Objective:To investigate the effects and underlying mechanisms of silent information regulator 1(SIRT1)on the dysfunction of umbilical vein endothelial cells(HUVECs)induced by oxidized low-density lipoprotein(ox-LDL).Methods:The impact of ox-LDL on the viability of HUVEC was assessed using the Cell Counting Kit-8(CCK-8)assay, which also facilitated the determination of the optimal ox-LDL concentration.Subsequent to ox-LDL treatment, several parameters were evaluated, including reactive oxygen species(ROS)production, apoptosis, migration, and angiogenesis, utilizing a ROS detection kit, flow cytometry, a Transwell migration assay, and an angiogenesis assay, respectively.The expression levels of apoptosis-related proteins, namely cleaved caspase-3(c-caspase-3), Bcl-2-associated X protein(Bax), B-cell lymphoma-2(Bcl-2), SIRT1, and peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α), were quantified using Western blot analysis.Adenoviral vectors were employed to either overexpress or silence SIRT1, while the ROS inhibitor N-acetylcysteine(NAC)was applied to assess its effects on cell function.Additionally, PGC-1α acetylation(Ac-Lys)was investigated through co-immunoprecipitation.Results:In the oxidative model of ox-LDL-stimulated HUVECs, compared to controls, we observed a significant increase in ROS-positive cells(35.9±3.1 vs.5.4±0.9), heightened apoptosis(16.3±0.9 vs.7.6±0.7), diminished endothelial cell migration capacity, and reduced angiogenic capacity.Additionally, there was an elevation in the pro-apoptotic protein c-caspase3 and Bax, alongside a decrease in the anti-apoptotic protein bcl-2.Furthermore, SIRT1 expression was increased, as was the expression of PGC-1α.In comparison to the GFP group(28.5±1.9), the reduction in SIRT1 expression resulted in an increase in apoptosis(37.0±1.9).Conversely, overexpression of SIRT1 mitigated ox-LDL-induced apoptosis(25.2±1.6)(all P<0.05).Notably, the expression levels of PGC-1α and SIRT1 exhibited consistent changes: PGC-1α expression increased with SIRT1 overexpression and decreased when SIRT1 expression was reduced(both P<0.05).The administration of NAC to the ox-LDL-treated group led to a reduction in ROS production( t=11.18, P<0.01)and a significant enhancement in cell function.Immunoprecipitation results indicated that SIRT1 overexpression decreased ox-LDL-induced PGC-1α acetylation( t=18.18, P<0.01), whereas silencing of SIRT1 further increased PGC-1α acetylation levels( t=-19.09, P<0.01). Conclusions:SIRT1 is shown to protect against ox-LDL-induced apoptosis and dysfunction in HUVECs by deacetylating and activating PGC-1α, thereby highlighting its therapeutic potential in the context of endothelial cell injury.

Background and Purpose:Previous studies have investigated the prognostic significance of skeletal muscle and adipose tissue composition and distribution in colorectal cancer patients,yet most have not differentiated between rectal and colon cancer patient cohorts.This study aimed to explore the relationship between body composition and long-term prognosis,and to develop a postoperative predictive model.Methods:Clinical data of rectal cancer patients who underwent surgical treatment at Qingdao University Affiliated Hospital from January 2018 to December 2021 were retrospectively collected.Inclusion criteria:①Age＞18 years;② Preoperative colonoscopy and pathological diagnosis of colorectal cancer;③ Complete surgical resection;④Abdominal computed tomography(CT)scan 1 month before surgery.Exclusion criteria:① Clinical data is missing;② Multiple metastases of tumors;③ Tumor T stage 0 or carcinoma in situ;④ Severe artifacts lead to poor quality CT imaging,making it difficult to distinguish between fat and muscle;⑤ Inability to obtain follow-up results.This study has been approved by the Medical Ethics Committee of the Affiliated Hospital of Qingdao University(approval number:QYFYWZLL30313),and informed consent has been waived in the ethical approval process.The skeletal muscle index(SMI)and subcutaneous adipose tissue index(SATI)were calculated by dividing the areas of skeletal muscle and subcutaneous fat observed on CT scans by the square of the patient's height.Univariate and multivariate COX regression analyses were conducted to identify risk factors influencing recurrence-free survival(RFS)and overall survival(OS)in rectal cancer patients.Based on the results of the multivariate analysis,a nomogram prediction model was developed,its predictive power and accuracy were assessed using the receiver operating characteristic(ROC)curve,calibration plots and decision curve analysis(DCA),and internal validation was conducted.Results:A total of 696 patients were included in this study,with 96(13.8%)patients experiencing postoperative recurrence and 89(12.8%)patients dying.Multivariate COX regression analysis showed that SMI,SATI,tumor T stage and N stage were independent factors affecting the postoperative RFS and OS of patients.Nomogram prediction models for RFS and OS in rectal cancer patients were constructed based on the above independent predictors.The area under ROC curve(AUC)for 3-,4-and 5-year RFS was 0.862,0.846 and 0.824,respectively;the AUC for 3-,4-and 5-year OS was 0.886,0.898 and 0.875,respectively.The models were evaluated using calibration curves and decision curves,and internal validation was performed,which showed that the prediction accuracy of the models was good.Conclusion:CT body composition is an independent predictor of RFS and OS in rectal cancer patients,and the nomogram model developed based on these factors demonstrates good predictive value for patient prognosis.

