Label-free and sensitive MiRNA detection based on turn-on fluorescence of DNA-templated silver nanoclusters coupled with duplex-specific nuclease-assisted signal amplification.

A novel strategy for microRNAs (miRNAs) detection has been developed utilizing duplex-specific nuclease-assisted signal amplification (DSNSA) and guanine-rich DNA-enhanced fluorescence of DNA-templated silver nanoclusters (AgNCs). The combination between target miRNA, DSNSA, and AgNCs is achieved by the unique design of DNA sequences. Target miRNA opens the hairpin structure of the Hairpin DNA probe (HP) by hybridizing with the HP and initiates the duplex-specific nuclease-assisted signal amplification (DSNSA) reaction. The DSNSA reaction generates the release of the guanine-rich DNA sequence, which can turn on the fluorescence of the dark AgNCs by hybridizing with the DNA template of the dark AgNCs. The fluorescence intensity of AgNCs corresponds to the dosage of the target miRNA. This is measured at 630 nm by exciting at 560 nm. The constructed method exhibits a low detection limit (~8.3 fmol), a great dynamic range of more than three orders of magnitude, and excellent selectivity. Moreover, it has a good performance for miR-21 detection in complex biological samples. A novel strategy for microRNAs (miRNAs) detection has been developed utilizing duplex-specific nuclease-assisted signal amplification (DSNSA) and guanine-rich DNA-enhanced fluorescence of DNA-templated silver nanoclusters (AgNCs).

Efficacy and Safety of Omalizumab for Chronic Spontaneous Urticaria: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Omalizumab has been proposed as a possible effective treatment of chronic spontaneous urticaria (CSU). STUDY QUESTION: We aimed to access the efficacy and safety of omalizumab in the treatment of CSU based on qualified, randomized controlled trials (RCTs). DATA SOURCES: PubMed, the Cochrane library, and Embase databases. STUDY DESIGN: Computerized search by index words was performed to identify qualified RCTs, and relevant literature sources were also searched. RESULT: Nine RCTs were included in the meta-analysis with 1612 patients in the omalizumab group and 1251 patients in the placebo group. Compared with the placebo group, omalizumab significantly decreased the weekly itch score after therapy [Weighted Mean Difference (WMD), -3.94; 95% confidence interval (CI), -4.64 to -3.24], the weekly hive score (WMD, -5.27; 95% CI, -6.17 to -4.38), the dermatology life quality index (DLQI; WMD, -3.58; 95% CI, -4.66 to -2.50), and the urticaria activity score over 7 days (UAS7; WMD, -9.51; 95% CI, -10.94 to -8.08). There was no significant difference in the incidence of adverse events (AE) [relative risk (RR), 1.01; 95% CI, 0.91-1.12], serious AE (RR, 0.85; 95% CI, 0.57-1.27), and severe AE (RR, 0.83; 95% CI, 0.60-1.14) between the 2 groups. Compared with the placebo, omalizumab significantly decreased the weekly itch score and weekly hive score after therapy in patients receiving 75, 150, and 300 mg omalizumab, respectively. DLQI was significantly reduced in patients receiving 150 and 300 mg of omalizumab, respectively. In all the subgroup of UAS7, omalizumab significantly decreased the score compared with the placebo. Only patients receiving 600-mg omalizumab had a significantly higher AE incidence versus placebo. There was no significant difference in serious and severe AE between the 2 groups. CONCLUSION: Omalizumab caused a significantly greater reduction in weekly itch score, weekly hive score, DLQI, and UAS7 in CSU patients than the placebo. However, high-quality, multicenter RCTs with a larger sample size are needed to confirm the safety of omalizumab, and whether AEs are caused by omalizumab or other factors.

Efficacy and safety of imiquimod 5% cream for basal cell carcinoma: a meta-analysis of randomized controlled trial.

Background: We conducted this meta-analysis to compare the efficacy and safety of imiquimod with other treatments in patients with basal cell carcinoma (BCC).Methods: A comprehensive literature search was performed in the database of PubMed, Embase, and Web of Science, focusing on studies that evaluated the efficacy and safety profile of BCC patients who were treated with imiquimod. The main outcome measures included histological/composite clearance rate, success rate, complete response rate, tumor free survival, and adverse events. Results were expressed as risk ratio (RR) with 95% confidence intervals (CIs). Pooled estimates were calculated using a fixed-effects or random-effects model according to the heterogeneity among studies.Results: A total of 13 studies involving 4256 patients were included in this meta-analysis. Imiquimod was associated with significantly higher histological clearance rate and composite clearance rate. Moreover, imiquimod also significantly increased complete response rate but had no effect in the success rate and probability of tumor free survival, as compared with other treatments. There were more patients with imiquimod developed adverse events than those with other treatments.Conclusion: The present meta-analysis indicated the effects of imiquimod in improving the histological/composite clearance rates as compared with other treatments.

