OBJECTIVES: This work aimed to investigate the molecular mechanism of cyclic tensile stress (CTS) stimulating autophagy in human periodontal ligament cells (hPDLCs). METHODS: hPDLCs were isolated and cultured from normal periodontal tissues. hPDLCs were loaded with tensile stress by force four-point bending extender to simulate the autophagy of hPDLCs induced by orthodontic force du-ring orthodontic tooth movement. XMU-MP-1 was used to inhibit the Hippo signaling pathway to explore the role of the Hippo-YAP signaling pathway in activating hPDLC autophagy by tensile stress. The expression levels of autophagy-related genes (Beclin-1, LC3, and p62) in hPDLCs were detected by real-time quantitative polymerase chain reaction. Western blot was used to detect the expression levels of autophagy-related proteins (Beclin-1, LC3-Ⅱ/LC3-Ⅰ, and p62) and Hippo-YAP pathway proteins (active-YAP and p-YAP) in hPDLCs. Immunofluorescence was used to locate autophagy-related proteins (LC3-Ⅱand p62) and Hippo-YAP pathway proteins (active-YAP) of hPDLCs. RESULTS: CTS-activated autophagy in hPDLCs and expression of autophagy-related proteins initially increased and then decreased; it began to increase at 30 min, peaked at 3 h, and decreased (P<0.05). CTS increased the expression of active-YAP protein and decreased the expression of p-YAP protein (P<0.05). When XMU-MP-1 inhibited the Hippo-YAP signaling pathway (P<0.05), active-YAP protein was promoted to enter the nucleus and autophagy expression was enhanced (P<0.05). CONCLUSIONS The Hippo-YAP signaling pathway is involved in the regulation of autophagy activation in hPDLCs under CTS.
Humans ; Hippo Signaling Pathway ; Periodontal Ligament/metabolism* ; Beclin-1/metabolism* ; Cells, Cultured ; Autophagy

To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected TrxR1 knockout sites were obtained. Next, we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector. After transfection of the plasmid into HCT-116 cells, TrxR1 knockout HCT-116 cells were selected using puromycin resistance. The TrxR1 knockout efficiency was identified and verified by DNA sequencing, immunoblotting, TRFS-green fluorescent probe, and cellular TrxR1 enzyme activity detection. Finally, the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay. The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116 (HCT116-TrxR1-KO) cells. The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation. Based on these results, we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.
CRISPR-Cas Systems/genetics* ; Gene Editing ; Gene Knockout Techniques ; HCT116 Cells ; Humans ; RNA, Guide/metabolism*

Objective:To analyse the relationship between serum electrolyte concentrations and risk of cardiovascular events in physical examination population.Methods:A cross-sectional study design was applied to survey 8 445 adults whose serum high-sensitivity cardiac tropon Ⅰ (hs-cTnⅠ) and serum electrolytes (chloride, phosphorus, calcium, sodium, potassium and magnesium) concentrations were measured at the health examination center of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from January 1, 2018 to February 28, 2022. The risk of cardiovascular events was classified into three levels according to the serum hypersensitive cardiac troponin Ⅰ(hs-cTnⅠ) concentration: low, middle or high risk group. One-way analysis of variance was applied to compare the differences in serum electrolyte concentrations of participants with different risk levels of cardiovascular events. Ordered multi-category logistic regression was performed to analyze the correlation between serum electrolyte levels and the risk of cardiovascular events.Results:The concentration of potassium and magnesium ion in the subjects with low risk of cardiovascular events were both higher than those in the middle and high risk group [potassium ion (4.28±0.29) vs (4.24±0.34), (4.23±0.36) mmol/L, magnesium ion (0.88±0.06) vs (0.87±0.07), (0.87±0.07) mmol/L](both P<0.05), while the concentration of sodium ion was lower [(140.54±1.75) vs (140.88±1.73), (140.81±2.20) mmol/L]( P<0.001); the concentration of phosphorus ion in the high-risk group was lower than those in the middle and low risk groups [(1.04±0.17) vs (1.08±0.16), (1.05±0.15) mmol/L]( P=0.001); no significant difference was found in the concentrations of chloride and calcium ion among the three groups (both P>0.05). Compared to subjects with normal concentrations of electrolyte, the risk level of cardiovascular events in subjects with hypokalemia ( OR=6.96, 95% CI: 3.67-13.10) and hypomagnesemia ( OR=5.00, 95% CI: 1.01-24.50) was higher(both P<0.05). Within the normal range, sodium concentration was positively correlated with the risk of cardiovascular events ( OR=1.08, 95% CI: 1.03-1.14; P<0.001). Conclusions:The serum sodium, potassium and magnesium concentrations in health examination subjects are correlated with the risk of cardiovascular events. Maintaining the balanced concentration of serum potassium and magnesium, as well as low sodium levels within normal limits may help prevent cardiovascular events.

