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Background: Microglia exist in two polarized states: a classical activation state (M1) and an alternative activation state (M2). Accumulating evidence has suggested an involvement of miR-181a in neuronal development, survival, function, and plasticity, but its exact functional role in microglial polarization still requires clarification. We investigated the impact of miR-181a on microglial polarization using a rat chronic social stress model of depression. Methods: N9 microglia were stimulated with lipopolysaccharide (LPS; 100 ng/mL ) or IL-4 (10 ng/mL ) for 12h. Expression of Nos2, Arg-1, miR-181a, and NDRG2 was determined using real-time PCR. NDRG2 and syntaxin-1A protein expressions were analyzed by western blotting. The cell viability of N2A neuron was measured by an MTT assay. Body weight gain and adrenal weight were also evaluated. Results: Five weeks of daily social defeat significantly increased Nos2 and NDRG2 mRNA expression and dramatically reduced miR-181a expression in the hippocampus. Microglia were also polarized into different functional phenotypes. LPS treatment significantly enhanced mRNA expression of Nos2 and dramatically inhibited the expression of miR-181a. Conversely, treatment with IL-4 markedly increased the mRNA levels of Arg-1 and the expression of miR-181a. Expression of miR-181a regulated microglia differentiation by targeting NDRG2, while inhibition of miR-181a expression in N9 microglia induced N2A neuron death, which could be reversed by NDRG2 interference. Overexpression of miR-181a overcame the impacts of chronic psychosocial stress on body weight gain, adrenal weight, and syntaxin-1A expression in the hippocampus. Conclusion: Down-regulation of miR-181a promoted microglial M1 polarization by enhancing NDRG2 expression. Keywords: miR-181a, microglial M1 polarization, NDRG2

Herein, we found that surface defects quench persistent luminescence in nanophosphors. Passivation of surface defects by thermal treatment or surface coating can effectively enhance the intensity and prolong the decay time of persistent luminescence. The surface passivated persistent nanophosphors are promising in autofluorescence-free bioimaging and time-gated steganography.

Hypochlorous acid (HClO) is a special kind of reactive oxygen species, which plays an important role in resisting pathogen invasion and maintaining cell redox balance and other physiological processes. In addition, HClO is commonly used in daily life as a bleaching and disinfectant agent. Its excessive use can also lead to death of water animals and serious respiratory and skin diseases in humans. Therefore, it is of great significance to develop a quick and convenient tool for detecting HClO in the environment and organisms. In this paper, we utilize the specific reaction of HClO with dimethylthiocarbamate to develop a novel naphthalene derivative fluorescent probe (BNA-HClO), it was designed and synthesized by using 6-(2-benzothiazolyl)-2-naphthol as the fluorophore and

Osteosarcoma represents one of the most devastating cancers due to its high metastatic potency and fatality. Osteosarcoma is insensitive to traditional chemotherapy. Identification of a small molecule that blocks osteosarcoma progression has been a challenge in drug development. Phillygenin, a plant-derived tetrahydrofurofuran lignin, has shown to suppress cancer cell growth and inflammatory response. However, how phillygenin plays functional roles in osteosarcoma has remained unveiled. In this study, we showed that phillygenin inhibited osteosarcoma cell growth and motility in vitro. Further mechanistic studies indicated that phillygenin blocked STAT3 signaling pathway. Phillygenin led to significant downregulation of Janus kinase 2 and upregulation of Src homology region 2 domain-containing phosphatase 1. Gene products of STAT3 regulating cell survival and invasion were also inhibited by phillygenin. Therefore, our studies provided the first evidence that phillygenin repressed osteosarcoma progression by interfering STAT3 signaling pathway. Phillygenin is a potential candidate in osteosarcoma therapy.

