Affinity binding-mediated fluorometric protein assay based on the use of target-triggered DNA assembling probes and aptamers labelled with upconversion nanoparticles: application to the determination of platelet derived growth factor-BB.

The target-triggered DNA assembling probe is presented for highly selective protein detection. Target-triggered DNA assembling is used in an amplification strategy based on affinity binding for identification and determination of proteins in general. Specifically, it was applied to the platelet derived growth factor-BB (PDGF-BB). A hairpin DNA (H-DNA) probe was designed containing (a) an aptamer domain for protein recognition and (b) a blocked DNAzyme domain for DNAzyme cleavage. An assistant DNA (A-DNA) probe containing aptamer and complementary domains was also employed to recognize protein and to induce DNA assembly. Once H-DNA and A-DNA recognize the same protein, H-DNA and A-DNA are in close proximity to each other. This induces DNA assembling for protein-triggered complex (Protein-Complex) with free DNAzyme domains. The free DNAzymes trigger the circular cleavage of molecular beacons for amplified signals. The assay is performed by fluorometry at an excitation wavelength of 980 nm and by collecting fluorescence at 545 nm. The platelet derived growth factor-BB (PDGF-BB) was accurately identified and selectively determined by this assay with a 22 pM detection limit (using the 3σ criterion). The responses for PDGF-BB is nearly 6-fold higher than for PDGF-AB, and 16-fold higher than PDGF-AA. This upconversion assay avoids any interference by the autofluorescence of biological fluids. Graphical abstractSchematic representation of the principle of the target-triggered DNA assembling probes mediated amplification strategy based on affinity binding for PDGF-BB. The UCNP probe is used for the quantitation of PDGF-BB with high selectivity.

Different Performance of Intravertebral Vacuum Clefts in Kümmell's Disease and Relevant Treatment Strategies.

OBJECTIVES: This study aimed to present the different pattern of intravertebral vacuum cleft (IVC) related to high risk of cement complications in minimally invasive treatments for Kümmell's disease (KD) and relevant treatment strategies. METHODS: A retrospective study from January 2016 to January 2018 was conducted at Wuhan Fourth Hospital and comprised 35 patients with Kümmell's disease. There were seven males and 28 females, and the mean age of the patients was 70.4 years. The patterns of IVC in KD were analyzed. These patients were divided into three groups based on the treatment method used. The treatment methods included long-segment fixation (LSF), posterior short-segment fixation (SSF), and percutaneous kyphoplasty (PKP). We retrospectively reviewed outcomes, including the Oswestry Disability Index (ODI), visual analog scale (VAS) score, anterior height of affected vertebrae, kyphotic Cobb angle, and complications. RESULTS: All patients were followed up for 12-38 months. According to their radiographic appearance we could observe two main patterns of clefts. Pattern I, clefts that were found to be near to the endplate and connected with intervertebral space, the endplate was incomplete. Pattern II, IVC traversed to anterior edge of the vertebral body affected. Both were related to high risk of cement complications in minimal invasive treatments for KD. Good results have been achieved in LSF and SSF groups, the VAS, ODI, anterior height of affected vertebrae and kyphotic Cobb angle showed statistically significant differences between pre- and post-operation and between pre- and final follow-up (P < 0.05). In PKP group, although the VSA and ODI showed statistically significant differences between pre- and post-operation and between pre- and final follow-up (P < 0.05), we could observe that the VSA and ODI rebounded a little at the final follow-up. Cement leakage into intervertebral space occurred in four (44.45%) patients of PKP group. CONCLUSIONS: PKP should be chosen carefully if the IVC of the patient presents to be pattern I or II. LSF and SSF are safe and effective, and can achieve satisfactory correction of kyphosis and vertebral height, with pain relief and improvement in patient's daily life, with few complications.

The proximal promoter region of the human vascular endothelial growth factor gene has a G-quadruplex structure that can be targeted by G-quadruplex-interactive agents.

