Aptamer/Graphene Quantum Dots Nanocomposite Capped Fluorescent Mesoporous Silica Nanoparticles for Intracellular Drug Delivery and Real-Time Monitoring of Drug Release.

Great challenges in investigating the release of drug in complex cellular microenvironments necessitate the development of stimuli-responsive drug delivery systems with real-time monitoring capability. In this work, a smart drug nanocarrier based on fluorescence resonance energy transfer (FRET) is fabricated by capping graphene quantum dots (GQDs, the acceptor) onto fluorescent mesoporous silica nanoparticles (FMSNs, the donor) via ATP aptamer for real-time monitoring of ATP-triggered drug release. Under extracellular conditions, the fluorescence of FMSNs remains in the "off" state in the low ATP level which is unable to trigger the release of drug. Once specifically recognized and internalized into the target tumor cells by AS1411 aptamer, in the ATP-rich cytoplasm, the conformation switch of the ATP aptamer causes the shedding of the GQDs from the nanocarriers, leading to the release of the loaded drugs and consequently severe cytotoxicity. Simultaneously, the fluorescence of FMSNs turns "on" along with the dissociation of GQDs, which allows real-time monitoring of the release of drug from the pores. Such a drug delivery system features high specificity of dual-target recognition with AS1411 and ATP aptamer as well as high sensitivity of the FRET-based monitoring strategy. Thus, the proposed multifunctional ATP triggered FRET-nanocarriers will find potential applications for versatile drug-release monitoring, efficient drug transport, and targeted cancer therapeutics.

[Revealing Driving Factors of Urban O3 Based on Explainable Machine Learning].

Driven by precursor emissions, meteorological conditions, and other factors, atmospheric ozone (O3) has become the main pollutant affecting urban air quality in summer. The current deductive models driven by physical and chemical mechanisms require a large number of parameters for the analysis of O3 pollution, and the calculation timeliness is poor. The data-driven inductive models are efficient but have problems such as poor explanation. In this study, an explainable model of data-driven Correlation-ML-SHAP was established to reveal the strongly correlated influencing factors of O3 concentration. Additionally, the machine learning ML module coupled with the explainable SHAP module was used to calculate the contributions of driving factors to O3 concentration, so as to realize the quantitative analysis of driving factors. The O3 pollution process in the summer of 2021 in Jincheng City was used as an example to carry out the application research. The results showed that the Correlation-ML-SHAP model could reveal and use strong driving factors to simulate O3 concentration and quantify influence contribution, and the ML module used the XGBoost model to achieve the best simulation accuracy. Air temperature, solar radiation, relative humidity, and precursor emission level were the strong driving factors of O3 pollution in Jincheng City in summer 2021, and the contribution weights were 32.1%, 21.3%, 16.5%, and 15.6%. The contribution weights of air temperature, solar radiation, and precursor emission level increased by 3.4%, 1.2%, and 1.2% on polluted days, respectively, and the contribution weights of precursor emission level rose to third place on polluted days. Each driving factor had a nonlinear interaction effect on O3 concentration. When the air temperature exceeded 24℃, or the relative humidity was lower than 70%, there was a 94.9% and 94.1% probability of positive contribution to O3 pollution, respectively. Under such meteorological conditions, ρ(NO2) exceeded 9 µg·m-3, or ρ(CO) exceeded 0.7 mg·m-3, and there was a 94.9% and 99.3% probability of positive contribution to O3 pollution, respectively. The southeast wind speed was lower than 5.8 m·s-1, or the south wind speed was lower than 5.3 m·s-1, both of which contributed positively to O3 pollution. The model quantitatively analyzed the influence contribution of various driving factors on urban O3 concentration, which could provide a basis for the prevention and control of urban atmospheric O3 pollution in summer.

Quantum dots-based immunofluorescent microfluidic chip for the analysis of glycan expression at single-cells.