Objective To investigate the effect and the mechanism of nifedipine on oxidative stress trip-ping by iron-overload of HK-2 cells. Methods The cells were divided into 4 groups,blank group,iron overload group,nifedipine group and FAC with nifedipine co-treatment group. Cells were treated with FAC or/and nifedipine for 24 hours,and then malonaldehyde (MDA) content,superoxide dismutase (T-SOD) activity,glutathione (GSH) content,intracellular iron content and expression of DMT1 and FPN1 protein were evaluated. Results Compared to the iron overload group,both nifedipine treatment and co-treatment decreased the content of MDA (P < 0.05),increased the activity of SOD(P < 0.05),increased the content of GSH(P < 0.05),reduced the intracellular iron content(P<0.05),increased the expression of DMT1(P<0.05),and increased the expression of FPN1(P < 0.05). Conclusion Nifedipine plays a protective role against oxidative stress induced by iron-overload in HK-2 cells,and it is related to promote DMT1 and FPN1 protein expression and reduce intracellular iron content.

Objective To investigate the effects of different upper limb abduction angles on the occurrence of malposition of PICC into internal jugular vein. Methods Totally 210 cases of patients treated with PICCs for unilateral breast cancer of our hospital from August,2015 to January,2017 were randomly assigned to three groups with 70 cases in each group. The abduction angle of the upper limb for placement was set at 45° ,90° and 160° , respectively. We chose the basilic vein of the uninfected arm using modified Seldinger technique under the guidance of ultrasound for PICC,and malposition was confirmed by detecting the tip of PICC in internal jugular vein. Results The incidence rate of internal jugular venous dislocation in 45° group was 7.14%(5 cases) and 8.57% in 90° group (6 cases),and no internal jugular venous dislocation in 160° group. There was no statistically significant difference among 45°,90° and 160° groups(χ2=5.95,P>0.05). Significant differences of group comparisons for 45° v.s. 160° and 160° v.s. 90° were found (P<0.05). There was no statistically significant difference between 45° and 90° groups (P>0.05). Conclusion Abduction angles of 45° ,90° and 160° can all be used for PICC placement. The abduction angle can be selected according to specific situation of patients instead of being limited to the standard abduction angle of 90°.

Cystic echinococcosis (CE) treatment urgently requires a novel drug. The p38 mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases, but still have to be characterized in Echinococcus granulosus. We identified a 1,107 bp cDNA encoding a 368 amino acid MAPK protein (Egp38) in E. granulosus. Egp38 exhibits 2 distinguishing features of p38-like kinases: a highly conserved T-X-Y motif and an activation loop segment. Structural homology modeling indicated a conserved structure among Egp38, EmMPK2, and H. sapiens p38α, implying a common binding mechanism for the ligand domain and downstream signal transduction processing similar to that described for p38α. Egp38 and its phosphorylated form are expressed in the E. granulosus larval stages vesicle and protoscolices during intermediate host infection of an intermediate host. Treatment of in vitro cultivated protoscolices with the p38-MAPK inhibitor ML3403 effectively suppressed Egp38 activity and led to significant protoscolices death within 5 days. Treatment of in vitro-cultivated protoscolices with TGF-β1 effectively induced Egp38 phosphorylation. In summary, the MAPK, Egp38, was identified in E. granulosus, as an anti-CE drug target and participates in the interplay between the host and E. granulosus via human TGF-β1.
Cloning, Molecular* ; DNA, Complementary ; Echinococcosis ; Echinococcus granulosus* ; Echinococcus* ; Humans ; In Vitro Techniques ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Phosphotransferases ; Protein Kinases* ; Signal Transduction

Auxotrophic strains of N1-37 (Phe-) and N2-27 (His-), screened from mutations of Paenibacillus polymyxa JSa-9 previously, were used as the parent strains to screen high-producing LI-F antibacterial lipopeptide fusion strain through protoplast fusion with polyethylene glycol as a promote agent. Fusion strain F5-15 was obtained. Then the product of LI-F antibacterial lipopeptide was quantified by HPLC, and the difference of expression of the key genes of lipopeptide synthase between wild strain JSa-9 and the fusion strain was analyzed by real-time PCR. LI-F antibacterial lipopeptide yield of the fusion strain F5-15 was 3.1-fold of the original strain JSa9's, and the expression levels of the target genes were 10.48, 2.48, 2.1 and 11.8 fold of the initial strain JSa-9, respectively.
Anti-Bacterial Agents ; biosynthesis ; Chromatography, High Pressure Liquid ; Lipopeptides ; biosynthesis ; Paenibacillus ; metabolism ; Protoplasts ; metabolism ; Real-Time Polymerase Chain Reaction

