3-Methyladenine ameliorates surgery-induced anxiety-like behaviors in aged mice by inhibiting autophagy-induced excessive oxidative stress.

BACKGROUND: Postoperative anxiety is a common surgical complication in older patients. Research has recently linked excessive autophagy to several neurological disorders, including anxiety. This study aimed to determine whether 3-Methyladenine (3-MA) administration reduced anxiety-like behaviors in a mouse model following abdominal exploratory laparotomy. METHODS: An abdominal exploratory laparotomy model of postoperative anxiety was established using male C57BL/6 mice aged 20 months. 3-MA (6, 30, and 150 mg/ml) was administered via intracerebroventricular immediately following surgery. The mice were assessed 14 days after surgery using the marble burying, elevated plus maze tests, and local field potential recording in the amygdala. The levels of expression of phosphorylated-Akt, Beclin-1, LC3B, nuclear factor erythroid 2-related factor 2 (Nrf2)-occupied regions in NeuN-positive cells, superoxide dismutase (SOD) activity, malondialdehyde (MDA), and glutathione (GSH) were measured at 24 h after surgery. RESULTS: The injection of 3-MA reversed the increased number of marbles buried, decreased time spent in the open arm, and enhanced θ oscillation power after 14 days of abdominal exploratory laparotomy. In addition, administration of 3-MA reduced the ratio of phosphorylated- to total-Akt, decreased expression in Beclin-1 and LC3B, attenuated MDA levels, and increased the ratio of Nrf2-occupied areas in NeuN-positive cells, SOD activity, and GSH levels under abdominal exploratory laparotomy conditions. CONCLUSIONS: 3-MA improved anxiety-like behaviors in aged mice undergoing abdominal exploratory laparotomy by inhibiting excessive autophagy-induced oxidative stress. These results suggest that 3-MA could be an effective treatment for postoperative anxiety.

Fc-specific and covalent conjugation of a fluorescent protein to a native antibody through a photoconjugation strategy for fabrication of a novel photostable fluorescent antibody.

Fluorophore-antibody conjugates with high photobleaching resistance, high chemical stability, and Fc-specific attachment is a great advantage for immunofluorescence imaging. Here, an Fc-binding protein (Z-domain) carrying a photo-cross-linker (p-benzoylphenylalanine, Bpa) fused with enhanced green fluorescent protein (EGFP), namely photoactivatable ZBpa-EGFP recombinant, was directly generated using the aminoacyl-tRNA synthetase/suppressor tRNA technique without any further modification. By employing the photoactivatable ZBpa-EGFP, an optimal approach was successfully developed which enabled EGFP to site-selectively and covalently attach to native antibody (IgG) with approximately 90% conjugation efficiency. After characterizing the Fc-specific and covalent manner of the EGFP-photoconjugated antibody, its excellent photobleaching resistance for immunofluorescence imaging was demonstrated in a model study by monitoring the toll-like receptor 4 (TLR4) expression in HepG2 cells. The proposed approach here for the preparation of a novel fluorescent antibody is available and reliable, which would play an important role in fluorescence immunoassay, and is expected to be extended to the generation of other biomolecule-photoconjugated antibodies, such as other fluorescent proteins for multiplex immunofluorescence imaging or reporter enzymes for highly sensitive enzyme immunoassays.Graphical abstract.

Chest Computed Tomography Image for Accurately Predicting the Optimal Insertion Depth of Left-Sided Double-Lumen Tube.

