Dynamics of an Excess Electron in Molten LiF, KF, MgF2, and BeF2.

Ab initio molecular dynamics simulations suggest that the dynamics of an excess electron in different types of molten salts are not always the same. In molten LiF, KF, and MgF2, the excess electron localizes in the cavity as a solvated electron for 10 ps, which agrees with the widely accepted theory of Pikaev. In molten BeF2, the excess electron shows a different localization pattern: it mostly exists in localized states but also occurs in many delocalized states. This "localize-delocalize" pattern originates from the high viscosity of BeF2 (16 000 cP at 900 °C), which will lead to slow ionic motion and finally result in slow solvent relaxation. Besides, the species formed by the localization of the excess electron in these four melts are also different. The spectral feature (broad peak in the vis-IR region) of the localized electron in molten alkaline halides was also observed in LiF, KF, MgF2, and BeF2. Both an excess electron and electrons in the bulk liquid could contribute to the spectra, but the excitation of the excess electron makes a bigger contribution to the broad vis-IR peak. Our predicted spectrum of molten LiF/KF qualitatively reproduces the major feature of the experimental spectrum, which partially validates our simulations.

Device-Free Wireless Sensing for Gesture Recognition Based on Complementary CSI Amplitude and Phase.

By integrating sensing capability into wireless communication, wireless sensing technology has become a promising contactless and non-line-of-sight sensing paradigm to explore the dynamic characteristics of channel state information (CSI) for recognizing human behaviors. In this paper, we develop an effective device-free human gesture recognition (HGR) system based on WiFi wireless sensing technology in which the complementary CSI amplitude and phase of communication link are jointly exploited. To improve the quality of collected CSI, a linear transform-based data processing method is first used to eliminate the phase offset and noise and to reduce the impact of multi-path effects. Then, six different time and frequency domain features are chosen for both amplitude and phase, including the mean, variance, root mean square, interquartile range, energy entropy and power spectral entropy, and a feature selection algorithm to remove irrelevant and redundant features is proposed based on filtering and principal component analysis methods, resulting in the construction of a feature subspace to distinguish different gestures. On this basis, a support vector machine-based stacking algorithm is proposed for gesture classification based on the selected and complementary amplitude and phase features. Lastly, we conduct experiments under a practical scenario with one transmitter and receiver. The results demonstrate that the average accuracy of the proposed HGR system is 98.3% and that the F1-score is over 97%.

Catalytic methane decomposition on CNT-supported Fe-catalysts.

Methane, either as natural gas or as a resource obtained from various bioprocesses (e.g., digestion, landfill) can be converted to carbon and hydrogen according to. CH4(g)→C(s)+2H2(g)ΔH298K=74.8kJ/mol. Previous research has stressed the growing importance of substituting the high-temperature Steam Methane Reforming (SMR) by a moderate temperature Catalytic Methane Decomposition (CMD). The carbon formed is moreover of nanotube nature, in high industrial demand. To avoid the use of an inert support for the active catalyst species, e.g., Al2O3 for Fe, leading to a progressive contamination of the catalyst by support debris and coking of the catalyst, the present research investigates the use of carbon nanotubes (CNTs) as Fe-support. Average CH4 conversions of 75-85% are obtained at 700 °C for a continuous operation of 40 h. The produced CNT from the methane conversion can be continuously removed from the catalyst bed by carry-over due to its bulk density difference (∼120 kg/m3) with the catalyst itself (∼1500 kg/m3). CNT properties are fully specified. No thermal regeneration of the catalyst is required. A tentative process layout and economic analysis demonstrate the scalability of the process and the very competitive production costs of H2 and CNT.

[Progress on historical evolution, clinical application, and pharmacological effects of Dachaihu Decoction and predictive analysis of its quality markers].

Dachaihu Decoction is a classic prescription with the function of harmonizing Shaoyang and purging away internal stasis of heat, which was specially developed by Master ZHANG Zhongjing for the concurrent disease of Shaoyang and Yangming. A large number of international studies have shown that Dachaihu Decoction has liver protection, gallbladder benefit, anti-inflammatory, and other pharmacological effects and is mostly used in modern clinical treatment of acute pancreatitis, acute cholecystitis, cholelithiasis, and other digestive diseases. This paper combined bibliography and statistics and selected the ancient book database and CNKI database to search the relevant literature on Dachaihu decoction, verify the composition and dosage, processing method, main diseases, and modern clinical application, and predict its quality markers(Q-markers) based on the "five principles" of Q-markers. The results suggest that saikosaponin a, baicalin, and 6-gingerol can be selected as potential Q-markers for Dachaihu Decoction, so as to provide a basis for the clinical research of traditional Chinese medicine and the development and application of compound preparations.