The rapid detection of pollutants with high sensitivity and selectivity is of considerable significance for security screening, environmental safety, and human health. In this study, we prepared persistent luminescence nanoparticles (PLNPs) and constructed a label-free sensor for sensitive and selective detection of pollutants in real samples and test papers. Following excitation, PLNPs could store absorbed light energy and release it in the form of luminescence. Compared with a fluorescence-based technique, a PLNPs-based measurement could effectively avoid background interference. Under optimal conditions, the limit of detection for TNP was found to be 10 nM, while for an antibiotic it was 5 nM. The nanoprobe was successfully applied for the detection of pollutants in real samples including milk and Dianchi Lake water samples. Due to the long-lasting afterglow nature of PLNPs, the signal-to-noise ratio could be greatly increased in complex real samples. By hand-writing with TNP solution as ink on filter paper, the photoluminescence (PL) of the part stained with TNP was immediately quenched. Moreover, after direct exposure under a UV lamp for 10 min and without further excitation, the luminescence of the test paper was investigated to avoid interferents. This PLNP material could be potentially employed as a multi-responsive luminescent sensor. In addition, these easy-to-use visual techniques could provide a powerful tool for a convenient POC assay of organic pollutants.

Superoxide anion radical (O 2 − ), the “primary” reactive oxygen species (ROS) in living systems, is linked to a variety of physiological and pathological processes. Therefore, developing an effective strategy to monitor the fluctuation of O 2 − in biological systems is of great importance. This paper describes a new turn-on two-photon fluorescent probe for endogenous O 2 − detection and imaging, which was rationally designed and synthesized via a non-redox strategy. In the presence of O 2 − , the probe exhibited notable fluorescence enhancement (∼235-fold) with a low detection limit down to 1 nM, indicating a high signal-to-background ratio and excellent sensitivity. In addition, short response time, good biocompatibility, low cytotoxicity, long-term stability against light illumination, specificity to O 2 − over general reductants, and pH stability were demonstrated, indicating that the requirements for cellular O 2 − determination are met. Furthermore, the probe was successfully applied in two-photon fluorescence imaging of endogenous O 2 − in living cells and tissues and showed high imaging resolution and a deep-tissue imaging depth of ∼150 μm, illustrating the promising potential for practical applications in complex biosystems and providing a valuable theoretical basis and technical support for the study of physiological and pathological functions of O 2 − .

The monitoring of K+ is very important and emergency because of their unique relationship in various disease diagnosis and treatment. G-quadruplex analogue is a classical recognition unit for K+ detection and has been widely applied in K+ relevant research. Common fluorescent dyes were employed for design of G-quadruplex structure-based K+ probes which suffered from the aggregation-caused quenching effect, and possibly limited the biological applications in living systems. Herein, we report an aggregation-induced emission (AIE) effect-based fluorescent probe for cellular K+ analysis and imaging. Benefitting from the K+ triggered AIE phenomenon, the designed TPE derivative modified guanine (G)-rich oligonucleotide fluorescent probe (TPE-oligonucleotide probe) exhibits high sensitivity (∼10-fold higher than most reported G-quadruplex-based probes) with extended photostability which facilitates the prolonged fluorescence observations of K+ in living cells. On the basis of these advantages, the TPE-oligonucleotide probe serves as a promising candidate for the functional study and analysis of K+.

Hydrogen sulfide (H2S), one of the biologically important gaseous signal molecules, plays an essential role in diverse normal biochemical functions and pathological processes. Herein, an efficient two-photon in and near-infrared out mitochondria-targeting dye has been designed, synthesized and characterized. It is easily synthesized by the condensation reaction (C˭C) of 4-hydroxybenzaldehyde and 6-(diethylamino)-1,2,3,4-tetrahydroxanthylium (mitochondria-targeting), which possesses large two-photon action absorption cross-section ~160g and high fluorescence quantum yield ~0.15. Encouraged by the results, we proceeded to conjugate this new dye with a H2S recognition moiety (4-dinitrobenzene-ether, DNB), on the basis of the intramolecular charge transfer (ICT) strategy, to construct a novel H2S fluorescent probe (TP-NIR-HS), which shows a targeting ability with high sensitivity and selectivity, and low cytotoxicity. This new probe was then applied for two-photon imaging of living cells and tissues and showed high imaging resolution and a deep-tissue imaging depth of ~350µm, thus demonstrating its practical application in biological systems, and providing a valuable theoretical basis and technical support for the study of physiological and pathological functions of H2S.