OBJECTIVE: The main objective of this study was to assess the feasibility and accuracy of measuring the distance between the vocal cord and carina using chest computer tomography (CT) as a guide for the intubation of a left-sided double-lumen tube (LDLT). DESIGN: Single-center, prospective, randomized study. SETTING: Local hospital in China. PARTICIPANTS: Sixty adult patients undergoing elective thoracic surgery requiring an LDLT for one lung ventilation were enrolled in this study. INTERVENTIONS: Patients were randomly allocated to the following 2 groups: blind intubation group (B group, n = 30) or chest computed tomography-guided group (C group, n = 30). The placement of the LDLT was accomplished using 1 of the 2 intubation methods. After intubation, an independent anesthesiologist evaluated the position of the LDLT and carina and bronchial injuries using fiber optic bronchoscopy. The number of optimal positions, the time for LDLT intubation, the time for fiber optic bronchoscope confirmation, and carina and bronchial injuries were recorded. RESULTS: Sixteen of 30 intubations in the B group were in optimal position, whereas 27 of 30 intubations in the C group were in optimal position; the difference was statistically significant (p < 0.01). The time for intubation of the LDLT took 118.0 ± 26.2 seconds in the B group and 71.5 ± 8.7 seconds in the C group (p < 0.01). The time for position confirmation using fiber optic bronchoscope took 40.8 ± 15.8 seconds in the B group and 18.7 ± 7.9 seconds in the C group (p < 0.05). The incidences of carina and bronchial injuries were obviously lower in the C group (occurred in 3 of 30 cases) than in the B group (11 of 30 cases) p < 0.05. The incidences of postoperative sore throat and hoarseness showed no significant differences between the 2 groups (p > 0.05). CONCLUSION: This study demonstrated that the method of measuring the distance between the vocal cord and carina according to the chest CT as a guide for the intubation of LDLT is more effective and more accurate than the blind intubation method.

Immobilization of unraveled immunoglobulin G using well-oriented ZZ-His protein on functionalized microtiter plate for sensitive immunoassay.

Highly efficient protein immobilization is extremely crucial for solid-phase immunoassays. We present a strategy for oriented immobilization of functionally intact immunoglobulin G (IgG) on a polystyrene microtiter plate via iminodiacetic acid (IDA)-Ni(2+) and ZZ-His protein interaction. We immobilized a ZZ-EAP (Escherichia coli alkaline phosphatase)-His fusion protein, which exhibits Fc binding, His tag, and intrinsic AP activities, and analyzed it against the interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP to investigate the specificity and efficacy of this method. We compared the IDA-Ni(2+)-(ZZ-His) method with ZZ-EAP random immobilization using sandwich enzyme-linked immunosorbent assay, and the results showed that the former method had an enhanced signal, 10-fold higher sensitivity, and a wider linear range. Thus, the proposed method allows a broad range of oriented immobilized functionally intact IgG antibodies on polystyrene plates using only one type of IDA-Ni(2+) chelate surface because the ZZ protein can bind to the Fc region of various IgGs.

Comparative characterization of recombinant ZZ protein-alkaline phosphatase and its application in enzyme immunoassays.

A functional fusion protein, which consists of an antibody and an enzyme that can be used in enzyme immunoassays, has been constructed. However, a quantitative comparison of the characteristics of fusion proteins and chemical conjugates of the parents, which are functionally produced in a uniform microbial system, has not been adequately achieved. In this study, a fusion protein between the ZZ protein and Escherichia coli alkaline phosphatase (AP) and the parental ZZ protein and AP for chemical conjugate was functionally produced in the same bacterial system. A detailed examination of the ZZ-AP fusion protein and the effect of the ZZ-AP chemical conjugate on IgG affinity and enzymatic activity were performed. Compared with the parents, the equilibrium dissociation constant of ZZ-AP conjugate decreased by 32 % and catalytic activity decreased by 24 %, whereas the ZZ-AP fusion retained full parental activities and exhibited an approximately tenfold higher sensitivity than that of ZZ-AP conjugate in enzyme-linked immunosorbent assay. Thus, ZZ-AP fusion is a promising immunoreagent for IgG detection and a potential biolinker between antibodies and reporter enzymes (i.e., IgG-ZZ-AP fusion complex) in immunoassays.

The efficacy and adverse effects of the Uniblocker and left-side double-lumen tube for one-lung ventilation under the guidance of chest CT.

