Effects of miR-214 on cervical cancer cell proliferation, apoptosis and invasion via modulating PI3K/AKT/mTOR signal pathway.

Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, "Effects of miR-214 on cervical cancer cell proliferation, apoptosis and invasion via modulating PI3K/AKT/mTOR signal pathway, by F. Wang, W.-H. Tan, W. Liu, Y.-X. Jin, D.-D. Dong, X.-J. Zhao, Q. Liu, published in Eur Rev Med Pharmacol Sci 2018; 22 (7): 1891-1898-DOI: 10.26355/eurrev_201804_14711-PMID: 29687840" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/14711.

Precise label-free leukocyte subpopulation separation using hybrid acoustic-optical chip.

Leukocyte subpopulations contain crucial physiological information; hence, precise and specific leukocyte separation is very important for leukemia diagnosis and analysis. However, conventional centrifugation and immunofluorescence-based separation methods are inaccurate and inconvenient due to the overlapping cell size and density or complex marking processes. Herein, we report a new label-free technology for precise leukocyte subpopulation separation by synergy of acoustic and optical technologies. Standing surface acoustic wave (SSAW) solved the problem of gentle and precise focusing of cells in optical systems. In addition, SSAW was used for the separation of granulocytes, which have evident size distinction from other components. In case of lymphocytes and monocytes, which have overlap in size/density, optical force could distinguish them accurately based on the RI difference, with the convenience of acoustic pre-focusing. In this experiment, separation of three types of leukocyte subtypes with considerable throughput and purity was conducted, through which we obtained 99% pure lymphocytes, 98% pure monocytes, and 95% pure granulocytes. Experimental results prove that the device has robust ability in separating leukocyte phenotypes and have the advantages of being non-invasive, label-free and precise. In the future, this convenient hybrid method will be a potential powerful tool for auxiliary clinical diagnosis and analysis.

[Application of multi-functional modularized prosthesis in reconstruction for sternal defect].

Objective: To investigate the feasibility and effectiveness of sternal reconstruction using a multi-functional modularized sternal reconstruction system. Methods: In June 2016 and August 2017, the modularized sternal prosthesis system was used on two patients for sternal reconstruction in the Department of Thoracic Surgery of Shanghai Pulmonary Hospital, Tongji University. Both patients were female aging 48 and 72 years, respectively, with the primary diagnosis of tumor of the lower sternal body and huge mediastinal tumor. Partial sternal resection and reconstruction was performed through median sternotomy. The multi-functional modularized sternal reconstruction system consisted of manubium, superior sternal body, inferior sternal body, rib and clavicle modules. Each module was designed into 3 to 6 sizes. Appropriate modules were chosen in each case to be assembled as a sternal reconstruction prosthesis. Results: Both operation were smooth, with operation time of 240 minutes and 280 minutes, intraoperative blood loss of 100 ml and 400 ml. The patients were followed up for 18 months and 4 months, respectively. In both cases, the sternal reconstruction was satisfactorily healed, without local infection, fluid accumulation, loose part or dislocation. No local recurrence or distant metastasis was found. Conclusion: The multi-functional modularized sternal reconstruction system can be safely and effectively applied for sternal reconstruction in 2 cases.

Real-time detection and monitoring of the drug resistance of single myeloid leukemia cells by diffused total internal reflection.