In contrast to one-photon microscopy, two-photon probe-based fluorescence imaging can provide improved three-dimensional spatial localization and increased imaging depth. Therefore, the development of new functional two-photon fluorescent dye has attracted great attention. Herein, we have adopted the fluorescence resonance energy transfer (FRET) strategy to design a unique type of two-photon ratiometric fluorescent probe platform. Specifically, a two-photon fluorophore (D-π-A-structured naphthalimide derivative) and a rhodamine B fluorophore are directly connected by a flexible piperidine linker. We further used this platform to develop a new type of two-photon ratiometric fluorescent probe, NRLys, for lysosomal pH detection and bioimaging. The experiments demonstrated that the NRLys is a reliable and specific probe for labeling lysosomes in living cells and tissues with two well-resolved emission peaks separated by 60 nm. The probe showed high ratiometric imaging resolution and deep-tissue imaging depth of over 180 μm. Based on the above results, we expect that the new platform may prompt the development of a wide variety of two-photon ratiometric fluorescent probes application in biological systems.

Hypochlorite anion, one of the biologically important reactive oxygen species, plays an essential role in diverse normal biochemical functions and abnormal pathological processes. Herein, an efficient high-resolution mitochondria-targeting ratiometric fluorescent probe for hypochlorous acid detection has been designed, synthesized and characterized. It is easily synthesized by the condensation reaction (C C) of a 2-(2-hydroxyphenyl) quinazolin-4(3H)-one fluorophore and a cyanine group (mitochondria-targeting), which made the whole molecular a large Stokes shift (210 nm) and the two well-resolved emission peaks separated by 140 nm. As a result, it is considered as a good candidate for high resolution hypochlorous acid imaging in live cells. The ratiometric fluorescent probe exhibited outstanding features of high sensitivity, high selectivity, rapid response time (within 50 s), and excellent mitochondria-targeting ability. Moreover, the probe can also be successfully applied to imaging endogenously hypochlorous acid in the mitochondria of living cells with low cytotoxicity, and high resolution.

Aptamers, which are selected in vitro by a technology known as the systematic evolution of ligands by exponential enrichment (SELEX), represent a crucial recognition element in molecular sensing. With advantages such as good biocompatibility, facile functionalization, and special optical and physical properties, various nanomaterials can protect aptamers from enzymatic degradation and nonspecific binding in living systems and thus provide a preeminent platform for biochemical applications. Coupling aptamers with various nanomaterials offers many opportunities for developing highly sensitive and selective sensing systems. Here, we focus on the recent applications of aptamer-assembled nanomaterials in fluorescent sensing and imaging. Different types of nanomaterials are examined along with their advantages and disadvantages. Finally, we look toward the future of aptamer-assembled nanomaterials.

Conjugation of DNA to defined locations on a protein surface will be a powerful tool for positioning functional groups and molecules in biological and biomedical studies. However, tagging protein with DNA is challenging in physiological environments, and requires a bioorthogonal approach. Here, we report a chemical solution to selectively conjugate DNA aptamers with a protein by protein–aptamer template (PAT)-directed reactions. Since protein–aptamer interactions are bioorthogonal, we exploit the PAT as a unique platform for specific DNA–protein cross-linking. We develop a series of modified oligonucleotides for PAT-directed reactions and find an F-carboxyl group as a suitable functionality for selective and site-specific conjugation. The functionality is incorporated into aptamers in our F-carboxyl phosphoramidite with an easy synthesis. We also demonstrate the necessity of a linker between the reactive functionality and the aptamer sequences.

DNA molecular machines show great promise in fields such as biomarker discovery and biological activity regulation, but operating DNA machines with specific functions within living systems remains extremely challenging. Although DNA machines have been engineered with exact molecular-level specifications, some intrinsic imperfections such as poor cell permeation and fragility in complex cytoplasmic milieu persist due to the well-established character of nucleic acid molecules. To circumvent these problems, we herein report a molecularly engineered, entropy-driven three-dimensional DNA amplifier (EDTD) that can operate inside living cells in response to a specific mRNA target. In particular, mRNA target/EDTD interaction can specifically initiate an autonomous DNA circuit inside living cells owing to the exclusive entropy-driven force, thus providing enormous signal amplification for ultrasensitive detection of the mRNA. Moreover, owing to molecular engineering of a unique DNA tetrahedral framework into the DNA amplifier, EDTD exhibits significantly enhanced biostability and cellular uptake efficiency, which are prerequisites for DNA machines used for in vivo applications. This programmable DNA machine presents a simple and modular amplification mechanism for the detection of intracellular biomarkers. Moreover, this study provides a potentially valuable molecular tool for understanding the chemistry of cellular systems and offers a design blueprint for further expansion of DNA nanotechnology in living systems.