One-lung ventilation (OLV) is essential in numerous clinical procedures, in which the left-sided double-lumen tube (LDLT) is the most commonly used device. The application of bronchial blockers, including the Uniblocker or Arndt blocker, has increased in OLV. The present study aimed to compare the efficacy and adverse effects of the Uniblocker and LDLT for OLV under the guidance of chest CT. A total of 60 adult patients undergoing elective left-side thoracic surgery requiring OLV were included in the study. The patients were randomly assigned to the Uniblocker group (U group, n=30) or the LDLT group (D group, n=30). The time for initial tube placement, the number of optimal positions of the tube upon blind insertion, the number of attempts to adjust the tube to the optimal position, incidence of airway device displacement, injury to the bronchi and carina, the duration until lung collapse and the occurrence of sore throat and hoarseness over 24 h following surgery were recorded. The time for successful placement of the LDLT was 83.9±19.4 sec and that for the Uniblocker was 84.3±17.1 sec (P>0.05). The degree of lung collapse 1 min following opening of the pleura was greater in the D group than that in the U group (P<0.01) and the time required for the lung to completely collapse was shorter in the D group (3.3±0.5 min) than that in the U group (8.4±1.2 min; P<0.01). On the contrary, the incidence of injury to the bronchi and carina was lower in the U group (2/30 cases) than in the D group (10/30 cases; P=0.02); the incidence of sore throat was also lower in the U group (2/30 cases) compared with that in the D group (9/30 cases). The mean arterial pressure of patients immediately following intubation was lower in the U group (122.0±13.4 mmHg) than that in the D group (129.2±12.1 mmHg; P<0.05). The results of the present study indicated that the extraluminal use of the Uniblocker under guidance of chest CT is an efficient method with few adverse effects in left-side thoracic surgery. The study was registered at ClinicalTrials.gov on 16th December 2017 (no. NCT03392922).

[The expression vector of murine secreting IL-1ß promotes proliferation and migration of Hepa1-6 hepatoma cells].

AIM: To establish a murine secreted mature peptide IL-1ß expression vector, transfect into Hepa1-6 hepatoma cells, and analyze the effect of recombinant IL-1ß on proliferation, migration, and its specific expression in Hepa1-6 hepatoma cells. METHODS: The murine AFP signal peptide encoding sequence and mature IL-1ß encoding fragment were linked together through overlapping PCR, and the chimeric DNA sequence was then inserted into a liver specific expression vector pLIVE(TM); to make a recombinant pLIVE-smIL-1ß which expressed secreted murine IL-1ß of classical pathway. pLIVE-smIL-1ß, pLIVE(TM); and pLIVE-lacZ were transfected into Hepa1-6 by jetPEI respectively. Transfection of the vectors were detected by ß-gal staining using pLIVE-lacZ transfectants. Cells treated with 5 µg/mL lipopolysaccharide were used as positive control and 3 µmol/L monesin was added into culture system to block classical pathway secretion, then sandwich ELISA was employed to detect the IL-1ß levels both in supernatant and in cytoplasm of each group of transfected cells. The proliferation of Hepa1-6 hepatoma cells was determined by MTT assay and migration of Hepa1-6 cells was assessed by scratch test in vitro. RESULTS: pLIVE-smIL-1ß vector successfully expressed murine IL-1ß in Hepa1-6 cells. Expression of the recombinant vector peaked at day 3 as indicated in a ß-gal staining method. After transfection, compared with Hepa1-6/mock cells, IL-1ß expression levels both in supernatant and in cytoplasm of Hepa1-6/smIL-1ß cell were significantly increased detected by ELISA. The proliferation of Hepa1-6/smIL-1ß group was markedly promoted in vitro detected by MTT assay. CONCLUSION: The recombinant expression vector can secret IL-1ß through classical pathway which significantly promoted proliferation and migration of hepatoma cells in vitro.

[IL-1ß can promote proliferation and migration of Hepa1-6 cells and impair IFNα-induced cell growth inhibition in vitro].

AIM: To analyze the effect of recombinant IL-1ß on proliferation, migration, and the effect on IFNα induced cell growth inhibtion. METHODS: The vector pLIVE-mIL-1ß was transfected into Hepa1-6 cells mediated by transIT-LT1. Gene expression level of IL-1ß was analyzed by RT-PCR and Sandwich ELISA. Cell migration was assessed using wound healing assay. RESULTS: IL-1ß significantly stimulated proliferation and migration of Hepa1-6 cells. However, expression of IL-1ß significantly down-regulated growth inhibition inducecd by IFNα. CONCLUSION: The recombinant vector could stably express IL-1ß and promote in vitro proliferation, migration, and impair IFNα-induced cell growth inhibition.