Effect of RNA Interference Targeting STAT3 Gene Combined with Ultrasonic Irradiation and SonoVue Microbubbles on Proliferation and Apoptosis in Keratinocytes of Psoriatic Lesions.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) was strongly expressed and activated in psoriatic keratinocytes (KCs) and correlated with the severity of psoriasis. The study aimed to investigate the effects of STAT3 small interfering RNA (siRNA) combined with ultrasonic irradiation and SonoVue microbubbles on the proliferation and apoptosis in KCs of psoriatic lesions and the relative mechanisms. METHODS: Psoriatic KCs were transfected under four experimental conditions: (1) STAT3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group); (2) STAT3 siRNA only carried by Lipofectamine 3000 (L group); (3) the negative control of siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (siRNA-NC); (4) not treated as Blank. Cell Counting Kit-8 assay was used to evaluate the cell proliferation. Cell cycle analysis was detected with cycle test Plus DNA reagent kit associated with flow cytometer. FITC Annexin V apoptosis detection kit associated with flow cytometer was applied for apoptosis analysis. Fluo calcium indicator associated with flow cytometer was used to analyze intracellular free calcium concentration ([Ca2+]i). The expressions of cyclin D1 and Bcl-xL were detected both at the mRNA level by real-time reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. The obtained data were statistically evaluated by two-way analysis of variance. RESULTS: STAT3 siRNA inhibited the growth of KCs in a time-dependent manner showing the highest proliferation inhibition in LUS group with proliferation ratio of 45.38% ± 5.85% at 72h (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced an altered cell cycle distribution of KCs showing the highest increases in G2/M-phase population up to 18.06% ± 0.36% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced late apoptosis of KCs with the highest late apoptosis percentage of 22.87% ± 1.28% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the elevation of [Ca2+]iof KCs with the highest calcium fluorescence intensity mean of 1213.67 ± 60.51 in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the downregulation of cyclin D1 and Bcl-xL expressions of KCs at mRNA and protein levels with the lowest expressions in LUS group with cyclin D1 expression of 51.81% ± 9.58% and 70.17% ± 4.22% at mRNA level and at protein level, respectively, and with Bcl-xL expression of 37.58% ± 4.92% and 64.06% ± 7.78% at mRNA level and at protein level, respectively (P < 0.05 vs. L group, siRNA-NC, or Blank). CONCLUSIONS: STAT3 siRNA inhibited the growth and induced the apoptosis in psoriatic KCs likely partly through altering cell cycle distribution, elevating [Ca2+]i, and downregulating cyclin D1 and Bcl-xL expressions. Silencing the target gene STAT3 in psoriatic KCs with siRNA combined with ultrasonic irradiation and microbubbles would contribute to a significant innovation as a new clinical therapy for psoriasis.

Inhibitory Effect of MiR-449b on Cancer Cell Growth and Invasion through LGR4 in Non-Small-Cell Lung Carcinoma / 华中科技大学学报（医学）（英德文版）

Non-small-cell lung carcinoma (NSCLC) is one of the most frequently diagnosed malignancies worldwide.Previous studies have shown that microRNA-449b (miR-449b) functions as a tumor suppressor in many cancers.However,the role of miR-449b in NSCLC is still unknown.In the present study,miR-449b was significantly downregulated in NSCLC samples and cell lines.Bioinformatics analysis revealed that 3'-UTR region of leucine rich repeat containing G protein-coupled receptor 4 (LGR4) mRNA had putative complementary sequences to miR-449b,which was further confirmed by the luciferase assay.Western blotting showed that restoration of miR-449b in NSCLC cells decreased the expression of LGR4.Interestingly,over-expression of miR-449b inhibited growth and invasion of NSCLC cells in vitro.Furthermore,ectopic expression of LGR4 reversed miR-449b-suppressed proliferation and invasion of NSCLC cells.Therefore,the data of the present study demonstrate that miR-449b inhibits tumor cell growth and invasion by targeting LGR4 in NSCLC.

