Genetically Encoded RNA Sensors for Ratiometric and Multiplexed Imaging of Small Molecules in Living Cells.

Genetically encoded sensors afford powerful tools for studying small molecules and metabolites in live cells. However, genetically encoded sensors with a general design remain to be developed. Here we develop genetically encoded RNA sensors with a modular design for ratiometric and multiplexed imaging of small molecules in live cells. The sensor utilizes aptazyme as a recognition module and the light-up RNA aptamer as a signal reporter. The conformation of light-up aptamers is abrogated by a blocking sequence, and aptazyme-mediated cleavage restores the correct conformation, delivering activated fluorescence for small molecule imaging. We first developed a genetically encoded ratiometric sensor using Mango aptamer as a reference and SRB2 as a reporter. It is shown that the sensor allows quantitative imaging and detection of theophylline in live cells. The generality of the design is further demonstrated for imaging other small molecules by replacing the aptazymes. Its ability for multiplexed imaging of small molecules is further explored via the integration of different small-molecule responsive aptazymes and light-up RNA aptamers. This modular design could offer a versatile platform for imaging diverse molecules in living cells.

Protein-Scaffolded DNA Nanostructures for Imaging of Apurinic/Apyrimidinic Endonuclease 1 Activity in Live Cells.

Nucleic acids are valuable tools for intracellular biomarker detection and gene regulation. Here we propose a new type of protein (avidin)-scaffolded DNA nanostructure (ADN) for imaging the activity of apurinic/apyrimidinic endonuclease 1 (APE1) in live cells. ADN is designed by assembling an avidin-displayed abasic site containing DNA strands labeled with a fluorophore or a quencher via a complementary linker strand. ADN is nonemissive due to the close proximity of fluorophores and quenchers. APE1-mediated cleavage separates the fluorophores from the quenchers, delivering activated fluorescence. In vitro assays show that ADN is responsive to APE1 with high sensitivity and high specificity. ADN can efficiently enter the cells, and its capability to visualize and detect intracellular APE1 activities is demonstrated in drug-treated cells and different cell lines. The modular and easy preparation of our nanostructures would afford a valuable platform for imaging and detecting APE1 activities in live cells.

Genetically Encoded Light-Up RNA Amplifier Dissecting MicroRNA Activity in Live Cells.

Live cell dissection of microRNA activities is crucial for basic and translational medicine, but current hybridization-based strategies may fail to dissect surrounding-dependent activities. Here, we develop a genetically encoded miRNA-induced light-up RNA amplifier (iLAMP) that enables fast-activated, signal-amplified, fluorogenic imaging of miRNA activities in live cells. iLAMP responds to miRNA targets in the mode of "activation upon cleavage", in which the light-up RNA aptamer restores its fluorescence rapidly upon cleavage by the RNA-induced silencing complex. We demonstrate that iLAMP affords substantial signal amplification of ∼100-fold and high specificity in single nucleotide discrimination because of the miRNA-mediated cyclic cleavage. Combined with a Mango RNA aptamer reference module and a pseudoknot terminal stabilizer, iLAMP is shown for quantitative ratiometric imaging and dynamic monitoring of miRNA activities under exogenous stimulations. iLAMP is featured by a modular "plug and play" design and can be readily adapted to the detection of other miRNAs, highlighting its potential in tracking cell differentiation and screening miRNA therapeutics.

Gold Nanoflares with Computing Function as Smart Diagnostic Automata for Multi-miRNA Patterns in Living Cells.

Investigating the multimolecule patterns in living cells is of vital importance for clinical and biomedical studies. Herein, we reported for the first time the engineering of gold nanoflares as smart automata to implement computing-based diagnosis in living mammalian cells. Defining the logic combinations of miR122 and miR21 as the detection patterns, the corresponding OR and AND diagnostic automata were designed. The results showed that they could recognize the correct patterns rapidly and sensitively. The automata could enter cells via self-delivery and have good biocompatibility. They enabled accurate diagnosis on miRNA signatures in different cell lines and differentiation of fluctuations in the same cell line at single cell resolution. Moreover, the automata afforded an innovative diagnostic mode. It simplified the complicated process of detecting, data-collecting, computing, and evaluating. The direct diagnosing result ("1" or "0") was exported according to the embedded computation code. It highlighted the new possibility of using smart automata for intelligent diagnostics and cancer therapy at single cell resolution.

