BACKGROUND: Current replacement procedures for stenosis or occluded arteries using prosthetic grafts have serious limitations in clinical applications, particularly, endothelialization of the luminal surface is a long-standing unresolved problem.METHOD: We produced a cell-based hybrid vascular graft using a bioink engulfing adipose-derived mesenchymal stromal cells (ADSCs) and a 3D bioprinting process lining the ADSCs on the luminal surface of GORE-Tex grafts. The hybrid graft was implanted as an interposition conduit to replace a 3-cm-long segment of the infrarenal abdominal aorta in Rhesus monkeys. RESULTS: Complete endothelium layer and smooth muscle layer were fully developed within 21 days post-implantation, along with normalized collagen deposition and crosslinking in the regenerated vasculature in all monkeys. The regenerated blood vessels showed normal functionality for the longest observation of more than 1650 days. The same procedure was also conducted in miniature pigs for the interposition replacement of a 10-cm-long right iliac artery and showed the same long-term effective and safe outcome. CONCLUSION This cell-based vascular graft is ready to undergo clinical trials for human patients.

OBJECTIVE: To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection. METHODS: The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs. RESULTS: A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated (P < 0.01). CONCLUSION DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.
Humans ; Autophagy ; Cell Death ; Cell Nucleus ; Human Umbilical Vein Endothelial Cells/virology* ; Dengue ; Proteome

OBJECTIVE: To compare the clinical efficacy of acupuncture, Chinese medication and combination of acupuncture and medication in the treatment of dry eye complicated with computer vision syndrome (CVS). METHODS: A total of 152 patients with dry eye complicated with CVS were randomly divided into an acupuncture-medication group (38 cases, 1 case was removed), an acupuncture group (38 cases, 1 case dropped off), a Chinese medication group (38 cases, 1 case was removed), and a western medication group (38 cases, 1 case dropped off). In the western medication group, sodium hyaluronate eye drop combined with esculin and digitalis glycosides eye drop were used. In the acupuncture group, acupuncture was applied at bilateral Taiyang (EX-HN 5)， Cuanzhu (BL 2), Fengchi (GB 20), Qimen (LR 14) , and Hegu (LI 4) etc., once a day. In the Chinese medication group, Yiqi Congming decoction formula ganule was given orally, one dose a day. In the acupuncture-medication group, acupuncture combined with Yiqi Congming decoction formula granule were used. All groups were treated for 14 d. The non-invasive first tear film break-up time (NIBUT f), non-invasive average tear film break-up time (NIBUT av), tear meniscus height (TMH), ocular surface disease index (OSDI) score, and CVS symptom score were compared between the patients of each group before and after treatment. RESULTS: After treatment, the NIBUT f, NIBUT av, and TMH were increased compared with those before treatment in the patients of the 4 groups (P<0.01); the NIBUT f and NIBUT av in the acupuncture-medication group and the acupuncture group were higher than those in the Chinese medication group and the western medication group (P<0.05), and the TMH in the acupuncture-medication group and the Chinese medication group were higher than those in the acupuncture group and the western medication group (P<0.05). After treatment, the OSDI scores, the various scores and total scores of CVS (except for head symptom score in the western medication group) were decreased compared with those before treatment in the patients of the 4 groups (P<0.01). The OSDI score, total score, eye symptom score, and body symptom score of CVS in the acupuncture-medication group were lower than those in the acupuncture group, the Chinese medication group, and the western medication group (P<0.01, P<0.05), the head symptom score of the acupuncture-medication group was lower than that in the western medication group (P<0.05), and the CVS physical symptom scores and mental cognitive symptom scores of the acupuncture-medication group and the acupuncture group were lower than those in the Chinese medication group and the western medication group (P<0.05). CONCLUSION Acupuncture has advantages in improving NIBUT f, NIBUT av, and CVS physical symptoms and cognitive symptoms, and the Chinese medication has advantage in improving TMH. The combination of acupuncture and Chinese medication has better effects compared with monotherapy.
Humans ; Dry Eye Syndromes/etiology* ; Acupuncture Therapy ; Computers ; Treatment Outcome ; Acupuncture Points ; Ophthalmic Solutions

Inductively coupled plasma mass spectrometry (ICP-MS) was applied to determine the concentrations of lead (Pb), cadmium (Cd) and arsenic (As) in Lindera aggregata (Sims) Kosterm. The physiologically based extraction test (PBET) digestion in vitro/Caco-2 cell model was established to investigate the bioaccessible contents of Pb, Cd and As in decoction of Lindera aggregata (Sims) Kosterm. The target-organ toxicity dose modification of HI method (TTD) was used to evaluate the cumulative risk caused by the combined exposure of the total levels of Pb, Cd and As in Lindera aggregata (Sims) Kosterm. and the bioaccessible contents in the decoction. The results showed that the total contents of Pb, Cd and As in 4 batches of samples were in the range of 2.901-3.872, 1.299-1.800 and 0.062-0.216 mg·kg^-1, respectively. After transportation by Cacco-2 cells, the bioaccessible contents of Pb, Cd, and As in the decoction were in the range of 0.045-0.080, 0.070-0.112 and 0.004-0.018 mg·kg^-1. The results of risk assessment showed that calculated by the total amounts of heavy metals in the Lindera aggregata (Sims) Kosterm., for the end points of nervous system, the cumulative risks of co-exposure of heavy metals in 3 batches of samples were of concern. After decoction and transportation by Caco-2 cells, for the end points of cardiovascular system, blood, nervous system, kidney and testis, the TTD modification of HI values of all batches of samples were less than 1, and the health risks were acceptable. The study provided methodology basis for a more objective assessment of the health risks of heavy metals and harmful elements in traditional Chinese medicine and for a more scientific limit standard of heavy metals and harmful elements.

ObjectiveTo investigate the value of the serum levels of Clusterin and sphingosine 1-phosphate （S1P） in assessing the prognosis of sepsis patients with acute liver injury. MethodsA total of 127 sepsis patients with acute liver injury who were admitted to Lianyungang Hospital， Xuzhou Medical University， from March 2019 to May 2022 were enrolled， and according to their prognosis after 28 days of treatment， they were divided into death group with 35 patients and survival group with 92 patients. The independent-samples t test was used for comparison of continuous data between two groups， and the chi-square test was used for comparison of categorical data between groups. A pearson correlation analysis was used to investigate the correlation. The prognostic value of serum Clusterin and S1P was analyzed by receiver operating characteristic curve. ResultsThere were significant differences between the two groups in the degree of liver injury， Acute Physiology and Chronic Health Evaluation Ⅱ （APACHEⅡ） score， Sequential Organ Failure Assessment （SOFA） score， the presence or absence of acute kidney injury， prothrombin time （PT）， international normalized ratio （INR）， Child-Pugh class， and C-reactive protein （all P<0.05）. The death group had significantly lower serum levels of Clusterin and S1P than the survival group （t=11.094 and 10.390， both P<0.05）. The patients with severe liver injury had significantly lower serum levels of Clusterin and S1P than those with mild or moderate liver injury （t=9.825 and 11.418， both P<0.05）. The multivariate regression analysis showed that the degree of liver injury （odds ratio ［OR］=1.260， 95% confidence interval ［CI］： 1.081 — 1.468， P<0.05）， APACHEII score （OR=1.031， 95%CI： 1.019 — 1.044， P<0.05）， SOFA score （OR=1.066， 95%CI： 1.039 — 1.094， P<0.05）， Clusterin （OR=0.899， 95%CI： 0.859 — 0.940， P<0.05）， and S1P （OR=0.824， 95%CI： 0.749 — 0.908， P<0.05） were independent risk factors for the prognosis of patients with sepsis. The ROC curve analysis showed that serum Clusterin and S1P used alone or in combination had an area under the ROC curve of 0.864， 0.861， and 0.949， respectively. Serum Clusterin and S1P were significantly negatively correlated with alanine aminotransferase， total bilirubin， PT， and INR in sepsis patients with acute liver injury （all P<0.05）. ConclusionThe sepsis patients with acute liver injury who died had significant reductions in serum Clusterin and S1P compared with those who survived， and the levels of Clusterin and S1P are closely associated with the degree of liver injury. The combination of Clusterin and S1P has a good value in predicting the prognosis of sepsis patients with acute liver injury and is expected to become a potential marker for predicting the prognosis of sepsis patients with acute liver injury.

【Objective】 To compare the bioactive ingredients in activated platelet-rich plasma (PRP) from cord blood and adult blood, explore its reasonable indicators reflecting the inflammatory regulation ability, in order to guide the preparation. 【Methods】 PRP was prepared and activated from 63 healthy adults (31 males, 32 females) and 61 neonates (30 males, 31 females), and 20 cytokines were measured using Luminex technology for assessing the age- and sex-based bioactive differences of PRP. High-sensitivity C-reactive protein(hs-CRP), procalcitonin and MMPs/TIMPs from each sample were measured for their correlations with the 10 inflammation-related cytokines. 【Results】 The activated cord blood PRP released 10 growth factors and chemokines more than the adult blood PRP, whereas IGF-1, HGF and 8 pro-inflammatory cytokines lower than the latter. Most cytokines of adult PRP were more in females than in males (P<0.05), except for IGF-1 and HGF, which showed no difference by gender. Compared with hs-CRP and PCT, MMPs/TIMPs ratio was more closely related with the inflammation-related cytokines, which can reflect the inflammatory regulation of PRP. 【Conclusion】 Due to the lower immunocompetence and no age or gender disturbance, cord blood PRP has the rational MMPs/TIMPs ratio and more cytokines which promote the inflammation and wound healing.

Phage antibody display technology is currently the most widely used in vitro antibody screening technology,which uses bacteriophages as a vector,and inserts exogenous antibody library genes into phage capsid protein genes,and expresses the capsid protein on the phage surface while also displays the antibody protein.Antibody drugs play an important role in tumor immunity and microbial immunity due to their targeting advantages,which is also an important driving force for them to become a hot spot in the field of pharmaceutical research and development.Therefore,this article reviews the background,basic principles,antibody library types and antibody fragment types of phage display technology,and looks forward to the latest progress and application prospects of fully human antibodies.

BACKGROUND: Patients with atrial fibrillation (AF) and prior stroke history have a high risk of cardiovascular events despite anticoagulation therapy. It is unclear whether catheter ablation (CA) has further benefits in these patients. METHODS: AF patients with a previous history of stroke or systemic embolism (SE) from the prospective Chinese Atrial Fibrillation Registry study between August 2011 and December 2020 were included in the analysis. Patients were matched in a 1:1 ratio to CA or medical treatment (MT) based on propensity score. The primary outcome was a composite of all-cause death or ischemic stroke (IS)/SE. RESULTS: During a total of 4.1 ± 2.3 years of follow-up, the primary outcome occurred in 111 patients in the CA group (3.3 per 100 person-years) and in 229 patients in the MT group (5.7 per 100 person-years). The CA group had a lower risk of the primary outcome compared to the MT group [hazard ratio (HR) = 0.59, 95% CI: 0.47-0.74, P < 0.001]. There was a significant decreasing risk of all-cause mortality (HR = 0.43, 95% CI: 0.31-0.61, P < 0.001), IS/SE (HR = 0.73, 95% CI: 0.54-0.97, P = 0.033), cardiovascular mortality (HR = 0.32, 95% CI: 0.19-0.54, P < 0.001) and AF recurrence (HR = 0.33, 95% CI: 0.30-0.37, P < 0.001) in the CA group compared to that in the MT group. Sensitivity analysis generated consistent results when adjusting for time-dependent usage of anticoagulants. CONCLUSIONS In AF patients with a prior stroke history, CA was associated with a lower combined risk of all-cause death or IS/SE. Further clinical trials are warranted to confirm the benefits of CA in these patients.