Objective:To investigate the impact of body composition and inflammatory nutritional indicators on postoperative complications in patients with obstructive colorectal cancer,and to develop and validate a nomogram model.Methods:This is a retrospective case series study. The clinical data of 293 patients with obstructive colorectal cancer who were treated at the Department of Gastrointestinal Surgery,the Affiliated Hospital of Qingdao University,between January 2016 and January 2024,were retrospectively collected. The cohort included 182 males and 111 females,aged (65.0±12.1) years (range: 18 to 80 years). The dataset was randomly divided into a training group ( n=196) and a validation group ( n=97) with a 7∶3 ratio. Independent sample t test and multivariate logistic regression analysis were employed to identify independent risk factors associated with postoperative complications in patients with obstructive colorectal cancer. A preoperative nomogram model was subsequently developed for predicting postoperative complications,which was further validated using a validation cohort. Results:The training group comprised 119 males and 77 females,with 68 cases experiencing postoperative complications and 128 cases without complications. The validation group included 63 males and 34 females,with 30 cases experiencing postoperative complications and 67 cases without complications.Univariate analysis and multivariate analysis revealed that low skeletal muscle index ( OR=0.867,95% CI: 0.795 to 0.947),high visceral fat index ( OR=1.058,95% CI: 1.028 to 1.089),high systemic immune inflammation index ( OR=1.002, 95% CI: 1.000 to 1.003), low prognostic nutritional index ( OR=0.847,95% CI: 0.782 to 0.917),and preoperative anemia ( OR=2.714,95% CI: 1.161 to 6.344) were independent risk factors for postoperative complications (all P<0.05). A nomogram prediction model based on these five indicators was established. The area under the receiver operating characteristic (ROC) curve for the prediction model was 0.878 (95% CI: 0.829 to 0.928) in the training group and 0.849 (95% CI:0.767 to 0.930) in the validation group. Conclusions:The preoperative nomogram model,which incorporates inflammatory and nutritional indicators,demonstrates a good accuracy in predicting postoperative complications for patients with obstructive colorectal cancer. This model can effectively assist in guiding treatment decisions.

Background and Purpose:Previous studies have investigated the prognostic significance of skeletal muscle and adipose tissue composition and distribution in colorectal cancer patients,yet most have not differentiated between rectal and colon cancer patient cohorts.This study aimed to explore the relationship between body composition and long-term prognosis,and to develop a postoperative predictive model.Methods:Clinical data of rectal cancer patients who underwent surgical treatment at Qingdao University Affiliated Hospital from January 2018 to December 2021 were retrospectively collected.Inclusion criteria:①Age＞18 years;② Preoperative colonoscopy and pathological diagnosis of colorectal cancer;③ Complete surgical resection;④Abdominal computed tomography(CT)scan 1 month before surgery.Exclusion criteria:① Clinical data is missing;② Multiple metastases of tumors;③ Tumor T stage 0 or carcinoma in situ;④ Severe artifacts lead to poor quality CT imaging,making it difficult to distinguish between fat and muscle;⑤ Inability to obtain follow-up results.This study has been approved by the Medical Ethics Committee of the Affiliated Hospital of Qingdao University(approval number:QYFYWZLL30313),and informed consent has been waived in the ethical approval process.The skeletal muscle index(SMI)and subcutaneous adipose tissue index(SATI)were calculated by dividing the areas of skeletal muscle and subcutaneous fat observed on CT scans by the square of the patient's height.Univariate and multivariate COX regression analyses were conducted to identify risk factors influencing recurrence-free survival(RFS)and overall survival(OS)in rectal cancer patients.Based on the results of the multivariate analysis,a nomogram prediction model was developed,its predictive power and accuracy were assessed using the receiver operating characteristic(ROC)curve,calibration plots and decision curve analysis(DCA),and internal validation was conducted.Results:A total of 696 patients were included in this study,with 96(13.8%)patients experiencing postoperative recurrence and 89(12.8%)patients dying.Multivariate COX regression analysis showed that SMI,SATI,tumor T stage and N stage were independent factors affecting the postoperative RFS and OS of patients.Nomogram prediction models for RFS and OS in rectal cancer patients were constructed based on the above independent predictors.The area under ROC curve(AUC)for 3-,4-and 5-year RFS was 0.862,0.846 and 0.824,respectively;the AUC for 3-,4-and 5-year OS was 0.886,0.898 and 0.875,respectively.The models were evaluated using calibration curves and decision curves,and internal validation was performed,which showed that the prediction accuracy of the models was good.Conclusion:CT body composition is an independent predictor of RFS and OS in rectal cancer patients,and the nomogram model developed based on these factors demonstrates good predictive value for patient prognosis.

