Objective To explore the mechanism of Gandou Fumu Decoction(GDFMD)for improving Wilson's disease(WD)in tx-J mice.Methods With 6 syngeneic wild-type mice as the control group,30 tx-J mice were randomized into WD model group,low-,medium-and high-dose GDFMD treatment groups,and Fer-1 treatment group.Saline(in control and model groups)and GDFMD(3.48,6.96 or 13.92 g/kg)were administered by gavage,and Fer-1 was injected intraperitoneally once daily for 14 days.Oil red and HE staining were used to observe lipid deposition and pathological conditions in the liver tissue;ALT,AST,albumin,AKP levels were determined to assess liver function of the mice.Western blotting and RT-qPCR were used to detect hepatic protein and mRNA expressions of GPX4,ACSL4,ALOX15,FTH1,FLT,TFR1,FAS,SCD1,and ACOX1,and Fe2+,MDA,ROS,SOD,GSH and 4-HNE levels were analyzed to assess oxidative stress.Results The mouse models of WD showed obvious fatty degeneration in the liver tissue significantly increased serum levels of ALT,AST and AKP,decreased albumin level,increased Fe2+,MDA,ROS,4-HNE levels,decreased SOD and GSH levels(P<0.05),lowered protein expressions of ACOX1,GPX4,FTH1,FLT,FAS,and SCD1,and increased protein contents of TFR1,ACSL4 and ALOX15 in the liver.Treatment with GDFMD and Fer-1 improved liver histopathology and liver function of the mouse models,decreased the levels of Fe2+,MDA and ROS,increased SOD and GSH levels,and reversed the changes in hepatic protein expressions.Conclusion GDFMD improves liver steatosis in mouse models of WD possibly by inhibiting hepatocyte ferroptosis through the GPX4/ACSL4/ALOX15 signaling pathway.

Objective·To analyze the root canal diameter and taper of mandibular first molars using cone-beam CT(CBCT),investigate age-related changes in these parameters,and propose optimized clinical protocols for root canal preparation.Methods·From October 2022 to October 2023,CBCT images of 240 healthy mandibular first molars(120 three-canal type cases and 120 four-canal type cases)were collected from patients aged 20?59 years at the Stomatology Center,Haikou Affiliated Hospital of Central South University Xiangya School of Medicine.The three-canal type and the four-canal type samples were respectively divided into four age groups(20?29,30?39,40?49,and 50?59 years),with 30 cases in each group.Root canal length was measured,and buccolingual and mesiodistal diameters were recorded at four levels(coronal,middle,apical,and foramen).The tapers of the coronal 1/3,middle 1/3,and apical 1/3 segments were calculated in both buccolingual and mesiodistal directions.Differences among the age groups were compared.Results·The buccolingual diameters of all root canals exceeded the corresponding mesiodistal diameters.At several levels of most root canals,the diameters in the<50-year-old groups were significantly larger than those in the 50?59-year-old group(P<0.05).The buccolingual tapers exceeded the corresponding mesiodistal tapers in all root canals of mandibular first molars.The mean mesiodistal tapers in different age groups ranged from 0.07 to 0.34 in the coronal 1/3 segment,0.03 to 0.09 in the middle 1/3 segment,and 0.05 to 0.11 in the apical 1/3 segment.At several levels of most root canals,the tapers in the<50-year-old groups were significantly larger than those in the 50?59-year-old group(P<0.05).Conclusion·Mandibular first molars exhibit age-related narrowing of root canal diameter and reduced taper in individuals aged≥50 years.For initial apical file selection,#15 files are recommended for the distal canals in the three-canal type,while#10 files are advised for all other canals in the three-canal type and all canals in the four-canal type.For patients aged 50 years or older,the files for the distal canals in the three-canal type can be adjusted to#10,and the files for the mesiobuccal and distallingual canals in the four-canal type to#8.Regarding preparation taper,a 0.08-taper orifice opener is recommended for the coronal 1/3 segment.During mid-lower segment preparation,0.04-taper master apical files are suggested for the mesiobuccal and mesiolingual canals in the three-canal type and the mesiolingual and distallingual canals in the four-canal type,0.06-taper files for the distal canals in the three-canal type and the mesiobuccal canals in the four-canal type,and 0.08-taper files for the distobuccal canals in the four-canal type.Root canal obturation is recommended to be performed using vertical compaction with large-taper gutta-percha cones combined with bioceramic sealer.

Objective To investigate the effects of Gandou Fumu Decoction on liver fibrosis,iron metabolism,and ferroptosis in patients with hepatolenticular degeneration(Wilson disease,WD).Methods Seventy-eight hospitalized patients with WD characterized by kidney and liver deficiency,with phlegm and blood stasis,from the Department of Neurology,the First Affiliated Hospital of Anhui University of Chinese Medicine,were randomly assigned to two groups using a random number table method.The control group(n=39)received sodium dimercaptosulfonate in combination with a low-copper and high-protein diet.The observation group(n=39)received the same treatment as the control group,with the addition of Gandou Fumu Decoction(one dose per day,taken twice daily,in the morning and evening).Both groups underwent six treatment cycles,each lasting eight days.Ultrasonographic parameters,including portal vein main trunk diameter(PVMD),portal vein velocity(PVV),shear wave velocity(SWV),liver stiffness measurement(LSM),serum liver fibrosis markers(hyaluronic acid[HA],laminin[LN],procollagen typeⅢN-terminal peptide[PⅢNP],collagen type Ⅳ[CⅣ],aspartate aminotransferase to platelet ratio index[APRI],fibrosis-4 index[FIB-4]),and iron metabolism indicators(serum iron[SI],ferritin[FT])were compared before and after treatment.The relationship between baseline iron metabolism markers and ultrasonographic parameters,as well as serum liver fibrosis markers,was analyzed.Clinical efficacy,traditional Chinese medicine(TCM)syndrome scores,and adverse reactions were also compared between the groups.Additionally,bioinformatics analysis was performed to identify potential targets of Gandou Fumu Decoction for WD treatment.Peripheral blood mononuclear cells were collected from a normal group of 20 healthy individuals,as well as from both the control and observation groups before and after treatment.Real time fluorogenic quantitative PCR was performed to validate the expression changes of these targets across the groups.Results Compared with pre-treatment values,no significant changes were observed in PVMD levels in either group after treatment.No significant change in PVV was observed in the control group,whereas a significant decrease was noted in the observation group(P＜0.01).SWV,LSM,HA,LN,PⅢNP,CⅣ,APRI,FIB-4,and FT levels were significantly reduced compared to pre-treatment levels(P＜0.05,P＜0.01),whereas SI remained unchanged.Compared with the control group,the observation group had no significant difference in PVMD but had significantly lower PVV,SWV,LSM,HA,LN,PⅢNP,CⅣ,APRI,FIB-4,and FT levels(P＜0.05,P＜0.01),whereas SI remained unchanged.The total effective rate of treatment in the observation group was significantly higher than that in the control group(P＜0.05).Both groups showed a significant reduction in TCM syndrome scores after treatment(P＜0.01),with a significantly greater reduction observed in the observation group(P＜0.01).No significant adverse reactions were reported during treatment.Before treatment,there was no significant correlation between the SI of both groups and PVMD,PVV,SWV,LSM,HA,LN,PⅢNP,CⅣ,APRI,and FIB-4.In the observation group,FT showed a positive correlation with SWV,LSM,LN,PⅢNP,CⅣ,APRI,and FIB-4(P＜0.05,P＜0.01),while in the control group,FT showed a positive correlation with HA,LN,PⅢNP,CⅣ,APRI,and FIB-4(P＜0.01).After treatment,in the control group,SI showed a positive correlation with APRI and FIB-4(P＜0.05,P＜0.01),but there was no significant correlation between SI in the observation group and FT in both groups with the above-mentioned indicators.Bioinformatics analysis identified four potential targets of Gandou Fumu Decoction for treating WD,namely heme oxygenase 1(HMOX1),peroxisome proliferator-activated receptor alpha(PPARα),small heat shock protein B1(HSPB1),and mitogen-activated protein kinase 3(MAPK3).Compared to the normal group,both the control and observation groups had significantly lower PPARαand HSPB1 expression and significantly higher HMOX1 and MAPK3 expression before treatment(P＜0.01).Compared to before treatment within the same group,both groups showed significantly increased PPARα and HSPB1 expression and significantly decreased HMOX1 and MAPK3 expression after treatment(P＜0.05,P＜0.01).After treatment,the observation group had significantly higher PPARα and HSPB1 expression and lower HMOX1 and MAPK3 expression than the control group(P＜0.05,P＜0.01).Conclusion Gandou Fumu Decoction demonstrates remarkable advantages in improving clinical efficacy,Chinese medicine syndrome scores,iron metabolism,liver fibrosis progression,ultrasound imaging parameters,and ferroptosis-related biomarkers expression in patients with WD,with a favorable safety profile.