Real-time detection and monitoring of the drug resistance of single cells have important significance in clinical diagnosis and therapy. Traditional methods operate a number of times for each individual concentration, and innovation is required for the design of more simple and efficient manipulation platforms with necessary higher sensitivity. Here, we have developed a novel diffused total internal reflection (TIR) method to perform drug metabolism and cytotoxicity analysis of trapped myeloid leukemia cells. Molm-13 cells, a type of acute myeloid leukemia cell, were chosen and injected into the device and fittingly captured by cell traps. Differing from previous studies, a series of different concentrations of azelaic acid (AZA) drug could be used from 0 mM to 50 mM through convection and diffusion processes in a single chip, with each concentration region featuring 50 cells, with a total of 549 cell trapping units. Thanks to the high sensitivity of the TIR method, only cells with the same drug concentration could be illuminated in the detection process. By adjusting the incident angle, we could exactly detect and monitor the drug resistance of the cells using different drug concentrations and the experimental resolution of the drug concentration was as small as 5 mM. Images of the membrane integrity and morphology of the cells in the bright field were measured and we also monitored the cell viabilities in the dark field over 2 hours. The effects of AZA on the Molm-13 cells were explored in different concentrations at the single cell level. Compared with the results of the traditional MTT assay method, the experimental results are more simple and accurate. A cell death of 5% at an AZA concentration of 5 mM was observed after 30 minutes, while a concentration of 40 mM corresponded to a 98% cell death. The designed method in this study provides a novel toolkit to control and monitor drug resistance at the single cell level more easily with higher sensitivity and we believe it has significant potential application in single cell quality assessment and medicine analysis in clinical practice.

Effects of miR-214 on cervical cancer cell proliferation, apoptosis and invasion via modulating PI3K/AKT/mTOR signal pathway.

OBJECTIVE: PI3K/AKT/mTOR pathway plays important roles in tumor pathogenesis. mTOR is up-regulated and miR-214 is down-regulated in cervical can-*cers. This study investigated whether miR-214 regulated mTOR expression and affected cervical cancer cell proliferation, apoptosis or invasion. PATIENTS AND METHODS: Cervical cancer tissues were collected in parallel with normal epithelium for measuring the expression of miR-214 and mTOR. Dual luciferase expression assay was performed to evaluate the targeted relationship between miR-214 and mTOR. In vitro cultured SiHa cells were treated with miR-214 mimic or si-mTOR followed by measuring mTOR, p-mTOR and Bcl-2 expression. Cell apoptosis, proliferation and invasion were measured by flow cytometry and transwell assay. RESULTS: Bioinformatics analysis showed targeted binding sites between miR-214 and 3'-UTR of mTOR mRNA. Dual luciferase reporter assay confirmed this regulatory relationship between miR-214 and mTOR mRNA. Compared to normal cervical epithelium, cancer tissues had lower expression of miR-214 and higher mTOR, both of which were correlated with TNM stage and tissue pathology grade. Compared to Ect1/E6E7 cells, SiHa cells had lower level of miR-214 and higher mTOR/p-mTOR and Bcl-2 expression. Transfection of miR-214 mimic or si-mTOR significantly decreased mTOR/p-mTOR or Bcl-2 expression, inhibited cell proliferation or invasion, and enhanced cell apoptosis. CONCLUSIONS: miR-214 down-regulation plays a role in elevating mTOR expression and in facilitating cervical cancer pathogenesis. Over-expression of miR-214 inhibits cervical cancer cell proliferation or invasion, and facilitates apoptosis via targeted inhibition of mTOR expression.

Field evidence for earlier leaf-out dates in alpine grassland on the eastern Tibetan Plateau from 1990 to 2006.

Worldwide, many plant species are experiencing an earlier onset of spring phenophases due to climate warming. Rapid recent temperature increases on the Tibetan Plateau (TP) have triggered changes in the spring phenology of the local vegetation. However, remote sensing studies of the land surface phenology have reached conflicting interpretations about green-up patterns observed on the TP since the mid-1990s. We investigated this issue using field phenological observations from 1990 to 2006, for 11 dominant plants on the TP at the levels of species, families (Gramineae-grasses and Cyperaceae-sedges) and vegetation communities (alpine meadow and alpine steppe). We found a significant trend of earlier leaf-out dates for one species (Koeleria cristata). The leaf-out dates of both Gramineae and Cyperaceae had advanced (the latter significantly, starting an average of 9 days later per year than the former), but the correlation between them was significant. The leaf-out dates of both vegetation communities also advanced, but the pattern was only significant in the alpine meadow. This study provides the first field evidence of advancement in spring leaf phenology on the TP and suggests that the phenology of the alpine steppe can differ from that of the alpine meadow. These findings will be useful for understanding ecosystem responses to climate change and for grassland management on the TP.

Alterations of spindle and microfilament assembly in aged cat oocytes.