Photoactivatable probe-based fluorescent imaging has become an efficient and attractive technique for spatiotemporal microscopic studies of biological events. However, almost all previously reported photoactivatable organic probes have been based on hydrosoluble precursors, which have produced water-soluble active fluorophores able to readily diffuse away from the photocleavage site, thereby dramatically reducing spatial resolution. Hydroxyphenylquinazolinone (HPQ), a small organic dye known for its classic luminescence mechanism through excited-state intramolecular proton transfer (ESIPT), shows strong light emission in the solid state, but no emission in solution. In this work, HPQ was employed as a precursor to develop a localizable, photoactivatable two-photon probe (PHPQ) for spatiotemporal bioimaging applications. After photocleavage, PHPQ releases a precipitating HPQ fluorophore which shows both one-photon and two-photon excited yellow-green fluorescence, thereby producing a localizable fluorescence signal that affords high spatial resolution for bioimaging, with more than 200-fold one-photon and 150-fold two-photon fluorescence enhancement.

Accurate detection and imaging of tumor-related mRNA in living cells hold great promise for early cancer detection. However, currently, most probes designed to image intracellular mRNA confront intrinsic interferences arising from complex biological matrices and resulting in inevitable false-positive signals. To circumvent this problem, an intracellular DNA nanoprobe, termed DNA tetrahedron nanotweezer (DTNT), was developed to reliably image tumor-related mRNA in living cells based on the FRET (fluorescence resonance energy transfer) "off" to "on" signal readout mode. DTNT was self-assembled from four single-stranded DNAs. In the absence of target mRNA, the respectively labeled donor and acceptor fluorophores are separated, thus inducing low FRET efficiency. However, in the presence of target mRNA, DTNT alters its structure from the open to closed state, thus bringing the dual fluorophores into close proximity for high FRET efficiency. The DTNT exhibited high cellular permeability, fast response and excellent biocompatibility. Moreover, intracellular imaging experiments showed that DTNT could effectively distinguish cancer cells from normal cells and, moreover, distinguish among changes of mRNA expression levels in living cells. The DTNT nanoprobe also exhibits minimal effect of probe concentration, distribution and laser power as other ratiometric probe. More importantly, as a result of the FRET "off" to "on" signal readout mode, the DTNT nanoprobe almost entirely avoids false-positive signals due to intrinsic interferences, such as nuclease digestion, protein binding and thermodynamic fluctuations in complex biological matrices. This design blueprint can be applied to the development of powerful DNA nanomachines for biomedical research and clinical early diagnosis.

A universal aptameric system based on the taking advantage of double-stranded DNA/perylene diimide (dsDNA/PDI) as the signal probe was developed for multiplexed detection of small molecules. Aptamers are single-stranded DNA or RNA oligonucleotides which are selected in vitro by a process known as systematic evolution of ligands by exponential enrichment. In this work, we synthesized a new kind of PDI and reported this aggregated PDI could quench the double-stranded DNA (dsDNA)-labeled fluorophores with a high quenching efficiency. The quenching efficiencies on the fluorescence of FAM, TAMRA and Cy5 could reach to 98.3%±0.9%, 97.2%±0.6% and 98.1%±1.1%, respectively. This broad-spectrum quencher was then adopted to construct a multicolor biosensor via a label-free approach. A structure-switching-triggered enzymatic recycling amplification was employed for signal amplification. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity towards small analytes. For other targets, changing the corresponding aptamer can achieve the goal. The quencher did not interfere with the catalytic activity of nuclease. The biosensor could be manipulated with similar sensitivity no matter in pre-addition or post-addition manner. Moreover, simultaneous and multiplexed analysis of several small molecules in homogeneous solution was achieved, demonstrating its potential application in the rapid screening of multiple biotargets.