Transcriptome analysis of the dimorphic transition induced by pH change and lipid biosynthesis in Trichosporon cutaneum.

Trichosporon cutaneum, a dimorphic oleaginous yeast, has immense biotechnological potential, which can use lignocellulose hydrolysates to accumulate lipids. Our preliminary studies on its dimorphic transition suggested that pH can significantly induce its morphogenesis. However, researches on dimorphic transition correlating with lipid biosynthesis in oleaginous yeasts are still limited. In this study, the unicellular yeast cells induced under pH 6.0-7.0 shake flask cultures resulted in 54.32% lipid content and 21.75 g/L dry cell weight (DCW), so lipid production was over threefold than that in hypha cells induced by acidic condition (pH 3.0-4.0). Furthermore, in bioreactor batch cultivation, the DCW and lipid content in unicellular yeast cells can reach 21.94 g/L and 58.72%, respectively, both of which were also more than twofold than that in hypha cells. Moreover, the activities of isocitrate dehydrogenase (IDH), malic enzyme (MAE), isocitrate lyase (ICL) and ATP citrate lyase (ACL) in unicellular cells were all higher than in the hyphal cells. In the meanwhile, the transcriptome data showed that the genes related to fatty acid biosynthesis, carbon metabolism and encoded Rim101 and cAMP-PKA signaling transduction pathways were significantly up-regulated in unicellular cells, which may play an important role in enhancing the lipid accumulation. In conclusion, our results provided insightful information focused on the molecular mechanism of dimorphic transition and process optimization for enhancing lipid accumulation in T. cutaneum.

Reduced protocadherin17 expression in leukemia stem cells: the clinical and biological effect in acute myeloid leukemia.

BACKGROUND: Leukemia stem cell (LSC)-enriched genes have been shown to be highly prognostic in acute myeloid leukemia (AML). However, the prognostic value of tumor suppressor genes (TSGs) that are repressed early in LSC remains largely unknown. METHODS: We compared the public available expression/methylation profiling data of LSCs with that of hematopoietic stem cells (HSCs), in order to identify potential tumor suppressor genes in LSC. The prognostic relevance of PCDH17 was analyzed on a cohort of 173 AML patients from The Cancer Genome Atlas (TCGA), and further validated in three independent cohorts (n = 339). RESULTS: We identified protocadherin17 (PCDH17) and demonstrated that it was significantly down-regulated and hypermethylated in LSCs compared with HSCs. Our analyses of primary AML patient samples also confirmed these deregulations. Clinically, low PCDH17 expression was associated with female sex (P = 0.01), higher WBC (P < 0.0001), higher percentages of blasts in bone marrow (BM) and peripheral blood (PB) (P = 0.04 and < 0.001, respectively), presence of FLT3-internal tandem duplications (P = 0.002), mutated NPM1 (P = 0.02), and wild-type TP53 (P = 0.005). Moreover, low PCDH17 expression predicted worse overall survival (OS) in four independent cohorts as well as in the molecularly defined subgroups of AML patients. In multivariable analyses, low PCDH17 expression retained independent prognostic value for OS. Biologically, PCDH17 expression-associated gene signatures were characterized by deregulations of EMT- and Wnt pathway-related genes. CONCLUSIONS: PCDH17 gene was silenced by DNA methylation in AML. Low PCDH17 expression is associated with distinct clinical and biological features and improves risk stratification in patients with AML.

Down-regulation of miR-29c is a prognostic biomarker in acute myeloid leukemia and can reduce the sensitivity of leukemic cells to decitabine.

BACKGROUND: MicroRNA-29c (miR-29c) is abnormally expressed in several cancers and serves as an important predictor of tumor prognosis. Herein, we investigate the effects of abnormal miR-29c expression and analyze its clinical significance in acute myeloid leukemia (AML) patients. In addition, decitabine (DAC) has made great progress in the treatment of AML in recent years, but DAC resistance is still common phenomenon and the mechanism of resistance is still unclear. We further analyze the influences of miR-29c to leukemic cells treated with DAC. METHODS: Real-time quantitative PCR (RQ-PCR) was carried out to detect miR-29c transcript level in 102 de novo AML patients and 25 normal controls. miR-29c/shRNA-29c were respectively transfected into K562 cells and HEL cells. Cell viability after transfection was detected by cell counting Kit-8 assays. Flow cytometry was used to detect apoptosis. RESULTS: MiR-29c was significantly down-regulated in AML (P < 0.001). Low miR-29c expression was frequently observed in patients with poor karyotype and high risk (P = 0.006 and 0.013, respectively). Patients with low miR-29c expression had a markedly shorter overall survival (OS) than those with high miR-29c expression (P < 0.001). Multivariate analysis confirmed the independent prognostic value of low miR-29c expression in both the whole cohort as well as the cytogenetically normal AML (CN-AML) subset. Over-expression of miR-29c in K562 treated with DAC inhibited growth, while silencing of miR-29c in HEL promoted growth and inhibited apoptosis. MiR-29c overexpression decreased the half maximal inhibitory concentration (IC50) of DAC in K562, while miR-29c silencing increased the IC50 of DAC in HEL. The demethylation of the miR-29c promoter was associated with its up-regulated expression. Although miR-29c demethylation was also observed in DAC-resistant K562 (K562/DAC), miR-29c expression was down-regulated. MiR-29c transfection also promoted apoptosis and decreased the IC50 of DAC in K562/DAC cells. CONCLUSIONS: Our results suggest that miR-29c down-regulation may act as an independent prognostic biomarker in AML patients, and miR-29c over-expression can increase the sensitivity of both non-resistant and resistant of leukemic cells to DAC.

Engineering Organelle-Specific Molecular Viscosimeters Using Aggregation-Induced Emission Luminogens for Live Cell Imaging.

Subcellular viscosity is essential for cell functions and may indicate its physiological status. We screen two fluorescent probes by engineering tetraphenylethene (TPE) for measuring viscosity in mitochondria and lysosomes, respectively. These two probes are only weakly emissive in nonviscous medium and the emission signals are greatly enhanced in viscous medium due to the restriction of intramolecular motion. The presence of pyridium has endowed one probe with mitochondrial specificity, while the presence of indole ring has granted the other probe with lysosome-targeting ability. Their optical properties are characterized in vitro and their applications in imaging viscosity variations in mitochondria and lysosomes are also demonstrated in living cells under different stimulated processes. In addition, an increase in both mitochondrial and lysosomal viscosity during mitophagy was revealed for the first time with our probes. To our knowledge, this is the first time that TPE is engineered to be fluorescent molecular viscosimeters that possess desirable aqueous solubility, red-shifted emission, and organelle specificity.

A Solution-Processed Transparent NiO Hole-Extraction Layer for High-Performance Inverted Perovskite Solar Cells.

A highly transparent NiO layer was prepared by a solution processing method with nickel(II) 2-ethylhexanoate in non-polar solvent and utilized as HTM in perovskite solar cells. Excellent optical transmittance and the matched energy level lead to the enhanced power conversion efficiency (PCE, 18.15 %) than that of conventional sol-gel-processed NiO-based device (12.98 %).

In Situ Imaging of Individual mRNA Mutation in Single Cells Using Ligation-Mediated Branched Hybridization Chain Reaction (Ligation-bHCR).

Ultrasensitive and specific in situ imaging of gene expression is essential for molecular medicine and clinical theranostics. We develop a novel fluorescence in situ hybridization (FISH) strategy based on a new branched hybridization chain reaction (bHCR) for efficient signal amplification in the FISH assay and a ligase-mediated discrimination for specific mutation detection. To our knowledge, this is the first time that HCR has been realized for mutation detection in the FISH assay. In vitro assay shows that the ligation-bHCR strategy affords high specificity in discriminating single-nucleotide variation in mRNA, and it generates a highly branched polymeric product that confers more efficient amplification or better sensitivity than HCR. Imaging analysis reveals that ligation-bHCR generates highly bright spot-like signals for localization of individual mRNA molecules, and spot signals of different colors are highly specific in genotyping point mutation of individual mRNA. Moreover, this strategy is shown to have the potential for quantitative imaging of the expression of mRNA at the single-cell level. Therefore, this strategy may provide a new promising paradigm in developing highly sensitive and specific FISH methods for various diagnostic and research applications.

Proton-Fueled, Reversible DNA Hybridization Chain Assembly for pH Sensing and Imaging.

Design of DNA self-assembly with reversible responsiveness to external stimuli is of great interest for diverse applications. We for the first time develop a pH-responsive, fully reversible hybridization chain reaction (HCR) assembly that allows sensitive sensing and imaging of pH in living cells. Our design relies on the triplex forming sequences that form DNA triplex with toehold regions under acidic conditions and then induce a cascade of strand displacement and DNA assembly. The HCR assembly has shown dynamic responses in physiological pH ranges with excellent reversibility and demonstrated the potential for in vitro detection and live-cell imaging of pH. Moreover, this method affords HCR assemblies with highly localized fluorescence responses, offering advantages of improving sensitivity and better selectivity. The proton-fueled, reversible HCR assembly may provide a useful approach for pH-related cell biology study and disease diagnostics.

A novel off-on fluorescent probe for sensitive imaging of mitochondria-specific nitroreductase activity in living tumor cells.

Sensitive and selective detection and imaging of nitroreductase (NTR) in cancer cells is of great importance for better understanding their biological functions. Since there are a few fluorescent probes concerning NTR activity specifically located in mitochondria, we developed a novel fluorescent benzoindocyanine probe (BICP) for mitochondrial NTR activity monitoring and imaging via extending a benzoindole moiety into a benzoindocyanine based fluorophore (BICF) with a strong intramolecular charge transfer (ICT) effect and incorporating 4-nitrobenzyl as a fluorescence-quenching and enzyme-responsive moiety. Live cell imaging of HeLa and A549 demonstrates that the developed BICP is able to realize sensitive and selective mitochondrial NTR activity probing with high-contrast "off-on" fluorescence. These findings implied the great potential of the developed probe for monitoring mitochondrial-specific NTR activities in living cells and related applications in cell biology.

Plasmon Coupling Enhanced Raman Scattering Nanobeacon for Single-Step, Ultrasensitive Detection of Cholera Toxin.

We report the development of a novel plasmon coupling enhanced Raman scattering (PCERS) method, PCERS nanobeacon, for ultrasensitive, single-step, homogeneous detection of cholera toxin (CT). This method relies on our design of the plasmonic nanoparticles, which have a bilayer phospholipid coating with embedded Raman indicators and CT-binding ligands of monosialoganglioside (GM1). This design allows a facile synthesis of the plasmonic nanoparticle via two-step self-assembly without any specific modification or chemical immobilization. The realization of tethering GM1 on the surface imparts the plasmonic nanoparticles with high affinity, excellent specificity, and multivalence for interaction with CT. The unique lipid-based bilayer coated structure also affords excellent biocompatibility and stability for the plasmonic nanoparticles. The plasmonic nanoparticles are able to show substantial enhancement of the surface-enhanced Raman scattering (SERS) signals in a single-step interaction with CT, because of their assembly into aggregates in response to the CT-sandwiched interactions. The results reveal that the developed nanobeacon provides a simple but ultrasensitive sensor for rapid detection of CT with a large signal-to-background ratio and excellent reproducibility in a wide dynamic range, implying its potential for point-of-care applications in preventive and diagnostic monitoring of cholera.

[Study on the Inorganic Components Elements in Rare and Endangered Plant Alsophila Spinulosa].