To obtain insights into the cytoplasmic maturation status of cat oocytes recovered from cat ovaries following hormone treatment, we first examined microtubule and microfilament assembly in cat oocytes recovered from hormone-treated ovaries at various stages of maturation. Additionally, we determined the alteration of spindle and microfilament assembly, as well as mitogen-activated protein kinase (MAPK) activity, in cat oocytes at 0, 6, 12 and 18 h of further maturation in vitro. We then looked at pronuclear formation and cleavage of these oocytes following parthenogenetic activation. Similar to other species, microtubules are present in germinal vesicle (GV) stage cat oocytes, and following GV breakdown, microtubules encompassed condensed chromatin particles to form the meiotic metaphase spindle. Microfilaments were located in the cortex and around the GV. A microfilament-rich area, in which the chromatin is located, was observed in the oocytes during meiotic maturation. Maturation rates in aged oocytes (cultured for 18 h) were increased when compared with that in relatively fresh oocytes (<12 h culture), and the number of oocytes with abnormal spindle shapes was also increased in aged oocytes. Furthermore, in aged oocytes, the incidence of the metaphase plate observed outside the thick microfilament domain was higher compared with that of young oocytes, and this seemed to result in an increase in the number of oocytes with two pronuclei and one polar body following activation. Western blot analysis revealed a decrease in MAPK activity in aged cat oocytes. Taken collectively, these results suggest that the optimum time for improved cytoplasmic maturation is <12 h in cat oocytes recovered from hormone-treated ovaries.

Germinal vesicle chromatin configuration and meiotic competence is related to the oocyte source in canine.

This study investigated the effect of deriving oocytes from different stages of the estrous cycle on oocyte diameter, germinal vesicle (GV) chromatin configuration, and in vitro meiotic competence in canine oocytes. Cumulus oocyte complexes (COCs) were recovered from both ovaries during anestrous, follicular, and luteal phases and in vivo ovulated oocytes. The diameter of canine oocyte was compared with or without the zona pellucida (ZP) before in vitro maturation (IVM). Also, GV chromatin configuration was evaluated before (0 h) or 72 h after IVM by fixation with 3.7% formaldehyde supplemented with 10 microg/ml Hoechst 33342 for 30 min. COCs were matured in TCM199 supplemented with 10% fetal bovine serum (FBS), 0.6 mM cysteine, 0.2 mM pyruvic acid, 50 microg/ml gentamycin sulfate, and 20 microg/ml 17beta-estradiol (E(2)) at 39 degrees C and 5% CO(2) in air for 72 h. The diameter of in vivo ovulated oocytes with the ZP (167.5+/-12.7 microm) or without ZP (133.9+/-5.3 microm) was significantly greater (p<0.05) than those of anestrous, follicular, and luteal oocytes (with ZP, 151.2+/-7.4, 153.1+/-8.8 and 152.8+/-5.4 microm, respectively; without ZP, 115.3+/-7.6, 122.1+/-4.9 and 114.3+/-6.6 microm, respectively). At 0 h, the GV-II configuration was more prevalent in oocytes from anestrual ovaries than from follicular or luteal ovaries or in vivo ovulated oocytes (63.6% versus 14.8%, 33.0%, and 0.0%; p<0.05), whereas the proportion of oocytes with the GV-V configuration was higher in follicular phase and ovulated oocytes than in oocytes from anestrus and luteal phase (57.4% and 100% versus 2.0% and 22.7%; p<0.05). However, oocytes in luteal phase exhibited diverse GV configurations (10.3%, 33.0%, 16.5%, 13.4%, and 22.7% in GV-I, GV-II, GV-III, GV-IV, and GV-V, respectively). After 72 h post-IVM, a greater percentage of in vivo ovulated oocytes progressed to MII than those oocytes collected during anestrous, follicular, and luteal phases (50.0% versus 5.5%, 11.5%, and 9.1%; p<0.05). In conclusion, the oocyte diameter, GV chromatin configuration, and meiotic maturation of canine COCs are related to the oocyte source. These results indicated that the oocyte source could be critical to nuclear progression to MII stage in canines.

[The analysis of rice total RNA by capillary electrophoresis].