Rich high-quality single-cell information from rare cell sample is very important for the quantitative systems biology description of cellular function. However, this type of data is often prohibited by the conventional analytical technology such as flow cytometry. In this paper, we described a microfluidic platform coupled with a quantum dots-based (QDs) immunofluorescence (IF) approach to measure the expression of glycans on the cell surface of single cells or cell population. Compared with conventional IF staining, the QDs-based IF probe exhibited higher brightness and stability against photobleaching. With the merits of the novel IF staining protocol and microfluidic platform, high-throughput IF staining was performed to measure the glycan expressions and the changes at single K562 cells after drug treatment. The protocol proposed here showed a high sensitivity on the glycan expression profile owing to the amplification of the signal in indirect IF staining. The size of cell sample was only 4 × 10(3) cells, which made the rare cell sample analysis accessible. This method may find widespread application for assessing cell-surface glycoprotein expression as well as analysis of the heterogeneity in cell populations in a high-throughput manner.

An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y.

A sensitive and easy technique has been developed for the negative detection of DNA following PAGE using eosin Y. After electrophoresis, gels are fixed and stained within 40 min to provide a detection limit of 0.1-0.2 ng of single DNA band, which appears as transparent and colorless under the opaque gel matrix background. The sensitivity of the new stain is fourfold better than zinc-imidazole negative and ethidium bromide stains. Furthermore, the newly developed staining method has been successfully applied to RNA visualization in polyacrylamide gels. In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency.

ARRB1-Promoted NOTCH1 Degradation Is Suppressed by OncomiR miR-223 in T-cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a type of aggressive leukemia with inferior prognosis. Although activating mutations of NOTCH1 are observed in most T-ALL cases, these mutations alone are not sufficient to drive the full development of T-ALL. ß-Arrestins (ARRB) are versatile and multifunctional adapter proteins that regulate diverse cellular functions, including promoting the development of cancer. However, the role of ARRBs in T-ALL has largely remained elusive. In this study, we showed that ARRB1 is expressed at low levels in assayed T-ALL clinical samples and cell lines. Exogenous ARRB1 expression inhibited T-ALL proliferation and improved the survival of T-ALL xenograft animals. ARRB1 facilitated NOTCH1 ubiquitination and degradation through interactions with NOTCH1 and DTX1. Mechanistically, the oncogenic miRNA (oncomiR) miR-223 targets the 3'-UTR of ARRB1 (BUTR) and inhibits its expression in T-ALL. Furthermore, overexpression of the ARRB1-derived miR-223 sponge suppressed T-ALL cell proliferation and induced apoptosis. Collectively, these results demonstrate that ARRB1 acts as a tumor suppressor in T-ALL by promoting NOTCH1 degradation, which is inhibited by elevated miR-223, suggesting that ARRB1 may serve as a valid drug target in the development of novel T-ALL therapeutics.Significance: These findings highlight a novel tumor suppressive function of the adaptor protein ß-arrestin1 in T-ALL.

A Targeted DNAzyme-Nanocomposite Probe Equipped with Built-in Zn(2+) Arsenal for Combined Treatment of Gene Regulation and Drug Delivery.

As catalytic nucleic acids, DNAzymes have been extensively used in the design of sensing platforms. However, their potentials as intelligent drug carriers for responsive drug release in gene therapy and chemotherapy were rarely explored. Herein, we report a dual-functional probe composed of gold nanoparticles (GNPs), catalytic Zn(2+)-dependent DNAzyme, anticancer drug doxorubicin (Dox), targeted AS1411 aptamer and acid-decomposable ZnO quantum dots (ZnO QDs) to achieve intracellular gene regulation and drug delivery in a controlled manner. By means of aptamer-guided targeting and receptor-mediated endocytosis, the probes were specifically internalized into the HeLa cells and trapped in the acidic endo-/lysosomes, where the ZnO QDs as the built-in Zn(2+) arsenal were promptly dissolved to offer Zn(2+), leading to the activation of DNAzyme to cleave the substrate strands, and subsequent drug release. Meanwhile, as designed, one part of the cleaved substrate, hybridized with the overexpressed miR-21 in the target cells, thereby declining its intracellular level. Taken together, the down-regulation of miR-21 has a synergistic effect with Dox to efficiently eradicate the cancer cells. Thus, the favorable biocompatibility, cancer cell specificity and combined treatment make the probe promising for therapy of multidrug-resistant cancer and in vivo application.

