Here we report a reduced graphene oxide-assisted rolling circle amplification for the detection of miRNA SNPs. The difference of the signal of a miRNA SNP reaches 100 fold, a value over 10 times larger than some current methodologies, which allows the discrimination of a SNP even with the naked eye.

Upregulation of the embryonic M2 isoform of pyruvate kinase (PKM2) emerges as a critical player in the cancer development and metabolism, yet the underlying mechanism of PKM2 overexpression remains to be elucidated. Here we demonstrate that IGF-1/IGF-IR regulates PKM2 expression by enhancing HIF-1α-p65 complex binding to PKM2 promoter. PKM2 expression is regulated by miR-148a/152 suppression. PKM2 directly interacts with NF-κB p65 subunit to promote EGR1 expression for regulating miR-148a/152 feedback circuit in normal cells, but not in cancer cells because of the DNA hypermethylation of miR-148a and miR-152 gene promoters. The silencing of miR-148a/152 contributes to the overexpression of PKM2, NF-κB or/and IGF-IR in some cancer cells. We show that disruption of PKM2/NF-κB/miR-148a/152 feedback loop can regulate cancer cell growth and angiogenesis, and is also associated with triple-negative breast cancer (TNBC) phenotype, which may have clinical implication for providing novel biomarker(s) of TNBC and potential therapeutic target(s) in the future.

Rational utilization of nanomaterials to construct electrochemical nucleic acid sensors has attracted large attention in recent years. In this work, we systematically interrogate the interaction between gold nanoparticles (GNPs) and single-strand DNA (ssDNA) immobilized on an electrode surface and then take advantage of the ultrahigh charge-transfer efficiency of GNPs to develop a novel DNA sensing method. Specifically, ssDNA modified gold electrode can adsorb GNPs because of the interaction between gold and nitrogen-containing bases; thus, the negative electrochemical species [Fe(CN)6]3–/4– may transfer electrons to electrode through adsorbed GNPs. In the presence of target DNA, the formed double-strand DNA (dsDNA) cannot capture GNPs onto the electrode surface and the dsDNA may result in a large charge-transfer resistance owing to the negatively charged phosphate backbones of DNA. So a simple but sensitive method for the detection of target DNA can be developed by using GNPs without any requirement of modification. Experimental results demonstrate that the electrochemical method we have proposed in this work can detect as low as 1 pM breast cancer gene BRCA1 in a 10 μL sample volume without any signal amplification process or the involvement of other synthesized complex, which may provide an alternative for cancer DNA detection. This method may also be generalized for detecting a spectrum of targets using functional DNA (aptamer, metal-specific oligonucleotide, or DNAzyme) in the future.Keywords: biosensor; charge transfer; DNA; electrochemical impedance spectroscopy; gold nanoparticle;

A lipase-based electrochemical biosensor has been fabricated for the quantitative determination of target DNA. It is based on a stem-loop nucleic acid probe labeled with ferrocene containing a butanoate ester that is hydrolyzed by lipase. The other end of the probe DNA is linked, via carboxy groups, to magnetic nanoparticles. The binding of target DNA transforms the hairpin structure of the probe DNA and causes the exposure of ester bonds. This results in the release of electro-active ferrocene after hydrolysis of the ester bonds, and in an observable electrochemical response. The quantity of target DNA in the concentration range between 1 × 10−12 mol·L−1 and 1 × 10−8 mol·L−1 can be determined by measuring the electrochemical current. The method can detect target DNA with rapid response (30 min) and low interference.

•A sensitive biosensor for Dicer activity is developed.•Methylene blue labeled DNA is used as the signal reporter.•It has potential utility for functional studies of Dicer and RNAi.Dicer cleaves long double strand RNA (dsRNA) into short ones and initiates RNA interference (RNAi), a sequence-specific post-transcriptional gene silencing process. Moreover, it is also involved in the development of cancers. Herein, we report an electrochemical method to study the ribonuclease activity of Dicer for the first time. The designed dsRNA which is thiolated at one end is firstly immobilized on a gold electrode. Square wave voltammetry is then employed to characterize the ribonuclease activity of Dicer after the enzyme digestion of the dsRNA and the subsequent hybridization between short denatured RNA on the electrode and DNA molecules labeled with electrochemical species. This proposed method shows desirable sensitivity and reproducibility, demonstrating the great potential utility for functional studies of Dicer and RNAi in the future.