Compared to traditional suturing, lung stapling using automatic staplers offers advantages such as smaller trauma, faster wound healing, ease of operation, and lower complication rates, making it widely used in clinical practice. However, there are significant differences in bronchial tissue thickness at different anatomical locations, and the market is flooded with various types of staplers. Currently, there is a lack of recommended stapling schemes for bronchial staplers at different anatomical locations. This article reviews the development and application of automatic staplers and summarizes some types of staplers that are currently used in clinical practice, with the aim of promoting the formation of individualized stapler selection protocols for minimally invasive thoracic surgery based on the Chinese population.

ObjectiveThis study aims to identify cuproptosis-related gene biomarkers in Alzheimer's disease（AD） using bioinformatics and machine learning methods， validate them at the clinical specimen level， and predict potential traditional Chinese medicine（TCM）. MethodsDifferentially expressed genes in the AD group and normal group were obtained using the Gene Expression Omnibus （GEO） database， and intersections were taken with reported cuproptosis-related genes to obtain differentially expressed cuproptosis-related genes. Machine learning methods were applied to identify core differential genes of cuproptosis in AD. Peripheral blood's single nucleated cells from clinical AD patients were collected， and the relative gene expression was clinically verified by real-time polymerase chain reaction（Real-time PCR）. Potential TCM regulating cuproptosis for AD were predicted by COREMINE database. ResultsA total of 12 cuproptosis-related genes were obtained， mainly involved in pathways of tricarboxylic acid cycle， 2-oxocarboxylic acid metabolism， and carbon metabolism. Five core cuproptosis-related genes， dihydrolipoamide dehydrogenase （DLD）， glutaminase （GLS）， pyruvate dehydrogenase E1 subunit beta （PDHB）， full name nuclear factor （erythroid-derived 2）-related factor 2 （NFE2L2）， and dihydrolipoamide branched-chain transacylase E2 （DBT） were finally screened using four machine methods. Thirty cases each of normal and AD patients were collected clinically. Compared with those in the normal group， minimum mental state examination （MMSE） and Montreal cognitive assessment （MoCA） were significantly decreased in the AD group （P<0.01）， Homocysteine（Hcy）， interleukin（IL）-6， C-reactive protein（CRP） ， and β amyloid protein（Aβ） indexes were significantly increased （P<0.01）， and malondialdehyde（MDA） indexes were decreased （P<0.05）. Superoxide dismutase（SOD） levels were significantly decreased （P<0.01）. The mRNA relative expressions of NFE2L2 and DBT were up-regulated （P<0.05）， and those of DLD， GLS， and PDHB were significantly down-regulated （P<0.01）. The TCM regulating cuproptosis-related genes for the treatment of AD were mainly based on the four Qi such as warmth， calmness， and cold， and the five flavors including bitterness， sweetness， and pungency， and it was attributed to the meridians of the liver， spleen， stomach， and kidney， with the efficacy of tonifying Qi， activating blood， eliminating phlegm， and resoling dampness. ConclusionDLD， GLS， NFE2L2， PDHB， and DBT can be used as novel diagnostic molecular markers for AD cuproptosis-related genes， and the corresponding potential therapeutic TCM can provide new ideas for the treatment of AD by TCM.

