Metal sulfide nanoparticle-based dual barcode-triggered DNAzyme cascade for multiplex miRNA detection in a single assay.

Single miRNAs are not specific and accurate enough to meet the strict diagnosis requirements in practice. Therefore, simultaneous monitoring of multiplexed miRNA in biological samples can not only improve the accuracy and specificity of bioassays but also avoid the squandering of valuable biological specimens. Herein, we designed a metal sulfide nanoparticle-based dual barcode-triggered DNAzyme cascade strategy for the sensitive and simultaneous multiplex miRNA detection in a single assay. Firstly, the capture probes (H1, H2) specifically recognize targets (miRNA-21, miRNA-141), exposing the stem of H1 and H2. Then, with the introduction of a detection probe (CuS-H3, ZnS-H4), the exposed H1 and H2 catalyze the hairpin assembly (CHA) reaction, realizing target miRNA recycling, and forming H1/H3-CuS and H2/H4-ZnS complexes. Subsequently, the formed H1/H3-CuS and H2/H4-ZnS complexes are encoded on magnetic beads through the biotin/streptavidin interaction. The CuS and ZnS nanoparticles captured by magnetic beads release thousands of Cu2+ and Zn2+via the cation exchange reaction. Finally, the released Cu2+ and Zn2+ specially activate the DNAzyme of the catalytic and molecular beacon (CAMB) system. The CAMB system affords an amplified fluorescence signal output by cycling and regenerating the metal ion-dependent DNAzyme to realize multiple enzymatic turnovers. Benefiting from target recycling, nanoparticle amplification, and catalytic and molecular beacon amplification, there is substantial amplification and the target miRNAs can be detected at 0.06 fM (miRNA-21) and 0.048 fM (miRNA-141) in a single assay. Furthermore, the high selectivity and accuracy of the assay were proved by practical analysis of different cancer cells, which exhibited good practicability in multiplex miRNA detection in clinical sera. The results indicate that the proposed strategy holds great potential for the sensitive detection of multiplex cancer biomarkers and offers the opportunity for future applications in clinical diagnosis.

Blockade of IL-17A/IL-17R Pathway Protected Mice from Sepsis-Associated Encephalopathy by Inhibition of Microglia Activation.

Sepsis-associated encephalopathy (SAE) is a poorly understood condition that leads to long-term cognitive impairment and increased mortality in survivors. Recent research revealed that IL-17A/IL-17R might serve as a checkpoint in microglia-mediated neuroinflammation. The present study was designed to determine the specific role of IL-17A-mediated microglia activation in the development of SAE. A mouse model of SAE was induced by cecal ligation and puncture (CLP), and behavior performance was evaluated by the inhibitory avoidance test and the open field test. Cytokine expression and microglia activation in brain tissue were determined at 6 h, 12 h, 24 h, 48 h, and day 7 post surgery. Further, septic mice were intracerebral ventricle- (i.c.v.-) injected with recombinant IL-17A, anti-IL-17A ab, anti-IL-17R ab, or isotype controls to evaluate the potential effects of IL-17A/IL-17R blockade in the prevention of SAE. Septic peritonitis induced significant impairment of learning memory and exploratory activity, which was associated with a higher expression of IL-17A, IL-1ß, and TNF-α in the brain homogenate. Fluorescence intensity of Iba-1 and IL-17R in the hippocampus was significantly increased following CLP. Treatment with recombinant IL-17A enhanced the neuroinflammation and microglia activation in CLP mice. On the contrary, neutralizing anti-IL-17A or anti-IL-17R antibodies mitigated the CNS inflammation and microglia activation, thus alleviating the cognitive dysfunction. Furthermore, as compared to the sham control, microglia cultured from CLP mice produced significantly higher levels of cytokines and expressed with higher fluorescence intensity of Iba-1 in response to IL-17A or LPS. Pretreatment with anti-IL-17R ab suppressed the Iba-1 expression and cytokine production in microglia stimulated by IL-17A. In conclusion, blockade of the IL-17A/IL-17R pathway inhibited microglia activation and neuroinflammation, thereby partially reversing sepsis-induced cognitive impairment. The present study suggested that the IL-17A/IL-17R signaling pathway had an important, nonredundant role in the development of SAE.