Objective:To investigate the effects and underlying mechanisms of silent information regulator 1(SIRT1)on the dysfunction of umbilical vein endothelial cells(HUVECs)induced by oxidized low-density lipoprotein(ox-LDL).Methods:The impact of ox-LDL on the viability of HUVEC was assessed using the Cell Counting Kit-8(CCK-8)assay, which also facilitated the determination of the optimal ox-LDL concentration.Subsequent to ox-LDL treatment, several parameters were evaluated, including reactive oxygen species(ROS)production, apoptosis, migration, and angiogenesis, utilizing a ROS detection kit, flow cytometry, a Transwell migration assay, and an angiogenesis assay, respectively.The expression levels of apoptosis-related proteins, namely cleaved caspase-3(c-caspase-3), Bcl-2-associated X protein(Bax), B-cell lymphoma-2(Bcl-2), SIRT1, and peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α), were quantified using Western blot analysis.Adenoviral vectors were employed to either overexpress or silence SIRT1, while the ROS inhibitor N-acetylcysteine(NAC)was applied to assess its effects on cell function.Additionally, PGC-1α acetylation(Ac-Lys)was investigated through co-immunoprecipitation.Results:In the oxidative model of ox-LDL-stimulated HUVECs, compared to controls, we observed a significant increase in ROS-positive cells(35.9±3.1 vs.5.4±0.9), heightened apoptosis(16.3±0.9 vs.7.6±0.7), diminished endothelial cell migration capacity, and reduced angiogenic capacity.Additionally, there was an elevation in the pro-apoptotic protein c-caspase3 and Bax, alongside a decrease in the anti-apoptotic protein bcl-2.Furthermore, SIRT1 expression was increased, as was the expression of PGC-1α.In comparison to the GFP group(28.5±1.9), the reduction in SIRT1 expression resulted in an increase in apoptosis(37.0±1.9).Conversely, overexpression of SIRT1 mitigated ox-LDL-induced apoptosis(25.2±1.6)(all P<0.05).Notably, the expression levels of PGC-1α and SIRT1 exhibited consistent changes: PGC-1α expression increased with SIRT1 overexpression and decreased when SIRT1 expression was reduced(both P<0.05).The administration of NAC to the ox-LDL-treated group led to a reduction in ROS production( t=11.18, P<0.01)and a significant enhancement in cell function.Immunoprecipitation results indicated that SIRT1 overexpression decreased ox-LDL-induced PGC-1α acetylation( t=18.18, P<0.01), whereas silencing of SIRT1 further increased PGC-1α acetylation levels( t=-19.09, P<0.01). Conclusions:SIRT1 is shown to protect against ox-LDL-induced apoptosis and dysfunction in HUVECs by deacetylating and activating PGC-1α, thereby highlighting its therapeutic potential in the context of endothelial cell injury.

Objective:To investigate the impact of body composition and inflammatory nutritional indicators on postoperative complications in patients with obstructive colorectal cancer,and to develop and validate a nomogram model.Methods:This is a retrospective case series study. The clinical data of 293 patients with obstructive colorectal cancer who were treated at the Department of Gastrointestinal Surgery,the Affiliated Hospital of Qingdao University,between January 2016 and January 2024,were retrospectively collected. The cohort included 182 males and 111 females,aged (65.0±12.1) years (range: 18 to 80 years). The dataset was randomly divided into a training group ( n=196) and a validation group ( n=97) with a 7∶3 ratio. Independent sample t test and multivariate logistic regression analysis were employed to identify independent risk factors associated with postoperative complications in patients with obstructive colorectal cancer. A preoperative nomogram model was subsequently developed for predicting postoperative complications,which was further validated using a validation cohort. Results:The training group comprised 119 males and 77 females,with 68 cases experiencing postoperative complications and 128 cases without complications. The validation group included 63 males and 34 females,with 30 cases experiencing postoperative complications and 67 cases without complications.Univariate analysis and multivariate analysis revealed that low skeletal muscle index ( OR=0.867,95% CI: 0.795 to 0.947),high visceral fat index ( OR=1.058,95% CI: 1.028 to 1.089),high systemic immune inflammation index ( OR=1.002, 95% CI: 1.000 to 1.003), low prognostic nutritional index ( OR=0.847,95% CI: 0.782 to 0.917),and preoperative anemia ( OR=2.714,95% CI: 1.161 to 6.344) were independent risk factors for postoperative complications (all P<0.05). A nomogram prediction model based on these five indicators was established. The area under the receiver operating characteristic (ROC) curve for the prediction model was 0.878 (95% CI: 0.829 to 0.928) in the training group and 0.849 (95% CI:0.767 to 0.930) in the validation group. Conclusions:The preoperative nomogram model,which incorporates inflammatory and nutritional indicators,demonstrates a good accuracy in predicting postoperative complications for patients with obstructive colorectal cancer. This model can effectively assist in guiding treatment decisions.

Objective To study the quality comparability of human coagulation factor Ⅷ(FⅧ)produced before and after the change of factory site.Methods A comparative study was carried out on quality quantitative indexes,related im-purities and stability data of FⅧ produced before and after the change of factory site.Results The FⅧ quantitative quality before and after the change of factory site all met the quality standard,and the related impurities including aluminum resi-due,tributyl phosphate residue,polysorbate 80 residue and PEG residue all met the quality standard.Other impurities in-cluding human fibrinogen,fibronectin,plasminogen,IgA,IgM and IgG were extremely low in content and equivalent in quality.The content of VWF(von Willebrand factor)had no obvious change before and after the change of factory site,but was significantly higher than that of other domestic manufacturers'commercial products.The results of accelerated stability and long-term stability tests showed that the titer of FⅧ fluctuated within the methodological error range,and the results all met the quality standard.Conclusion The change of factory site of FⅧ has no effect on the quality.

Objective:To explore the effect of the etoposide (VP-16) on the reactivation of human immunodeficiency virus (HIV-1) from latent infection and study its potential molecular mechanism.Methods:The HIV-1 latently infected cell line J-Lat was treated with DMSO, VP-16 and vorinostat (SAHA), flow cytometry was used to detect the positive ratio of green fluorescent protein (GFP) in J-Lat, which can reflect the efficacy of high concentration VP-16 on reactivation. Then the reactivation efficiency of VP-16 on HIV-1 latently infected cells at different concentration and treatment time was measured by flow cytometry. The expression of CD25, CD69 and interleukin-6 (IL-6) of the VP-16-treated J-Lat cells were also detected in the same way. The effect of VP-16 on the transcription of long terminal repeat (LTR) was tested using a dual-luciferase reporter assay. The protein expression of the silent information regulator 1 (SIRT1) and the acetylated nuclear factor κB (NF-κB) p65 under the effect of VP-16 was checked by western blot (WB). Lentivirus-mediated SIRT1 short-hairpin RNA (SIRT1-shRNA) was employed to knock down SIRT1, and then tested for efficiency of reactivation.Results:The results of flow cytometry showed that VP-16 specifically reactivated HIV-1 latently infected J-Lat cells in a concentration and time-dependent manner. Meanwhile, the expression of CD25 and CD69 was upregulated to a certain extent, but the expression of IL-6 was not affected. Dual-luciferase reporter assay suggested that VP-16 positively regulated the transcription of HIV-1 LTR through NF-κB signaling pathway. WB results indicated that VP-16 can inhibit the protein expression of SIRT1 and promote the acetylation of NF-κB p65. Lentivirus-mediated SIRT1-shRNA successfully knocked down the mRNA and protein expression of SIRT1, and reactivated the HIV-1 latency effectively.Conclusions:VP-16 upregulates the acetylation of NF-κB p65 by inhibiting the expression of SIRT1, which subsequently promotes the transcription of HIV-1 LTR and reactivates the latent HIV-1 infection.

An improved method for extracting high quality and quantity RNA from a jelly mushroom and a dimorphic fungus—Tremella fuciformis which is especially rich in polysaccharides, is described. RNA was extracted from T. fuciformis mycelium M1332 and its parental monokaryotic yeast-like cells Y13 and Y32. The A260/280 and A260/230 ratios were both approximately 2, and the RNA integrity number was larger than 8.9. The yields of RNA were between 108 and 213 µg/g fresh wt. Downstream molecular applications including reverse transcriptional PCR and quantitative real-time PCR were also performed. This protocol is reliable and may be widely applicable for total RNA extraction from other jelly mushrooms or filamentous fungi rich in polysaccharides.
Agaricales* ; Fungi ; Humans ; Methods* ; Mycelium ; Parents ; Polymerase Chain Reaction ; Polysaccharides* ; Real-Time Polymerase Chain Reaction ; RNA*