The liver regenerative capacity is crucial for patients with end-stage liver disease following partial hepatectomy (PHx). The specific bacteria and mechanisms regulating liver regeneration post-PHx remain unclear. This study demonstrated dynamic changes in the abundance of Parabacteroides distasonis (P. distasonis) post-PHx, correlating with hepatocyte proliferation. Treatment with live P. distasonis significantly promoted hepatocyte proliferation and liver regeneration after PHx. Targeted metabolomics revealed a significant positive correlation between P. distasonis and β-hydroxybutyric acid (BHB), as well as hyodeoxycholic acid and 3-hydroxyphenylacetic acid in the gut after PHx. Notably, treatment with BHB, but not hyodeoxycholic acid or 3-hydroxyphenylacetic acid, significantly promoted hepatocyte proliferation and liver regeneration in mice after PHx. Moreover, STAT3 inhibitor Stattic attenuated the promotive effects of BHB on cell proliferation and liver regeneration both in vitro and in vivo. Mechanistically, P. distasonis upregulated the expression of fatty acid oxidation-related proteins, and increased BHB levels in the liver, and then BHB activated the STAT3 signaling pathway to promote liver regeneration. This study, for the first time, identifies the involvement of P. distasonis and its associated metabolite BHB in promoting liver regeneration after PHx, providing new insights for considering P. distasonis and BHB as potential strategies for promoting hepatic regeneration.

OBJECTIVES: To explore the mechanism of Gandou Fumu Decoction (GDFMD) for improving Wilson's disease (WD) in tx-J mice. METHODS: With 6 syngeneic wild-type mice as the control group, 30 tx-J mice were randomized into WD model group, low-, medium- and high-dose GDFMD treatment groups, and Fer-1 treatment group. Saline (in control and model groups) and GDFMD (3.48, 6.96 or 13.92 g/kg) were administered by gavage, and Fer-1 was injected intraperitoneally once daily for 14 days. Oil red and HE staining were used to observe lipid deposition and pathological conditions in the liver tissue; ALT, AST, albumin, AKP levels were determined to assess liver function of the mice. Western blotting and RT-qPCR were used to detect hepatic protein and mRNA expressions of GPX4, ACSL4, ALOX15, FTH1, FLT, TFR1, FAS, SCD1, and ACOX1, and Fe²⁺, MDA, ROS, SOD, GSH and 4-HNE levels were analyzed to assess oxidative stress. RESULTS: The mouse models of WD showed obvious fatty degeneration in the liver tissue significantly increased serum levels of ALT, AST and AKP, decreased albumin level, increased Fe²⁺, MDA, ROS, 4-HNE levels, decreased SOD and GSH levels (P<0.05), lowered protein expressions of ACOX1, GPX4, FTH1, FLT, FAS, and SCD1, and increased protein contents of TFR1, ACSL4 and ALOX15 in the liver. Treatment with GDFMD and Fer-1 improved liver histopathology and liver function of the mouse models, decreased the levels of Fe²⁺, MDA and ROS, increased SOD and GSH levels, and reversed the changes in hepatic protein expressions. CONCLUSIONS GDFMD improves liver steatosis in mouse models of WD possibly by inhibiting hepatocyte ferroptosis through the GPX4/ACSL4/ALOX15 signaling pathway.
Animals ; Ferroptosis/drug effects* ; Mice ; Signal Transduction/drug effects* ; Drugs, Chinese Herbal/therapeutic use* ; Hepatolenticular Degeneration/drug therapy* ; Hepatocytes/metabolism* ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Fatty Liver/metabolism* ; Arachidonate 15-Lipoxygenase/metabolism* ; Coenzyme A Ligases/metabolism* ; Liver/metabolism* ; Male

Objective:To investigate the efficacy of Xingnaojing injection combined with Angong Niuhuang pill in the treatment of cerebral hemorrhage and their effects on neuronal injury. Methods:A prospective study was conducted on 117 patients with cerebral hemorrhage admitted to Department of Neurosurgery, Shaoxing Second Hospital, from January 2023 to August 2024. The patients were randomly divided into a control group and an observation group using a random number table method. The control group ( n = 59) was treated with Xingnaojing injection, while the observation group ( n = 58) was treated with Angong Niuhuang pill and Xingnaojing injection for 1 month. Clinical efficacy, neuronal cytokines (S-100B protein, neuron-specific enolase, and brain-derived neurotrophic factor), oxidative stress indicators (superoxide dismutase, malondialdehyde, and glutathione peroxidase), inflammatory factor levels (interleukin-1β, C-reactive protein, and interleukin-6), and adverse reactions (itching, chest tightness, gastrointestinal discomfort, rash, and increased heart rate) were compared