A new mitochondria-targeted fluorescent probe for exogenous and endogenous superoxide anion imaging in living cells and pneumonia tissue.

As a precursor of all reactive oxygen species (ROS), superoxide anions play an important role in organisms. However, excessive superoxide anions can cause various diseases. Thus, it is highly urgent to develop efficient tools for in situ superoxide anion detection. In this work, a novel boric acid-based, mitochondria-targeted fluorescent probe Mito-YX for superoxide anion detection was designed by regulating its intramolecular charge transfer (ICT) effect. The probe exhibited turn-on fluorescence enhancement within 4 min of reaction with the superoxide anion. In addition, Mito-YX also exhibited high selectivity and a low detection limit down to 0.24 µM with good mitochondrial targeting characteristics, which provided a necessary basis for in vivo detection of superoxide anions. What is more, Mito-YX was successfully applied for the in situ monitoring of superoxide anions in living MCF-7 cells, RAW 264.7 cells and a mouse model of lung inflammation stimulated by LPS. This work provided an important and promising tool for rapid in situ diagnosis and research of the progression of pneumonia.

A novel fast-response and highly selective AIEgen fluorescent probe for visualizing peroxynitrite in living cells, C. elegans and inflammatory mice.

Most of the ONOO- fluorescent probes have restricted applications because of their aggregation-caused quenching (ACQ) effect, long response time and low fluorescence enhancement. Herein, we developed a novel AIEgen fluorescent probe (PE-XY) based on a benzothiazole and quinolin scaffold with high sensitivity and selectivity for imaging of ONOO-. The results indicated that probe PE-XY exhibited fast response towards ONOO- with 2000-fold enhancement of fluorescence intensity ratio in vitro. Moreover, PE-XY exhibited a relatively high sensitivity (limit of detection: 8.58 nM), rapid response (<50 s), high fluorescence quantum yield (δ = 0.81) and excellent selectivity over other analytes towards ONOO-in vitro. Furthermore, PE-XY was successfully applied to detect endogenous ONOO- levels in living HeLa cells, C. elegans and inflammatory mice with low cytotoxicity. Overall, this work provided a novel fast-response and highly selective AIEgen fluorescent probe for real-time monitoring ONOO- fluctuations in living systems.

A rapid cell-permeating turn-on probe for sensitive and selective detection of sulfite in living cells.

A rapid cell-permeating probe NJUXJ-1 was introduced for sensitive and selective detection of sulfite in living cells. It generated a turn-on response to sulfite with high sensitivity (detection limit 13.0 nM) and selectivity (at a physiological level) and low toxicity. The fluorescence of the detecting system was steady for a wide pH range (5-8) and a long period of time (over 12 h). The most attractive point, its rapid cell-permeating ability, made it suitable for bioimaging with a 2 min incubation time and shortened the whole detecting period (cell-permeation and reaction), and thus could decrease background interference. It offered a convenient approach for determining exogenous or endogenous sulfite levels in living cells and further applications.

A Bibliometric Analysis: Current Perspectives and Potential Trends of Enzyme Thermostability from 1991-2022.

Thermostability is considered a crucial parameter to evaluate the viability of enzymes in industrial applications. Over the past 31 years, many studies have been reported on the thermostability of enzymes. However, there is no systematic bibliometric analysis of publications on the thermostability of enzymes. In this study, 16,035 publications related to the thermostability of enzymes were searched and collected, showing an increasing annual trend. China contributed the most publications, while the United States had the highest citation count. International Journal of Biological Macromolecules is the most productive journal in the research field. Moreover, Chinese acad sci and Khosro Khajeh are the most active institutions and prolific authors in the field, respectively. Analysis of references with the strongest citation bursts and keyword co-occurrences, magnetic nanoparticles, metal-organic frameworks, molecular dynamics, and rational design are current hot spots and significant future research directions. This study is the first comprehensive bibliometric analysis summarizing trends and developments in enzyme thermostability research. Our findings could provide scholars with an understanding of the fundamental knowledge framework of the field and identify recent potential hotspots and research trends that could facilitate the discovery of collaboration opportunities.

A mitochondria-targeted fluorescent sensor for imaging endogenous peroxynitrite changes in acute lung injury.

Acute lung injury (ALI) is a serious pulmonary inflammatory disease resulting from excessive reactive oxygen species (ROS) which could cause the damage of the alveolar epithelial cells and capillary endothelial cells. Peroxynitrite, as one of short-lived reactive oxygen species, is closely related to the process of ALI. Thus, it is important to monitor the fluctuation of peroxynitrite in living system for understanding the process of ALI. Herein, the novel mitochondria-targeted fluorescent probe BHMT was designed to respond to peroxynitrite and pH with distinct fluorescence properties respectively. The absorption spectrum of the probe BHMT exhibited a notable red shift as the pH value declined from 8.8 to 2.6. Upon reaction with peroxynitrite, BHMT had a significant increase of fluorescence intensity (63-fold) with maintaining a detection limit of only 43.7 nM. Furthermore, BHMT could detect the levels of endogenous peroxynitrite and image the intracellular pH in ratiometric channels utilizing cell imaging. In addition, BHMT was successfully applied to revealing the relationship between the peroxynitrite and the extent of ALI. Thus, these results indicated the probe BHMT could be a potential tool for diagnosing the early stage of ALI and revealed the peroxynitrite was likely to be a crucial therapeutic target in ALI treatment.

A highly chromogenic selective Rhodamine-chloride-based fluorescence probe activated by cysteine and application in living cells and zebrafish.

Cysteine (Cys), one of the biological thiols, which plays critical roles in biological system regulating the balance of redox homeostasis. In order to monitor the level of Cys in the living cells and organisms, a chromogenic fluorescence probe Rhocl-Cys based on Rhodamine chloride exhibiting the preferable performance of fluorescence turn-on response reacting with Cys was presented. Rhocl-Cys responded rapidly to Cys within 20 min, and had stable fluorescence intensity within pH 6.0-10.0, high selectivity towards Cys and the anti-inference capability with a low detection limit of 0.80 µM. In particular, Rhocl-Cys could qualitatively and quantitatively monitor the level of endogenous and exogenous Cys in living cells and successfully apply to zebrafish detecting Cys. Therefore, these results might further provide the basis exploring the role of Cys in biological system and facilitate as clinical diagnostic molecular tools.

Preparation of optically active ß-hydroxy-α-amino acid by immobilized Escherichia coli cells with serine hydroxymethyl transferase activity.

In this research, an improved method for preparation of optically pure ß-hydroxy-α-amino acids, catalyzed by serine hydroxymethyl transferase with threonine aldolase activity, is reported. Using recombinant serine hydroxymethyl transferase (SHMT), an enzymatic resolution process was established. A series of new substrates, ß-phenylserine, ß-(nitrophenyl) serine and ß-(methylsulfonylphenyl) serine were used in the resolution process catalyzed by immobilized Escherichia coli cells with SHMT activity. It was observed that the K (m) for L: -threonine was 28-fold higher than that for L: -allo-threonine, suggesting that this enzyme can be classified as a low-specificity L: -allo-threonine aldolase. The results also shows that SHMT activity with ß-phenylserine as substrate was about 1.48-fold and 1.25-fold higher than that with ß-(methylsulfonylphenyl) serine and ß-(nitrophenyl) serine as substrate, respectively. Reaction conditions were optimized by using 200 mmol/l ß-hydroxy-α-amino acid, and 0.1 g/ml of immobilized SHMT cells at pH 7.5 and 45°C. Under these conditions, the immobilized cells were continuously used 10 times, yielding an average conversion rate of 60.4%. Bead activity did not change significantly the first five times they were used, and the average conversion rate during the first five instances was 84.1%. The immobilized cells exhibited favourable operational stability.

[Studies on the interaction of gatifloxacin with bovine serum albumin in the presence of carbon nanotubes by fluorescence spectroscopy].

Fluorescence spectroscopy was used to investigate the influences of carbon nanotubes (CNTs) on the fluorescence of bovine serum albumin (BSA), the influences of CNTs on that of gatifloxacin (GFLX), and the influences of GFLX on that of BSA with or without coexisting CNTs under imitated physiological condition. The experimental results demonstrate that both gatifloxacin and carbon nanotubes could quench the intrinsic fluorescence of BSA, and the quenching mechanism is mainly static quenching. The fluorescence quenching action of GFLX on BSA was weakened in the presence of CNTs. The fluorescence quenching data were analyzed according to Stern-Volmer equation and double-reciprocal Lineweaver-Burk equation. It was shown that the quenching constant (K(sv)) and the binding constant (K) are decreased with the concentration of carbon nanotubes increasing. The effects of coexisting CNTs on GFLX-BSA interactions were discussed to offer a reference for the studies on the action mechanism of CNTs or GFLX with albumins in vivo.

Covalently immobilize crude d-amino acid transaminase onto UiO-66-NH2 surface for d-Ala biosynthesis.

Enzyme reaction has been accepted widely in numerous applications owing to the high efficiency and stereo-selectivity, as well as simple preparation by gene engineering. However, the fragility and complex purification process of the enzyme are long-standing problems which limit the large-scale application. One possible solution may be the enzyme immobilization. As one type of porous material with high loading capacity and designable functionality, Metal-Organic Frameworks (MOFs) are ideal choices for the immobilization of enzyme with a considerable interest in recent years. In this study, d-amino acid transaminase (DAT), an important enzyme for industrial synthesis of d-Ala, was covalently immobilized on the surface of a star MOFs material, UiO-66-NH2. Interestingly, we found that the nanoscale hybrid enzyme UiO-66-NH2-Gd-DAT not only maintained the high catalytic efficiency but also got rid of the interference of polluting enzymes, which meant that we could obtain efficient and stereo-selective immobilized enzyme without complex purification process. In general, our findings demonstrated that using UiO-66-NH2 might be a promising strategy to immobilize enzyme and produce effective biocatalyst with high activity and stereo-selectivity.

Glutathione-responsive prodrug conjugates for image-guided combination in cancer therapy.

Theranostic prodrug was highly desirable for precise diagnosis and anti-cancer therapy to decrease side effects. However, it is difficult to conjugate chemo-drug and molecular probe for combined therapy due to the complex pharmacokinetics of different molecules. Here, a novel anticancer theranostic prodrug (BTMP-SS-PTX) had been designed and synthesized by conjugating paclitaxel (PTX) with 2-(benzo[d]thiazol-2-yl)-4-methoxyphenol (BTMP) through a disulphide (-S-S-) linkage, which was redox-sensitive to the high concentration of glutathione in tumors. Upon activation with glutathione in weakly acid media, the BTMP-SS-PTX can be dissociated to release free PTX and visible BTMP, which realized the visual tracking of free drug. The cytotoxicity study demonstrated that soluble prodrug BTMP-SS-PTX displayed more outstanding anticancer activity in HepG2, MCF-7 and HeLa cells, lower toxicity to non-cancer cells (293 T) than free drugs. Furthermore, BTMP-SS-PTX was still able to induce apoptosis of HeLa cells and significantly inhibited tumor growth in HeLa-xenograft mouse model. On the basis of these findings, BTMP-SS-PTX could play a potential role in cancer diagnosis and therapy.

A new fluorescently labeled bisphosphonate for theranostics in tumor bone metastasis.

Bone metastasis of malignant solid tumors has become one of the most serious complications, especially in breast cancer, which was particularly challenging for early detection and treatment in clinical practice. In this work, we reported a new fluorescently labeled bisphosphonate for bone metastasis detection of breast cancer. The designed probes were based on Rhodamine B and bisphosphonate as recognition group, which can specifically target hydroxyapatite (HA) existed in bone tissue. After the osteoclasts were adsorbed on the bone surface, the surrounding microenvironment was acidified, causing the HA to locally dissolve. The probe bound to the HA was then released, and realized the fluorescence turn on under acidic conditions. In vitro experiments showed that G0 was more excellent than G2 owing to shorter connecting arm. Subsequently, we proved that G0 could combine with HA rapidly and exhibit excellent response in solid state. More importantly, we established a model of bone metastasis with MDA-MB-231 cells which was similar to the clinical cases and evaluated the theranostics value of G0 prospectively, which provide the potential application prospect in clinical.

Enzymatic synthesis of theanine from glutamic acid γ-methyl ester and ethylamine by immobilized Escherichia coli cells with γ-glutamyltranspeptidase activity.

Theanine (γ-glutamylethylamide) is the main amino acid component in green tea. The demand for theanine in the food and pharmaceutical industries continues to increase because of its special flavour and multiple physiological effects. In this research, an improved method for enzymatic theanine synthesis is reported. An economical substrate, glutamic acid γ-methyl ester, was used in the synthesis catalyzed by immobilized Escherichia coli cells with γ-glutamyltranspeptidase (GGT) activity. The results show that GGT activity with glutamic acid γ-methyl ester as substrate was about 1.2-folds higher than that with glutamine as substrate. Reaction conditions were optimized by using 300 mmol/l glutamic acid γ-methyl ester, 3,000 mmol/l ethylamine, and 0.1 g/ml of immobilized GGT cells at pH 10 and 50°C. Under these conditions, the immobilized cells were continuously used ten times, yielding an average glutamic acid γ-methyl ester to theanine conversion rate of 69.3%. Bead activity did not change significantly the first six times they were used, and the average conversion rate during the first six instances was 87.2%. The immobilized cells exhibited favourable operational stability.

Synthesis, biological evaluation, and molecular docking studies of 2-chloropyridine derivatives possessing 1,3,4-oxadiazole moiety as potential antitumor agents.

A series of new 2-chloropyridine derivatives possessing 1,3,4-oxadiazole moiety were synthesized. Antiproliferative assay results indicated that compounds 6o and 6u exhibited the most potent activity against gastric cancer cell SGC-7901, which was more potent than the positive control. Especially, compound 6o exhibited significant telomerase inhibitory activity (IC(50)=2.3±0.07µM), which was comparable to the positive control ethidium bromide. Docking simulation was performed to position compound 6o into the active site of telomerase (3DU6) to determine the probable binding model.

Synthesis, biological evaluation and molecular docking studies of amide-coupled benzoic nitrogen mustard derivatives as potential antitumor agents.

A series of amide-coupled benzoic nitrogen mustard derivatives as potential EGFR and HER-2 kinase inhibitors were synthesized and reported for the first time. Some of them exhibited significant EGFR and HER-2 inhibitory activity. Of all the studied compounds, compounds 5b and 5t exhibited the most potent inhibitory activity, which was comparable to the positive control erlotinib. Docking simulation was performed to position compounds 5b and 5t into the EGFR active site to determine the probable binding model. Antiproliferative assay results indicated that some of the benzoic nitrogen mustard derivatives possessed high antiproliferative activity against MCF-7. In particular, compounds 5b and 5t with potent inhibitory activity in tumor growth inhibition may function as potential antitumor agents.

Synthesis of theanine from glutamic acid gamma-methyl ester and ethylamine catalyzed by Escherichia coli having gamma-glutamyltranspeptidase activity.

Glutamic acid gamma-methyl ester (GAME) was used as substrate for theanine synthesis catalyzed by Escherichia coli cells possessing gamma-glutamyltranspeptidase activity. The yield was about 1.2-fold higher than with glutamine as substrate. The reaction was optimal at pH 10 and 45 degrees C, and the optimal substrate ratio of GAME to ethylamine was 1:10 (mol/mol). With GAME at 100 mmol, 95 mmol theanine was obtained after 8 h.

[Study on the mechanism of color changes of Azur A and sodium dodecyl sulphate before micelle formation].

The mechanism of color changes of sodium dodecyl sulphate(SDS) and Azur A (AA) before micelle formation was studied by spectral probe. The changes in the absorption spectra of SDS-AA and chondroitin sulfate(CS)-AA complexes were compared. The influence of ethanol and NaCl on the absorption spectra of SDS-AA complex was investigated. It was found that SDS-AA system showed color changes from blue to amaranth to blue due to the difference in the spatial orientation aggregative degree of AA binding on SDS regular aggregate. The critical concentration of premicelle formation (CPC) was found to be 1.0 x 10(-4) mol x L(-1) by the relation between c(SDS) and deltaA498/deltac(SDS).

A new selective fluorescence probe with a quinoxalinone structure (QP-1) for cysteine and its application in live-cell imaging.

A new selective probe with a quinoxalinone structure, QP-1, has been developed for detection of Cys from biothiols. QP-1 features superb selectivity to Cys and a wide pH range. QP-1 has selectivity to Cys over Hcy, GSH, other amino acids and ions. HRMS spectra confirmed that the detection process was a conjugate addition-addition-elimination reaction. Moreover, QP-1 has been successfully applied in the imaging of Cys in living cells. Finally, QP-1 has been used to detect Cys in rat urine samples.

[Fluorescence characteristic of terbium-ciprofloxacin complex and its application].

On binding to deoxyribonucleic acid, the complex of terbium-ciprofloxacin (Tb(3+)-CIP) increases its fluorescence quantum efficiency. Based on this, an easy, rapid and sensitive method for the determination of DNA was developed. Like ethidium bromide (EB), the complex can be intercalated into DNA bases, but it is non-poisonous. Determination can be made at pH 7.0, where the native structure of DNA is not disrupted. The maximum emission is at 545 nm with excitation at 325 nm. This method has good sensitivity (2.8 x 10(-9) mol x L(-1) of ctDNA), high selectivity and a wide linear range (4.3 x 10(-7)-3. 0 x 10(-5) mol x L(-1) of ctDNA). This complex was also employed for the clinical examination of DNA in whole blood of tumor patients. Ten tumor patients and 10 normal persons were enrolled in this study. It was showed that significant difference existed between control group and tumor group (p < 0.05).

[Study on the interaction of lomefloxacin terbium complex and bovine serum albumin by fluorescence spectra].

Using lomefloxacin-terbium as a fluorescent probe, the interaction of LFLX-Tb3+ and bovine serum albumin (BSA) was investigated by fluorescence spectra. The interaction of LFLX and BSA is quite strong. LFLX can affect the conformation of BSA to some degree. Meanwhile, the electrostatic binding of the BSA with Tb3+ in LFLX-Tb3+ complex could decease the nonradiative energy loss through O-H vibration of H2O molecule in Tb3+ complex. Under the optimum conditions, BSA can enhance the fluorescence intensity of the lomefloxacin-terbium complex at 545 nm, and the enhanced fluorescence intensity of Tb3+ is proportional to the concentration of BSA. Based on that, a new method is constructed for the determination of BSA. The linear ranges for BSA are 16.5-148.5 microg x mL(-1) with a detection limit of 68.8 ng x mL(-1) and relative standard deviations of 1.4%. This method is simple, practical and relatively free of interference from coexistent substances.

[Spatial orientation interaction mechanism of three component complex of protein--BSA/SDS/Azur a system].

The interaction of bovine serum albumin (BSA), sodium dodecyl sulphate (SDS) and Azur A(AA) was studied by spectral probe. The influence of the molar ratio of SDS/BSA on the absorption spectra of BSA-SDS-AA complex was investigated. It was found that the new absorption peak and color changes generated by BSA-SDS-AA complex arose from the difference in the spatial orientation aggregative degree of AA binding on BSA-SDS-AA complex.