Previous studies on the functional analysis of the human vascular endothelial growth factor (VEGF) promoter using the full-length VEGF promoter reporter revealed that the proximal 36-bp region (-85 to -50 relative to transcription initiation site) is essential for basal or inducible VEGF promoter activity in several human cancer cells. This region consists of a polypurine (guanine) tract that contains four runs of at least three contiguous guanines separated by one or more bases, thus conforming to a general motif capable of forming an intramolecular G-quadruplex. Here, we show that the G-rich strand in this region is able to form an intramolecular propeller-type parallel-stranded G-quadruplex structure in vitro by using the electrophoretic mobility shift assay, dimethyl sulfate footprinting technique, the DNA polymerase stop assay, circular dichroism spectroscopy, and computer-aided molecular modeling. Two well-known G-quadruplex-interactive agents, TMPyP4 and Se2SAP, stabilize G-quadruplex structures formed by this sequence in the presence of a potassium ion, although Se2SAP is at least 10-fold more effective in binding to the G-quadruplex than TMPyP4. Between these two agents, Se2SAP better suppresses VEGF transcription in different cancer cell lines, including HEC1A and MDA-MB-231. Collectively, our results provide evidence that specific G-quadruplex structures can be formed in the VEGF promoter region, and that the transcription of this gene can be controlled by ligand-mediated G-quadruplex stabilization. Our results also provide further support for the idea that G-quadruplex structures may play structural roles in vivo and therefore might provide insight into novel methodologies for rational drug design.

Cooperative catalysis with chiral Brønsted acid-Rh2(OAc)4: highly enantioselective three-component reactions of diazo compounds with alcohols and imines.

An asymmetric three-component reaction of diazo compounds and alcohols with imines catalyzed cooperatively by a rhodium complex and a chiral Brønsted acid provides a general and efficient entry to beta-amino-alpha-hydroxyl acid derivatives in high yields with excellent stereoselectivities.

Comparison of Anterior Cervical Discectomy and Fusion versus Posterior Cervical Foraminotomy in the Treatment of Cervical Radiculopathy: A Systematic Review.

Controversy remains over whether anterior cervical discectomy and fusion (ACDF) or posterior cervical foraminotomy (PCF) is superior for the treatment of cervical radiculopathy. We therefore performed a systematic review including three prospective randomized controlled trails (RCT) and seven retrospective comparative studies (RCoS) by searching PubMed and EMBASE. These studies were assessed on risk of bias according to the Cochrane Handbook for Systematic Reviews of Interventions, and the quality of evidence and level of recommendation were evaluated according to the GRADE approach. Clinical outcomes, complications, reoperation rates, radiological parameters, and cost/cost-utility were evaluated. The mean complication rate was 7% in the ACDF group and 4% in the PCF group, and the mean reoperation rate was 4% in the ACDF group and 6% in the PCF group within 2 years of the initial surgery. There was a strong level of recommendation that no difference existed in clinical outcome, complication rate and reoperation rate between the ACDF and the PCF group. There was conflicting evidence that the ACDF group had better clinical outcomes than the PCF group (one study with weak level of recommendation). PCF could preserve the range of motion (ROM) of the operated segment but did not increase the ROM of the adjacent segment (weak level of recommendation). Meanwhile, the average cost or cost-utility of the PCF group was significantly lower than that of the ACDF group (weak level of recommendation). In conclusion, the PCF was just as safe and effective as the ACDF in the treatment of cervical radiculopathy. Meanwhile, PCF might have lower medical cost than ACDF and decrease the incidence of adjacent segment disease. Based on the available evidence, PCF appears to be another good surgical approach in the treatment of cervical radiculopathy.

Fabrication of MPEG-b-PMAA capped YVO4:Eu nanoparticles with biocompatibility for cell imaging.

A novel nanoparticle with multilayer core-shell architecture for cell imaging is designed and synthesized by coating a fluorescent YVO4:Eu core with a diblock copolymer, MPEG-b-PMAA. The synthesis of YVO4:Eu core, which further makes MPEG-b-PMAA-YVO4:Eu NPs adapt for cell imaging, is guided by the model determined upon the evaluation of pH and CEu%. The PMAA block attached tightly on the YVO4:Eu core forms the inner shell and the MPEG block forms the biocompatible outermost shell. Factors including reaction time, reaction temperature, CEu% and pH are optimized for the preparation of the YVO4:Eu NPs. A precise defined model is established according to analyzing the coefficients of pH and CEu% during the synthesis. The MPEG-b-PMAA-YVO4:Eu NPs, with an average diameter of 24 nm, have a tetragonal structure and demonstrate luminescence in the red region, which lies in a biological window (optical imaging). Significant enhancement in luminescence intensity by MPEG-b-PMAA-YVO4:Eu NPs formation is observed. The capping copolymer MPEG-b-PMAA improves the dispersibility of hydrophobic YVO4:Eu NPs in water, making the NPs stable under different conditions. In addition, the biocompatibility MPEG layer reduces the cytotoxicity of the nanoparticles effectively. 95% cell viability can be achieved at the NPs concentration of 800 mgL(-1) after 24h of culture. Cellular uptake of the MPEG-b-PMAA-YVO4:Eu NPs is evaluated by cell imaging assay, indicating that the NPs can be taken up rapidly and largely by cancerous or non-cancerous cells through an endocytosis mechanism.

