High-Fidelity Spatiotemporal Recognition of Golgi ALP through an Initial-Accumulation and Postactivation Strategy.

Various signal molecules mediate complex physiological processes collectively in the Golgi. However, most currently accessible probes are questionable in illuminating the functions of these reactive species in Golgi because of the inability to irradiate these probes only at the desired Golgi location, which compromises specificity and accuracy. In this study, we rationally designed the first photocontrollable and Golgi-targeted fluorescent probe to in situ visualize the Golgi alkaline phosphatase (ALP). The designed probe with natural yellow fluorescence can provide access into Golgi and monitor the exact timing of accumulation in Golgi. On-demand photoactivation at only the desired Golgi location affords a significant emission response to ALP with illuminating red fluorescence at 710 nm. Through the photocontrollable fluorescence responsiveness to ALP, precise spatiotemporal recognition of Golgi ALP fluctuations is successfully performed. With this probe, for the first time, we revealed the Golgi ALP levels during cisplatin-induced acute kidney injury (AKI), which will further facilitate and complement the comprehensive exploration of ALP kinetics during physiological and pathological processes.

Depletion and Downregulation of Hydrogen Sulfide Using an Activatable Probe for Promoting Photothermal Therapy toward Colorectal Cancers.

Since hydrogen sulfide (H2S) is an important endogenous gaseous mediator, therapeutic manipulation of H2S is promising for anticancer treatment. In this work, we develop a novel theranostic nanoplatform with H2S-specific and photocontrolled synergistic activation for imaging-guided H2S depletion and downregulation along with promoted photothermal therapy. Such a nanoplatform is fabricated by integration of a H2S-responsive molecule probe that can generate a cystathionine-ß-synthase (CBS) inhibitor AOAA and a photothermal transducer into an NIR-light-responsive container. Our nanoplatform can turn on NIR fluorescence specifically in H2S-rich cancers, guiding further laser irradiation. Furthermore, prominent conversion of photoenergy into heat guarantees special container melting with controllable AOAA release for H2S-level downregulation. This smart regulation of the endogenous H2S level amplifies the PTT therapeutic effect, successfully suppressing colorectal tumor in living mice under NIR fluorescence imaging guidance. Thus, we believe that this nanoplatform may provide a powerful tool toward H2S-concerned cancer treatment with an optimized diagnostic and therapeutic effect.

RSS and ROS Sequentially Activated Carbon Monoxide Release for Boosting NIR Imaging-Guided On-Demand Photodynamic Therapy.

Carbon monoxide shows great therapeutic potential in anti-cancer. In particular, the construction of multifunctional CO delivery systems can promote the precise delivery of CO and achieve ideal therapeutic effects, but there are still great challenges in design. In this work, a RSS and ROS sequentially activated CO delivery system is developed for boosting NIR imaging-guided on-demand photodynamic therapy. This designed system is composed of a CO releaser (BOD-CO) and a photosensitizer (BOD-I). BOD-CO can be specifically activated by hydrogen sulfide with simultaneous release of CO donor and NIR fluorescence that can identify H2S-rich tumors and guide light therapy, also depleting H2S in the process. Moreover, BOD-I generates 1O2 under long-wavelength light irradiation, enabling both PDT and precise local release of CO via a photooxidation mechanism. Such sequential activation of CO release by RSS and ROS ensured the safety and controllability of CO delivery, and effectively avoided leakage during delivery. Importantly, cytotoxicity and in vivo studies reveal that the release of CO combined with the depletion of endogenous H2S amplified PDT, achieving ideal anticancer results. It is believed that such theranostic nanoplatform can provide a novel strategy for the precise CO delivery and combined therapy involved in gas therapy and PDT.

Specific imaging of intracellular hydrogen sulfide by a positively charged NIR fluorescent probe.