•A sensitive biosensor for clenbuterol is developed using platinum nanoparticles.•It can be used to unequivocally diagnose clenbuterol food poisoning.•It has potentials to develop next-generation strategies for pharmacokinetic study.Clenbuterol is a member of β2 adrenergic agonists, which is widely used not only as a food additive for livestocks, but also a kind of stimulant for athletes; however, the abuse of clenbuterol may pose a significant negative impact on human health. Since it is highly required to develop fast, sensitive and cost-effective method to determine clenbuterol level in the suspected urine or blood, we herein have fabricated an electrochemical biosensor for the determination of clenbuterol. Measurement of the species with the proposed biosensor can also have the advantages of simplicity, high sensitivity and selectivity. Moreover, the sensor can be directly used for clenbuterol determination in rat urine. We have further studied the pharmacokinetics of clenbuterol by using this proposed electrochemical biosensor, so a new tool to investigate pharmacokinetic is developed in this work.Graphical abstract

Proteases are involved in a large number of serious disease processes, while the assay of proteolytic activity can be used for clinical diagnostics. In this paper we report a simple electrochemical method to assay protease activity. This method makes use of an unlabeled peptide that comprises the specific substrate domain of a protease, which can be easily operated and generalized for assay of various kinds of proteases. Specifically, the peptide is immobilized onto a gold electrode surface via the chemical adsorption of the C-terminal cysteine residue, forming a positively charged interface derived from the N-terminal cationic residue. Therefore, the positive electrochemical probes [Ru(NH3)5Cl]2+ cannot get across to the electrode to generate signal. Nevertheless, the proteolytic digestion of the peptide will decrease the number of positive charges on the electrode surface and weaken the blocking effect against the positive electrochemical species, resulting in an increased electrochemical signal. Under optimized conditions, the activity of the model protease, trypsin, can be assayed with a detection limit of 0.026 U/mL. The method may also allow the determination of trypsin activity in serum samples. Moreover, since this approach can be used for the assay of other proteases by simply changing the substrate domain of the peptide, it may have great potential in biomedical applications in the future.Highlights► A simple electrochemical biosensor is proposed for the assay of protease activity. ► The detection bases on proteolytic digestion of unlabeled peptide substrates. ► Trypsin is detected as a model analyte with a detection limit of 0.05 U/mL. ► This method is applicable to other proteases with a suitable peptide substrate.

Cytokine-induced killer (CIK) cells exert high impact on adoptive immunotherapeutic approaches for malignant tumors. This study aimed to evaluate the effect of adjuvant immunotherapy with CIK cells on the prognosis of solid tumor.Peripheral blood mononuclear cells were collected by a blood cell separator from 40 patients, then expanded by priming them with interferon-gamma followed by monoclonal antibody against CD3 and interleukin-2 the next day. The phenotypic patterns of CIK cells were characterized by flow cytometry on days 0, 7, 10, 14 and 21 of incubation, respectively. Then, 5 ml of venous blood was obtained from 40 patients before and after CIK cells were transfused into patients to assess the influence of CIK cells on the percentages of effector cells.After 14 days of incubation in vitro, the percentages of CD3+, CD8+, CD3+ and CD56+ increased significantly (P < 0.05). The clinical symptoms of 40 patients were improved apparently. The 6-month, 1-year and 3-year overall survival rates were 70.0, 60.0 and 57.5%, respectively.Our results indicated that CIKs immunotherapy can be an effective adjuvant instrument of the routine therapy of malignancy.

In this work we have developed a novel electrochemical biosensor for the detection of alkaline phosphatase (AP) by the use of two complementary DNA probes (DNA 1 and DNA 2) coupled with λ exonuclease (λ exo). Firstly, the 5′-phosphoryl end of DNA 1 is dephosphorylated by AP. Then DNA 1 hybridizes with DNA 2, previously modified on a gold electrode surface. In this double-strand DNA, DNA 2 strand will be promptly cleaved by λ exo with its phosphoryl at the 5′ end. After the DNA 2 strand is completely digested, DNA 1 will be released from the double strands and then hybridizes with another DNA 2 strand on the electrode surface, thus the cycle of the release of DNA 1 and the digestion of DNA 2 continues. Since the DNA probes may absorb hexaammineruthenium(III) chloride, the electrochemical species, and the removal of the DNA 2 strand from the electrode surface will result in the decrease of the detected electrochemical signal, which is initially activated by AP, an electrochemical biosensor to assay the activity of AP is proposed in this work. This method may have a linear detection range from 1 to 20 unit/mL with a detection limit of 0.1 unit/mL, and the detection of the enzymatic activity in complex biological fluids can also be realized.