Cu-Co Dual-Atom Catalysts Supported on Hierarchical USY Zeolites for an Efficient Cross-Dehydrogenative C(sp2)-N Coupling Reaction.

A cross-coupling reaction via the dehydrogenative route over heterogeneous solid atomic catalysts offers practical solutions toward an economical and sustainable elaboration of simple organic substrates. The current utilization of this technology is, however, hampered by limited molecular definition of many solid catalysts. Here, we report the development of Cu-M dual-atom catalysts (where M = Co, Ni, Cu, and Zn) supported on a hierarchical USY zeolite to mediate efficient dehydrogenative cross-coupling of unprotected phenols with amine partners. Over 80% isolated yields have been attained over Cu-Co-USY, which shows much superior reactivity when compared with our Cu1 and other Cu-M analogues. This amination reaction has hence involved simple and non-forceful reaction condition requirements. The superior reactivity can be attributed to (1) the specifically designed bimetallic Cu-Co active sites within the micropore for "co-adsorption-co-activation" of the reaction substrates and (2) the facile intracrystalline (meso/micropore) diffusion of the heterocyclic organic substrates. This study offers critical insights into the engineering of next-generation solid atomic catalysts with complex reaction steps.

3D-Printed Piezoelectric Scaffolds with Shape Memory Polymer for Bone Regeneration.

The application of piezoelectric nanoparticles with shape memory polymer (SMP) to 3D-printed piezoelectric scaffolds for bone defect repair is an attractive research direction. However, there is a significant difference in dielectric constants between the piezoelectric phase and polymer phase, limiting the piezoelectric property. Therefore, novel piezoelectric acrylate epoxidized soybean oil (AESO) scaffolds doped with piezoelectric Ag-TMSPM-pBT (ATP) nanoparticles (AESO-ATP scaffolds) are prepared via digital light procession 3D-printing. The Ag-TMSPM-pBT nanoparticles improve the piezoelectric properties of the AESO scaffolds by TMSPM covalent functionalization and conductive Ag nanoparticles. The AESO scaffolds doped with 10 wt% Ag-TMSPM-pBT nanoparticles (AESO-10ATP scaffolds) exhibit promising piezoelectrical properties, with a piezoelectric coefficient (d33) of 0.9 pC N-1 and an output current of 146.4 nA, which are close to the piezoelectric constants of bone tissue. In addition, these scaffolds exhibit good shape memory function and can quickly recover their original shape under near-infrared (NIR) light irradiation. The results of osteogenesis capability evaluation indicate that the AESO-10ATP scaffolds can promote osteogenic differentiation of BMSCs in vitro and bone defect repair in vivo, indicating the 3D-printed AESO-10ATP piezoelectric scaffolds may have great application potential for bone regeneration.

Antibacterial and antibiofilm efficacy of repurposing drug hexestrol against methicillin-resistant Staphylococcus aureus.

There has been an explosion in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) because of the indiscriminate use of antibiotics. In this study, we repurposed hexestrol (HXS) as an antibacterial agent to fight planktonic and biofilm-related MRSA infections. HXS is a nonsteroidal synthetic estrogen that targets estrogen receptors (ERα and ERß) and has been used as a hormonal antineoplastic agent. In our work, the minimum inhibitory concentrations (MICs) were determined using the antimicrobial susceptibility of MSSA and MRSA strains. Anti-biofilm activity was evaluated using biofilm inhibition and eradication assays. Biofilm-related genes were analyzed with or without HXS treatment using RTqPCR analysis of S. aureus. HXS was tested using the checkerboard dilution assay to identify antibiotics that may have synergistic effects. Measurement of ATP and detection of ATPase allowed the determination of bacterial energy metabolism. As shown in the results, HXS showed effective antimicrobial activity against S. aureus, including both type strains and clinical isolations, with MICs of 16 µg/mL. Sub-HXS strongly inhibited the adhesion of S. aureus. The content of extracellular polymeric substances (EPS) and the relative transcription levels of eno, sacC, clfA, pls and fnbpB were reduced after HXS treatment. HXS showed antibacterial effects against S. aureus and synergistic activity with aminoglycosides by directly interfering with cellular energy metabolism. HXS inhibits adhesion and biofilm formation and eradicates biofilms formed by MRSA by reducing the expression of related genes. Furthermore, HXS increases the susceptibility of aminoglycosides against MRSA. In conclusion, HXS is a repurposed drug that may be a promising therapeutic option for MRSA infection.

Triple combination of SPR741, clarithromycin, and erythromycin against Acinetobacter baumannii and its tolerant phenotype.

AIMS: Extensively drug-resistant (XDR) Acinetobacter baumannii poses a severe threat to public health due to its ability to form biofilms and persister cells, which contributes to critical drug resistance and refractory device-associated infections. A novel strategy to alleviate such an emergency is to identify promising compounds that restore the antimicrobial susceptibility of existing antibiotics against refractory infections. METHODS AND RESULTS: Here, we found a significant synergy among three combinations of SPR741, clarithromycin and erythromycin with a potent antimicrobial activity against XDR A. baumannii (SPR741/CLA/E at 8/10/10 µg ml-1 for XDR AB1069 and at 10/16/10 µg ml-1 for XDR AB1208, respectively). Moreover, the triple combination therapy exhibits a significant antipersister and antibiofilm effect against XDR strains. Mechanistic studies demonstrate that SPR741 may promote intracellular accumulation of macrolides by permeabilizing the outer membrane as well as disrupting membrane potential and further enhance the quorum sensing inhibition activity of the macrolides against XDR A. baumannii and its biofilms. In addition, the triple combination of SPR741 with clarithromycin and erythromycin was not easy to induce resistance in A. baumannii and had effective antimicrobial activity with low toxicity in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: Collectively, these results reveal the potential of SPR741 in combination with clarithromycin and erythromycin as a clinical therapy for refractory infections caused by XDR A. baumannii.

Juglone promotes antitumor activity against prostate cancer via suppressing glycolysis and oxidative phosphorylation.

The treatments currently used for prostate cancer (PC) do not meet clinical needs, and thus, new therapies with greater effectiveness are urgently required. Metabolic reprogramming of tumor cells is emerging as an exciting field for cancer therapy. Although the Warburg effect is a common feature of glucose metabolism in many cancers, PC cells have a unique metabolic phenotype. Non-neoplastic prostate cells show reduced oxidative phosphorylation (OXPHOS) because large, accumulated zinc inhibits citrate oxidation. During transformation, there are low levels of zinc in PC cells, and the tricarboxylic acid (TCA) cycle is reactivated. However, metastatic PC exhibits the Warburg effect. Due to metabolic differences in prostate tissue, targeting metabolic alterations in PC cells is an attractive therapeutic strategy. In this study, we investigated the effect of juglone on energy metabolism in PC cells. We found that juglone inhibited cell proliferation and induced apoptosis. Mechanistically, we demonstrated that juglone suppressed OXPHOS and glycolysis due to its inhibition of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) activity. Furthermore, downregulation of PFK and PK, but not HK contributed to the inhibition of these enzyme activities. The current study indicates that further development of juglone for PC treatment would be beneficial.

Gallium Triggers Ferroptosis through a Synergistic Mechanism.

The gallium ion (Ga3+ ) has long been believed to disrupt ferric homeostasis in the body by competing with iron cofactors in metalloproteins, ultimately leading to cell death. This study revealed that through an indirect pathway, gallium can trigger ferroptosis, a type of non-apoptotic cell death regulated by iron. This is exemplified by the gallium complex of the salen ligand (Ga-1); we found that Ga-1 acts as an effective anion transporter that can affect the pH gradient and change membrane permeability, leading to mitochondrial dysfunction and the release of ferrous iron from the electron transfer chain (ETC). In addition, Ga-1 also targeted protein disulfide isomerases (PDIs) located in the endoplasmic reticulum (ER) membrane, preventing the repair of the antioxidant glutathione (GSH) system and thus enforcing ferroptosis. Finally, a combination treatment of Ga-1 and dietary polyunsaturated fatty acids (PUFAs), which enhances lipid peroxidation during ferroptosis, showed a synergistic therapeutic effect both in vitro and in vivo. This study provided us with a strategy to synergistically induce Ferroptosis in tumor cells, thereby enhancing the anti-neoplastic effect.

Postmortem detection of COL gene family variants in two aortic dissection cases.

Aortic dissection (AD) usually remains undiagnosed, but its manifestation is abrupt and is associated with high morbidity and poor prognosis, leading to sudden cardiac death. Variants in COL family genes are associated with AD. In case 1, a 32-year-old Chinese man was admitted to the hospital with complaints of abdominal pain and died on the next day. In case 2, a 36-year-old Chinese woman was admitted to the hospital because of waist pain and died the next afternoon. According to autopsy findings, the cause of death in both cases was an acute cardiac tamponade, which was attributed to AD rupture. Whole-exome sequencing was performed on the blood collected from the hearts of the two deceased patients. Positive variants in COL family genes were found in both cases, without positive variants in other AD-associated genes. In case 1, a novel, likely pathogenic, missense variant was identified in COL6A1. In case 2, we identified one novel, likely pathogenic, frameshift deletion in COL23A1 and one novel, likely pathogenic, missense mutation in COL1A2. Based on these two cases, physicians should consider the role and significance of COL family gene mutations in AD in young patients. Furthermore, molecular anatomy is clearly necessary and significant in cases of sudden cardiac death attributed to AD, particularly in younger individuals.

Anti-hepatitis C virus drug simeprevir: a promising antimicrobial agent against MRSA.

Staphylococcus aureus is a major human pathogen, and the appearance of methicillin-resistant S. aureus (MRSA) renders S. aureus infections more challenging to treat. Therefore, new antimicrobial drugs are urgently needed to combat MRSA infections. Drug repurposing is an effective and feasible strategy. Here, we reported that the clinically approved anti-hepatitis C virus drug simeprevir had strong antibacterial activity against MRSA, with a minimum inhibitory concentration of 2-8 µg/mL. Simeprevir did not easily induce in vitro resistance. In addition, simeprevir significantly prevented S. aureus biofilm formation. Furthermore, simeprevir displayed limited toxicity in in vitro and in vivo assays. Moreover, simeprevir showed synergistic antimicrobial effects against both type and clinical strains of S. aureus. Simeprevir combined with gentamicin effectively reduced the bacterial burden in an MRSA-infected subcutaneous abscess mouse model. Results from a series of experiments, including membrane permeability assay, membrane potential assay, intracellular ATP level assay, and electron microscope observation, demonstrated that the action of simeprevir may be by disrupting bacterial cell membranes. Collectively, these results demonstrated the potential of simeprevir as an antimicrobial agent for the treatment of MRSA infections. KEY POINTS: • Simeprevir showed strong antibacterial activity against MRSA. • The antibacterial mechanism of simeprevir was mediated by membrane disruption and intracellular ATP depletion. • In vitro and in vivo synergistic antimicrobial efficacy between simeprevir and gentamicin was found.

A novel bactericidal small molecule, STK-35, and its derivative, STK-66, as antibacterial agents against Gram-negative pathogenic bacteria in vitro and in vivo.

Due to the increasing rate of antibiotic resistance and the emergence of persister cells of Gram-negative pathogenic bacteria, the development of new antibacterial agents is urgently needed to deal with this problem. Our results indicated that both newly identified small molecule STK-35 and its derivative STK-66 exhibited effective antibacterial properties against a variety of Gram-negative pathogens including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. The minimal inhibitory concentrations and minimal bactericidal concentrations ranges were 0·0625-8 µg ml-1 and 0·125-16 µg ml-1 , respectively, while no haemolytic activity and mammalian cell cytotoxicity were observed. The time-killing assays showed STK-35/66 had strong bactericidal activity against Gram-negative pathogens. STK-35/66 also showed different degrees of synergistic antibacterial activity with conventional antibiotics and exhibited persister cells killing activity. Moreover, STK-35/66 effectively eradicated the pre-formed biofilms of P. aeruginosa and A. baumannii. In addition, STK-35/66 significantly increased the survival rate of E. coli infected mice and induced a decrease in bacterial load of the peritonitis model. In nutshell, these results suggested that STK-35/66 possessed antimicrobial activity against Gram-negative pathogenic bacteria in vitro and in vivo, which could be considered as potential substitutes for the treatment of Gram-negative pathogenic infections after further structure optimization.

Regulation of the Volume Flow Rate of Aqueous Methyl Blue Solution and the Wettability of CuO/ZnO Nanorods to Improve the Photodegradation Performance of Related Microfluidic Reactors.

Six CuO/ZnO nanorod (CuO/ZnONR)-based microfluidic reactors were constructed for different UV irradiation durations, with which an aqueous methylene blue (MB) solution was photodegraded at varied volume flow rate Q. Via numerical and experimental routes, the effects of the Q on the kinetic adsorption rate constant Ka and the initial rate constant KA of the CuO/ZnONR-based microfluidic reactors were discussed. Moreover, a reverse contacting angle (CA) trend of CuO/ZnONRs to the reaction constant K curve of corresponding CuO/ZnONR-based microfluidic reactor suggested that the CA of CuO/ZnONRs was another key influencing factor that affected greatly the photodegradation performance of the microfluidic reactors. The Q of the aqueous MB solution and the UV irradiation duration for the photodeposition of CuO/ZnONRs were optimized to be 125 µL/min and 1.0 h, the K of the CuO/ZnONR-based microfluidic reactors reached 4.84 min-1, and the related ΔKA/K was less than 6%. Similarly, these methods and results can be employed not only to enhance the mass transport and adsorption of specific species within other nanostructured matrix material-coated microchannels but also to enlarge the actual contacting surface areas between these microchannels and the related solution, which further improve the performance of other nanostructured catalyst-based microfluidic reactors, rGO microfluidic voltage generation, and a GOx/AuNW enzymatic glucose microfluidic sensor.

Repurposing Sitafloxacin, Prulifloxacin, Tosufloxacin, and Sisomicin as Antimicrobials Against Biofilm and Persister Cells of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous bacterium found in hospitals and the surrounding environment. The ability of P. aeruginosa to form biofilms confers high-level resistance to antibiotics, and the persister cells formed in the presence of high antibacterial drug concentrations make P. aeruginosa-related infections more refractory. Further, there rarely is an effective antimicrobial alternative when biofilm- and persister cell-targeting treatment fails. Using a high-throughput screening assay, we previously identified fluoroquinolones sitafloxacin, prulifloxacin, and tosufloxacin as well as aminoglycoside sisomicin among FDA-approved drugs with significant bactericidal activity against P. aeruginosa. In addition, in our current study, these antibiotics exhibited an effective time- and dose-dependent eradication effects against the preformed biofilms of P. aeruginosa at the concentrations of 2-4 µM. These agents also exhibited bactericidal efficacy against CCCP-induced P. aeruginosa persister cells with the viable cell count decreased from 9.14 log10 CFU/mL to 6.15 (sitafloxacin), 7.59 (prulifloxacin), 4.27 (tosufloxacin), and 6.17 (sisomicin) log10 CFU/mL, respectively, following 4 h of treatment. Furthermore, sisomicin was also effective against conventional antibiotics induced persister cells in a time-dependent manner within 24 h. In addition, we confirmed the in vivo anti-biofilm efficacy of the identified antibiotics in a subcutaneous implantation biofilm-related infection model. Tosufloxacin exhibited the greatest in vivo bactericidal activity against P. aeruginosa biofilms with a reduction of 4.54 ΔLog10 CFU/mL compared to the vehicle group, followed by prulifloxacin, sitafloxacin, and sisomicin. Taken together, our results indicate that sitafloxacin, prulifloxacin, tosufloxacin, and sisomicin have great potential as alternatives for the treatment of refractory infections caused by P. aeruginosa biofilms and persister cells.

Tomographic Proximity Imaging Using Conductive Sheet for Object Tracking.

This paper proposes a proximity imaging sensor based on a tomographic approach with a low-cost conductive sheet. Particularly, by defining capacitance density, physical proximity information is transformed into electric potential. A novel theoretical model is developed to solve the capacitance density problem using the tomographic approach. Additionally, a prototype is built and tested based on the model, and the system solves an inverse problem for imaging the capacitance density change that indicates the object's proximity change. In the evaluation test, the prototype reaches an error rate of 10.0-15.8% in horizontal localization at different heights. Finally, a hand-tracking demonstration is carried out, where a position difference of 33.8-46.7 mm between the proposed sensor and depth camera is achieved at 30 fps.

Construction of a Stable Expression Cell Line of Human Phospholamban. / 人源性受磷蛋白稳定表达细胞系的构建.

OBJECTIVES: To construct a cell line that can stably express human phospholamban(PLN) and initially explore its application in the study of myocardial toxicity mechanism. METHODS: FastCloning method was used to insert the open reading frame sequence of target gene PLN into eukaryotic expression vector pcDNA5/FRT/TO(hereinafter referred to as pDFT) to construct the pDFT-PLN-Flag plasmid. The Flp-InTM T-RExTM 293 cells were generated by cotransfection of the constructed plasmid and pOG44 plasmid to express the target gene. Successfully recombined monoclonal cell lines were screened by hygromycin B resistance. Western blot and indirect immunofluorescence (IFA) were used to examine the expression of the target protein in recombinant cells. After the cell line was exposed to aconitine, it was verified by Western blot to detect changes in PLN protein phosphorylation. RESULTS: After PCR amplification of the recombinant plasmid and DNA electrophoresis, the length of the amplified product is the same as the known PLN gene fragment, which is consistent with the open reading frame (ORF) sequence of the human PLN gene after sequencing. IFA and Western blot showed that the constructed proliferation cell line can stably express high levels of human PLN under induction and regulation. Preliminary results showed that the phosphorylation level of Thr17-PLN decreased after two hours of exposure to 1 µmol/L aconitine. CONCLUSIONS: This human cell line can stably express PLN and can be used to study the mechanism of action of aconitine on the cell at molecular level.

Exploring the genetic pathogenicity of aortic dissection from 72 Han Chinese individuals using next-generation sequencing.

Aortic dissection (AD) is a heterogeneous genetic disease with high morbidity and mortality. Although many genes predispose patients to AD, the pathogenic spectrum remains incomplete. This study aims to (a) investigate whether genotype differences exist between Stanford A and B AD individuals, and (b) broaden the pathogenic genetic spectrum of AD and reported novel variants of AD-associated genes. The DNA of 72 unrelated Han Chinese individuals with AD was tested by whole-exome sequencing. Of 142 AD-associated genes, 10 pathogenic variants, and 48 likely pathogenic variants in 36 genes were identified among 39 cases. The diagnostic yield was 54.2%. Of the 58 positive variants, 27 were novel. FBN1 was the most frequently positive gene in both Stanford A and Stanford B. Twenty-seven positive variants from 18 COL family genes were distributed in 36.8% of Stanford A and 6.7% of Stanford B cases. We emphasize that positive variants of COL family genes show distribution predominance and strong pathogenicity in Stanford A, while positive variants of smooth muscle cell pathway genes present distribution advantages mainly in Stanford B cases. Our findings provide a new perspective for both the pathogenic mechanism and the treatment of AD.

Probing DNA Hybridization Equilibrium by Cationic Conjugated Polymer for Highly Selective Detection and Imaging of Single-Nucleotide Mutation.

Hybridization-based probes emerge as a promising tool for nucleic acid target detection and imaging. However, the single-nucleotide selectivity is still challenging because the specificity of hybridization reaction is typically low at room temperature. We disclose an effective and simple method for highly selective detection and in situ imaging of single-nucleotide mutation (SNM) by taking the advantages of the specific hybridization of short duplex and the signal amplifying effect of cationic conjugated polymer (CCP). Excellent discrimination of the nucleic acid strands only differing by single nucleotide was achieved enabling the sensitive detection of SNM at the abundance as low as 0.1%. Single-molecule fluorescence resonance energy transfer (smFRET) study reveals that the presence of CCP enhances the perfect matched duplex and the mismatched duplex to a different extent, which can be an explanation for the high single-nucleotide selectivity. Due to the simple design of the probe and the stable brightness of CCP, highly selective mRNA in situ imaging was achieved in fixed cells. Melanoma cell line A375 with BRAF V600E point mutation exhibits higher FRET efficiency than liver cancer cell line HegG2 that was not reported having the mutation at this point.

Telomerase Activity Detection with Amplification-Free Single Molecule Stochastic Binding Assay.

Because the elongation of telomeres has been associated with tumorigenesis, it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis. Currently, amplification-based strategies have been extensively explored for telomerase detection in vitro and in vivo. However, amplification is typically associated with poor reproducibility and high background, which hamper further applications of the strategies, particularly for real sample assays. Here, we demonstrate a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy. The dynamic stochastic binding of a short fluorescent DNA probe with a genuine target yields a distinct kinetic signature from the background noise, allowing us to identify telomerase reaction products (TRPs) at the single molecule level. A limit-of-detection as low as 0.5 fM and a dynamic range of 0.5-500 fM for TRP detection were readily achieved. With this method, telomerase extracted from cancer cells was determined with sensitivity down to 10 cells. Moreover, the length distribution of TRPs was also determined by multiple stochastic probing, which could provide deep insight into the mechanistic study of telomerase catalysis.