 ± 0.28, 1.74 ± 0.26, 9.78 ± 0.59) ( t values were 4.91-31.61, all P<0.01). One month after discharge, the scores of mental health, physical pain, physiological function, physiological function, emotional function, life vitality, social function and overall health of SF-36 in the observation group were significantly higher than those in the control group (the control group: 62.83 ± 6.31, 68.94 ± 5.91, 61.99 ± 5.98, 64.85 ± 6.13, 43.28 ± 5.74, 64.85 ± 6.12, 61.21 ± 5.74, 62.31 ± 6.85; the observation group: 69.81 ± 5.74, 76.12 ± 6.02, 70.84 ± 6.08, 71.74 ± 5.99, 50.93 ± 6.12, 70.52 ± 5.94, 69.89 ± 5.53, 68.41 ± 4.99)( t values were 4.18-7.77, all P<0.01). One month after discharge, the scores of visual space and executive function, attention, language, delayed recall, orientation, abstraction and total score of MoCA in the observation group were significantly higher than those in the control group (the control group: 4.48 ± 0.37, 5.23 ± 0.29, 2.43 ± 0.27, 3.37 ± 0.31, 5.01 ± 0.33, 5.27 ± 0.26, 25.79 ± 1.17; the observation group:4.95 ± 0.31, 5.68 ± 0.27, 2.67 ± 0.24, 3.98 ± 0.19, 5.47 ± 0.28, 5.64 ± 0.23, 28.39 ± 1.09)( t values were 4.17-10.51, all P<0.01). One month after discharge, the levels of FVC, FEV 1 and FEV 1/FVC in the observation group were significantly higher than those in the control group, the control group: (2.59 ± 0.18) L, (1.60 ± 0.14) L, (61.78 ± 4.01)%; the observation group: (2.89 ± 0.19) L, (1.99 ± 0.17) L, (68.86 ± 3.99)% ( t = 7.21, 11.14, 7.87, all P<0.05). Conclusions:TCM combined with resistance breathing training can effectively improve the hypoxic response of respiratory center in elderly obese patients with OSAHS.

Background Tin and its compounds can cause serious harm to human respiratory system and nervous system, but there is no corresponding national standard method for the determination of tin in PM_2.5. Objective To establish a method for the determination of tin and its compounds in PM_2.5 by atomic fluorescence spectrometry (AFS) after ultrasonic extraction with concentrated hydrochloric acid. Methods We extracted a fixed volume of air at a constant speed through a sampler with preset cutting characteristics to trap PM_2.5 in the ambient air on quartz filter membranes. By selecting extraction solvent, comparing extraction temperature and time, and adjusting the acidity of solution to be measured, the sample pretreatment process was optimized, and a method for the determination of tin and its compounds in PM_2.5 by AFS was proposed, and its performance indexes such as linearity, detection limit, and lower limit of quantification were obtained. The accuracy and precision of the method were evaluated by the standard addition recovery test with blank quartz filter membranes, and the interference test was carried out by adding standard urban particulate samples. The proposed method and the method recommended by the “Handbook on Monitoring and Protection of Air Pollution (Haze) Effects on Population Health (2020)” (the Handbook) were applied to actual samples, and the results were compared. Results This experiment used concentrated hydrochloric acid as the extraction solvent. The higher the reaction temperature and the longer the reaction time, the higher the recovery rate. Therefore, 70 ℃ water bath ultrasonic extraction for 3 h was selected. In terms of the proposed method, the linear range of detection was from 5.00 μg·L⁻¹ to 50.00 μg·L⁻¹, with a correlation coefficient ≥0.999 and a detection limit of 0.27 μg·L⁻¹. When the quantitative detection of the lower limit was 0.90 μg·L⁻¹，and the sampling volume was 144 m³, the limit of quantification was 1.25 ng·m⁻³. The recovery rate of standard addition of blank quartz filter membranes was 94.1%-97.5%, with a relative standard deviation ≤3.2%; the recovery rate of standard addition of standard urban particulate matter samples was 93.5%-103.0%, and the relative standard deviation was ≤2.1%, indicating that coexisting components in PM_2.5 samples would not affect the determination of tin. For the 10 quartz filter membrane samples of PM_2.5 monitoring, the results of tin by the established method (extraction with concentrated hydrochloric acid) were higher than those of the Handbook recommended method (extraction with nitric acid), and the difference is (3.61±0.54) ng·m⁻³(t=21.303, P<0.05). Conclusion The established method for the determination of tin and its compounds in PM_2.5 by AFS after ultrasonic extraction with concentrated hydrochloric acid is simple, accurate, and suitable for laboratory determination of tin and its compounds in large quantities of PM_2.5 samples.