Versatile Microfluidic Platform for the Assessment of Sialic Acid Expression on Cancer Cells Using Quantum Dots with Phenylboronic Acid Tags.

This work describes a versatile microfluidic platform for evaluation of cell-surface glycan expression at the single-cell level using quantum dots (QDs) tagged with phenylboronic acid. The platform was integrated with dual microwell arrays, allowing the introduction of cells in two states using the same cell culture chamber. The simultaneous analysis of cells in the same environment minimized errors resulting from different culture conditions. As proof-of-concept, the expressions of sialic acid (SA) groups on K562 cells, with or without 3'-azido-3'-deoxythymidine (AZT) treatment, were evaluated in the same chamber. 3-Aminophenylboronic acid functionalized CdSeTe@ZnS-SiO2 QDs (APBA-QDs) were prepared as probes to recognize SA groups on K562 cells with only one-step labeling. The results showed that the expression of SA moieties on K562 cells was increased by 18% and 31% after treatment with 20 and 40 µM AZT, respectively. Performing the drug treatment and control experiments simultaneously in the same chamber significantly improved the robustness and effectiveness of the assay. The strategy presented here provides an alternative tool for glycan analysis in a sensitive, high-throughput, and effective manner.

Multi-shell structured fluorescent-magnetic nanoprobe for target cell imaging and on-chip sorting.

In this paper, we have developed a core-triple-shell structured multi-functional nanoprobe Fe3O4/SiO2/CdSeTe@ZnS-SiO2/polydopamine with strong fluorescence and a fast magnetic response for specifically recognizing, fluorescently labeling, and magnetically sorting target tumor cells on a microfluidic chip. The outer polydopamine layer not only effectively alleviated the quenching effect of the interlayer quantum dots but also provided a convenient and versatile functional interface to readily conjugate with the recognizing model molecules of aptamer KH1C12 with amine, thiol, or carboxyl groups. Moreover, the polydopamine isolation and PEG decoration equipped the as-fabricated nanoprobes with little cytotoxicity and nonspecific affinity, leading to the effective and specific profiling of the protein epitopes expressed on the target tumor cells. Taking advantage of the magnetic property and specific recognition, the modified nanoprobe was utilized to label and isolate HL-60 cells from a homogeneous cell mixture of HL-60 and K562 cells on a microfluidic chip. Combining with the high throughput of the microfluidic chip, 1.0 × 10(4) HL-60 cells were readily separated from 2.0 × 10(4) cells in only 10 min with 98% separation efficiency, markedly improved in comparison with conventional strategies. This study presents an innovative strategy for developing highly integrated nanoprobes of strong fluorescence and magnetic controllability, opening up a promising probe-based avenue for biological imaging and separation.

Fabrication of graphene quantum dots and hexagonal boron nitride nanocomposites for fluorescent cell imaging.

Graphene quantum dots (GQDs) with green fluorescence were incorporated onto hexagonal boron nitride sheets (HBN) through electrostatic interaction by using poly(diallyldimethylammonium) (PDDA) as the bridge to fabricate the novel nanocomposites (HBN-GQDs). The HBN-GQDs nanocomposites exhibited strong green fluorescent property, high stability, water solubility, very low cytotoxicity on Hela cells. These properties make the HBN-GQDs nanocomposites as good candidate materials for biological applications. The results for the imaging of live cells indicated that the cell-penetrating HBN-GQDs could be a promising nanoprobe for intracellular imaging and therapeutic applications.

A practical therapeutic protocol for cervical tuberculosis.