Down-regulation of telomerase activity via protein phosphatase 2A activation in salvicine-induced human leukemia HL-60 cell apoptosis.

Salvicine is a novel topoisomerase II inhibitor possessing significant antitumor activity, both in vitro and in vivo. The antitumor effect of salvicine is associated with its ability to induce tumor cell apoptosis. Telomerase plays an important role in the apoptotic pathway. However, little is known about the mechanisms of telomerase regulation during apoptosis induced by anticancer drugs. This study investigated the regulation of telomerase activity in salvicine-induced human leukemia HL-60 cell apoptosis. Salvicine treatment resulted in HL-60 cell apoptosis and down-regulation of telomerase activity in a time- and concentration-dependent manner. Repression of telomerase activity preceded a decrease in expression of the telomerase catalytic subunit (hTERT) and telomerase-associated protein (TP1) at the mRNA level, suggesting that the salvicine-induced decrease in telomerase activity may be additionally regulated by mechanisms other than telomerase subunit transcription. We observed that okadaic acid (OA), a protein phosphatase inhibitor, prevented the induction of apoptosis and the down-regulation of telomerase activity by salvicine. The significant increase in protein phosphatase 2A (PP2A) activity induced by salvicine treatment was blocked completely by OA. Moreover, although salvicine induced HL-60 cell apoptosis in a caspase-3-dependent manner, a specific caspase-3 inhibitor, Z-DEVD-FMK, did not prevent a decrease in telomerase activity or an increase in PP2A activity in apoptotic HL-60 cells, ruling out a role for caspase-3 in PP2A activation by salvicine. The results collectively suggest that the salvicine-induced decline in telomerase activity is not a consequence of HL-60 cell apoptosis and that it may be caused principally by the dephosphorylation of telomerase components mediated by PP2A activation.

[Protein phosphatase 2A: its structure, function and activity regulation].

Protein phosphatase 2A (PP2A) is a major kind of serine/threonine protein phosphatase in eukaryotic cells. PP2A has many subunits and isozymes encoded by different genes to form many different PP2A holoenzymes. PP2A plays important roles in regulation of cell cycle, signal transduction, cell differentiation, and transformation. Its activity is related to several diseases, including neurodegenerative diseases and cancer. The regulation of PP2A activity is accomplished by its subunits' expression, cellular localization, phosphorylation and methylation. The second messenger, ceramide, also plays an important role in the regulation of PP2A activity, as well as a plenty of low molecular mass inhibitors of PP2A and other protein phosphatases.

[Preliminary studies on the mechanisms of a new anti-tumor agent PH II-7 with special preference to multidrug resistant tumor cells].

OBJECTIVE: To determine the anti-tumor activity of PH II-7 in vitro and explore preliminarily its mechanisms. METHODS: The anti-tumor activity was measured using colorimetric MTT assay. Apoptosis was determined with fluorescence-activated cell sorter (FACS), electron microscopy and agarose gel electrophoresis. The expressions of mdr1 and sorcin genes were determined by Northern blot assay. RESULTS: PH II-7 inhibited the proliferation of various human tumor cells derived from different tumor cell lines. The IC50 values varied from 0.34-18.61 mumol/L. Especially, PH II-7 had strong inhibitory effect on multidrug resistant tumor cells, whereas adriamycin (ADR) was resistant. Apoptosis was induced in HL60 and HL60/ADR cells treated with 1 microgram/ml PH II-7, while PH II-7 inhibited the expressions of mdr1 and sorcin genes. CONCLUSIONS: PH II-7 is a new potential agent which has strong inhibitory effect on both multidrug resistant cells and their parental cells. PH II-7 may increase the intracellular drug concentration in MDR cells by inhibiting the expressions of the MDR-related genes mdr1 and sorcin and induce the apoptosis of MDR cells and their parental tumor cells.

5-Aza-2<-deoxycytidine induces hepatoma cell apoptosis via enhancing methionine adenosyltransferase 1A expression and inducing S-adenosylmethionine production.