We cloned the lipoxygenase gene (ana-LOX) from Anabaena sp. PCC 7120 and expressed it in Escherichia coli BL21 (DE3) pLysS. We determined the active site of the recombinant ana-LOX through site-directed gene mutagenesis and obtained the shortest length of the functional gene. Meanwhile, we studied the properties of recombinant ana-LOX after purification. The C-terminal of the Aos (allene oxide synthase)-LOX fusion gene in Anabaena sp. PCC 7120 genome was found belonging to LOXs family by bioinformatics analysis. Further results of site-directed gene mutagenesis confirmed that the active sites of ana-LOX were His197, His202, His369, Asn373and Ile455. The shortest length of functional gene was identified to be 1 254 bp based on the strategy of shortening the gene length gradually. The highest activity of recombinant ana-LOX of 6 750 U/mL could be achieved when constructed to pET-32a vector and expressed at low temperature 16 degrees C. We purified the enzyme by Ni-NTA chelating affinity chromatography, with 60.89% yield and specific activity of 11.4 x 10(4) U/mg. The optimum reaction temperature and pH for ana-LOX were 45 degrees C and 6.0, respectively. Furthermore, the obtained ana-LOX was stable at room temperature. The effect of metal ions on ana-LOX was determined also. Fe2+, Mg2+ Ca2+ could markedly promote the activity of this enzyme whereas Fe3+ and Cu2+ had a strong inhibitory effect on it. Finally, the ana-LOX could improve the microscopical structure of dough. The results of this study will provide a basis for future improvements and food industrial applications of ana-LOX.
Anabaena ; enzymology ; genetics ; Catalytic Domain ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; metabolism ; Lipoxygenase ; chemistry ; genetics ; Metals, Heavy ; chemistry ; Mutagenesis, Site-Directed ; Recombinant Proteins ; chemistry ; genetics

Objective To explore the relationship between polymorphism of catechol-O-methyltransferase (COMT) gene valine (Val) 158 methionine (Met) (G to A transition)and the distribution in population and esophageal squamous cell carcinoma (ESCC) in Yili prefecture of Xinjiang.Methods A hospital based case-control study was adopted, a total of 622 subjects, which including 214 ESCC patients and 408 age, gender and ethnicity-matched normal control individuals.The polymorphism of COMT gene G to A transition was analyzed with PCR-restriction fragment length polymorphism approaches.Results The COMT genotype frequencies in 622 subjects in Yili prefecture were GG genotype accounted for 47.3%, GA type for 42.3% and AA type for 10.4%, G allele was 68.4% and A allele was 31.6%.There was no statistical difference in the COMT genotype and frequencies of allele distribution between ESCC group and control group.Furthermore, stratified analysis indicated that there was statistical difference between ESCC group and control group in subjects less than 60 years old.There was statistical difference in the allele distribution among Kazak,Uygur and Han ESCC groups.The COMT genotype and frequency of allele distribution among normal control groups of the three ethnic groups were statistically different.After corrected age and gender,there was no statistical difference in COMT Val158Met polymorphisms among Kazakh, Uygur and Han ethnic groups in both ESCC and control groups in Yili Prefecture of Xinjiang.Conclusion COMT gene Val158Met single nucleotide polymorphism may not be the genetic markers of ESCC risk in Yili Prefecture of Xinjiang.

With the genomic DNA of strain EJS-3 as the template, we amplified the gene of fibrinolytic enzyme from Paenibacillus polymyxa (PPFE-I) by PCR. We purified the PCR product and ligated it into pMD19-T. After DNA sequencing, we cloned the PPFE-I gene into expression vector pET-DsbA and transformed it into Escherichia coli BL21(DE3). Upon induction of IPTG, we found that the activity of recombinant fibrinolytic enzyme fused with DsbA expressed in Escherichia coli was 228 IU/mL. SDS-PAGE analysis showed that the recombinant enzyme was soluble and accounted for about 18.4% of total cell protein. Western blotting demonstrated that the recombinant protein was DsbA-PPFE-I. We purified the recombinant enzyme by Ni affinity chromatography, thrombin digestion and sephadex G-100 gel-filtration, and identified the molecular weight of purified product to be 66.3 kDa with MALDI-TOF mass spectrometry. The purified enzyme exhibited distinct fibrinolytic activity on fibrin plate.
Antifibrinolytic Agents ; pharmacology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fibrinolytic Agents ; metabolism ; Genetic Vectors ; genetics ; Paenibacillus ; chemistry ; enzymology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology