Carbazole-based mitochondria-targeted fluorescent probes for in vivo viscosity and cyanide detection in cells and zebrafish.

Cells of most eukaryotic species contain mitochondria, which play a role in physiological processes such as cellular senescence, metabolism, and autophagy. Viscosity is considered a key marker for many illnesses and is involved in several crucial physiological processes. Cyanide (CN-) can target cytochrome-c oxidase, disrupting the mitochondrial electron transport chain and causing cell death through asphyxiation. In this study, a fluorescent probe named HL-1, which targets mitochondria and measures viscosity and CN- levels, was designed and synthesized. HL-1 is viscosity-sensitive, with a linear correlation coefficient of up to 0.992. In addition, HL-1 was found to change color substantially during a nucleophilic addition reaction with CN-, which has a low detection limit of 47 nM. HL-1 not only detects viscosity and exogenous CN- in SKOV-3 cells and zebrafish but also monitors viscosity changes during mitochondrial autophagy in real time. Furthermore, HL-1 has been used successfully to monitor changes in mitochondrial membrane potential during apoptosis. Endogenous CN- in plant samples was quantified. HL-1 provides new ideas for studying viscosity and CN-.

Mechanisms involved in the sequestration and resistance of cadmium for a plant-associated Pseudomonas strain.

Understanding Cd-resistant bacterial cadmium (Cd) resistance systems is crucial for improving microremediation in Cd-contaminated environments. However, these mechanisms are not fully understood in plant-associated bacteria. In the present study, we investigated the mechanisms underlying Cd sequestration and resistance in the strain AN-B15. These results showed that extracellular Cd sequestration by complexation in strain AN-B15 was primarily responsible for the removal of Cd from the solution. Transcriptome analyses have shown that the mechanisms of Cd resistance at the transcriptional level involve collaborative processes involving multiple metabolic pathways. The AN-B15 strain upregulated the expression of genes related to exopolymeric substance synthesis, metal transport, Fe-S cluster biogenesis, iron recruitment, reactive oxygen species oxidative stress defense, and DNA and protein repair to resist Cd-induced stress. Furthermore, inoculation with AN-B15 alleviated Cd-induced toxicity and reduced Cd uptake in the shoots of wheat seedlings, indicating its potential for remediation. Overall, the results improve our understanding of the mechanisms involved in Cd resistance in bacteria and thus have important implications for improving microremediation.

A Small-Molecule Fluorescent Probe for the Detection of Mitochondrial Peroxynitrite.

Reactive oxygen species (ROS) are pivotal signaling molecules that control a variety of physiological functions. As a member of the ROS family, peroxynitrite (ONOO-) possesses strong oxidation and nitrification abilities. Abnormally elevated levels of ONOO- can lead to cellular oxidative stress, which may cause several diseases. In this work, based on the rhodamine fluorophore, we designed and synthesized a novel small-molecule fluorescent probe (DH-1) for ONOO-. Upon reaction with ONOO-, DH-1 exhibited a significant fluorescence signal enhancement (approximately 34-fold). Moreover, DH-1 showed an excellent mitochondria-targeting capability. Confocal fluorescence imaging validated its ability to detect ONOO- changes in HeLa and RAW264.7 cells. Notably, we observed the ONOO- generation during the ferroptosis process by taking advantage of the probe. DH-1 displayed good biocompatibility, facile synthesis, and high selectivity, and may have potential applications in the study of ONOO--associated diseases in biosystems.

An Oxazine-Based Fluorogenic Probe with Changeable π-Conjugation to Eliminate False-Positive Interference of Albumin and Its Application to Sensing Aminopeptidase†N.

Many fluorophores/probes suffer from the interference of albumin in biosystems. Herein, we propose an effective strategy to overcome this interference by virtue of both an albumin-insensitive fluorophore and its changeable π-conjugation, and demonstrate the strategy by designing an oxazine-based fluorogenic probe for aminopeptidase N (APN). The modification on the N atom in the oxazine fluorophore with alanine through a cleavable linker locks the resulting probe in a non-conjugated, colorless and non-fluorescent state, so the non-specific interaction of albumin produces no spectroscopic response. APN can selectively cleave the alanine moiety, restoring the large π-conjugation and strong fluorescence. The capability of the probe to eliminate the albumin influence has been demonstrated by imaging APN in different cell lines, and by quantitatively determining APN in human serum and mouse urine. The present strategy may be useful for developing more specific fluorogenic probes for other enzymes.

Using bioenergy crop cassava (Manihot esculenta) for reclamation of heavily metal-contaminated land.

Heavy metal contamination of agricultural lands may give rise to health risks by cultivation and consumption of food crops from such lands, as well as result in economic loss. Phytoremediation is an eco-friendly and cost-effective approach to restore contaminated soil. However, the restoration process is slow and its sustainability is difficult to maintain. Bioenergy crops may provide alternative economic benefits to agriculture sector and reduce the risks associated with transfering heavy metals into food webs. In this study, a field experiment was carried out to determine the level of reclamation that would be attained in severely heavy metal-contaminated land by planting cassava (Manihot esculenta), a bioenergy crop. The results showed that cassava could grow well on the derelict land, with a fresh tuber yield of 23.13-26.22 t ha-1 in one growing season, which could potentially produce 3680-4160 L ha-1 bioethanol. The economic income of the cassava was estimated to be 11.6-13.1 × 103 CNY ha-1. Among the cassava tissues, metal concentrations were lowest in the tuber. The soil fertility and acidity were ameliorated after cassava plantation, and the mobile and bioavailable metal fractions in the soils were decreased. The cultivation of cassava as a renewable energy crop appears applicable for sustainable utilization and reclamation of heavy metal-contaminated land.

Characterization of an Hg(II)-volatilizing Pseudomonas sp. strain, DC-B1, and its potential for soil remediation when combined with biochar amendment.

Hg contamination is a critical environmental problem, and its remediation using cost-effective and environmentally friendly methods is highly desirable. In this study, a multi-metal-resistant bacterium showing strong Hg(II) volatilization ability, Pseudomonas sp. DC-B1, was isolated from heavy metal-contaminated soils. DC-B1 volatilized 81.1%, 79.2% and 74.3% of the initial Hg2+ from culture solutions with initial Hg2+ concentrations of 5.1, 10.4, and 15.7 mg/L, respectively, within 24 h. Microcosm experiments were performed to investigate the remediation of Hg(II)-spiked soils inoculated with DC-B1 coupled with sawdust biochar amendment. The efficiency of Hg removal from two types of soil samples with different properties and an initial Hg(II) content of approximately 100 mg/kg was enhanced 5.7-13.1% by bio-augmentation with inoculation of the bacterial strain DC-B1, 5.4-10.7% by amendment of 4% (w/w) biochar, and 10.7-23.2% by the combination of DC-B1 and biochar amendments over an incubation period of 24 d over the efficiency in the control treatment under flooded conditions. Longer root lengths were observed in lettuce grown in the treated soils than in lettuce from the control soil, confirming the bioremediation efficacy of the two bioagents for soil Hg contamination.

Arsenic mobility in the arsenic-contaminated Yangzonghai Lake in China.

Coagulation-precipitation techniques have been used successfully in the remediation of arsenic (As) contamination, but insufficient data exist to evaluate the side effects on lake environments (especially secondary pollution). Yangzonghai Lake, a deep lake located in southwest China that was treated with flocculants after an accident resulted in As-contamination, was selected as a case study. We attempted to elucidate how As migrates and transforms in the lake based on water simulation experiments. The results were expected to facilitate evaluation of the suitability and safety of the technology when used in a natural water body. The results showed that the As that had already been precipitated into the sediment by FeCl3 would be released again into the water body due to the increasing activity of anaerobic microorganisms, thereby causing secondary pollution. This phenomenon was especially evident during summer because water temperature stratification reduced the dissolved oxygen (DO) at the sediment surface and led to anaerobic conditions, which enhanced the anaerobic activity at the bottom of the lake. In summer, the concentration of As in the water column increased with increasing depth. In contrast, during winter, the concentration of As was quite similar at all depths of the lake because the water temperature was uniform during this period. As was released from sediments to the aqueous phase in the form of trivalent As [As(III)] upon anaerobic incubation and was oxidized gradually into pentavalent As [As(V)] by the higher DO in the upper layers of the lake water. Contrary to expectation, the disturbance (turnover in the fall) did not increase, but rather decreased the As concentration in the lake, which might result in further coagulation and precipitation through intensive mixture of the unsaturated flocculants from the sediments.

A novel pyrazoline-based selective fluorescent probe for detecting reduced glutathione and its application in living cells and serum.

A new fluorescent probe, N-(4-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-2,4-dinitrobenzenesulfonamide (probe 3), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting glutathione among biological thiols in aqueous media. Probe 3 is a nonfluorescent compound. On being mixed with biothiols under neutral aqueous conditions, the 2,4-dinitrobenzenesulfoyl moiety can be cleaved off by glutathione, and the blue emission of the pyrazoline at 464 nm is switched on, with a fluorescence enhancement of 488-fold for glutathione. Furthermore, probe 3 was highly selective for glutathione without interference from some biologically relevant analytes. The detection limit of glutathione was 4.11 × 10(-7) M. The emission of the probe is pH independent in the physiological pH range. Moreover, the probe can be used for fluorescent imaging of cellular glutathione and can be used for detecting glutathione in calf serum.

Dual-response near-infrared fluorescent probe for detecting cyanide and mitochondrial viscosity and its application in bioimaging.

Viscosity has a significant impact on aerobic respiration in mitochondria. Many foods contain cyanide (CN-) and can cause serious toxicity when consumed in excess. This study discusses the design and synthesis of a dual-response coumarin-based near-infrared fluorescent probe (CCB) for the simultaneous detection of mitochondrial viscosity and CN-. CCB and viscosity have a strong log-linear relationship with a correlation coefficient of 0.997. Additionally, CN- detection can be visualized using a colorimetric method with a detection limit as low as 0.22 µM. Test strips were prepared to facilitate CN- detection in plants. Additional studies have shown the remarkable biocompatibility of CCB, which may be used for real time detection of exogenous CN- and intracellular mitochondrial viscosity and in vivo bioimaging of viscosity in mice. The probe is crucial for understanding disorders connected with mitochondrial viscosity and identifying CN- in daily living.

Combination of changeable π-conjugation and hydrophilic groups for developing water-soluble small-molecule NIR-II fluorogenic probes.

Small-molecule probes emitting in the second near-infrared window (NIR-II) are attracting great attention because of their deep-tissue imaging ability. However, developing NIR-II fluorogenic (off-on) probes with good water solubility remains a great challenge due to the lack of a facile approach. Herein we first report the combination of changeable π-conjugation and hydrophilic groups as an effective strategy for developing water-soluble NIR-II fluorogenic probes. With the strategy, new water-soluble NIR-II fluorophores are prepared, among which NIR-II-F2 and NIR-II-F3 show superior stability and bright fluorescence in aqueous media, and are thus used to design two water-soluble NIR-II fluorogenic probes for leucine aminopeptidase (LAP). The excellent performance in real aqueous bio-environments is demonstrated by imaging mouse vasculatures and organs with NIR-II-F2, and LAP in drug-induced liver injury mice with one of the enzymatic probes; however, water-insoluble dyes cannot achieve such in vivo imaging under the same conditions. Our strategy may be helpful for further developing water-soluble organic NIR-II fluorogenic probes for in vivo imaging of other analytes.

A highly sensitive NIR fluorescence probe for hypoxia imaging in cells and ulcerative colitis.

Ulcerative colitis, a kind of inflammatory bowel disease (IBD), is caused by dysregulated immune response of intestinal bacteria. This chronic disorder can lead to a deficiency of O2 (hypoxia) in the colon microenvironment. Nitroreductase (NTR) is a highly expressed endogenous enzyme under hypoxia, so the detection of NTR can provide diagnostic information about ulcerative colitis. Herein, an ultrasensitive NTR-triggered fluorescence probe (WS-1-NO2) is developed for hypoxia imaging in ulcerative colitis. The probe shows a significant fluorescence enhancement (45-fold) after reacting with NTR, with an extremely low detection limit of 0.096 ng/mL. Furthermore, we apply it for fluorescence imaging of hypoxia in living cells, tumors and dextran sulphate sodium (DSS)-induced ulcerative colitis mouse models. We believe that the probe may be investigated as an effective potential tool for gaining insight into the hypoxia-relevant diseases, such as cancer and ulcerative colitis.

Reduced cadmium toxicity in rapeseed via alteration of root properties and accelerated plant growth by a nitrogen-fixing bacterium.

Cd accumulation in crops has become a global environmental problem because it endangers human health. Screening for microorganisms that can reduce Cd accumulation in crops is a possible measure to address this issue. However, success has been limited, and most previous work did not involve bacteria. In the present study, a strain of N-fixing bacteria (Burkholderia spp.) that exhibits high levels of Cd tolerance was screened. The ability of this bacterium to reduce Cd in rapeseed was then assessed in sterile hydroponic and open soil culture systems. In the hydroponic system, the Burkholderia inoculum promoted Cd fixation in rapeseed roots and thus reduced Cd enrichment in aboveground edible tissues (leaves). The mechanisms were related to increased activity of pectin methylesterase in root cell walls, and the transformation of the chemical form of root Cd from "active" (NaCl-extracted) to "inert" (HCl-extracted and residual Cd) states. Additionally, Burkholderia accelerated plant growth, thus shortening the period in which the plant is available for Cd absorption. In the soil culture system, Burkholderia also reduced Cd enrichment in rapeseed leaves in the presence of other microorganisms. Thus, the bacterial strain shows potential for broad application for reducing the accumulation of Cd in crops.

Visual monitoring of the mitochondrial pH changes during mitophagy with a NIR fluorescent probe and its application in tumor imaging.

Mitophagy, a mitochondria-selective autophagy process, plays critical roles in maintaining intracellular homeostasis by removing the damaged mitochondria and recycling the nutrients in a lysosome-dependent manner. Mitophagy process could result in the changes of mitochondrial pH. So fluorescent probes for detecting mitochondrial pH during mitophagy are highly needed for exploring the functions of mitochondria. Herein, a series of near-infrared pH probes were designed based on the rhodamine framework. The probes showed high sensitivity for pH with the tunable pKa from 4.74 to 6.54. Particularly, for probe 5 (with the pKa of 6.54), a linear relationship between fluorescence intensity and pH in the range of 5.6-7.2 was observed, which was suitable for mitochondrial pH detection. The probe displayed excellent mitochondria-targeting ability. It was applied to monitor pH changes during mitophagy caused by starvation. Besides, in vivo non-invasive visualization of tumor pH variations was achieved via the fluorescence imaging in the near-infrared region. We anticipate that the probe may be a useful tool for revealing essential information about mitophagy-related research and clinical tumor diagnosis.

A sensitive NIR mitochondria-targeting fluorescence probe for visualizing viscosity in living cells and mice.