[Preparation and characterization of monoclonal antibodies for human BCSC-1 protein].

AIM: To obtain monoclonal antibodies against human BCSC-1 protein for further study of the structure and function of human BCSC-1 protein. METHODS: pET-30a-BCSC-1 plasmid was constructed and transformed into E.coli BL21 (DE3) to express recombinant protein. BALB/c mice were immunized with recombinant human BCSC-1 protein, hybridoma cell lines secreting monoclonal antibodies against human BCSC-1protein were screened by regular cell fusion and subcloning approach. The specificity of these monoclonal antibodies were determined by ELISA and Western blotting. Expression of BCSC-1 protein in pcDNA3.1/v5-HisB-BCSC-1transfected MCF-7 cells was identified by Immunohistochemistry. RESULTS: Successfully constructed a prokaryotic expression vector pET30a-BCSC-1.BCSC-1 protein was expressed in E.coli BL21 (DE3). One hybridoma cell line 1B3 stable in secreting specific monoclonal antibodies wa successfully obtained. It could bind specifically to BCSC-1 protein proved by Western blotting and Immunohistochemistry assay. CONCLUSION: Monoclonal antibodies of high specificity against human BCSC-1 protein has been successfully prepared, which laid the foundation for the further study of human BCSC-1 protein.

[Establishment of AFP promoter operated murine IL-1beta recombinant vector and its expression in H22 cells].

AIM: To establish a hepatoma specific murine IL-1beta (mIL-1beta) expression vector operated by AFP promoter and analysis its expression in H22 cell. METHODS: The chimeric operating sequence composed of the minimal AFP promoter and CMV enhancer(ECMV) was prepared through SOE-PCR. The sequence was inserted to replace the conventional enhancer and promoter in pIRES2-EGFP to establish the novel hepatoma specific vector p(afp)IRES2-EGFP. Full length of murine IL-1beta was amplified through RT-PCR by pfu DNA polymerase followed by cloning to establish the recombinant pIRES2-EGFP-mIL-1beta expression vector verified through PCR, restriction enzyme assay, DNA sequencing and cell transfection. p(afp)IRES2-EGFP-mIL-1beta was tranfected into H22 hepatoma cells and YAC-1 lymphoma cells in a transient transfection system mediated by jetPEI. Expression of the vector was observed under fluorescent microscope 48 h after transfection. Expression level of mIL-1beta was detected by RT-PCR. RESULTS: A 537 bp chimeric AFP promoter and ECMV was yield and inserted to establish a novel hepatoma specific vector p(afp)IRES2-EGFP proved by restriction enzyme assay, DNA sequencing and transfection. Full length murine IL-1beta was then amplified and cloned to establish the recombinant expression vector p(afp)IRES2-EGFP-mIL-1beta verified through repeated clony PCR, restriction enzyme assay by EcoR I and Xho I, DNA sequencing and transfection. Purified p(afp)IRES2-EGFP-mIL-1beta was transiently transfected into H22 cells and YAC-1 cells by jetPEI, and bright green fluorescence was only seen on the surface of H22 cells, indicating that p(afp)IRES2-EGFP-mIL-1beta can specifically express target gene within the murine hepatoma cells. Simutaneously, the expression level of mIL-1beta was markedly elevated in H22/mIL-1beta in RT-PCR assay. CONCLUSION: We successfully prepared a hepatoma specific expression vector named p(afp)IRES2-EGFP-mIL-1beta that could expression high level of murine IL-1beta in a transient transfection system.

[IL-1beta antisense RNA enhanced sensitivity of HepG2 cells to NK cell mediated cytotoxicity].