Background::Helicobacter pylori ( H. pylori) infection is an infectious disease with a prevalence rate of up to 50% worldwide. It can cause indigestion, gastritis, peptic ulcer, and gastric cancer. H. pylori eradication treatment can effectively control disease progression and reduce the risk of the above conditions. However, the escalating trend of antibiotic resistance presents a global challenge for H. pylori eradication. We aim to provide guidance on pharmacological treatment of H. pylori infection. Methods::This clinical practice guideline is developed following the World Health Organization’s recommended process, adopting Grading of Recommendations Assessment, Development and Evaluation in assessing evidence quality, and utilizing Evidence to Decision framework to formulate clinical recommendations, minimizing bias and increasing transparency of the clinical practice guideline development process. We used the Reporting Items for practice Guidelines in HealThcare (RIGHT) statement and The Appraisal of Guidelines for Research and Evaluation II (AGREE II) as reporting and conduct guides to ensure the guideline’s completeness and transparency.Results::Though decreasing in developed countries, the prevalence of H. pylori remains high in developing countries, causing a major public health burden. This clinical practice guideline contains 12 recommendations concerning pharmacological treatment for H. pylori eradication. Among them, it is worth highlighting that bismuth preparations are inexpensive, safe, and effective, consequently making bismuth quadruple therapy a preferred choice for initial and rescue treatment. In empirical treatment, high-dose dual therapy is equally effective compared with bismuth quadruple therapy. Conclusions::The 12 recommendations in this clinical practice guideline are formed with consideration for stakeholders’ values and preferences, resource use, feasibility, and acceptability. Recommendations are generalizable to resource limited settings with similar antibiotic resistance pattern as China, and lower middle-income countries facing comparable sociological and technical challenges.Registration::Guidelines International Network (GIN) website, https://guidelines.ebmportal.com/node/69996.

Klebsiella pneumoniae(K.pneumoniae)is an important pathogen that can cause severe hospital-and community-acquired infections.To systematically investigate its methylation features,we determined the whole-genome sequences of 14 K.pneumoniae strains covering varying serotypes,multilocus sequence types,clonal groups,viscosity/virulence,and drug resistance.Their methy-lomes were further characterized using Pacific Biosciences single-molecule real-time and bisulfite technologies.We identified 15 methylation motifs[13 N6-methyladenine(6mA)and two 5-methylcytosine(5mC)motifs],among which eight were novel.Their corresponding DNA methyl-transferases were also validated.Additionally,we analyzed the genomic distribution of GATC and CCWGG methylation motifs shared by all strains,and identified differential distribution pat-terns of some hemi-/un-methylated GATC motifs,which tend to be located within intergenic regions(IGRs).Specifically,we characterized the in vivo methylation kinetics at single-base resolu-tion on a genome-wide scale by simulating the dynamic processes of replication-mediated passive demethylation and MTase-catalyzed re-methylation.The slow methylation of the GATC motifs in the replication origin(oriC)regions and IGRs implicates the epigenetic regulation of replication initiation and transcription.Our findings illustrate the first comprehensive dynamic methylome map of K.pneumoniae at single-base resolution,and provide a useful reference to better understand epigenetic regulation in this and other bacterial species.