The poor water solubility of traditional activatable organic molecular probes usually limits their detection ability in physiological environment. In this work, a positively charged H2S probe was designed, which exhibited a significantly enhanced responsiveness to H2S in the aggregated state due to the increased positive charge density on the aggregate surface. Under physiological conditions, the probe could be activated by H2S with specificity and sensitivity to release near-infrared fluorescence signal. Moreover, endogenous H2S levels in living cells were successfully monitored by using this probe. We expect that this probe can provide a new strategy for the design of activatable probes to break the limitation of poor water solubility of conventional organic molecular probes.

Molecular Engineering of Luminogens for High-Integrity Imaging of Hydrogen Polysulfides via Activatable Aggregation-Induced Dual-Color Fluorescence.

Understanding biological events associated with H2Sn rather than mediated by H2S is of great significance but remains to be solved due to a lack of high-integrity imaging tools. In this study, we report a chemoselective probe for H2Sn over H2S through the molecular engineering of luminogens. Based on our search for H2Sn-activatable probes with high selectivity, we fabricate water-soluble and biocompatible nanoprobes. Such a designed nanoprobe shows rare aggregation-induced dual-color fluorescence responses to H2Sn, lighting up bright emissions at 588 and 750 nm, respectively. By use of this activatable dual-color fluorescence, high-integrity identification of intracellular H2Sn was successfully realized. Thus, our approach to H2Sn-activated multicolor fluorescent probes could provide valuable insight into interrogating H2Sn-mediated biological events.

Precise Spatiotemporal Identification of Mitochondrial H2S Fluctuations through Exploiting an On-Demand Photoactivated Probe.

Various signal molecules participate in complex biological processes in mitochondria. However, most currently available probes have problems in elucidating the functions of these active species in mitochondria due to the inability to light up these probes exclusively at the desired mitochondrial location, thereby compromising the specificity and accuracy. In this study, we present an on-demand photoactivation approach to the molecular design of optimized probes for precise spatiotemporal identification of mitochondrial H2S fluctuations. The designed probe with native yellow fluorescence can monitor the process into mitochondria but maintains nonfluorescent response to H2S during cellular delivery, providing the accurate timing of accumulation in mitochondria. On-demand photoactivation exclusively at the desired mitochondrial location affords a significant aggregation-enhanced and emissive response to H2S with lighting up red fluorescence at 690 nm, which is the only way to get such an emissive phenomenon and greatly improves the specificity and accuracy of targeting mitochondrial H2S. By using this photocontrolled fluorescence responsiveness to H2S, precise spatiotemporal identification of mitochondrial H2S fluctuations is successfully performed. Our work could facilitate advances toward interrogating the physiological and pathological consequences of mitochondrial H2S in various biological events.

An activatable endoplasmic reticulum-targeted probe for NIR imaging-guided photothermal therapy.

An H2O2-activated, endoplasmic reticulum-targeted theranostic probe was developed. This designed probe could be activated by H2O2, resulting in increased NIR fluorescence and photothermal signals, thus achieving specific recognition of H2O2 and further photothermal therapy in the endoplasmic reticulum of H2O2-overexpressing cancer cells.

High-efficiency adsorption removal of CR and MG dyes using AlOOH fibers embedded with porous CoFe2O4 nanoparticles.