AIM: To investigate the inhibitory effect of IL-1beta antisense RNA on the sensitivity of HepG2 cells to the NK cell mediated cytotoxicity. METHODS: Two gene segments of IL-1beta [IL-1beta1(17-331, 315 bp) and IL-1beta2(246-505, 260 bp)] were selected for antisense RNA. Total RNA was extracted from PBMC of a healthy donor treated with LPS. IL-1beta1 and IL-1beta2 were prepared by RT-PCR. PCR products were cloned into pMD18-T-simple vector and then sub-cloned to construct the pcDNA3.0-antiIL-1beta1 and pcDNA3.0-antiIL-1beta2 antisense RNA expression vectors. HepG2 cells were transfected by jetPEI, the expression of antisense RNA in HepG2 cells was assayed by RT-PCR, level of IL-1beta was analyzed by intracellular staining. The response of HepG2 cells to NK-92 cells was assessed by MTT assay. RESULTS: Two gene fragments of 260 bp and 315 bp products were obtained by RT-PCR. The purified gene fragments were cloned to construct pMD18 T-IL-1beta1 and pMD18 T-IL-1beta2 which were verified by PCR, restriction enzyme assay (Xho I) and DNA sequencing. The PCR products using Pfu DNA polymerase from cloning vectors were sub-cloned to create the antisense RNA expression vectors of pcDNA3.0-antiIL-1beta1 and pcDNA3.0-antiIL-1beta2 which were confirmed by PCR, restriction enzyme assay (Pst I) and DNA sequencing. When transfected into HepG2 cells, HepG2 cells expressed high level of antisense RNA, and simultaneously expression of IL-1beta was markedly suppressed which rendered HepG2 cells to be more sensitive to NK-92 cell mediated cytotoxicity compared with the cells transfected by pcDNA3.0. The cytolytic activity of NK-92 cells to HepG2 cells increase about 20% at the effector to target ratio of 10:1. CONCLUSION: Inhibiting of proinflammatory cytokine IL-1beta can reduce the sensitivity of hepatoma cells to the NK cell mediated cytolysis which provide an useful way of rendering NK cell activity against hepatoma.

[Expression of recombinant mouse IL-1beta significantly down-regulates NK cell mediated cytolysis [corrected] against H22 hepatoma cells].

AIM: To investigate the effect of the expression of recombinant IL-1beta in H22 hepatoma cells on its response to NK cell mediated cytotoxicity. METHODS: BALB/c mouse was stimulated by 6% of starch. Total RNA was prepared from peripheral blood monocytes (PBMCs). IL-1beta gene (843 bp) was obtained by RT-PCR. The purified PCR product digested by Xho I and EcoR I was cloned into pIRES2-EGFP to construct the recombinant pIRES2-EGFP-mIL-1beta expression vector which was verified by PCR, restriction enzyme assay (Xho I and EcoR I) and DNA sequencing. Then the purified pIRES2-EGFP-mIL-1beta plasmid was transfected into H22 hepatoma cells by jetPEI. The expression level of recombinant IL-1beta was detected by RT-PCR and confocal microscopy. The cytotoxicity of wild-type spleenic NK cells against H22 cells was assessed by MTT assay. RESULTS: After the total RNA isolated from the starch stimulated BALB/c mouse PBMC, 843 bp IL-1beta gene in length was prepared by RT-PCR. The purified PCR product digested by EcoR I and Xho I was ligated by pIRES2-EGFP to create pIRES2-EGFP-mIL-1beta expression plasmid which was verified by PCR, restriction enzyme assay and DNA sequencing. Then pIRES2-EGFP-mIL-1beta was transfected into H22 hepatoma cells by jetPEI. RT-PCR and confocal microscopy assay showed these cells expressed high level of recombinant IL-1beta expression vector. In a 4-hour based MTT assay, IL-1beta in H22 cells was more resistant to NK92 cell mediated cytotoxicity compared with the cells transfected with pIRES2-EGFP. Meanwhile, the cytolytic capacity of the spleenic NK cells separated from wild-type mouse decreased about 10% when the ratio of effector to target was 40:1. CONCLUSION: The expression of proinflammatory cytokine IL-1beta can significantly down-regulate the cytolytic activity of NK cells against H22 hepatoma cells. It plays a crucial role in the immune escape of hepatoma from NK cell mediated innate immunity.