Isoalantolactone Inhibits Esophageal Squamous Cell Carcinoma Growth Through Downregulation of MicroRNA-21 and Derepression of PDCD4.

BACKGROUND: This study was designed to explore the anticancer potential of isoalantolactone, a sesquiterpene lactone, on esophageal squamous cell carcinoma (ESCC) cells and associated molecular mechanisms. METHODS: ESCC cell lines were treated with isoalantolactone or vehicle and tested for viability, proliferation, cell cycle distribution, and apoptosis. Xenograft tumor studies in nude mice were done to examine the in vivo anticancer effect of isoalantolactone. RESULTS: Isoalantolactone treatment reduced ESCC cell viability and proliferation in vitro, which was coupled with induction of G0/G1 cell cycle arrest and apoptosis. In vivo studies confirmed the growth-suppressive effect of isoalantolactone on ESCC cells. Mechanistically, isoalantolactone reversed microRNA-21-mediated repression of programmed cell death 4 (PDCD4). Overexpression of microRNA-21 and knockdown of PDCD4 blocked the growth suppression and apoptosis induction by isoalantolactone in ESCC cells. CONCLUSIONS: Isoalantolactone shows growth-suppressive activity against ESCC cells, which is ascribed to upregulation of PDCD4 via downregulation of microRNA-21.

Measurement error in hydrostatic leveling system due to temperature effect and their reduction method.

Temperature is a crucial factor influencing the accuracy of the hydrostatic leveling system (HLS), necessitating a temperature compensation test for HLS. This paper investigates HLS temperature compensation through theoretical correction and experimental verification. The influence of temperature on the accuracy of hydrostatic level products is determined through temperature tests on 34 hydrostatic level products. The optimal temperature compensation formula is derived using the non-linear curve fitting method. The HLS is enhanced with a temperature compensation algorithm and temperature sensor, resulting in a new, high-precision, and high-stability hydrostatic leveling product with temperature compensation. Experimental findings reveal that the stability of the improved hydrostatic leveling product exceeds 30% compared to products on the market and surpasses 70% compared to the original products.

Identification of nonessential regions of the nsp2 protein of an attenuated vaccine strain (HuN4-F112) of highly pathogenic porcine reproductive and respiratory syndrome virus for replication in marc-145 cell.

BACKGROUND: The regions in the middle of nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) have been shown to be nonessential for PRRSV replication, and these nonessential regions are different in various viral strains. FINDING: In this study, the nonessential regions of the nsp2 of an attenuated vaccine strain (HuN4-F112) of highly pathogenic porcine reproductive and respiratory syndrome virus were identified based on an infectious cDNA clone of HuN4-F112. The results demonstrated that the segments of nsp2 [amino acids (aa) 480 to 667] tolerated deletions. Characterization of the mutants demonstrated that those with small deletions did not affect the viral growth on Marc-145 cells, but deletion of these regions led to earlier PRRSV replication increased (before 36 h after infectious in vitro). CONCLUSION: The functional roles of nsp2 variable middle region for PRRSV HuN4-F112 replication have been identified. Our results also suggested that none-essential region might be an ideal insertion region to express foreign gene in PRRSV genome.

A Propensity-Matched Analysis of Outcomes of Patients with Clinical Stage I Non-Small Cell Lung Cancer Treated surgically or with stereotactic radiotherapy: A Meta-Analysis.

OBJECTIVE: The aim of this study was to compare the efficacy between SBRT and surgery based on the Propensity-Matched Analysis. METHODS: Publications on comparison SBRT and Surgery for early stage non- small cell lung cancer (NSCLC) from 2011 to 2017 were collected. Propensity score matching was used to achieve comparable treatment hazard ratios of the overall survival (OS), local control survival (LC), regional control survival (RC), loco-regional control survival (LRC), distant control survival (DC), disease-free survival (DFS), and progression-free survival (PFS) between SBRT and Surgery. The major outcomes measures were hazard ratios (HRs). Meta-analysis Revman 5.3 software was used to analyze the combined Pooled HRs using fixed- or random-effects models according to the heterogeneity. RESULT: Eleven studies met our inclusion criteria. The LC, L-R C, DC, DFS and PFS rates of patients with early-stage lung cancer who were treated with SBRT are equal to surgical results. While, patients with surgery achieved superior OS compared with SBRT. CONCLUSION: In this study we carried out a meta-analysis, which controls the acceptable level of the efficacy in the propensity score to match patients. The surgery had obvious OS advantages in this meta-analysis. However, these conclusions would be proven by further studies incorporating comorbidity data, and outcomes from randomized control study. The final decision for the optimal treatment of a patient with early-stage NSCLC can be substantiated by a personalized treatment model.