Photodynamic therapy (PDT) has recently emerged as an effective, noninvasive, and economical treatment for diseases including cancers. Traditional PDT suffers mainly from having an insufficient number of photons penetrating the tissue and preferentially targeting cancerous tissues with photosensitizers. Therefore, it is necessary to construct a method for controllable singlet-oxygen (O2) generation (SOG) with high selectivity and accurate localization to provide more efficient PDTwith fewer side effects. Several research groups have developed novel selective PDT agents, such as peptide or protein conjugates, and photosensitizer encapsulated nanocarriers. In recent years, single-stranded oligonucleotides called aptamers have emerged as a novel class of molecules which rival antibodies in both therapeutic and diagnostic applications. Compared to antibodies, aptamers offer significant advantages, such as flexible design, synthetic accessibility, easy modification, chemical stability, and rapid tissue penetration. Therefore, aptamers can potentially endow traditional PDT with high selectivity and accurate localization. The formation of a G-quadruplex (GQ) structure is results from a type of DNA self-assembly mode. This structure can be stabilized by a central monovalent metal cation such as potassium or sodium ions and small-molecule ligands including porphyrins. Cationic porphyrins usually bind to G-quadruplexes through p–p interactions with G-quartets and through electrostatic interactions with the anionic phosphate groups on G-quadruplexes. According to previous studies, some photosensitizers are porphyrin derivatives and are broadly used in PDT, such as 5,10,15,20-tetrakis-(1-methyl-4-pyridyl)-21H, 23H-porphine (TMPyP4). Therefore, the G-quadruplex DNA sequence can be a carrier for photosensitizers with porphyrin molecular structures. By taking advantage of the loading function of the G-quadruplex structure and the recognition function of aptamers, the photosensitizer can be delivered to a target cell with high affinity and selectivity. Up to now, most photosensitizers have been activated by visible light. As a consequence, the shallow penetration depth of incident light has limited their otherwise wide applications. If the photosensitizers are linked to a visible-light generator and the generator is remotely controlled (turned on/off) by near-infrared (NIR) light, then the problem of limited depth penetration by visible light could be easily overcome. To solve the problem of limited depth penetration of current PDT techniques, we used upconversion nanoparticles (UCNPs), in particular, lanthanide-doped rare-earth nanocrystals. These nanomaterials are able to emit shorter-wavelength photons under excitation by NIR light, thus making them good visiblelight generators with the ability to be remotely controlled by NIR light. Furthermore, owing to the ladderlike arrangement of energy levels in lanthanide ions, UCNPs show high efficiency of photon upconversion with a distinct set of sharp emission peaks under moderate excitation densities, thereby enabling targeted bioimaging when functionalized with biomolecular recognition moieties. Herein, we report a specific aptamer-guided G-quadruplex DNA nanoplatform for targeted bioimaging and PDT, and it is capable of selective recognition and imaging of cancer cells, controllable and effective activation of the photosensitizer, and improvement of the therapeutic effect. In particular, a guanine-rich DNA segment is linked to an aptamer to form a bifunctional DNA sequence, termed a G4aptamer. The G4-aptamer not only loads the photosensitizer but also specifically recognizes target cells. As shown in Scheme 1, the G4-aptamer is bioconjugated to a UCNP, thus placing the photosensitizer TMPyP4 at position near the UCNP for energy transfer between the UCNP and TMPyP4. Once the nanoplatform is delivered into cancer cells, the UCNPs are excited by NIR light to emit visible light to image cancer cells and, in turn, to activate TMPyP4, which, finally, generates sufficient ROS to efficiently kill cancer cells. [*] Prof. Q. Yuan, Y. Wu, D. Lu, Dr. Z. Zhao, T. Liu, Prof. X. Zhang, Prof. W. Tan Molecular Science and Biomedicine Laboratory State Key Laboratory for Chemo/Bio-Sensing and Chemometrics College of Chemistry and Chemical Engineering, College of Biology, and Collaborative Research Center for Chemistry and Molecular Medicine Hunan University, Changsha 410082 (China) E-mail: tan@chem.ufl.edu Prof. Q. Yuan, J. Wang Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education) College of Chemistry and Molecular Sciences Wuhan University, Wuhan 430072 (China) [] These authors contributed equally to this work.