Inhibitory Effect of MiR-449b on Cancer Cell Growth and Invasion through LGR4 in Non-Small-Cell Lung Carcinoma / 华中科技大学学报（医学）（英德文版）

Non-small-cell lung carcinoma (NSCLC) is one of the most frequently diagnosed malignancies worldwide.Previous studies have shown that microRNA-449b (miR-449b) functions as a tumor suppressor in many cancers.However,the role of miR-449b in NSCLC is still unknown.In the present study,miR-449b was significantly downregulated in NSCLC samples and cell lines.Bioinformatics analysis revealed that 3'-UTR region of leucine rich repeat containing G protein-coupled receptor 4 (LGR4) mRNA had putative complementary sequences to miR-449b,which was further confirmed by the luciferase assay.Western blotting showed that restoration of miR-449b in NSCLC cells decreased the expression of LGR4.Interestingly,over-expression of miR-449b inhibited growth and invasion of NSCLC cells in vitro.Furthermore,ectopic expression of LGR4 reversed miR-449b-suppressed proliferation and invasion of NSCLC cells.Therefore,the data of the present study demonstrate that miR-449b inhibits tumor cell growth and invasion by targeting LGR4 in NSCLC.

Effects of RNA interference combined with ultrasonic irradiation and SonoVue microbubbles on expression of STAT3 gene in keratinocytes of psoriatic lesions.

The most effective sequence of small interfering RNA (siRNA) silencing STAT3 of psoriatic keratinocytes (KCs) was screened out, and the effects of the most effective siRNA combined with ultrasonic irradiation and SonoVue microbubbles on the expression of STAT3 of KCs and the dose- and time-response were investigated. Three chemically-synthetic siRNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs, and the effects on STAT3 expression were detected, then the most effective siRNA was selected for the subsequent experiments. The negative controls of siRNA (siRNA-NC) labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles were transfected into KCs, then the optimal parameters of ultrasonic irradiation were determined. The most effective siRNA carried by Li-pofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and SonoVue microbubbles was transfected into KCs, and the dose- and time-response of RNA interference was determined. The effect of RNA interference by the most effective siRNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group) was compared with that only carried by Li-pofectamine 3000 (L group). The results showed that siRNA-3 achieved the highest silencing efficacy. 0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation. The siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3 expression at mRNA and protein levels in dose- and time-dependent manners determined at 100 nmol/L with maximum downregulation on mRNA at 48 h, and on protein at 72 h after transfection. The LUS group achieved the highest silencing efficacy. It was concluded that siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs, and the optimized transfection condition and the sequence of siRNA-3 could serve for further research on gene therapy of psoriasis.

Effects of RNA Interference Combined with Ultrasonic Irradiation and SonoVue Microbubbles on Expression of STAT3 Gene in Keratinocytes of Psoriatic Lesions / 华中科技大学学报（医学）（英德文版）

The most effective sequence of small interfering RNA (siRNA) silencing STAT3 of psoriatic keratinocytes (KCs) was screened out,and the effects of the most effective siRNA combined with ultrasonic irradiation and SonoVue microbubbles on the expression of STAT3 of KCs and the dose-and time-response were investigated.Three chemically-synthetic siRNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs,and the effects on STAT3 expression were detected,then the most effective siRNA was selected for the subsequent experiments.The negative controls of siRNA (siRNA-NC) labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles were transfected into KCs,then the optimal parameters of ultrasonic irradiation were determined.The most effective siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and SonoVue microbubbles was transfected into KCs,and the dose-and time-response of RNA interference was determined.The effect of RNA interference by the most effective siRNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group) was compared with that only carried by Lipofectamine 3000 (L group).The results showed that siRNA-3 achieved the highest silencing efficacy.0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation.The siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue mierobubbles could effectively knock down the STAT3 expression at mRNA and protein levels in dose-and time-dependent manners determined at 100 nmol/L with maximum downregulation on mRNA at 48 h,and on protein at 72 h after transfection.The LUS group achieved the highest silencing efficacy.It was concluded that siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs,and the optimized transfection condition and the sequence of siRNA-3 could serve for further research on gene therapy of psoriasis.

[Treatment of Guishen Zhiyang Recipe for Senile Pruritus Patients with Blood Deficiency and Hy- peractivity of Gan-yang Syndrome].