Owing to the toxicity and difficulty in degradation, how to the effective separation for the residual dyes in the aqueous solution is still an issue with great challenge in the area of environmental protection. Now, to high-efficiency removal of organic dyes from the aqueous solution, we design a unique AlOOH/CoFe2O4 adsorbent with porous CoFe2O4 nanoparticles embedded on the AlOOH fibers using a simple hydrothermal technique and calcination process. The structural properties and surface characteristics of the AlOOH/CoFe2O4 composites are detailedly analyzed by XRD, FTIR, XPS, TEM and SEM. Here, the high SBET and specific porous structure are beneficial to improve the adsorption performance of AlOOH/CoFe2O4 adsorbents. Especially, when the molar ratio of AlOOH to CoFe2O4 in the AlOOH/CoFe2O4 fibers is 1:1, an optimal performance on adsorbing anionic Congo red (CR) and cationic methyl green (MG) dyes can be obtained at pH = 6.29, where the corresponding maximum adsorption capacities reach up to 565.0 and 423.7 mg g-1, respectively. Factors leading to the change in the ability of adsorbing CR and MG dyes are systematically discussed, including contact time, temperature, initial concentrations, and pH values of the solutions. Meanwhile, the uptake of CR and MG dyes can best conform to Langmuir isotherm model and pseudo-second-order adsorption kinetics. The thermodynamic analysis verifies that the dye adsorption process is spontaneous and endothermic. Moreover, from the point view of practical application, the good reusability further makes the as-synthesized magnetic AlOOH/CoFe2O4 composite be a perfect adsorbent with efficiently removing both anionic and cationic dyes from aqueous solutions.

Activatable photothermal agents with target-initiated large spectral separation for highly effective reduction of side effects.

Photothermal agents (PTAs) with minimized side effects are critical for transforming cancer photothermal therapy (PTT) into clinical applications. However, most currently available PTAs lack true selective activation to reduce side effects because of heavy spectral overlap between photothermal agents and their corresponding products. This study reports the construction of activatable PTAs with target-initiated large spectral separation for highly effective reduction of side effects. Such designed probes involve two H2O2-activatable PTAs, aza-BOD-B1 (single activatable site) and aza-BOD-B2 (multiple activatable site). After interacting with H2O2, aza-BOD-B1 only displays a mild absorption redshift (60 nm) from 750 nm to 810 nm with serious spectral overlap, resulting in a mild photothermal effect on normal tissues upon 808 nm light irradiation. In contrast, aza-BOD-B2 displays a large absorption spectral separation (150 nm) from 660 nm to 810 nm, achieving true selective activation to minimize side effects during PTT of cancer. Besides, in vitro and in vivo investigations demonstrated that aza-BOD-B2 can specifically induce photothermal ablation of cancer cells and tumors while leaving normal sites undamaged, whereas aza-BOD-B1 exhibits undesirable side effects on normal cells. Our study provides a practical solution to the problem of undesired side effects of phototherapy, an advance in precision medicine.

The role of biochar in the photocatalytic treatment of a mixture of Cr(VI) and phenol pollutants: Biochar as a carrier for transferring and storing electrons.

Biochar (BC) is widely used to remove environmental pollutants due to its photocatalytic activity. However, the mechanism of BC in photocatalysis remains unclear. In this study, soybean straw biochar (D500), dewatered sludge biochar (S500) and TiO2/BC composite catalysts were prepared to test their photocatalytic activity in the photocatalysis-dark reaction using phenol and Cr(VI) as the representative pollutants. D500 had a good graphitized structure, layered structure and more active sites, which led to good photocatalytic activity. Compared with D500, S500 did not have a similar structure, resulting in a lack of photocatalytic activity. In addition, the efficiency of Cr(VI) and phenol removal using D500/TiO2 as a catalyst was higher than that obtained using D500 and TiO2, respectively. TiO2 coupled with D500 increased the generation of photoexcited electrons and reduced the recombination of e--h+ pairs. The removal efficiency of TiO2/D500 for Cr(VI) (80.4 %) and phenol (77.7 %) in the hybrid systems was higher than that of Cr(VI) and phenol in unitary systems. This difference was mainly attributed to the inhibition of e--h+ pair recombination by phenol and Cr(VI), which function as electron quenchers and hole quenchers, respectively. Furthermore, D500 stored electrons under light and released these electrons under dark conditions. When D500 was combined with TiO2, the electrons on the biochar activated the catalytic redox activity of TiO2, thereby removing pollutants under dark conditions. Meanwhile, TiO2/D500 also exhibited good reusability and stability. In summary, this study provides new insight into the role of biochar in photocatalysis.