Mitochondria are the powerhouses in cells, providing the energy needed for cellular activities. However, the abnormalities in the mitochondrial microenvironment (e.g., the increased viscosity) can lead to mitochondrial dysfunctions and diseases. Herein, we develop a series of near-infrared (NIR) fluorescence probes for the detection of viscosity. After screening, probe CQ-4 is selected since it shows a great fluorescence enhancement (89-fold) in the NIR window. Its specific response to viscosity is not influenced by pH, polarity and biological species. Under stimulation with monensin or nystatin, CQ-4 can measure the cellular viscosity changes with good biocompatibility. In addition, we can observe an increase of viscosity during starvation. CQ-4 is applied to distinguish cancer cells from normal cells based on the viscosity differences. Furthermore, the probe has been successfully applied to image viscosity in inflamed and tumor-bearing mice in vivo. Therefore, CQ-4 may contribute to the future study about viscosity in the physiological and pathological processes.

A near-infrared fluorescent probe based on the hemicyanine skeleton for the detection of hydrogen peroxide in vivo.

As a member of the reactive oxygen species, hydrogen peroxide (H2O2) plays critical roles in oxidative stress and cell signaling. Intracellular abnormal levels of H2O2 production are closely related to many diseases. Therefore, the real-time monitoring of H2O2 in the cells is important. In this work, we designed a novel fluorescent probe (Mito-H2O2) for the specific detection of H2O2 based on the hemicyanine skeleton, with bright near-infrared fluorescence emission. Mito-H2O2 displayed fast response, excellent water-solubility and great fluorescence intensity enhancement after the addition of H2O2. Furthermore, Mito-H2O2 has been successfully applied to image both of the exogenous and endogenous H2O2 in cells and mice with negligible cytotoxity.

Activatable near-infrared fluorescent probe triggered by nitroreductase for in vivo ulcerative colitis hypoxia imaging.

Ulcerative colitis is a prevalent inflammatory disease caused by the intestinal bacterial infection. And it is related to the hypoxic degrees in the colon microenvironment. Hypoxia, a condition of imbalance in O2 supply and consumption, is accompanied by the overexpressed level of nitroreductase (NTR). Therefore, the NTR detection has been widely applied for the diagnosis of hypoxia-related diseases. In this study, we developed a novel near-infrared fluorescent probe (IW-1) for NTR. Upon reaction with NTR, IW-1 exhibited a significant fluorescence off-on response at 740 nm with a low detection limit of 0.043 µg/mL. Confocal fluorescence imaging verified its ability to detect the overexpression of NTR in cancer cells. More significantly, IW-1 was applied for in vivo hypoxia imaging in tumors and dextran sulphate sodium (DSS)-induced ulcerative colitis mouse model. We expect that the probe may present a new tool for better understanding the biological functions of NTR as well as revealing essential information about hypoxia-related pathological processes, including cancer and ulcerative colitis.

Benzoindoxazine derivatives containing carbazole for detection of CN- and its application in plant seed extracts and cell imaging.

Cyanide (CN-) is a highly toxic compound that exists in many substances and is harmful to the environment and human health. Therefore, it is of great significance to develop excellent CN- ion probes, especially solvent-induced on-off fluorescent probes. Based on the condensation reaction of indolo[2,1-b][1,3]oxazine molecules with aldehydes, probes (E)-13a-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)-14,14-dimethyl-10-nitro-13a,14-dihydro-8H-benzo[e]benzo[5,6][1,3]oxazino[3,2-a]indole (NCO) and (E)-13a-(2-(9-benzyl-9H-carbazol-3-yl)vinyl)-14,14-dimethyl-10-nitro-13a,14-dihydro-8H-benzo[e]benzo[5,6][1,3]oxazino[3,2-a]indole (NBO) were synthesized to detect CN-. Compared with other cyanogen ion probes, NCO and NBO have special carbazole ring structures and large conjugate systems. When CN- is added to the probe-detection solution, color changes that are visible to the naked eye can occur. The UV-vis spectrum test using differential spectroscopy shows that the probe (i) has excellent solvent-induced switching characteristics and stability (CH3OH-H2O) and (ii) high selectivity, anti-interference ability, and sensitivity for the detection of CN-. The fluorescence limit of detections (LODs) are 1.05 µM for NCO and 1.34 µM for NBO. The UV LODs are 0.83 µM for NCO and 0.87 µM for NBO. Fluorescence spectroscopy shows that the probe has remarkable fluorescence properties. Fluorescence titration experiments, liver cancer cell (Hep G2) imaging, and cytotoxicity experiments all show that the probes have high biocompatibility, low toxicity, high cell permeability, and high sensitivity for the detection of CN- in cells. In addition, NCO and NBO have been successfully used for the detection of cyanogenic glycosides in the seeds of ginkgo, crabapple, apple, and cherry. Test strips were fabricated to detect CN-. After adding CN-, the color of the test strip changed significantly-from brown to light yellow; thus, the test strips have a high application value in the fields of drug quality control, drug safety testing, and pharmacological research.