Objective To observe the clinical efficacy of Guishen Zhiyang Recipe (GZR) in trea- ting senile pruritus patients with blood deficiency and hyperactivity of Gan-yang syndrome (BDHGYS) and to study the mechanism in nervous-endocrine-immune systems. Methods Ninety senile pruritus pa- tients were assigned to the treatment group and the control group by complete randomized lot, 45 in each group. Totally 41 patients in the treatment group and 38 patients in the control group completed the trial. Patients in the treatment group took GZR, while those in the control group took Fuyang Granule (FG). Ex- ternal application of Binghuang Fule Soft Ointment was performed to all patients. The therapeutic course for all was 8 weeks. Symptoms and efficacy changes in skin lesion were observed in the two groups. Lev- els of stem cell factor (SCF) , dynorphin (DYN) , testosterone (T) , estradiol (E2) , and IL-4 were detec- ted in the two groups using double antibody sandwich ELISA. Results Compared with before treatment, pruritus degree, scratching area, scaling, lichenoid lesion, irritability and restlessness, dysphoria, dry pharynx, dizziness, and tinnitus all decreased in the two groups after 8 weeks of treatment (P <0. 05). Scores of Chinese medical syndromes all decreased after 4 and 8 weeks of treatment (P <0. 05). Levels of SCF and DYN obviously decreased in the two groups after 4 and 8 weeks of treatment (P <0. 05,P < 0. 01). Compared with the control group, obvious improvement was seen in pruritus degree, scratching number, scaling, irritability and restlessness , dysphoria, dizziness , and tinnitus(P <0. 05) ; total scores of Chinese medical syndromes decreased (P <0. 05) ; SCF also decreased (P <0. 05) in the treatment group after 8 weeks of treatment. Conclusions Based on external application of Binghuang Fule Soft Ointment, GZR showed better efficacy than that of the control. It had certain roles in regulating related mediator levels that might affect nervous-endocrine-immune systems.

Association of toll-like receptor 4 polymorphisms with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is characterized by a chronic low-grade inflammatory state. Toll-like receptor 4 (TLR4) is a critical mediator of innate immunity. Polymorphisms in TLR4 gene have been shown to be associated with impaired inflammatory response. Here, we investigated the association of TLR4 polymorphisms with T2DM. Four TLR4 polymorphisms (+986A/G, +1196C/T, +3725G/C, and +11367G/C) were genotyped in a total number of 822 T2DM patients and 835 healthy controls. Results showed that the +986A/G and +1196C/T polymorphisms did not exist in the Han Chinese population. The prevalence of TLR4 +3725GC and CC genotypes were significantly decreased in T2DM cases than in controls (odds ratio (OR) = 0.62, 95 % confidence interval (CI) = 0.50-0.78, p = 3.48 × 10(-5), and OR = 0.36, 95 % CI = 0.22-0.59, p = 1.55 × 10(-5), respectively). Also, the frequency of TLR4 +3725C allele was significantly lower in T2DM patients (p = 2.46 × 10(-9)). When analyzing the TLR4 +11367G/C polymorphism, the +11367CC genotype revealed lower numbers in patients compared to healthy controls (OR = 0.46, 95 % CI = 0.27-0.78, p = 0.0032). Analysis of the clinical features on the control subjects demonstrated no correlations between these TLR4 polymorphisms and sex, age, body mass index, etc. (p > 0.05). In conclusion, these data indicate that TLR4 +3725G/C and +11367G/C polymorphisms may be novel protective factors against T2DM in the Chinese population.

Clinical experience in the use of marginal donor hearts / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Although heart transplantation has become a standard therapy for end-stage heart disease, there are few published studies regarding the use of transplant organs from marginal donors. Here we describe the clinical outcome we have obtained using marginal donor hearts.</p><p><b>METHODS</b>We analyzed 21 cases of orthotopic heart transplantation for end-stage heart disease performed in our department between September 2008 and July 2010. Of these patients, six received hearts from marginal donors and the remainder received standard-donor hearts. The two groups were compared in terms of both mortality and the incidence of perioperative complications such as infection, acute rejection, and right heart insufficiency.</p><p><b>RESULTS</b>The 1-year survival rate of both groups was 100%. Only one death was recorded in standard-donor group during follow-up. Patients who received marginal donor hearts (83%) experienced more early complications than did the standard-donor-heart group (13%), but the mortality of the two groups was the same. The duration of post-ICU stay was greater in the marginal donor group than in the standard-donor group, (35.5 ± 17.4) days and (21.7 ± 2.6) days, respectively (P < 0.05).</p><p><b>CONCLUSIONS</b>The use of marginal donor hearts increases the number of patients who can receive and benefit from transplants. However, it may introduce an increased risk of early complications, thus care should be taken both in the choice of patients who will receive marginal donor hearts and in the perioperative treatment of those for whom the procedure is performed.</p>