Hydrogen sulfide (H

We review advances in the development and the application of optical biosensing systems based on aptamers. Aptamers exhibit advantages as molecular recognition elements for biosensors when compared to traditional antibodies. Among different detection modes that have been employed, optical methods are among the most used, and the combination of aptamers with novel optical nanomaterials has significantly improved the performance of aptamer-based sensors. The review briefly introduces the unique optical properties of nanoscale materials and the urgency of research on aptamer-conjugated optical nanomaterials in bioanalysis. We then discuss current research activities with typical examples of fluorescence, surface-plasmon resonance and quencher nanomaterials for different detection methods (e.g., fluorescence resonance transfer, colorimetry, and surface-enhanced Raman scattering spectroscopy). The conclusion summarizes this exciting realm of study and offers perspectives for future developments.

Herein a photon-manipulated mesoporous release system was constructed based on azobenzene-modified nucleic acids. In this system, the azobenzene-incorporated DNA double strands were immobilized at the pore mouth of mesoporous silica nanoparticles. The photoisomerization of azobenzene induced dehybridization/hybridization switch of complementary DNA, causing uncapping/capping of pore gates of mesoporous silica. This nanoplatform permits holding of guest molecules within the nanopores under visible light but releases them when light wavelength turns to the UV range. These DNA/mesoporous silica hybrid nanostructures were exploited as carriers for the cancer cell chemotherapy drug doxorubicin (DOX) due to its stimuli-responsive property as well as good biocompatibility via MTT assay. It is found that the drug release behavior is light-wavelength-sensitive. Switching of the light from visible to the UV range uncapped the pores, causing the release of DOX from the mesoporous silica nanospheres and an obvious cytotoxic effect on cancer cells. We envision that this photocontrolled drug release system could find potential applications in cancer therapy.

Lysosomes are acidic organelles (approximately pH 4.5–5.5) and tracking the changes in lysosomal pH is of great biological importance. To address this issue, quite a few of fluorescent probes have been developed. However, few of these probes can realize the tracking of dynamic changes in lysosomal pH. Herein, we report a new lysosome-targeted ratiometric fluorescent probe (FR-Lys) by hybridizing morpholine with a xanthane derivative and an o-hydroxy benzoxazole group. In this probe, the morpholine group serves as a targeting unit for lysosome, the xanthane derivative exhibits a pH-modulated open/close reaction of the spirocycle, while the o-hydroxy benzoxazole moiety shows a pH modulated excited-state intramolecular proton transfer (ESIPT) process. Such a design affords the probe a ratiometric fluorescence response towards pH with pH values ranging from 4.0 to 6.3. The response of the probe to pH was fast and reversible with high selectivity. Moreover, this probe possesses further advantages such as easy synthesis, high photostability and low cytotoxicity. These features are favorable for tracking dynamic pH changes in biosystems. It was then applied for dynamic imaging pH changes in lysosomes with satisfactory results.

The water-soluble CP was conjugated with a rhodamine spirolactam for the first time to develop a new FRET-based ratiometric fluorescence sensing platform (CP 1) for intracellular metal-ion probing. CP 1 exhibits excellent water-solubility with two well-resolved emission peaks, which benefit ratiometric intracellular imaging applications.

The use of a nanoscale DNA–Au dendrimer as a signal amplifier was proposed for the universal design of functional DNA-based ultra-sensitive SERS biosensors. This novel design combines the high specificity of functional DNA with the high sensitivity of surface-enhanced Raman scattering (SERS) spectroscopy, resulting in sensitivity superior to that of previously reported sensors.

This work reports the development of a new molecular beacon-based junction sensing system with highly sensitive DNA detection and a strong capability to identify SNPs. The single linear probe typically labels the midsection of the oligonucleotide, but our next-generation junction sensing system uses a hairpin-structured MB with labels on each end of the oligonucleotide to maintain the cleaving activity of our newly designed ssDNA-cleaved endonuclease, Nt.BbvCI, rather than the typical dsDNA-cleaved endonuclease. These design improvements guarantee a true and efficient target-triggered enzymatic recycling amplification process in our sensing system. They also afford a faster and more sensitive response toward target DNA than the first-generation junction sensing system.