Fluorescence-enhanced p19 proteins-conjugated single quantum dot with multiplex antenna for one-step, specific and sensitive miRNAs detection.

Ideal methods for miRNA detection should be rapid and amplification-free in the mix-and-measure format. Here we report a novel FRET strategy based on fluorescence-enhanced p19 proteins-conjugated single quantum dot (QD) with multiplex antenna for one-step, specific and sensitive miRNAs quantitative analysis. It is simple in design and operation, and possesses high sensitivity and selectivity. In the strategy, RNA-binding viral p19 proteins-conjugated quantum dot (p19-QD) with high fluorescence and multiplex antenna was designed, and exploited for the capture recognition element and the donor. In the presence of target miRNAs, the p19-QD would tightly capture the miRNA-21-antimiRNA-21 duplex (dsRNA) formed in solution by hybridization of the specific antimiRNA-21-Cy3 probe (receptor) to the single-stranded miRNA-21 target. The FRET detection system between QD and Cy3 was constructed, and the signal read-out was measured. Conversely, in the absence of target miRNA-21, the p19-QD refuse to capture the free single-stranded antimiRNA-21-Cy3 probe (receptor), the FRET between quantum dot and Cy3 is deterred, thereby providing a low-background signal to improve sensitivity. Benefiting from the high affinity and specificity of p19 protein for duplex RNA and the fluorescence enhancement of donor p19-QD by the passivation and protection from p19 protein, a detection limit as low as 0.6 fM was achieved without employing any amplification techniques and pre-concentration or purification steps. The performance can be achieved by only one-step incubation without additional reagents and washing steps, thus greatly reducing the operating difficulty and saving time. Moreover, the fluorescence-enhanced p19-QD conjugate-based FRET strategy avoids the specific design in the number of the bases for interval in the DNA-QD conjugate-based FRET system using DNA strands as the FRET linker, and improves the FRET efficiency. Furthermore, it has been successfully applied to analyze the content of miRNA-21 in breast cancer cell. The results indicated that, the strategy will become a sensitive and reliable miRNAs quantification method in biomedical research and early clinical diagnostics.

Expression of calcium sensing receptor and E-cadherin correlated with survival of lung adenocarcinoma.

BACKGROUND: It has been reported that the calcium sensing receptor (CaSR), a widely expressed G protein-coupled receptor, can stimulate cell differentiation and proliferation. However, in malignant tumors, loss of CaSR expression has been associated with tumorigenesis, metastasis, and progression. Recent studies have indicated that the CaSR could promote the expression of E-cadherin, which was considered a tumor suppressor. However, in human lung adenocarcinoma, the importance of the CaSR and E-cadherin has not been sufficiently investigated. METHODS: Expression levels of CaSR and E-cadherin in paraffin sections from 117 resected lung adenocarcinoma patients were evaluated by immunohistochemistry. We analyzed the correlation between our target proteins and clinical variables. Clinical significance was analyzed by multivariate Cox regression analysis, Kaplan-Meier curve, and log-rank test. RESULTS: Expression of the CaSR in lung adenocarcinoma tissue was significantly lower than in the normal sample (P = 0.003). Kendall tau-b analysis showed that, in a lung adenocarcinoma sample, the expression of CaSR positively correlated with a high level of E-cadherin (P < 0.001). Lung adenocarcinoma patients with a strong expression of CaSR (P = 0.034) or E-cadherin (P = 0.001) had longer overall survival. Multivariate Cox proportional hazards model analysis showed that the combined marker was an independent prognostic indicator of overall survival (hazard ratio = 0.440, confidence interval = 0.249-0.779, P = 0.005). CONCLUSIONS: We identified the CaSR as a new prognostic biomarker in lung adenocarcinoma. These results also suggested that the CaSR may become a new therapeutic target of lung adenocarcinoma.