The vascular endothelial growth factor expression and vascular regeneration in infarcted myocardium by skeletal muscle satellite cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Myocardial infarction results in tissue necrosis, leading to cell loss and ultimately to cardiac failure. Implantation of skeletal muscle satellite cells into the scar area may compensate for the cell loss and provides a new strategy for infarct therapy. Vascular endothelial growth factor (VEGF) is a promising reagent for inducing myocardial angiogenesis. Skeletal myoblast transplantation has been shown to improve cardiac function in chronic heart failure models by regenerating muscle. We hypothesized that VEGF expression and vascular regeneration increased in infarcted myocardium by skeletal muscle satellite cells, which can promote vascular producing and improve survival environment in infarcted myocardium.</p><p><b>METHODS</b>The skeletal muscle satellite cells were implanted into the infarcted myocardium in a model through ligated left anterior artery in Louis Inbrad Strain rat. Specimens were got for identifying the expression of VEGF and the density of vascular by immunochemical method at two weeks after implantation.</p><p><b>RESULTS</b>The proliferation and differentiation of the skeletal muscle satellite cell was very well. The expression of VEGF was higher in the implanted group (146.83 +/- 2.49) than that in the control group (134.26 +/- 6.84) (P < 0.05). The vascular density in the implanted group (13.00 +/- 1.51) was also higher than that in the control (10.68 +/- 1.79) (P < 0.05).</p><p><b>CONCLUSION</b>The implanted satellite cell could excrete growth factor that would induce angiogenesis and improve cell survival environment in infarcted myocardium.</p>

[Construction and expression of a fusion protein containing extracellular domain of human Jagged1 and Fc fragment of human IgG1].

AIM: To construct a eukaryotic expression vector containing the extracellular domain of human Jagged1 and Fc fragment of human IgG1 fusion gene and to express the hJagged1(ext)-Fc fusion protein in mammalian cells. METHODS: The extracellular domain of human Jagged1 gene was cloned from normal human bone marrow cells. In order to obtain hJagged1(ext)-Fc fusion gene, the extracellular domain of Jagged1 gene was inserted into pBluescript-sk II-hc gamma 1, and then the fusion gene was inserted into pEF-BOSneo to get the eukaryotic expression vector pEF-BOSneo-hJagged1(ext)-Fc. The recombination plasmid was transiently transfected into COS7 cells and the expression of the fusion protein was identified by RT-PCR, immunofluorescent assay and sandwich ELISA. RESULTS: The extracellular domain of human Jagged1 gene was effectively amplified. The DNA sequencing result showed that the constructed plasmid containing hJagged1(ext)-Fc fusion gene was the same as designed. The fusion protein was successfully expressed in mammalian cells. CONCLUSION: The hJagged1(ext) gene has been successfully cloned and expressed, which provides a new fusion protein for further experiments, for example, the Jagged1(ext)-Fc fusion protein can be used as a new stimulator for proliferation of hematopoietic stem/progenitor cells in-vitro.

[Construction of eukaryotic expression vector of hdll1(ext)-Fc and its expression in COS-7 cells].

AIM: To construct an eukaryotic expression vector pEF-BOSneo-hdll1(ext)-Fc, and to express the fusion protein consisting of the extracellular region of delta-like1 and Fc fragment of human IgG1 in COS-7 cells. METHODS: The extracellular region of human delta-like1 was amplified from a human brain cDNA library by PCR. The expression vector was constructed by DNA recombination. The recombinant plasmid was transfected into COS-7 cells via liposome mediation. The expression of the fusion protein was detected by RT-PCR, immunofluorescence assay and sandwich ELISA. RESULTS: DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector pEF-BOSneo-hdll1(ext)-Fc had been constructed successfully. After recombinant plamid had been transfected into COS-7 cells, RT-PCR and DNA sequencing verified that the dll1(ext) gene and IgG1Fc gene were fused correctly. The results of immunofluorescence assay were positive and the fusion protein could be detected by sandwich ELISA in culture supernatant of transfected COS-7 cells. CONCLUSION: hdll1(ext) was successfully cloned and expressed in the form of Fc fusion protein, which is helpful for further study of the function of Notch pathway.