In hepatocellular cancer (HCC), lack of response to chemotherapy and radiation treatment can be caused by a loss of epigenetic modifications of cancer cells. Methionine adenosyltransferase 1A is inactivated in HCC and may be stimulated by an epigenetic change involving promoter hypermethylation. Therefore, drugs releasing epigenetic repression have been proposed to reverse this process. We studied the effect of the demethylating reagent 5-aza-2<-deoxycitidine (5-Aza-CdR) on MAT1A gene expression, DNA methylation and S-adenosylmethionine (SAMe) production in the HCC cell line Huh7. We found that MAT1A mRNA and protein expression were activated in Huh7 cells with the treatment of 5-Aza-CdR; the status of promoter hypermethylation was reversed. At the same time, MAT2A mRNA and protein expression was significantly reduced in Huh7 cells treated with 5-Aza-CdR, while SAMe production was significantly induced. However, 5-Aza-CdR showed no effects on MAT2A methylation. Furthermore, 5-Aza-CdR inhibited the growth of Huh7 cells and induced apoptosis and through down-regulation of Bcl-2, up-regulation of Bax and caspase-3. Our observations suggest that 5-Aza- CdR exerts its anti-tumor effects in Huh7 cells through an epigenetic change involving increased expression of the methionine adenosyltransferase 1A gene and induction of S-adenosylmethionine production.

Direct assembly of aldehydes, amino esters, and anilines into chiral imidazolidines via Brønsted acid catalyzed asymmetric 1,3-dipolar cycloadditions.

A chiral Brønsted acid catalyzed 1,3-dipolar cycloaddition reaction directly assembles aldehydes, amino esters, and anilines into synthetically useful chiral imidazolidines with high levels of stereoselectivity (up to 91/9 dr and 98% ee).

Alternative splicing of human telomerase reverse transcriptase may not be involved in telomerase regulation during all-trans-retinoic acid-induced HL-60 cell differentiation.

Alternative splicing of the human telomerase reverse transcriptase subunit (hTERT) suppresses telomerase activity during the development of human fetal kidney cells into mature cells. Tumor cell differentiation is the process of turning abnormal tumor cells into 'normal' cells accompanied by down-regulation of telomerase activity. However, the precise mechanism of the regulation of telomerase activity in differentiated cells is not fully understood. In this study, we observed the role of alternative splicing of hTERT in the regulation of telomerase activity in all-trans-retinoic acid (ATRA)-induced, differentiated HL-60 cells. ATRA-induced down-regulation of telomerase activity in differentiated HL-60 cells was associated with a decrease in hTERT and an increase in human telomerase-associated protein-1 (hTP1) transcription. Expression of full length variant hTERT alpha+ beta+ mRNA decreased in a dose- and time-dependent manner. The drop of hTERT beta- mRNA was time-dependent. hTERT alpha- and hTERT alpha- beta- mRNA were reduced dramatically after ATRA treatment. In the dose-effect study, hTERT alpha+ beta+ and hTERT beta- maintained a relatively stable ratio when telomerase activity decreased largely from treatment with 1 to 5 microM ATRA. Although the splicing pattern of hTERT mRNA was altered in time-effect research, the change was not related to the ATRA-treated decline of telomerase activity. The expression of alternative splicing variants of hTERT also decreased at the protein level. All these results suggested that alternative splicing of hTERT mRNA may not contribute to the suppression of telomerase activity during ATRA-induced HL-60 leukemia cell differentiation.

Telomerase inhibition is a specific early event in salvicine-treated human lung adenocarcinoma A549 cells.

The telomere and telomerase have been suggested as targets for anticancer drug discovery. However, the mechanisms by which conventional anticancer drugs affect these targets are currently unclear. The novel topoisomerase II inhibitor, salvicine, suppresses telomerase activity in leukemia HL-60 cells. To further determine whether this activity of salvicine is specific to the hematological tumor and distinct from those of other conventional anticancer agents, we studied its effects on telomere and telomerase in a solid lung carcinoma cell line, A549. Differences in telomerase inhibition and telomere erosion were observed between salvcine and other anticancer agents. All anticancer agents (except adriamycin) induced shortening of the telomere, which was identified independent of replication, but only salvicine inhibited telomerase activity in A549 cells under conditions of high concentration and short-term exposure. At the low concentration and long-term exposure mode, all the tested anticancer agents shortened the telomere and inhibited telomerase activity in the same cell line. Notably, salvicine inhibited telomerase activity more severely than the other agents examined. Moreover, the compound inhibited telomerase activity in A549 cells indirectly in a concentration- and time-dependent manner. Salvicine did not affect the expression of hTERT, hTP1, and hTR mRNA in A549 cells following 4 h of exposure. Okadaic acid protected telomerase from inhibition by salvicine. These results indicate specificity of salvicine and diversity of anticancer agents in the mechanism of interference with telomerase and the telomere system. Our data should be helpful for designing the study in the development of agents acting on telomere and/or telomerase.