PURPOSE: Cervical tuberculosis (CTB) is a relatively rare entity, even in endemic countries. Currently, management ranges from conservative to radical surgical approaches. We report our experience in diagnosing and treating 66 cases of CTB in the past eight years using our CTB therapeutic protocol. METHODS: All patients diagnosed with CTB were followed up over a 3.5-year period. Patients were divided into three grades using clinicoradiological criteria designed to evaluate the initial severity of the disease. Overall performance status was assessed based on the American Spinal Injury Association (ASIA) scale. Neurological recovery was evaluated with the ASIA scale as well as using X-rays and computed tomography every four weeks for the initial three months and every three months thereafter. RESULTS: The mean follow-up duration was 38.2 ± 6.2 months. No mortality occurred. One case of recurrence due to irregular antitubercular treatment (ATT) was cured by abscess clearing and regular ATT. All other patients had good clinicoradiological outcomes, regardless of grading. CONCLUSIONS: The use of our proposed scoring system and management protocol allowed speedy management of CTB.

Elevated ß-arrestin1 expression correlated with risk stratification in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the main subtype of childhood leukemia. Risk stratification is pivotal for ALL prognosis and individualized therapy. The current factors for risk stratification include clinical and laboratory features, cytogenetic characteristics of the blast, early response to chemotherapy, and genetic factors. Analyses of gene expression are becoming increasingly important in ALL risk stratification. ß-Arrestin1, a multifunctional scaffold protein mediating many intracellular signaling networks, has been shown to be involved in many tumors. However, little is known of ß-arrestin1 in leukemia. In this study, we found that ß-arrestin1 was significantly elevated in 155 newly diagnosed ALL patients, compared with 51 controls. Further analysis showed that ß-arrestin1 expression was positively related with risk classification and white blood cell count in ALL. Moreover, expression of Notch1, an essential gene for developing hematological cells and T-ALL, was found to be negatively correlated with ß-arrestin1 in ALL. In conclusion, ß-arrestin1 may be a useful predictor of risk stratification and prognosis of ALL, and thus of potential use in the design of individualized therapy strategies.

[Study on macrophages infected with Mycobacterium tuberculosis H37Ra in vitro].

AIM: To study the production of nitric oxide and secretion of cytokines after infection of macrophages with Mycobacterium tuberculosis H37Ra. METHODS: 24 hours after infection of RAW264.7 cells with Mycobacterium tuberculosis H37Ra, the production of NO and H(2);O(2); as well as the secretion levels of IL-12 and TNF-α in the supernatants of culture were determined by Griess method, chemical method and ELISA assay respectively. The expression of IL-12 and TNF-α mRNA in macrophages was detected by reverse transcription polymerase chain reaction (RT- PCR). RESULTS: Macrophages infected with Mycobacterium tuberculosis H37Ra produced effectively more NO, H(2);O(2);, and enhanced the release of IL-12, TNF-α and the expression of IL-12, TNF-α mRNA (P<0.05). CONCLUSION: Mycobacterium tuberculosis H37Ra can induce the production of more nitric oxide and cytokines which play important roles in the host immune response.

RNAi-hTERT inhibition hepatocellular carcinoma cell proliferation via decreasing telomerase activity.

BACKGROUND: RNA interference (RNAi), which has been demonstrated as having great potentional in the fields of gene function and gene therapy, was applied to inhibit the expression of some endogenous genes. Human telomerase reverse transcriptase (hTERT) is highly expressed in hepatocellular carcinoma cells. MATERIALS AND METHODS: In combination with DNA vector-based RNAi, quantitative real-time reverse transcription polymerase chain reaction, telomeric repeat amplification protocol-enzyme-linked immunosorbent assay, 3-(4,5 dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay, and xenograft tumor animal techniques, we first constructed three pTZU6 + 1-shRNA-hTERT vectors and their corresponding site-mutated vectors, then transfected them into hepatocarcinoma HepG2, SMMC-7721 cells, and normal liver L02 cells, respectively, injected them into xenograft hepatocarcinoma tumor tissues to induce RNAi, and then detected the alteration of cell and tumor proliferation, telomerase activity, hTERT, and c-myc expression in each treatment. RESULTS: The cell proliferation of hepatocellular carcinoma cells both in vitro and in vivo was significantly inhibited by ph1-shRNA, the most effective segment targeted hTERT gene. ph1-shRNA could inhibit telomerase activity, hTERT, and c-myc expression in hepatocarcinoma cells and xenograft tumor tissues compare with the cells treated with empty vector pTZU6 + 1. However, there were no obvious effects on normal liver L02 cells. Moreover, even a single base mutation in siRNAs transcription template would significantly reduce the ability of siRNAs to induce RNA silencing. CONCLUSIONS: RNAi-hTERT could inhibit the proliferation of hepatocarcinoma cells specifically via the suppression of telomease activity, hTERT, and c-myc expression. Therefore, hTERT and c-myc play key roles in hepatocarcinoma tumorgenesis, and an RNAi-targeted hTERT strategy would be a potential approach for hepatocarcinoma therapy.