This study compared miRNA expression patterns in primary squamous cell lung carcinoma specimens with those of matched normal lung tissue in order to determine their potential relevance to clinicopathological factors and patient postoperative survival times.Locked nucleic acids miRNA microarray expression profiling was performed on four matched pairs of tissues. After microarray validation by quantitative real-time reverse transcription polymerase chain reaction assays (qRT-PCR) (real-time PCR), miR-21 was selected for further TaqMan real-time PCR study in 30 matched tissue pairs.Seven miRNAs of hsa-miR-21, hsa-miR-31, hsa-miR-34a, hsa-miR-22*, hsa-miR-504, hsa-miR-18a, and hsa-miR-412 were observed to be upregulated greater than twofold in the squamous cell lung carcinoma tissues compared with normal tissues, whereas 23 miRNAs of hsa-miR-30a, hsa-miR-30d, hsa-miR-126, hsa-miR-652, hsa-miR-100, hsa-miR-143, hsa-miR-130a, hsa-miR-145, hsa-miR-30e, hsa-miR-126*, hsa-miR-181a, hsa-miR-125b, hsa-miR-886-3p, hsa-miR-451, hsa-miR-29c, hsa-miR-26b, hsa-miR-101, hsa-miR-320, hsa-miR-30b, hsa-miR-886-5p, hsa-miR-29a, hsa-miR-26a, and hsa-miR-99a were found to be downregulated greater than twofold. MiR-21 was overexpressed in 73.3% of the squamous cell lung carcinoma specimens examined (P = 0.022). The relationship between the miR-21 expression level and various clinicopathologic factors was also analyzed. High-level expression of miR-21 was significantly correlated with shortened survival time (P = 0.022, log-rank test; Kaplan–Meier). Multivariate Cox proportional hazard regression analysis revealed this significant prognostic impact (P = 0.000; HR 1.293; 95% CI 1.123–1.489) to be independent of clinical disease stage (P = 0.013; HR 2.660; 95% CI 1.229–5.758) and other clinicopathologic factors.Expression patterns of miRNAs were found to be systematically altered in squamous cell lung carcinoma tissue. High miR-21 expression is associated with shortened survival time, indicating that miR-21 may serve as a molecular diagnostic and prognostic marker for patients with squamous cell lung carcinoma.

Rab GTPases, which belong to the Ras superfamily, represent a group of small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. We first identified mRabL5 (GenBank Accession No. NP_080349), a novel Mus musculus Rab-like protein, present as a Golgi-associated protein. Here we presented the results of the cloning, prokaryotic expression, purification, and polyclonal antibody production of the novel Rab-like protein. In order to obtain a specific antibody against mRabL5, we prepared two GST fusion proteins, full-length mRabL5 GST fusion protein and mRabL5 C terminus GST fusion protein, to immunize rabbits. Western blot analysis showed that both antibodies prepared against full length of mRabL5 and its C terminus, respectively, can recognize mRabL5 protein. Immunofluorescence of mRabL5 in NIH3T3 cells using the two antibodies showed its perinuclear clustering distribution pattern. The polyclonal antibodies preparation against mRabL5 provided a good tool for us to study the functional involvement of mRabL5.

The purpose of this study was to evaluate the outcome of patients with unresectable hepatocellular carcinoma (HCC) treated by sequential therapy of transcatheter arterial chemoembolization (TACE), three-dimensional con-formal radiotherapy (3-DCRT) and high-intensity focused ultrasound (HIFU). From October, 2005 to September, 2010, 120 patients with unresectable HCC received the sequential treatments of several courses of TACE followed in 2-4 weeks by 3-DCRT and then a single session of HIFU with a curative intent. The median tumor irradiation dose was 40 Gy. Tumor response, toxicity and overall survival rate were analyzed. Clinicopathologic factors af-fecting the primary technique effectiveness and overall survival rates were investigated by univariate analysis or multivariate analysis. All 120 HCC patients were followed up by the last follow-up time. Among these patients, hepatic toxicities due to treatment were notable in 9 cases. Gastrointestinal bleeding after the overall treatment occurred in 2 cases, leukopenia of grade III was detected in 1 case, radiation-induced liver disease (RILD) was observed in 2 patients, and first- and second-degree skin burn around the HIFU treatment zone were observed in 2 patients and 1 patient, respectively. Among 120 patients, 23, 83 and 14 cases achieved partial response, stable disease and progressive disease, respectively. The overall survival rates at 1 year, 3 years and 5 years were 70%, 35% and 15%, respectively, with a median survival time of 26 months. Both Child-Pugh liver function grading and radiation dose were determined to be independent predictors for overall survival revealed by the multivariate analysis. It is concluded that the sequential therapy of TACE, 3-DCRT and HIFU is a promising therapeutic regi-men for unresectable HCC