When the RNase was eliminated through backing at 200 degrees C or treated by diethylpyrocarbonate (DEPC), the total RNA of rice could be separated within 15 min using 1.0%T, 0%C linear polyacrylamide as sieving matrix and 7 mol/L urea as denaturant. The tRNA of rice can be separated into two classes and about nine peaks when high concentrated polyacrylamide sieving matrix (5.0%T, 0%C) was used. This technique could provide one of the rapid and accurate methods for the determination of RNA in plants.

[Determination of human PDGF-B promoter binding nuclear protein by capillary electrophoresis].

With the use of 1.0% T, 0% C linear polyacrylamide as sieving matrix, 0.25 x TBE(Tris 89 mmol/L, boric acid 89 mmol/L, EDTA 2 mmol/L) as running buffer and 15 degrees C as column temperature, the human PDGF-B promoter binding nuclear protein can be determined within 50 min with good resolution. The results proved that there are two proteins having strong ability binding human PDGF-B promoter, which similar to that in slab gel electrophoresis. This technique can provide one of the rapid and accurate separation methods in the studying of the formation and repression behavior of DNA binding protein based on PDGF gene as the target.

Homology Modeling of Bacillus subtilis Tryptophanyl-tRNA Synthetase.

Tryptophanyl-tRNA synthetase (TrpRS) plays a pivotal role in protein synthesis. However, till now no stereostructural data of Bacillus subtilis TrpRS were reported. Here, by using the homology modeling using Bacillus stearothermophilus TrpRS as a template, it is demonstrated that the synthetase has 16alpha helices and 5beta sheets. The only tryptophan Trp(92) is located on the interface of subunits. Also the modeling presents the ligand binding site, active site and the putative binding of tRNA(Trp).

TGF-beta1 inhibits protracted-relapsing experimental autoimmune encephalomyelitis by activating dendritic cells.

Protracted-relapsing experimental autoimmune encephalomyelitis (PR-EAE) in DA rats is an animal model closely related to multiple sclerosis (MS). Previous studies showed that nasal administration of TGF-beta1 suppressed the development and relapse of PR-EAE clinically and pathologically. Here we demonstrate that this suppressive effect was associated with activation of dendritic cells (DC), showing elevated proliferative response and IFN-gamma and nitric oxide (NO) production by DC. DC derived from TGF-beta1-treated rats with PR-EAE also showed high expression of nitric oxide synthase 2 (NOS2) at both mRNA and protein levels. Apoptotic cells were increased in spleen sections of TGF-beta1-treated rats compared to control rats. In studying mechanisms of apoptosis in TGF-beta1-treated rats, in vitro experiments demonstrated that TGF-beta1-treated DC induced apoptosis of CD4(+)T cells by a NO pathway after co-culture with T cells. These results support the hypothesis that TGF-beta1-induced suppression of PR-EAE is associated with apoptosis of CD4(+)T cells induced by DC-derived NO.

Antisense oligodeoxynucleotide inhibits vascular endothelial growth factor in human glioma cells.

AIM: To study the effects of antisense vascular endothelial growth factor (VEGF) oligodeoxynucleotide (ODN) on the expression of VEGF in human glioma cell line (A172 cells). METHODS: VEGF mRNA level was measured by semiquantification reverse transcriptase polymerase chain reaction (RTPCR). VEGF protein expression in the cells was determined by immunohistochemistry. VEGF protein level in the media was measured by ELISA. RESULTS: When the cells were treated with antisense VEGF ODN (6.25-50 mumol.L-1), VEGF mRNA level in the cells decreased remarkably in a concentration-dependent manner. No change was found when the cells were treated with sense or missense ODN. When the cells were treated with antisense VEGF ODN 25 mumol.L-1, VEGF protein level decreased greatly both in the cells and the media. CONCLUSIONS: Antisense VEGF ODN inhibited VEGF expression specifically in A172 cells in vitro and thus the results provided the basis for the further studies in vivo.

Preparation and identification of activity of anti-HPV-6b/11E1 universal ribozyme--Rz1198 in vitro.