[Effect of Montmorillonite on Fractions and Availability of Phosphorus in Soils Applied with Organic Fertilizer].

An incubation experiment was carried out to study the effect of montmorillonite on pH, the fractions and availability of phosphorus in a sandy yellow soil (SY) and a mineral flavicant soil (MF) applied with biogas residues of swine manure (BSR). The rates of montmorillonite applied were 0%, 5%, 7.5% and 10%, respectively. The results indicated that soil pH was not affected by BSR, but was significantly increased by montmorillonite. In both soils, application of BSR caused an increase in the content and proportion of Al-P during the incubation, and an increase in the content but a slight decrease in the proportion of Fe-P in later period of incubation. The concentration of O-P was not markedly changed, but its proportion was decreased by applying BSR in both soils. The changes of Ca-P varied with soils. BSR had no significant influence on Ca-P content, but decreased the proportion of Ca-P in SY, meanwhile, it increased both Ca-P content and proportion in MF. Addition of montmorillonite in soils applied with BSR decreased the contents and proportions of Al-P and O-P, and the proportion of Fe-P did not change the contents of Fe-P, but increased the content and proportion of Ca-P in SY. In MF, montmorillonite could decrease the contents and proportions of Fe-P and O-P, and the proportion of Al-P did not affect the content of Al-P, but increased the contents and proportions of Ca-P greatly. The contents of available phosphorus (A-P) were enhanced by 17.9%-38.0% and 17.1%-33.7% in SY and MF respectively, and the phosphorus activation coefficient (PVC) was significantly improved in both soils by applying BSR. The contents of A-P were reduced by 8.8%-35.5% and 1.1%-11.6% in SY and MF respectively. Correlation analysis showed that montmorillonite reduced the availability of phosphorus through increasing pH and contents of Ca-P, decreasing the content of Al-P in SY, and through increasing pH and decreasing content of Fe-P in MF. The availability of phosphorus in SY was influenced more obviously than that in MF by montmorillonite.

[Study on using NSP2 protein of porcine reproductive and respiratory syndrome virus (HuN4-F112) to express E2 neutralizing epitope of classical swine fever virus].

Establishment of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) with co-expression E2 Epitope of Classical Swine Fever virus (CSFV) is a crucial step to develop a genetic engineered vaccine against PRRSV and CSFV. Reverse genetic manipulation could be adopted as a com monly used technique. In this study, we focus on using nonessential regions of NSP2 (aa480-532 and aa508-532) as viral vector to express E2 Epitope of CSFV. A neutralizing epitope of classical swine fever virus (CSFV) E2 protein was inserted into the two nonessential region of nsp2 by the method of mutant PCR, basing on the infectious clone of HuN4-F112 vaccine strain. The co-expressed full-length cDNA clones (psk-HuN4-F112-delta508-532 + E2 and psk-HuN4-F112-delta480-532 + E2) were assembled by cloning and splice of the gene fragments. The completely assembled full-length cDNA clones were confirmed by sequence and Swa I enzyme digestion. Capped RNAs were transcribed in vitro from a full-length cDNA clone of the viral genome and transfected into BHK-21 cells by liposome to acquire the rescued virus. The rescued recombinant viruses were passaged on MARC-145 cells. The successfully rescued viruses were tested by RT-PCR, digestion, and genome sequence. The results showed that these rescued viruses could be distinguished from the parental virus (HuN4-F112) with the mutant genetic marker (Mlu I enzyme site of virual genome at 14 667nt was created by synonymous mutation) and the inserted nsp2 gene region. The results of IFA showed that the inserted E2 epitope could be expressed by the recombinant viruses and the E2 epitope gene was stable during the viral serial passage. The results of plaque assay and viral growth curve showed that the recovery viruses possessed similar characterses of viral growth to those of the parental virus. In summary, the full-length infectious cDNA clones containing the marker gene were constructed and the marker recombinant viruses were rescued. The results suggested that these stable infectious clones could be used as an important tool for development of novel vaccine against PRRSV.

Stable expression of foreign gene in nonessential region of nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus: applications for marker vaccine design.