A water-soluble fluorescent probe for real-time visualization of γ-glutamyl transpeptidase activity in living cells.

γ-glutamyl transpeptidase (GGT) is a kind of cell-surface enzyme that is overexpressed in many cancer cells. It is of great significance to develop an ideal tool for the diagnosis of GGT-rich cancer cells. Here, we reported a simple-structured but effective imaging probe for the detection of GGT activity. In the presence of GGT, the γ-glutamyl linkage could be cleaved specifically to produce amino-substituted product, resulting in significant fluorescence enhancement at 578 nm. Moreover, we successfully employed the probe to monitor GGT activity in HepG2 cells. We envisaged that such a simple but effective imaging tool could improve the practical applications for bioimaging.

Real-Time Monitoring Renal Impairment Due to Drug-Induced AKI and Diabetes-Caused CKD Using an NAG-Activatable NIR-II Nanoprobe.

Real-time in vivo optical imaging of kidney function is important for the diagnosis of renal diseases, such as acute kidney injury (AKI) and chronic kidney disease (CKD), with high morbidity and mortality worldwide. However, the reported optical imaging agents still have limitations for identifying AKI or CKD in the early stage due to their low sensitivity, poor tissue penetration, and significant background interference. Herein, an N-acetyl-ß-d-glucosaminidase (NAG)-activatable second near-infrared (NIR-II) fluorescent nanoprobe (BOD-II-NAG-NP) is developed for monitoring the progression of drug-induced AKI and in vivo imaging of diabetes-caused CKD. NAG, as a biomarker of renal diseases, is able to specifically activate BOD-II-NAG-NP to release NIR-II fluorescence signals, enabling in vivo imaging of kidney dysfunctions in living mice. Importantly, such an active imaging mechanism allows BOD-II-NAG-NP to noninvasively detect the onset of drug-induced AKI at least 32 h earlier than the most existing assays, which indicates that BOD-II-NAG-NP has the potential to be an optical imaging agent for the early diagnosis of AKI. Moreover, NIR-II fluorescence produced by BOD-II-NAG-NP could deeply penetrate into the relatively thick layers of fat in diabetic nephropathy mice and provide in vivo imaging with high resolution, indicating that BOD-II-NAG-NP has clinical potential for precision diagnosis of CKD.

Probing the Intracellular Dynamics of Nitric Oxide and Hydrogen Sulfide Using an Activatable NIR II Fluorescence Reporter.

Understanding the complex interplay among gasotransmitters is of great significance but remains technically challenging. In this study, we present the design and synthesis of a dually responsive BOD-NH-SC reporter for probing the dynamic and alternating existence of NO and H2 S in living cells. This designed reporter can repeatedly cycle S-nitrosation and transnitrosation reactions when successively treated with NO and H2 S, thus affording the interchange of NIR fluorescence at 645 nm (NO) and NIR II fluorescence at 936 nm (H2 S). In light of this unique fluorescence alternation between two colors, we synthesized water-soluble BOD-NH-SC dots to visualize the intracellular dynamics of NO and H2 S. These molecular probes thus provide a toolbox to elucidate the interplaying roles of NO and H2 S in the complex interaction networks of various signal transduction pathways.

An electron-deficiency-based framework for NIR-II fluorescence probes.

Fluorescent probes in the NIR-II region provide high bioimaging quality. Optimizing the probe structure to achieve NIR-II imaging is ongoing, but remains challenging. Herein, increasing the electron withdrawing ability of the substituent in monochlorinated BODIPY greatly adjusted the emission wavelength from the NIR-I to NIR-II region, giving an efficient design strategy of NIR-II probes.

Aggregation Enhanced Responsiveness of Rationally Designed Probes to Hydrogen Sulfide for Targeted Cancer Imaging.