The cell cycle governs the proliferation and growth of cells and is strictly controlled by some regulators including cyclins, CDKs and CKIs. Germ-line and somatic mutations in cell cycle genes were frequently observed in a subset of cancers including non-small cell lung cancer (NSCLC). In this study, we hypothesized that potentially functional single nucleotide polymorphisms (SNPs) in cell cycle genes may contribute to the prognosis of NSCLC in China. 54 potentially functional polymorphisms in key cell cycle genes (CDK1, CDK2, CDK4, CDK6, CDK7, CCND1, CCND2, CCND3, CCNE1, CCNA1, CCNA2, CCNB1, CCNH, p15, p16, p18, p19, p21, p27, Cdc25A and Cdc25B) were genotyped by using Illumina SNP genotyping platform to evaluate their associations with survival of NSCLC in a clinical cohort of 568 patients. We found that p18 rs3176447 variant genotypes were significantly associated with the decreased risk of death of NSCLC patients (adjusted HR = 0.74, 95% CI = 0.57–0.97 in an additive model; adjusted HR = 0.76, 95% CI = 0.55–0.97 in a dominant model); however, p21 rs2395655 variant genotypes were significantly associated with the increased risk of death (adjusted HR = 1.21, 95% CI = 1.02–1.42 in an additive model; adjusted HR = 1.38, 95% CI = 1.07–1.78 in a recessive model). Furthermore, the combined effect of unfavorable genotypes for these two SNPs was more prominent in patients with squamous cell carcinoma, late stage and without chemo- or radio-therapy. Although the exact biological function remains to be explored, our findings suggest possible association of polymorphisms of p18 and p21 with the prognosis of NSCLC in a Chinese population. Further large and functional studies are needed to confirm our findings.

Lung cancer is the most common cancer in the world. It is a highly lethal disease in women and men, and new treatments are urgently needed. Several studies have implicated a role of estrogens and estrogen receptors in lung cancer progression. This review will investigate the biological significance of estrogens in lung cancer cells, the expression and molecular mechanisms of estrogen receptors(ER β, elucidate the prognostic significance of estrogens and their receptors in lung carcinomas and provide new options for patients afflicted with lung malignancies.

BackgroundNonsteroidal anti-inflammatory drugs (NSAIDs), especially aspirin, have emerged as the most potential chemopreventive agents. However, epidemiologic studies reported a controversial association between NSAID use and lung cancer risk. We conducted a meta-analysis to summarize the evidence for such relationship.MethodsEligible studies were identified by searching the electronic literature PubMed, Medline, Embase, and ScienceDirect databases for relevant reports and bibliographies. Studies were included if they designed as cohort study, case-control study, or clinical trial on the NSAID exposure and lung cancer with sufficient raw data to analyzes. Relative risk (RR) or odds ratio (OR) was used to evaluate the association between NSAIDs and lung cancer. Stratified analysis was also performed.ResultsA total of 19 studies including 20,266 lung cancer cases met the inclusion criteria. To the effect of aspirin on lung cancer, the combined RR for cohort studies was 0.96 (95%confidence interval [CI]: 0.78-1.19) and OR for case-control studies was 0.87 (95%CI: 0.69-1.09). When restricted in exposure of aspirin use to 7 tablets per week, the OR was 0.80 (95%CI: 0.67-0.95). The summary risk estimates showed no significant association between non-aspirin NSAID or overall NSAID use and lung cancer risk.ConclusionsAspirin use with a dose of 7 tablets per week can significantly reduce lung cancer risk, whereas non-aspirin NSAIDs showed no chemopreventive value. Greater attention should be paid to identifying appropriate individuals for this new indication of aspirin and the optimal dose and duration as a chemopreventive agent.

ObjectiveTo investigate the effects of CD137 signaling on the regulation of CD3−CD56+NK cells function.MethodsCD3− CD56+NK cells were treated with CD137 mAb or mouse IgG1 isotype control to study the effects of CD137 signaling on the function of CD3−CD56+NK cells. Cytotoxicity was measured by LDH activity in the supernatants of cell cultures; NKG2D and LFA-1 expression on CD3−CD56+NK cells were analyzed by flow cytometry.ResultsCD137 was expressed on activated CD3−CD56+NK cells. The CD137 mAb enhanced the ability of CD3−CD56+NK cells to kill lung cancer cells(A549); Further studies revealed that the expression of NKG2D and LFA-1 was significantly increased in activated cells, and blockade of NKG2D and LFA-1 dramatically attenuated CD3−CD56+NK cytolysis of A549 cancer cells.ConclusionCD137 signaling increases the ability of CD3−CD56+NK cells to kill cancer cells via up-regulating the expression of NKG2D and LFA-1.

Here we report a reduced graphene oxide-assisted rolling circle amplification for the detection of miRNA SNPs. The difference of the signal of a miRNA SNP reaches 100 fold, a value over 10 times larger than some current methodologies, which allows the discrimination of a SNP even with the naked eye.