AIM: To study the preparation and cleavage activity of Rz1198 directed against HPV-6bE1 and HPV-11E1 (HPV-6b/ 11E1) transcripts in vitro. METHODS: HPV-6b/11E1 gene fragments were cloned into T-vector under the control of T7 promoter. 32P-labeled HPV-6b/11E1 transcripts as target-RNAs were transcribed in vitro and purified by PAGE. Rz1198 gene designed as a universal ribozyme for both HPV-6b/11E1 transcripts was cloned into vector p1.5 between 5'-cis-Rz and 3'-cis-Rz. 32P-labeled Rz1198 transcript was gel-purified, incubated with target-RNAs at different conditions and autoradiographed after denaturing gel-electrophoresis. RESULTS: Rz1198 was active at 37 degrees C. The optimal temperature was 50 degrees C. For HPV-6bE1, km = 12.2 nmol/L, kcat = 0.18 min(-1); For HPV-11E1, km = 14.7 nmol/L, kcat = 0.14 min(-1). All these revealed that the design of Rz1198 was correct. It could be a universal ribozyme for the two substrates--HPV-6bE1 and HPV-11E1 transcripts. CONCLUSION: Rz1198 prepared in vitro possesses the perfect specific catalytic cleavage activity. It leads to the expectation that, in the future, it will be possible to develop a new nucleic acid drug from Rz1198 which can efficiently inhibit the replication of HPV-6b/11 DNA in vivo.

Regulation of beta-chemokine mRNA expression in adult rat astrocytes by lipopolysaccharide, proinflammatory and immunoregulatory cytokines.

Astrocytes constitute a part of the blood-brain barrier. Chemokine expression by astrocytes may contribute to leucocyte infiltration within the central nervous system (CNS) during inflammation. To investigate factor(s) regulating chemokine expression by astrocytes, we studied the induction of beta-chemokine mRNA expression in adult rat astrocytes. Astrocyte-derived monocyte chemoattractant protein- (MCP-1), RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta mRNA were induced by interferon-gamma (IFN-gamma). Tumour necrosis factor-alpha (TNF-alpha) induced MCP-1, RANTES and MIP-1beta mRNA expression, and lipopolysaccharide (LPS) induced MCP-1, MIP-1alpha and MIP-1beta mRNA expression in astrocytes. LPS-induced MCP-1, MIP-1alpha and MIP-1beta mRNA expression by astrocytes was antagonized by transforming growth factor (TGF)-beta1 and interleukin (IL)-10. TGF-beta1 and IL-10 also down-regulated MCP-1 and RANTES mRNA expression induced by TNF-alpha. IL-10, but not TGF-beta1, inhibited MIP-1beta mRNA expression induced by TNF-alpha. The results of this in vitro study suggest that beta-chemokine mRNA expression by adult rat astrocytes can be induced by LPS or proinflammatory cytokines, while regulatory cytokines, such as TGF-beta1 and IL-10, down-regulate astrocyte-derived beta-family chemokine mRNA expression induced by LPS, IFN-gamma and TNF-alpha. Further study of CNS chemokines will enhance our understanding of leucocyte recruitment to the CNS and suggest therapeutic strategies for neurological disorders.

Pleiotropic effects of intron removal on base modification pattern of yeast tRNAPhe: an in vitro study.

Cell-free yeast extract has been successfully used to catalyze the enzymatic formation of 11 out of the 14 naturally occurring modified nucleotides in yeast tRNAPhe(anticodon GAA). They are m2G10, D17, m22G26, Cm32, Gm34,psi39, m5C40, m7G46, m5C49, T54 andpsi55. Only D16, Y37 and m1A58 were not formed under in vitro conditions. However, m1G37was quantitatively produced instead of Y37. The naturally occurring intron was absolutely required for m5C40formation while it hindered completely the enzymatic formation of Cm32, Gm34and m1G37. Enzymatic formation of m22G26,psi39, m7G46, m5C49, T54 andpsi55were not or only slightly affected by the presence of the intron. These results allow us to classify the different tRNA modification enzymes into three groups: intron insensitive, intron dependent, and those requiring the absence of the intron. The fact that truncated tRNAPheconsisting of the anticodon stem and loop prolonged with the 19 nucleotide long intron is a substrate for tRNA: cytosine-40 methylase demonstrates that the enzyme is not only strictly intron dependent, but also does not require fully structured tRNA.