between the two groups. Results:The total response rate in the observation group was significantly higher than that in the control group [94.83% (55/58) vs. 81.36% (48/59), Z = 2.27, P = 0.023]. After treatment, the levels of S-100B and neuron-specific enolase in the observation group were (0.43 ± 0.16) μg/L and (6.79 ± 1.28) μg/L, respectively, which were significantly lower than those in the control group [(0.62 ± 0.15) μg/L, (8.37 ± 1.15) μg/L, t = -6.62, -7.02, both P < 0.001]. Additionally, the level of brain-derived neurotrophic factor in the observation group was significantly higher than that in the control group [(63.58 ± 4.05) ng/L vs. (57.13 ± 4.39) ng/L, t = 8.26, P < 0.001]. After treatment, the levels of superoxide dismutase and glutathione peroxidase in the observation group were significantly higher than those in the control group ( t = 6.39, 6.94, both P < 0.001). The level of malondialdehyde in the observation group was significantly lower than that in the control group ( t = -5.95, P < 0.001). The levels of interleukin-1β, C-reactive protein and interleukin-6 in the observation group were significantly lower than those in the control group group ( t = -6.64, -8.29, -7.06, all P < 0.05). There was no significant difference in incidence of adverse reactions between the observation and control groups [13.79% (8/58) vs. 22.03% (13/59), χ2 = 1.35, P > 0.05]. Conclusions:The combination of Xingnaojing injection and Angong Niuhuang pill shows good efficacy in the treatment of cerebral hemorrhage, as it can reduce neuronal damage, oxidative stress reactions, and inflammatory responses. This combined treatment is worthy of clinical application.

Objective To investigate the effects of Gandou Fumu Decoction on liver fibrosis,iron metabolism,and ferroptosis in patients with hepatolenticular degeneration(Wilson disease,WD).Methods Seventy-eight hospitalized patients with WD characterized by kidney and liver deficiency,with phlegm and blood stasis,from the Department of Neurology,the First Affiliated Hospital of Anhui University of Chinese Medicine,were randomly assigned to two groups using a random number table method.The control group(n=39)received sodium dimercaptosulfonate in combination with a low-copper and high-protein diet.The observation group(n=39)received the same treatment as the control group,with the addition of Gandou Fumu Decoction(one dose per day,taken twice daily,in the morning and evening).Both groups underwent six treatment cycles,each lasting eight days.Ultrasonographic parameters,including portal vein main trunk diameter(PVMD),portal vein velocity(PVV),shear wave velocity(SWV),liver stiffness measurement(LSM),serum liver fibrosis markers(hyaluronic acid[HA],laminin[LN],procollagen typeⅢN-terminal peptide[PⅢNP],collagen type Ⅳ[CⅣ],aspartate aminotransferase to platelet ratio index[APRI],fibrosis-4 index[FIB-4]),and iron metabolism indicators(serum iron[SI],ferritin[FT])were compared before and after treatment.The relationship between baseline iron metabolism markers and ultrasonographic parameters,as well as serum liver fibrosis markers,was analyzed.Clinical efficacy,traditional Chinese medicine(TCM)syndrome scores,and adverse reactions were also compared between the groups.Additionally,bioinformatics analysis was performed to identify potential targets of Gandou Fumu Decoction for WD treatment.Peripheral blood mononuclear cells were collected from a normal group of 20 healthy individuals,as well as from both the control and observation groups before and after treatment.Real time fluorogenic quantitative PCR was performed to validate the expression changes of these targets across the groups.Results Compared with pre-treatment values,no significant changes were observed in PVMD levels in either group after treatment.No significant change in PVV was observed in the control group,whereas a significant decrease was noted in the observation group(P＜0.01).SWV,LSM,HA,LN,PⅢNP,CⅣ,APRI,FIB-4,and FT levels were significantly reduced compared to pre-treatment levels(P＜0.05,P＜0.01),whereas SI remained unchanged.Compared with the control group,the observation group had no significant difference in PVMD but had significantly lower PVV,SWV,LSM,HA,LN,PⅢNP,CⅣ,APRI,FIB-4,and FT levels(P＜0.05,P＜0.01),whereas SI remained unchanged.The total effective rate of treatment in the observation group was significantly higher than that in the control group(P＜0.05).Both groups showed a significant reduction in TCM syndrome scores after treatment(P＜0.01),with a significantly greater reduction observed in the observation group(P＜0.01).No significant adverse reactions were reported