A method for simultaneously analysis of inorganic elements in rare and endangered plant spinulose tree fern was established. Alsophila Spinulosa samples were pretreated by using the hermetic microwave digestion. Six elements Na, Mg, Al, P, K and Ca were determined by using the inductively coupled plasma optical emission spectrometry (ICP-OES) and ten elements V, Cr, Mn, Fe, Ni, Cu, Zn, Cd, Hg and Pb were determined by using inductively coupled plasma mass spectrometry (ICP-MS). The working conditions were optimized as well. In order to keep the stability of the experiment, low concentration of HCl was added into the sample during testing. Multicomponent Spectral Fitting (MSF) and dynamic reaction cell (DRC) were used to correct the spectral interference and mass interference. Under optimized conditions, the standard linear relationships were satisfactory and the detection limit ranged from 1.87 ng·L-1～12.31 µg·L-1. Finally, the standard reference material NIST SRM 1 547 (peach leaves) was used to verify the proposed method. The obtained results were consistent with the reference method with the sameaccuracy and precision. Two Alsophila Spinulosa samples analysis results showed that Alsophila Spinulosa have higher concentrations of Na, Mg, K, Ca, Al, and P, and concentration of K is the highest. These results indicated that Alsophila Spinulosa have strong capability in K-enrichment. Alsophila Spinulosa have low concentrations of V, Cr, Mn, Fe, Cu, and Zn. Concentrations of Ni, Cd, Hg, and Pb are at very low trace levels, which indicated that the wild living environment is very healthy. This method has features of fast analyzing, high accuracy and convenience in operation, which can provide scientific basis for Alsophila Spinulosa inorganic elements contents measure.

[Study on the Inorganic Components Elements in Rare and Endangered Plant Alsophila Spinulosa].

A method for simultaneously analysis of inorganic elements in rare and endangered plant spinulose tree fern was established. Alsophila Spinulosa samples were pretreated by using the hermetic microwave digestion. Six elements Na, Mg, Al, P, K and Ca were determined by using the inductively coupled plasma optical emission spectrometry (ICP-OES) and ten elements V, Cr, Mn, Fe, Ni, Cu, Zn, Cd, Hg and Pb were determined by using inductively coupled plasma mass spectrometry (ICP-MS). The working conditions were optimized as well. In order to keep the stability of the experiment, low concentration of HCl was added into the sample during testing. Multicomponent Spectral Fitting (MSF) and dynamic reaction cell (DRC) were used to correct the spectral interference and mass interference. Under optimized conditions, the standard linear relationships were satisfactory and the detection limit ranged from 1.87 ng·L-1～12.31 µg·L-1. Finally, the standard reference material NIST SRM 1 547 (peach leaves) was used to verify the proposed method. The obtained results were consistent with the reference method with the sameaccuracy and precision. Two Alsophila Spinulosa samples analysis results showed that Alsophila Spinulosa have higher concentrations of Na, Mg, K, Ca, Al, and P, and concentration of K is the highest. These results indicated that Alsophila Spinulosa have strong capability in K-enrichment. Alsophila Spinulosa have low concentrations of V, Cr, Mn, Fe, Cu, and Zn. Concentrations of Ni, Cd, Hg, and Pb are at very low trace levels, which indicated that the wild living environment is very healthy. This method has features of fast analyzing, high accuracy and convenience in operation, which can provide scientific basis for Alsophila Spinulosa inorganic elements contents measure.

[Association of CCR2 gene rs1799864 polymorphism with hemophagocytic lymphohistiocytosis in children].

OBJECTIVE: To investigate the association between rs1799864 single nucleotide polymorphism (SNP) of the C-C chemokine receptor 2 (CCR2) gene and susceptibility of hemophagocytic lymphohistiocytosis (HLH) in children. METHODS: The clinical and laboratory data of 86 children diagnosed with HLH between January 2007 and December 2013 were retrospectively reviewed. The CCR2 gene rs1799864 was genotyped by SNaPshot technique in 86 HLH children and 128 healthy controls. The genotypic and allelic frequencies in the two groups were comparatively analyzed. RESULTS: No significant difference either in genotypic or allelic frequencies of rs1799864 polymorphism of the CCR2 gene was observed between HLH patients and controls (P>0.05), but there were significant differences in the age of onset and the periods of temperature and platelet returning to normal after treatment (P<0.05). CONCLUSIONS: There is no association between CCR2 gene rs1799864 polymorphism and the risk for HLH in children. However, the genotypic differences of this polymorphism might be associated with clinical characteristics and prognosis of HLH.

[Association between gene polymorphisms of Perforin 1 and hemophagocytic lymphohistiocytosis].

OBJECTIVE: To investigate frequency distribution of gene polymorphisms of PRF1 gene in children with hemophagocytic lymphohistiocytosis (HLH), and to explore whether the possible gene polymorphisms of PRF1 gene confer an increased risk of susceptibility to HLH. METHODS: Forty-eight children who were diagnosed with HLH between January 2009 and December 2013 (HLH group) and 100 healthy children (control group) were enrolled in this study. The gene polymorphisms in the coding region of PRF1 gene, which consists of three exons and two introns, were genotyped by PCR, followed by direct sequencing. RESULTS: Three single nucleotide polymorphisms (SNPs) were revealed in the coding sequence of PRF1 in the 48 children with HLH. Seven SNPs were detected in the noncoding sequence. Other two SNPs in the noncoding sequence including rs10999426 and rs10999427 were detected only in 5 healthy children (5%). There was no significant difference in allelic frequencies of all the SNPs above between the HLH and control groups (P>0.05). Haplotype analysis showed there was a pair-wise linkage disequilibrium between rs10999426 and rs10999427 (D=1, r2=1), but there was no significant difference in the distribution of A-T haplotype between the HLH and control groups (P>0.05). CONCLUSIONS: There is no association between gene polymorphisms of PRF1 gene and the susceptibility to HLH. There is a pair-wise linkage disequilibrium between rs10999426 and rs10999427, but a low detection rate of A-T haplotype in healthy children indicates that it might not play a protective role in the development of HLH.

Terminal protection of small molecule-linked DNA for small molecule-protein interaction assays.

Methods for the detection of specific interactions between diverse proteins and various small-molecule ligands are of significant importance in understanding the mechanisms of many critical physiological processes of organisms. The techniques also represent a major avenue to drug screening, molecular diagnostics, and public safety monitoring. Terminal protection assay of small molecule-linked DNA is a demonstrated novel methodology which has exhibited great potential for the development of simple, sensitive, specific and high-throughput methods for the detection of small molecule-protein interactions. Herein, we review the basic principle of terminal protection assay, the development of associated methods, and the signal amplification strategies adopted for performance improving in small molecule-protein interaction assay.

[Optimization of Preparation of Cutting-processed Arecae Pericarpium with Multi-index Orthogonal Method].

OBJECTIVE: 10 optimize the preparation of cutting-processed Arecae Pericarpium by the orthogonal method. METHODS: With four alkaloids such as arecoline, arecaidine, guvacoline, guvacine and yield of water-soluble decoction as indexes, a L9 (3(4)) orthogonal test was adopted to compare the effect of different factors on cutting-processed Arecae Pericarpium. RESULTS: According to the finalized optimal process, Arecae Pericarpium was washed quickly, moisturized, chopped lengthwise in 1 cm segment and then dried for 2.5 h at 50 °C. CONCLUSION: The optimal process method is reasonable and reliable, it can provide basis for the preparation of cutting-processed Arecae Pericarnium.

t-SMILES: a fragment-based molecular representation framework for de novo ligand design.

Effective representation of molecules is a crucial factor affecting the performance of artificial intelligence models. This study introduces a flexible, fragment-based, multiscale molecular representation framework called t-SMILES (tree-based SMILES) with three code algorithms: TSSA (t-SMILES with shared atom), TSDY (t-SMILES with dummy atom but without ID) and TSID (t-SMILES with ID and dummy atom). It describes molecules using SMILES-type strings obtained by performing a breadth-first search on a full binary tree formed from a fragmented molecular graph. Systematic evaluations using JTVAE, BRICS, MMPA, and Scaffold show the feasibility of constructing a multi-code molecular description system, where various descriptions complement each other, enhancing the overall performance. In addition, it can avoid overfitting and achieve higher novelty scores while maintaining reasonable similarity on labeled low-resource datasets, regardless of whether the model is original, data-augmented, or pre-trained then fine-tuned. Furthermore, it significantly outperforms classical SMILES, DeepSMILES, SELFIES and baseline models in goal-directed tasks. And it surpasses state-of-the-art fragment, graph and SMILES based approaches on ChEMBL, Zinc, and QM9.