Objective: To construct a myosin light chain kinase ( MLCK) knockout hypopharyngeal carcinoma FaDu cell line and discuss the knockout effect of MLCK on apoptosis of FaDu cells. Methods: MLCK knockout FaDu cell lines were constructed by liposome transfection of sgRNA and Cas9 2NLS Nuclease , and the knockout cell lines were identified by DNA sequencing and divided into control group , MLCK KO 1 group and MLCK KO 2 group. The knockout efficiency of MLCK was assessed by RT⁃qPCR and Western blot , and the effect of MLCK knockout on cell cycle and apoptosis was detected by flow cytometry while Western bolt was conducted to detect the effect of MLCK knockout on cell apoptosis. Results: DNA sequencing showed that MLCK base sequences were missing or substituted at the sgRNA sequence recognition , and RT⁃qPCR and Western blot showed lower mRNA and protein levels in MLCK knockout cell lines than those in the control group(P < 0. 000 1) . Furthermore , the flow cytometry assay showed that MLCK knockout did not significantly change the cycle of FaDu cells , but the rate of apoptosis increased (P < 0. 000 1) . Western blot showed that Bax/Bcl⁃2 (P < 0. 000 1) and Cleaved Caspase⁃3/Caspase⁃3 (P = 0. 000 7) ratios raised in the MLCK knockout group. Conclusion The knockout of MLCK induced apoptosis in FaDu cells , but the exact mechanism needs further study.

Objective:To explore the mechanism of tetrahydroxynonene(4-HNE)in the androgen antagonistic effect of prostate cancer through the androgen receptor(AR)-mitogen activated protein kinase(MAPK)signaling pathway.Method:Prostate canc-er LNCaP cells were divided into wild-type group(NC,control group)and transfection group.The transfection group was further divid-ed into empty vector transfection group(NC-L7 group)and GSTA4 gene transfection group(A0718,GSTA4-OE group).The GSTA4-OE group received LNCaP cell culture and GSTA4 plasmid transfection to construct LNCaP stable 4-HNE cell lines,while the control group received LNCaP cell culture without GSTA4 plasmid transfection.Stimulating prostate cancer cells with different concentrations of 4-HNE(0,40,80,120μmol/L)to activate the AR signaling pathway,Western blot was used to detect the expression of AR,MAKp,AKT,and PKCα proteins.Sixty cases of prostate cancer tissues and sixty cases of benign prostatic hyperplasia tissues were selected.Immunohistochemical staining was used to determine the positive expression rate of 4-HNE in the aforementioned tissues.The correla-tion between the positive expression of 4-HNE and tumor Gleason grade,as well as the progression of prostate cancer to CRPC,was an-alyzed.Result:The level of 4-HNE in the GSTA4-OE group cells was inhibited.Western blot analysis showed that compared with the control group,the GSTA4-OE group had PKC in cells α The protein expression level significantly decreased(P＜0.05),while the expression levels of AR and AKT proteins significantly increased(P＜0.05).After treating prostate cancer cells with 40,80,and 120μmol/L 4-HNE,compared with the control group,the expression level of AKT in the treatment group was significantly reduced(P＜0.01),while the expression levels of MAKP(P＜0.01),PKC(P＜0.01),and AR(P＜0.01)were significantly increased.The immunohistochemical results showed that the positive rate of 4-HNE was 5.0％in 60 cases of benign prostatic hyperplasia tissue and 63.3％in 60 cases of prostate cancer tissue,with a statistically significant difference(P＜0.01).The positive rates of 4-HNE in Gl-eason grades 1-5 were 41.2％,50.0％,63.6％,81.8％,and 100.0％,respectively.The higher the Gleason grade,the higher the positive rate of 4-HNE,and the difference was statistically significant(P＜0.05).During a follow-up period of 10-35 months,33 pa-tients advanced to CRCP,while 27 patients did not.The positive expression rate of 4-HNE in the two groups showed a statistically sig-nificant difference(P＜0.01).Conclusion:Under the action of 4-HNE,the expression of AR-MAPK pathway related proteins in-crease.4-HNE may promote the progression of prostate cancer through the AR-MAPK pathway,and 4-HNE is expected to become a new therapeutic target for CRPC.