The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to be highly heterogeneous and variable among PRRSV strains and some sequences in the middle region of the nsp2 are not essential to viral replication. Recent studies have attempted to insert foreign genes in the nsp2 nonessential regions but the foreign genes were not stably expressed by recombinant viruses in vitro. In the present study, we first constructed an infectious cDNA clone with deletion of 75 nucleotides (25 amino acids) in the nsp2 region (rHuN4-F112-Δ508-532) of the attenuated vaccine virus HuN4-F112 derived from a highly pathogenic PRRSV HuN4 and then inserted a gene fragment encoding a immunodominant B-cell epitope (49 amino acids) of Newcastle disease virus (NDV) nucleoprotein (NP) in-frame into the deletion site. The viable recombinant virus was rescued from the full-length cDNA infectious clone in vitro. The engineered viruses rescued from the cDNA clone indicated that the deletions of 75 nucleotides and insertion of NDV NP gene in the nsp2 region did not affect viral replication; they had similar growth kinetics to its parental virus. The inserting gene could be expressed consistently when the recombinant virus was passaged up to twenty times in cell cultures as determined by immunofluorescence assay (IFA) and genomic sequencing. To investigate the potential application of the NDV NP gene-inserted PRRSV as a marker vaccine, piglets were immunized with the recombinant virus and then challenged with lethal dose of highly pathogenic PRRSV. The immunized piglets produced specific antibodies against both the NDV NP and PRRSV, and lacked antibodies against the deleted 25aa nsp2 epitope. After challenge, all immunized piglets were protected from clinical disease or death, while all piglets in control group died (5/5) by ten days post challenge. The results of the present study indicated that the recombinant PRRSV (rHuN4-F112-Δ508-532) could be used as a potential marker vaccine against PRRS.

A novel dendrimeric peptide induces high level neutralizing antibodies against classical swine fever virus in rabbits.

The amino acid sequence (TAVSPTTLR, 829-837aa) on the glycoprotein E2 of classical swine fever virus (CSFV) is a conserved and linear neutralizing epitope. In the present study, two peptides were constructed based the core sequence of this neutralizing epitope, the dendrimeric peptide (Th-B(4)) containing four copies of B cell epitope fused to one copy of promiscuous T helper (Th) cell epitope and the peptide Th-B containing a single copy of B cell epitope fused to one copy of Th cell epitope. The dendrimeric peptide Th-B(4) elicited high titers of neutralizing antibodies as detected in an indirect ELISA, blocking ELISA and neutralization test and induced a complete protection against CSFV C strain in rabbits. The Th-B elicited low titers of neutralizing antibodies and did not induce a protection in rabbits. These results suggest that the dendrimeric peptide Th-B(4) may be a promising marker vaccine candidate against CSFV and the multimerization is a requirement for development of a peptide vaccine.

Synergistic anti-tumor effect of obatoclax and MG-132 in esophageal cancer cell line CaES-17 / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore whether MG-132 could enhance the anti-tumor activity of obatoclax against esophageal cancer cell line CaES-17.</p><p><b>METHODS</b>MTT assay was used to determine the cytotoxicity of obatoclax and MG-132 in CaES-17 cells. The IC(50) of obatoclax and MG-132 were used to determine the molar ratio (1:2.4) of the two drugs for combined treatment of the cells. The concentrations of obatoclax and MG-132 ranged from 1/8 IC(50) to 4 IC(50) after serial dilution, and their combination index (CI) was calculated using CompuSyn software. The expression of ubiquitin and the cleavage of PARP, caspase-9, phospho-histone H3 and phospho-aurora A/B/C in the exposed cells were examined with Western blotting; the cell apoptosis was measured by flow cytometry with Annexin V staining, and the percentage of cells in each cell cycle phase was also determined by flow cytometry.</p><p><b>RESULTS</b>The CI of obatoclax and MG-132 was 0.296 for a 50% inhibition of Caes-17 cells and was 0.104 for a 95% inhibition. The cells treated with obatoclax or MG-132 alone showed increased expression of ubiquitin and cleavage of PARP and caspase-9. Compared with the cells treated with obatoclax or MG-132 alone, the cells with a combined treatment exhibited significantly increased expression of ubiquitin, cleavage of PARP and caspase-9, and expression of phospho-Histone H3 (P<0.05). The combined treatment of the cells also resulted in significantly increased expression of phospho-Aurora A/B/C compared with obatoclax treatment alone. The cells with the combined treatment showed significantly higher percentages of apoptotic cells and cells in sub-G(1) and G(2)/M phases compared with the cells treated with either of the drugs (P<0.05).</p><p><b>CONCLUSION</b>Obatoclax combined with MG-132 shows a significant synergistic anti-tumor effect against esophageal cancer CaES-17 cells by inducing apoptosis and cell cycle arrest.</p>

Up-regulation of integrin beta3 expression in porcine vascular endothelial cells cultured in vitro by classical swine fever virus.