Enzymatic conversion of adenosine to inosine and to N1-methylinosine in transfer RNAs: a review.

Inosine (6-deaminated adenosine) is a characteristic modified nucleoside that is found at the first anticodon position (position 34) of several tRNAs of eukaryotic and eubacterial origins, while N1-methylinosine is found exclusively at position 37 (3' adjacent to the anticodon) of eukaryotic tRNA(Ala) and at position 57 (in the middle of the psi loop) of several tRNAs from halophilic and thermophilic archaebacteria. Inosine has also been recently found in double-stranded RNA, mRNA and viral RNAs. As for all other modified nucleosides in RNAs, formation of inosine and inosine derivative in these RNA is catalysed by specific enzymes acting after transcription of the RNA genes. Using recombinant tRNAs and T7-runoff transcripts of several tRNA genes as substrates, we have studied the mechanism and specificity of tRNA-inosine-forming enzymes. The results show that inosine-34 and inosine-37 in tRNAs are both synthesised by a hydrolytic deamination-type reaction, catalysed by distinct tRNA:adenosine deaminases. Recognition of tRNA substrates by the deaminases does not strictly depend on a particular "identity' nucleotide. However, the efficiency of adenosine to inosine conversion depends on the nucleotides composition of the anticodon loop and the proximal stem as well as on 3D-architecture of the tRNA. In eukaryotic tRNA(Ala), N1-methylinosine-37 is formed from inosine-37 by a specific SAM-dependent methylase, while in the case of N1-methylinosine-57 in archaeal tRNAs, methylation of adenosine-57 into N1-methyladenosine-57 occurs before the deamination process. The T psi-branch of fragmented tRNA is the minimalist substrate for the N1-methylinosine-57 forming enzymes. Inosine-34 and N1-methylinosine-37 in human tRNA(Ala) are targets for specific autoantibodies which are present in the serum of patients with inflammatory muscle disease of the PL-12 polymyositis type. Here we discuss the mechanism, specificity and general properties of the recently discovered RNA:adenosine deaminases/editases acting on double-stranded RNA, intron-containing mRNA and viral RNA in relation to those of the deaminases acting on tRNAs.

Effects of electroacupuncture of "zusanli" acupoint on high blood pressure and blood hyperviscosity in stress rats.

Elevation of blood pressure (BP) and blood viscosity (BV) was induced in unanesthetized Wistar rats by fixing and hanging. Electroacupuncture of "Zusanli" acupoint or microinjection of GABA (60 micrograms/10 microliters) into the IV ventricle of the brain could lower the high BP and BV induced by fixed-hanging, which could be blocked by a microinjection of GABAA receptor antagonist bicuculline (60 micrograms/10 microliters). The results showed that the depressant effect of electroacupuncture of "Zusanli" acupoint on high BP and blood hyperviscosity induced by fixed-hanging might be mediated by the activation of GABAA receptors in the brain.

Biological function of modified nucleotides in tRNA molecules--synthesis and biological activity of the analogues of yeast alanyl tRNA with I34 replaced by A34 or G34.

Analogues of yeast alanyl tRNA with I34 replaced by A34 or G34 were synthesized. Synthetic analogues of yeast alanyl tRNT occupy the same position as the natural yeast alanyl tRNA on polyacrylamide gel electrophoresis, and their purity is about 95% after electrophoresis on a 10% or 20% polyacrylamide gel. The two terminal and nearest neighbour nucleotides of the analogues are all correct. The accepting activity of the synthetic analogues is similar to that of the reconstituted natural yeast alanyl tRNA. The incorporation activity of alanine into proteins of the synthetic analogues is about 30% of that of the natural of reconstituted natural yeast alanyl tRNA when I34 is replaced by A, and is 90% when I34 is replaced by G. The reason of the variation in biological function of the analogues of yeast alanyl tRNA after I34 replaced by A or G was discussed.