Transcript regulation of human telomerase reverse transcriptase by c-myc and mad1.

Telomerase activity is highly positive correlated to most malignant neoplasms. Human telomerase reverse transcriptase (hTERT) is the rate-limiting factor of telomerase activity. Recent studies have shown that the expression of hTERT is mainly determined by its transcript regulation. Among the transcript regulation factors of hTERT, c-myc and mad1 are well known. Here, we constructed c-myc and mad1 eukaryotic expression vectors, then co-transfected them with the wild-type (Tw) or mutant hTERT promoter (Td) luciferase reporter plasmid, which were double-mutated in the E-box sequences from CACGTG to CACCTG of Tw. The change of luciferase activity in different cells was detected. The results showed that Tw was obviously activated in T24 and EJ bladder cancer cells, but not in normal fibrocytes. c-myc and mad1 had positive and negative effects respectively on the Tw transcript in a dose-dependent manner, while the roles of c-myc and mad1 in regulating the Td transcript were reversed. c-myc combined with mad1 can down-regulate Tw but not Td. These observations indicate that c-myc and mad1 can regulate the hTERT transcript in a different manner in hTERT positive cells, but not in normal cells. This may provide an insight into some telomerase-related carcinogenesis mechanisms.

[Experimental research of targeting hTERT gene inhibited in hepatocellular carcinoma therapy by RNA interference].

BACKGROUND & OBJECTIVE: RNA interference (RNAi) is a new gene blocking technology that silences target gene at post-transcription level induced by the small interference RNA (siRNA). RNAi has been demonstrated great prospect in gene functional research and gene therapy areas. Nowadays, RNAi has been reported to be used to inhibit the expression of endogenous genes including cyclophilin, GAPDH, p53, and c-myc; and there were some progresses in the therapy of the diseases caused by AIDS and hepatitis viruses with RNAi. However, hTERT gene, which was highly expressed in hepatocellular carcinoma and other malignant neoplasm, has not been researched by RNAi. In present research, we utilized RNAi to inhibit hTERT gene expression in vitro and in vivo, investigated the feasibility and specificity of gene therapy for hepatocellular carcinoma. METHODS: Small interference RNAs homologous to hTERT gene were designed,pTZU6+1-shRNA-hTERT vector was constructed and transfected into hepatocellular carcinoma SMMC-7721 cells and transplanted SMMC-7721 tumor in nude mice to induce RNAi. The changes of hTERT gene expression and tumor cell proliferation in both siRNA treatment groups and control group were determined by flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), immunochemistry in vitro and in vivo. RESULTS: The expression of hTERT had been obviously inhibited by RNAi in vitro. The inhibition rate of cell growth was 37.5% after pTZU6+1-shRNA-hTERT vector was transfected to hepatocellular carcinoma SMMC-7721 cells; the phase of cell cycle indicated the reduction of S phase, while G(1)/G(0) phase increased. The mRNA level of hTERT decreased from 99.4% to 53.1%, its protein expression reduced from 86.3% to 46.6%. The tumor size reduced after treated with pTZU6+1-shRNA-hTERT vector in vivo; hTERT mRNA level decreased from 99.1% to 76.2%, and its protein expression decreased from 87.2% to 61.8% in siRNA treatment group. In contrast, there were no changes in control groups in vitro and in vivo. CONCLUSION: RNAi inhibits the hTERT gene expression and proliferation of hepatocellular carcinoma SMMC-7721 cells with specificity, and is a possible new approach for neoplasm gene therapy.