Rapid and selective visualization of mitochondrial hypochlorite by a red region water-soluble fluorescence probe.

Hypochlorite (-OCl) has long been recognized as an effective microbicidal agent in immune system. Herein, we report the design, preparation and spectral characteristics of a -OCl fluorescent probe (FI-Mito). The probe exhibited remarkable fluorescence turn-on signal in the red region upon -OCl titration with the detection limit as low as 0.9 nM. FI-Mito displayed specific response for -OCl in completely aqueous solution. Meanwhile, the introduction of quaternized pyridine realized mitochondria-targeting ability. FI-Mito was further applied to monitor the generation of endogenous -OCl in the mitochondria of macrophage cells and mice. Therefore, it was established that FI-Mito may serve as a useful molecular tool for -OCl detection in vivo.

Promotion of bioremediation performance in constructed wetland microcosms for acid mine drainage treatment by using organic substrates and supplementing domestic wastewater and plant litter broth.

Gravel-based subsurface-flow constructed wetlands (CWs) amended with a walnut shell (WS) substrate were established to treat synthetic acid mine drainage (AMD) in this study, and artificial domestic wastewater (DW) and plant litter broth (PLB) were supplemented to enhance the performance. The CW media rapidly reached adsorption saturation with respect to metals (except Fe and Cr) without an external carbon source, while the addition of DW and PLB stimulated sulfate reduction activity and achieved efficient biogenic metal removal, primarily by the formation of hydroxide and sulfide precipitates and concomitant co-precipitation. The WS-amended CWs performed notably better than the control systems, not only in sequestering more metals and rapidly establishing favourable environments for biogenic metal abatement but also in supporting better growth of plants and functional microbes. The external organic carbon input greatly shaped the bacterial community compositions in the CWs, with substantial increases in the proportions of core functional populations involved in AMD biotreatment. Cooperation among Cellulomonas, Propioniciclava and sulfate-reducing bacteria (SRB), dominated by Desulfobulbus and Desulfatirhabdium, was the primary biogenic mechanism of AMD remediation in the CWs. Cellulosic waste-amended CWs with DW and PLB addition offer a promising eco-technology for AMD remediation.

A near-infrared rhodamine-based lysosomal pH probe and its application in lysosomal pH rise during heat shock.

Heat shock is a potentially fatal condition characterized by high body temperature (>40 °C), which may lead to physical discomfort and dysfunctions of organ systems. Acidic pH environment in lysosomes can activate enzymes, thus facilitating the degradation of proteins in cellular metabolism. Owing to the lack of a practical research tool, it remains difficult to exploit relationship between heat shock and lysosome. Herein, a NIR lysosomal pH chemosensor (NRLH) was developed. One typical lysosome-locating group, morpholine, was incorporated into NRLH. The fluorescence intensity showed pH-dependent characteristics and responded sensitively to pH fluctuations in the pH range of 3.0-5.5. NRLH with a pKa of 4.24 displayed rapid response and high selectivity for H+ among common species. We also demonstrated NRLH was capable of targeting lysosomes. Importantly, NRLH was applied in cellular imaging and the data revealed that lysosomal pH increased but never decreased during the heat shock. Therefore, NRLH may act as an effective molecular tool for exploring the mechanisms of heat-related pathology in bio-systems.