Classical swine fever (CSF) caused by virulent strains of classical swine fever virus (CSFV) is a haemorrhagic disease of pigs, characterized by disseminated intravascular coagulation, thrombocytopenia and immunosuppression. The cell adhesion molecule, integrin beta3, plays a central role in maintaining and regulating vascular permeability. In view of the haemorrhagic pathology of the disease, the effect of CSFV infection on integrin beta3 expression was investigated using the swine umbilical vein endothelial cell (SUVEC) line, in conjunction with quantitative PCR and Western blotting techniques. Following infection, the expression levels of integrin beta3 were significantly up-regulated along with corresponding transcription levels. The infected endothelial cells adhered onto immobilized extracellular matrix (ECM) with more extensive spreading than that of the control, and such interaction was strongly inhibited by an anti-integrin beta3 monoclonal antibody (mAb). This study revealed the up-regulation of integrin beta3 in vascular endothelial cells by CSFV infection, and cell adhesion molecules of this kind possibly play an important role in the changes of haemostatic balance in haemorrhagic pathology of CSF.

[Construction and immunogenicity of a recombinant adenovirus co-expressing the E2 protein of classical swine fever virus and the porcine interleukin 2 in rabbits].

To construct a recombinant adenovirus co-expressing the E2 protein of classical swine fever virus (CSFV) and the porcine interleukin 2 (pIL-2), the CSFV E2 gene and pIL-2 gene were amplified respectively from the plasmids pMD19-T-E2 and pMD19-T-pIL-2 by PCR. E2-pIL-2 fusion gene was obtained by using 5 consecutive glycine codons as a linker and cloned into the adenoviral shuttle plasmid AdTrack. The AdTrack-E2-pIL-2 was linearized and transformed into E. coli BJ5183 with the backbone plasmid AdEasy1. The resultant recombinant plasmid AdEasy-E2-pIL-2 was transfected into the 293 cells where the recombinant adenovirus rAd-E2-pIL-2 was produced. The immunogenicity of rAd-E2-pIL-2 was evaluated in rabbits. The results of RT-PCR and Western-blotting showed that rAd-E2-pIL-2 could carry and express E2 and pIL-2 proteins. The titer of the rAd-E2-pIL-2 was 10(8.12) PFU/mL. After immunized with rAd-E2pIL-2, The injected rabbits developed a high level of CSFV specific antibodies. Regular fever was not detected in the rAd-E2-pIL-2-immunized rabbits upon challenge with CSFV C stain, and specific lymphoproliferative responses to the CSFV was detected in the lymphocytes from the immunized rabbits. In conclusion, rAd-E2-pIL-2 was constructed successfully and it could be an attractive vaccine candidate against CSFV.

Frequency- and state-dependent blockade of human ether-a-go-go-related gene K+ channel by arecoline hydrobromide / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The rapidly activating delayed rectifier potassium current (I(Kr)), whose pore-forming alpha subunit is encoded by the human ether-a-go-go-related gene (hERG), is a key contributor to the third phase of action potential repolarization. The aim of this study was to investigate the effect and mechanism of arecoline hydrobromide induced inhibition of hERG K(+) current (I(hERG)).</p><p><b>METHODS</b>Transient transfection of hERG channel cDNA plasmid pcDNA3.1 into the cultured HEK293 cells was performed using Lipofectamine. A standard whole-cell patch-clamp technique was used to record the I(hERG) before and after the exposure to arecoline.</p><p><b>RESULTS</b>Arecoline decreased the amplitude and the density of the I(hERG) in a concentration-dependent manner (IC(50) = 9.55 mmol/L). At test potential of +60 mV, the magnitude of I(hERG) tail at test pulse of -40 mV was reduced from (151.7 ± 6.2) pA/pF to (84.4 ± 7.6) pA/pF (P < 0.01, n = 20) and the magnitude of I(hERG) tail at test pulse of -110 mV was reduced from (-187.5 ± 9.8) pA/pF to (-97.6 ± 12.6) pA/pF (P < 0.01, n = 20). The blockade of arecoline in the open and inactivated state was significant in a state-dependent manner. The maximal blockade was achieved in the inactivated state. Studies of gating mechanism showed that the steady-state activation curve of I(hERG) was significantly negatively shifted by arecoline. Time constants of activation were shortened. Steady-state inactivation curve and time constants of fast inactivation were not significantly affected by arecoline. Furthermore, the inhibition of I(hERG) by arecoline was characterized markedly by a frequency-dependent manner from 0.03 to 1.00 Hz pulse.</p><p><b>CONCLUSION</b>Arecoline could potently block I(hERG) in both frequency and state-dependent manner.</p>