during treatment.Before treatment,there was no significant correlation between the SI of both groups and PVMD,PVV,SWV,LSM,HA,LN,PⅢNP,CⅣ,APRI,and FIB-4.In the observation group,FT showed a positive correlation with SWV,LSM,LN,PⅢNP,CⅣ,APRI,and FIB-4(P＜0.05,P＜0.01),while in the control group,FT showed a positive correlation with HA,LN,PⅢNP,CⅣ,APRI,and FIB-4(P＜0.01).After treatment,in the control group,SI showed a positive correlation with APRI and FIB-4(P＜0.05,P＜0.01),but there was no significant correlation between SI in the observation group and FT in both groups with the above-mentioned indicators.Bioinformatics analysis identified four potential targets of Gandou Fumu Decoction for treating WD,namely heme oxygenase 1(HMOX1),peroxisome proliferator-activated receptor alpha(PPARα),small heat shock protein B1(HSPB1),and mitogen-activated protein kinase 3(MAPK3).Compared to the normal group,both the control and observation groups had significantly lower PPARαand HSPB1 expression and significantly higher HMOX1 and MAPK3 expression before treatment(P＜0.01).Compared to before treatment within the same group,both groups showed significantly increased PPARα and HSPB1 expression and significantly decreased HMOX1 and MAPK3 expression after treatment(P＜0.05,P＜0.01).After treatment,the observation group had significantly higher PPARα and HSPB1 expression and lower HMOX1 and MAPK3 expression than the control group(P＜0.05,P＜0.01).Conclusion Gandou Fumu Decoction demonstrates remarkable advantages in improving clinical efficacy,Chinese medicine syndrome scores,iron metabolism,liver fibrosis progression,ultrasound imaging parameters,and ferroptosis-related biomarkers expression in patients with WD,with a favorable safety profile.

Objective·To analyze the root canal diameter and taper of mandibular first molars using cone-beam CT(CBCT),investigate age-related changes in these parameters,and propose optimized clinical protocols for root canal preparation.Methods·From October 2022 to October 2023,CBCT images of 240 healthy mandibular first molars(120 three-canal type cases and 120 four-canal type cases)were collected from patients aged 20?59 years at the Stomatology Center,Haikou Affiliated Hospital of Central South University Xiangya School of Medicine.The three-canal type and the four-canal type samples were respectively divided into four age groups(20?29,30?39,40?49,and 50?59 years),with 30 cases in each group.Root canal length was measured,and buccolingual and mesiodistal diameters were recorded at four levels(coronal,middle,apical,and foramen).The tapers of the coronal 1/3,middle 1/3,and apical 1/3 segments were calculated in both buccolingual and mesiodistal directions.Differences among the age groups were compared.Results·The buccolingual diameters of all root canals exceeded the corresponding mesiodistal diameters.At several levels of most root canals,the diameters in the<50-year-old groups were significantly larger than those in the 50?59-year-old group(P<0.05).The buccolingual tapers exceeded the corresponding mesiodistal tapers in all root canals of mandibular first molars.The mean mesiodistal tapers in different age groups ranged from 0.07 to 0.34 in the coronal 1/3 segment,0.03 to 0.09 in the middle 1/3 segment,and 0.05 to 0.11 in the apical 1/3 segment.At several levels of most root canals,the tapers in the<50-year-old groups were significantly larger than those in the 50?59-year-old group(P<0.05).Conclusion·Mandibular first molars exhibit age-related narrowing of root canal diameter and reduced taper in individuals aged≥50 years.For initial apical file selection,#15 files are recommended for the distal canals in the three-canal type,while#10 files are advised for all other canals in the three-canal type and all canals in the four-canal type.For patients aged 50 years or older,the files for the distal canals in the three-canal type can be adjusted to#10,and the files for the mesiobuccal and distallingual canals in the four-canal type to#8.Regarding preparation taper,a 0.08-taper orifice opener is recommended for the coronal 1/3 segment.During mid-lower segment preparation,0.04-taper master apical files are suggested for the mesiobuccal and mesiolingual canals in the three-canal type and the mesiolingual and distallingual canals in the four-canal type,0.06-taper files for the distal canals in the three-canal type and the mesiobuccal canals in the four-canal type,and 0.08-taper files for the distobuccal canals in the four-canal type.Root canal obturation is recommended to be performed using vertical compaction with large-taper gutta-percha cones combined with bioceramic sealer.