Activatable molecular probes hold great promise for targeted cancer imaging. However, the hydrophobic nature of most conventional probes makes them generate precipitated agglomerate in aqueous media, thereby annihilating their responsiveness to analytes and precluding their practical applications for bioimaging. This study reports the development of two small molecular probes with unprecedented aggregation enhanced responsiveness to H2S for in vivo imaging of H2S-rich cancers. The subtle modulation of the equilibrium between hydrophilicity and lipophilicity by N-methylpyridinium endows these designed probes with the capability of spontaneously self-assembling into nanoprobes under physiological conditions. Such probes in an aggregated state, rather than a molecular dissolved state, show NIR fluorescence light up and photoacoustic signals turn on upon H2S specific activation, allowing in vivo visualization and differentiation of cancers based on differences in H2S content. Thus, our study presents an effective design strategy which should pave the way to molecular design of optimized probes for precision cancer diagnostics.

Rational design of water-dispersible and biocompatible nanoprobes with H2S-triggered NIR emission for cancer cell imaging.

We present an approach for constructing a H2S-specific nanoprobe by the entrapment of a small molecule probe within the hydrophobic interior of surface cross-linked micelles (SCMs), endowing the designed nanoprobes with good water solubility and biocompatibility. Importantly, the obtained nanoprobes displayed good responsiveness to H2S in both ratiometric fluorescence and light-up NIR emission modes, thus enabling accurate identification of H2S-rich colorectal cancer cells.

Precise imaging of mitochondria in cancer cells by real-time monitoring of nitroreductase activity with a targetable and activatable fluorescent probe.

An activatable and mitochondrial-targetable fluorescent probe was developed. This designed probe showed ratiometric fluorescence and light-up near-infrared emission responsiveness to nitroreductase, achieving precise imaging of mitochondria in cancer cells by real-time monitoring of nitroreductase activity.

Homeobox transcription factor OsZHD2 promotes root meristem activity in rice by inducing ethylene biosynthesis.

Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2, which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin.

Positional effects on efficiency of CRISPR/Cas9-based transcriptional activation in rice plants.

The nuclease-dead Cas9 (dCas9) has been reprogrammed for transcriptional activation by fusing dCas9 to a transcriptional activation domain. In the presence of a guide RNA (gRNA), the dCas9 fusions specifically bind to regions of a promoter to activate transcription. Significant amount of effort has been directed toward the identification and optimization of the fusions of dCas9-activation domain, but very little is known about the impact of gRNA target positions within a promoter in plants on transcriptional activation efficiency. The dCas9-6TAL-VP128 system (dCas9-TV) has been optimized to activate transcription in plants. Here we use the dCas9-TV to activate transcription of OsWOX11 and OsYUC1, two genes that cause dramatic developmental phenotypes when overexpressed. We designed a series of gRNAs targeting the promoters of the two genes. We show that gRNAs that target regions within 350 bp upstream of the transcription start site were most effective in transcriptional activation. Moreover, we show that using two gRNAs that simultaneously target two discrete sites in a promoter can further enhance transcription. This work provides guidelines for designed transcriptional activation through CRISPR/dCas9 systems.

Repurposing of Anthocyanin Biosynthesis for Plant Transformation and Genome Editing.

CRISPR/Cas9 gene editing technology has been very effective in editing genes in many plant species including rice. Here we further improve the current CRISPR/Cas9 gene editing technology in both efficiency and time needed for isolation of transgene-free and target gene-edited plants. We coupled the CRISPR/Cas9 cassette with a unit that activates anthocyanin biosynthesis, providing a visible marker for detecting the presence of transgenes. The anthocyanin-marker assisted CRISPR (AAC) technology enables us to identify transgenic events even at calli stage, to select transformants with elevated Cas9 expression, and to identify transgene-free plants in the field. We used the AAC technology to edit LAZY1 and G1 and successfully generated many transgene-free and target gene-edited plants at T1 generation. The AAC technology greatly reduced the labor, time, and costs needed for editing target genes in rice.